J Biol Chem, 1994 Nov 25, 269(47), 29468 - 73 The 65-kDa protein derived from the internal translational initiation site of the clpA gene inhibits the ATP-dependent protease Ti in Escherichia coli; Seol JH et al.; The clpA gene that encodes the ATPase subunit of the ATP-dependent protease Ti (Clp) in Escherichia coli contains a putative internal translational initiation site . Here we show that mutagenesis of its 5'-end AUG codon resulted in an exclusive synthesis of the 65-kDa protein (ClpA65), while mutation at the internal 169th AUG codon (Met) to ACG (Thr) produced only the 84-kDa protein (ClpA84T) . On the other hand, the cells carrying the wild-type clpA gene produced both the 84- and 65-kDa proteins (ClpA84/65) . While the purified ClpA84T and ClpA84/65 hydrolyzed ATP nearly as well as the 84-kDa ClpA alone (ClpA84), ClpA65 cleaved ATP at a rate less than 5% of that by ClpA84 . Unlike ClpA84 and ClpA84T, ClpA65 could not support the casein-degrading activity of ClpP . Furthermore, ClpA65 inhibited the proteolysis by the mixture of ClpP with ClpA84 or ClpA84T but not that with ClpA84/65, which could support the proteolytic activity of ClpP only about 40% as well as ClpA84 . Nevertheless, ClpA65 showed little or no effect on the basal or protein-activated ATPase activity of ClpA84, ClpA84T, or ClpA84/65 alone or in the presence of ClpP . These results suggest that ClpA65 may interfere the interaction of ClpA84 or ClpA84T with ClpP and, hence, impair their assembly into an active form of the ATP-dependent protease Ti. J Biol Chem, 1994 Nov 25, 269(47), 29423 - 9 Cooperative binding of the feedback modifiers isoleucine and valine to biosynthetic threonine deaminase from Escherichia coli; Eisenstein E et al.; Control of the regulatory enzyme threonine deaminase from Escherichia coli is achieved by isoleucine inhibition and valine activation . The mechanism by which these heterotropic effectors regulate the enzyme was investigated by measuring the binding of isoleucine and valine by spectroscopic, kinetic, calorimetric and equilibrium dialysis techniques . The addition of isoleucine or valine to threonine deaminase resulted in large changes in the intrinsic fluorescence of the two tryptophans per polypeptide chain . Slightly cooperative binding isotherms for isoleucine were obtained in potassium phosphate, pH 7.5, yielding an average dissociation constant of 4.91 microM, which was confirmed by equilibrium dialysis measurements . Valine binding was much more cooperative, and yielded an average dissociation constant of 122 microM . Titration calorimetry experiments indicated that cooperative heterotropic ligand binding was exothermic, and yielded a stoichiometry of four isoleucine bound per tetrameric enzyme, with an average enthalpy of -10.70 kcal/mol . Valine also bound to four sites per tetramer, with an average enthalpy of -7.45 kcal/mol . The effect of ligands on the fluorescence and circular dichroism spectra of the essential pyridoxal phosphate cofactor indicates that isoleucine and valine bind to effector sites that are distinct from the active sites in threonine deaminase . Shifts in the kinetic properties of threonine deaminase promoted by isoleucine and valine binding are to a first approximation consistent with analyses of effector binding isotherms in terms of a simple two-state model, and suggest that isoleucine regulates threonine deaminase by preferentially binding to the low activity T state, whereas valine binds preferentially to the high activity R state . Finally, analyses of heterotropic effector binding isotherms suggest that active site ligands may have significant affinity for the regulatory sites, which gives rise to underestimates for the allosteric equilibrium constants determined from substrate analog binding isotherms. J Biol Chem, 1994 Nov 25, 269(47), 29416 - 22 Energetics of cooperative ligand binding to the active sites of biosynthetic threonine deaminase from Escherichia coli; Eisenstein E; The sigmoidal kinetics of alpha-ketobutyrate production catalyzed by threonine deaminase are shifted in the presence of the feedback inhibitor isoleucine and the activator valine to control carbon flow through branched-chain amino acid biosynthesis in Escherichia coli . As an initial effort toward developing a molecular mechanism for cooperativity and feedback inhibition in this enzyme, the binding of the substrate analogs 2-aminobutyrate and alanine were measured . Binding isotherms were determined in potassium phosphate at pH 7.5 by detection of an 8-10-fold increase in intrinsic fluorescence of the external aldimine Schiff base of these analogs with the essential pyridoxal phosphate cofactor of the enzyme . Both 2-aminobutyrate and alanine bind cooperatively to four sites on threonine deaminase, with an average dissociation constant of 12.7 and 43.8 mM, respectively . The feedback inhibitor isoleucine decreases the average affinity for the ligands and increases the degree of cooperativity . The activator valine decreases the degree of cooperativity, but gives rise to a slight increase in the average dissociation constant for 2-aminobutyrate and alanine, possibly due to a competitive effect with active site ligands . The temperature dependence of the average affinity of ligands for the active sites indicates that binding is entropically controlled, with average values for delta H0 of +6.0 kcal/mol for alanine and +4.87 kcal/mol for 2-aminobutyrate . Since none of the ligands under investigation had any effect on the tetrameric quaternary structure of the enzyme as judged by sedimentation equilibrium, an initial attempt to describe cooperative ligand association with threonine deaminase was undertaken by analysis of the binding isotherms in terms of a two-state model in which the enzyme equilibrates between two conformations, T and R, that differ in their affinity for ligands . This analysis suggests that cooperative binding to the enzyme is largely the result of the high preferential affinity of ligands for the R state . In addition, estimates for the allosteric equilibrium constant L range from 2 to 5, in the absence of modifiers, to 35 in the presence of isoleucine, and 0.9 in the presence of valine . To a first approximation, the data are consistent with isoleucine and valine regulating threonine deaminase by shifting the allosteric equilibrium between the different affinity forms. J Biol Chem, 1994 Nov 25, 269(47), 29405 - 8 Superoxide and peroxynitrite inactivate aconitases, but nitric oxide does not; Hausladen A et al.; The Escherichia coli and recombinant human cytosolic aconitases are inactivated by O2-., with a rate constant of approximately 3 x 10(7) M-1 s-1; the corresponding value for the porcine mitochondrial aconitase is approximately 0.8 x 10(7) M-1 s-1 . Nitric oxide, which is reported to inactivate aconitase, did not do so at a perceptible rate, while incubation with peroxynitrite led to a rapid loss of aconitase activity . We propose that the reported inactivation of aconitase by nitric oxide in vivo is actually mediated through peroxynitrite, the product of the reaction between O2- . and NO.. J Biol Chem, 1994 Nov 25, 269(47), 29355 - 8 The murine interleukin 8 type B receptor homologue and its ligands . Expression and biological characterization; Bozic CR et al.; KC, the product of an immediate early gene induced in mouse fibroblasts by platelet-derived growth factor, was synthesized as a recombinant protein in Escherichia coli and binds with 0.8 nM affinity to mouse neutrophils . Human neutrophils also bind recombinant KC at a site competitive with human interleukin (IL8) and Gro-alpha/MGSA, consistent with binding at the IL8 type B receptor (IL8RB) . The cDNA corresponding to human IL8RB hybridizes strongly with two restriction fragments in murine genomic DNA, representing candidate receptor genes for KC . Molecular cloning of both mouse genomic DNA and neutrophil exudate cell cDNA libraries yielded a receptor with approximately 68% sequence identity to both the human IL8 type A and B receptors . Transient expression of the murine receptor cDNA in COS cells conferred binding ability to KC and a related gene product, macrophage inflammatory protein-2 (MIP-2) with high affinity (approximately 5 nM) . Human IL8 was a poor agonist for this expressed receptor (Kd = approximately 400 nM) . The potent activity of human IL8 on mouse polymorphonuclear neutrophils is not consistent with binding on the cloned receptor and suggests that murine homologues of IL8 and an IL8 type A receptor remain to be identified . Our data indicate that KC is the murine homologue of human Gro-alpha, and the KC receptor is an IL8 type B receptor homologue capable of binding both KC and macrophage inflammatory protein-2 with high affinity. Nucleic Acids Res, 1994 Nov 25, 22(23), 5054 - 9 Molecular sequestration stabilizes CAP-DNA complexes during polyacrylamide gel electrophoresis; Fried MG et al.; The gel electrophoresis mobility shift assay is widely used for qualitative and quantitative characterization of protein complexes with nucleic acids . Often it is found that complexes that are short-lived in free solution (t1/2 of the order of minutes) persist for hours under the conditions of gel electrophoresis . We have investigated the influence of polyacrylamide gels on the pseudo first-order dissociation kinetics of complexes containing the E.coli cyclic AMP receptor protein (CAP) and lactose promoter DNA . Within the gel matrix, kdiss decreased with increasing {polyacrylamide} and the order of the reaction was changed . In free solution, kdiss was proportional to {DNA}2, while in 5% gels, kdiss was proportional to {DNA}0.3 . In gels of {polyacrylamide} > or = 10%, kdiss was nearly independent of {DNA} until fragment concentrations exceeded 0.1 microM . Even in the absence of competing DNA, kdiss(gel) < kdiss(solution) . These results suggest that the lifetime of CAP-DNA complexes in free solution is limited by their encounter frequency with molecules of DNA or with protein-DNA complexes; some or all of the stabilization observed in gels may be due to a reduction in this frequency. Nucleic Acids Res, 1994 Nov 25, 22(23), 5024 - 30 CD and DNA binding studies of a proline repeat-containing segment of the replication arrest protein Tus; Nedved ML et al.; Tus is a sequence-specific DNA binding protein that regulates its own transcription and can arrest Escherichia coli replication when bound to Ter sites on the chromosome . In order to identify segments of Tus that may be involved in DNA binding interactions we have analyzed the Tus amino acid sequence with respect to secondary structure motifs and similarity to other protein sequences . A twenty amino acid segment containing several basic residues and a proline repeat motif with a periodicity of five residues was identified . The motif was common to several other nucleic acid binding proteins, including histone H1-3, Xenopus laevis ribosomal protein L1, and the single-stranded DNA binding protein (DBP) from adenovirus . A 22 amino acid peptide, TPPI, having a sequence similar to the Tus segment binds non-specifically and non-cooperatively to double- and single-stranded DNA with a binding constant of 1.5 +/- 0.2 x 10(6) M-1 . The estimated binding site size was 4.3 +/- 0.5 base pairs . Circular dichroism studies indicated that the peptide was a random coil in buffer but adopted a helical structure in 50% trifluroethanol and in sodium dodecyl sulfate at concentrations above the critical micellar concentration . Several helical models of the TPPI sequence were constructed graphically and minimized . One of them, an amphiphilic, left-handed, 5.1(19) helical model was best able to account for the observed structural properties of TPPI in the presence of structure-promoting additives. Nucleic Acids Res, 1994 Nov 25, 22(23), 5011 - 5 Introduction of YACs into intact yeast cells by a procedure which shows low levels of recombinagenicity and co-transformation; Heale SM et al.; Yeast artificial chromosomes (YACs) enable the cloning and analysis of large segments of genomic DNA and permit the isolation of sequences which are impossible to maintain in Escherichia coli . However, the construction of genome libraries in YAC vectors is beset by a number of technical problems, not least of which is the creation of cloned fragments which are not true representatives of the donor genome . These artefactual clones arise mainly due to intra-fragment rearrangements or inter-fragment chimaera formation, both phenomena resulting from the activity of the host yeast's mitotic recombination system . We demonstrate that this system is significantly stimulated by the spheroplasting step of the standard YAC transformation system . In contrast, the transformation of intact yeast cells by either the lithium method or a new lithium-free protocol is much less recombinagenic . It is not possible to introduce high molecular weight YACs into yeast using the lithium protocol, but we find that such molecules may be introduced into pde2-mutants using the lithium-free approach . Since intact cells are transformed by this method, automation of post-transformation steps in the construction of YAC libraries is facilitated . Moreover, the frequency of cotransformation (and, therefore, chimera formation) is significantly reduced . However, these advantages do incur a penalty . Yields of YAC transformants by this simplified intact cell approach are reduced some 25- to 30-fold compared to those obtained by the spheroplast transformation route . Nevertheless, the considerable advantages of the new system recommend it for a number of applications. Nucleic Acids Res, 1994 Nov 25, 22(23), 4890 - 7 Kinetic and equilibrium binding studies of the human papillomavirus type-16 transcription regulatory protein E2 interacting with core enhancer elements; Sanders CM et al.; The human papillomaviruses (HPVs) are a family of DNA viruses which cause benign tumours of the skin and mucosa that infrequently progress to malignant carcinoma . The E2 open reading frame of HPV is thought to encode a papillomavirus-specific transcription factor which also has a role in viral replication . The E2 proteins of all papillomaviruses studied to date have been shown to bind specifically to the common conserved sequence ACC(N)6GGT found at multiple locations in their genomes . In the case of HPV-16, a 'high risk' genital papillomavirus, the E2 protein is thought to negatively regulate expression of the major viral transforming genes E6 and E7, which have been directly implicated in the oncogenic process . However, little information exists concerning the relative or absolute affinities of the native HPV-16 protein for its palindromic recognition sequences; moreover, interpretation of any transcription or replication phenomena attributed to this protein is more complicated in the absence of such data . Here we describe the overexpression, purification and characterisation of the C-terminal 89 amino acids of the protein encompassing the DNA binding/dimerisation domain . We show that the recombinant protein purified from E.coli by a combination of non-group-specific chromatography steps retains high biological activity and is able to bind to all sites in the HPV-16 genome with high affinity (approximately 8 x 10(-11) M) . In addition, kinetic studies show that the E2-DNA complexes are very stable, with half-lives ranging from 2.15 to greater than 240 min, and that nucleotides internal and external to the conserved palindrome appear to influence stability. J Biol Chem, 1994 Nov 25, 269(47), 29367 - 70 Regulation of p68 RNA helicase by calmodulin and protein kinase C; Buelt MK et al.; Human p68 RNA helicase is a nuclear RNA-dependent ATPase that belongs to a family of putative helicases known as the DEAD box proteins . These proteins have been implicated in aspects of RNA function including translation initiation, splicing, and ribosome assembly in a variety of organisms ranging from Escherichia coli to humans . While members of this family are believed to function in the manipulation of RNA secondary structure, little is known about the regulation of these enzymes . By immunological methods and sequence comparison, we have found that p68 possesses a region of sequence similarity to the conserved protein kinase C phosphorylation site and calmodulin binding domain (also known as the IQ domain) of the neural-specific proteins neuromodulin (GAP-43) and neurogranin (RC3) . We report that p68 is phosphorylated by protein kinase C in vitro and binds calmodulin in a Ca(2+)-dependent manner . Both phosphorylation and calmodulin binding inhibited p68 ATPase activity, suggesting that the RNA unwinding activity of p68 may be regulated by dual Ca2+ signal transduction pathways through its IQ domain. Mol Cell Biochem, 1994 Nov 23, 140(2), 147 - 62 Are most transporters and channels beta barrels? Fischbarg J, Cheung M, Li J, Iserovich P, Czegledy F, Kuang K, Garner M. Given the sequence of transporters or channels of unknown secondary structure, it is usual to predict their putative transmembrane regions as alpha-helical . However, recent evidence for a facilitative glucose transporter (GLUT1) appears inconsistent with such predictions, which has led us to propose an alternative folding model for GLUTs based on the 16-stranded antiparallel beta-barrel of porins . Here we apply the same predictive algorithms we used for GLUTs to several other membrane proteins . For some of them, a high-resolution structure has been derived (beta-barrels: Rhodobacter capsulatus and Escherichia coli porins; multihelical: colicin A, bacteriorhodopsin, and reaction center L chain); we use them to test the prediction procedures . The other proteins we analyze (GLUT1, CHIP28, acetylcholine receptor alpha subunit, lac permease, Na(+)-glucose cotransporter, shaker K+ channel, sarcoplasmic reticulum Ca(2+)-ATPase) are representative of classes of similar membrane proteins . As with GLUTs, we find that the predicted transmembrane segments of these proteins are consistently shorter than expected for transmembrane spanning alpha-helices, but are of the correct length and number for the proteins to fold instead as porin-like beta-barrels. Proc Natl Acad Sci U S A, 1994 Nov 22, 91(24), 11655 - 9 The secondary structure of the ets domain of human Fli-1 resembles that of the helix-turn-helix DNA-binding motif of the Escherichia coli catabolite gene activator protein; Liang H et al.; The ets family of eukaryotic transcription factors is characterized by a conserved DNA-binding domain of approximately 85 amino acids for which the three-dimensional structure is not known . By using multidimensional NMR spectroscopy, we have determined the secondary structure of the ets domain of one member of this gene family, human Fli-1, both in the free form and in a complex with a 16-bp cognate DNA site . The secondary structure of the Fli-1 ets domain consists of three alpha-helices and a short four-stranded antiparallel beta-sheet . This secondary structure arrangement resembles that of the DNA-binding domain of the catabolite gene activator protein of Escherichia coli, as well as those of several eukaryotic DNA-binding proteins including histone H5, HNF-3/fork head, and the heat shock transcription factor . Differences in chemical shifts of backbone resonances and amide exchange rates between the DNA-bound and free forms of the Fli-1 ets domain suggest that the third helix is the DNA recognition helix, as in the catabolite gene activator protein and other structurally related proteins . These results suggest that the ets domain is structurally similar to the catabolite gene activator protein family of helix-turn-helix DNA-binding proteins. Proc Natl Acad Sci U S A, 1994 Nov 22, 91(24), 11552 - 6 PapD chaperone function in pilus biogenesis depends on oxidant and chaperone-like activities of DsbA; Jacob-Dubuisson F et al.; Adhesive P pili of uropathogenic Escherichia coli were not assembled by a strain that lacks the periplasmic disulfide isomerase DsbA . This defect was mostly attributed to the immunoglobulin-like pilus chaperone PapD, which possesses an unusual intrasheet disulfide bond between the last two beta-strands of its CD4-like carboxyl-terminal domain . The DsbA-dependent formation of this disulfide bond was critical for PapD's proper folding in vivo . Interestingly, the absence of the disulfide bond did not prevent PapD from folding in vitro or from forming a complex with the pilus adhesin in vitro . We suggest that DsbA maintains nascently translocated PapD in a folding-competent conformation prior to catalyzing disulfide bond formation, acting both as an oxidant and in a chaperone-like fashion . Disulfide bond formation in pilus subunits was also mediated by DsbA even in the absence of PapD . However, the ability of pilus subunits to achieve native-like conformations in vivo depended on PapD . These results suggest that a productive folding pathway for subunits requires sequential interactions with DsbA and the PapD chaperone. Proc Natl Acad Sci U S A, 1994 Nov 22, 91(24), 11547 - 51 Restoring allosterism with compensatory mutations in hemoglobin; Kim HW et al.; Abnormal human hemoglobins (HBs) with amino acid substitutions in the alpha 1 beta 2 interface have very high oxygen affinity and greatly reduced cooperativity in O2 binding compared to normal human Hb . In such abnormal Hbs with mutations at position beta 99, the intersubunit hydrogen bonds between Asp-beta 99 and Tyr-alpha 42 and between Asp-beta 99 and Asn-alpha 97 are broken, thus destabilizing the deoxyquaternary structure of these Hbs . A molecular dynamics method has been used to design compensatory amino acid substitutions in these Hbs that can restore their allosteric properties . We have designed a compensatory mutation in a naturally occurring mutant Hb, Hb Kempsey (Asp-beta 99-->Asn), and have produced it using our Escherichia coli expression plasmid pHE2 . We have determined the O2 binding properties of this recombinant double mutant Hb, Hb(Asp-beta 99-->Asn and Tyr-alpha 42-->Asp) and have used 1H NMR spectroscopy to investigate the tertiary structures around the heme groups and the quaternary structure in the alpha 1 beta 2 subunit interface . Our results clearly show that the Tyr-alpha 42-->Asp replacement can substantially compensate for the functional defect of Hb Kempsey caused by the Asp-beta 99-->Asn substitution . The structural and functional information derived from this recombinant Hb provides insights into the structural basis of allosterism and the design of compensatory amino acid substitutions to restore the functional properties of other abnormal HBs associated with hemoglobinopathies. Proc Natl Acad Sci U S A, 1994 Nov 22, 91(24), 11497 - 501 Chloroplast molecular chaperone-assisted refolding and reconstitution of an active multisubunit coupling factor CF1 core; Chen GG et al.; The chloroplast coupling factor 1 (CF1) is composed of five kinds of subunits with a stoichiometry of alpha 3 beta 3 gamma delta epsilon . Reconstitution of a catalytically active alpha 3 beta 3 gamma core from urea-denatured subunits at a physiological pH is reported here . A restoration of approximately 90% of the CF1 ATPase activity has been observed . The reconstitution was achieved by using subunits overexpressed in Escherichia coli, purified, and combined in the presence of MgATP, K+, and a mixture of several chloroplast molecular chaperones at pH 7.5 . The combination of chaperonin 60 and chaperonin 24 failed to reconstitute the active CF1 core, as did the GroEL/GroES pair (E . coli chaperonin 60/10 homologues) . Characteristics of the reconstituted ATPase were very close to those of the native complex, including methanol-reversible inhibition by the purified epsilon subunit of CF1 and sensitivity to inhibition by azide and by tentoxin . In reconstitution with a mixture of tentoxin-resistant and -sensitive beta subunits, the extent of inhibition by tentoxin depended on the proportion of sensitive subunits in the reconstitution mixture . Finally, a model for the assembly of the CF1 core alpha 3 beta 3 gamma structure is proposed. Proc Natl Acad Sci U S A, 1994 Nov 22, 91(24), 11467 - 71 NMR analysis of tRNA acceptor stem microhelices: discriminator base change affects tRNA conformation at the 3' end; Puglisi EV et al.; An important step in initiation of protein synthesis in Escherichia coli is the specific formylation of the initiator methionyl-tRNA (Met-tRNA) by Met-tRNA transformylase . The determinants for formylation are clustered mostly in the acceptor stem of the initiator tRNA . Here we use NMR spectroscopy to characterize the conformation of two RNA microhelices, which correspond to the acceptor stem of mutants of E . coli initiator tRNA and which differ only at the position corresponding to the "discriminator base" in tRNAs . One of the mutant tRNAs is an extremely poor substrate for Met-tRNA transformylase, whereas the other one is a much better substrate . We show that one microhelix forms a structure in which its 3'-ACCA sequence extends the stacking of the acceptor stem . The other microhelix forms a structure in which its 3'-UCCA sequence folds back such that the 3'-terminal A22 is in close proximity to G1 . These results highlight the importance of the discriminator base in determining tRNA conformation at the 3' end . They also suggest a correlation between tRNA structure at the 3' end and its recognition by Met-tRNA transformylase. Proc Natl Acad Sci U S A, 1994 Nov 22, 91(24), 11442 - 6 PTR1: a reductase mediating salvage of oxidized pteridines and methotrexate resistance in the protozoan parasite Leishmania major; Bello AR et al.; Trypanosomatid protozoans are pterin auxotrophs, a finding noted decades ago which heralded the discovery of key metabolic roles played by pteridines in eukaryotes . We have now identified the enzyme mediating unconjugated pteridine salvage in the human parasite Leishmania major, PTR1 (pteridine reductase 1, formerly hmtxr or ltdh) . PTR1 is the gene in the amplified H region responsible for methotrexate (MTX) resistance, and belongs to a large family of oxidoreductases with diverse substrates and roles . We generated Leishmania lacking PTR1 by homologous gene targeting, and these ptr1- mutants required reduced biopterin (dihydro- or tetrahydrobiopterin) for growth . PTR1 purified from engineered Escherichia coli exhibited a MTX-sensitive, NADPH-dependent biopterin reductase activity . PTR1 showed good activity with folate and significant activity with dihydrofolate and dihydrobiopterin, but not with quinonoid dihydrobiopterin . PTR1 thus differs considerably from previously reported pteridine reductases of trypanosomatids and vertebrates . Pteridine reductase activity was diminished in ptr1- Leishmania and was elevated in transfected parasites bearing multiple copies of PTR1; correspondingly, ptr1- was MTX-hypersensitive whereas the multicopy transfectant was MTX-resistant . The concordance of the biochemical and genetic properties of PTR1 suggests that this is the primary enzyme mediating pteridine salvage . These findings suggest several possible mechanisms for PTR1-mediated MTX resistance and should aid in the design of rational chemotherapy. Proc Natl Acad Sci U S A, 1994 Nov 22, 91(24), 11427 - 31 Polysialic acid engineering: synthesis of polysialylated neoglycosphingolipids by using the polysialyltransferase from neuroinvasive Escherichia coli K1; Cho JW et al.; The CMP-sialic acid:poly alpha 2,8sialosyl sialyltransferase (polyST) in neurotropic Escherichia coli K1 inner membranes catalyzes synthesis of the alpha 2,8-linked polysialic acid capsule . The capsule is a neurovirulent determinant associated with neonatal meningitis in humans . A functionally similar polyST in human neuroblastomas polysialylates neural cell adhesion molecules . While bacteria do not synthesize glycosphingolipids (GSLs), we report here that the E . coli K1 polyST can selectively polysialylate several structurally related GSLs, when added as exogenous sialyl acceptors . A structural feature common to the preferred sialyl acceptors (GD3 > GT1a > GQ1b = GT1b > GD2 = GD1b = GD1a > GM1) was the disialyl glycotope, Sia alpha 2,8Sia, alpha 2,3-linked to galactose (Sia is sialic acid) . A linear tetrasaccharide with a terminal Sia residue (e.g., GD3) was the minimum length oligosaccharide recognized by the polyST . Endo-N-acylneuraminidase was used to confirm the alpha 2,8-specific polysialylation of GSL . Ceramide glycanase was used to release the polysialyllactose chains from the ceramide moiety . Size analysis of these chains showed that 60-80 Sia residues were transferred to the disialyllactose moiety of GD3 . The significance of these findings is two-fold . (i) The E . coli K1 polyST can be used as a synthetic reagent to enzymatically engineer the glycosyl moiety of GSL, thus creating oligo- or polysialylated GSLs . Such "designer" GSLs may have potentially important biological and pharmacological properties . (ii) The use of GSLs as exogenous sialyl acceptors increases the sensitivity of detecting polyST activity . The practical advantage of this finding is that polyST activity can be identified and studied in those eukaryotic cells that express low levels of this developmentally regulated enzyme and/or its acceptor. Proc Natl Acad Sci U S A, 1994 Nov 22, 91(24), 11343 - 7 NMR structure determination of the Escherichia coli DnaJ molecular chaperone: secondary structure and backbone fold of the N-terminal region (residues 2-108) containing the highly conserved J domain; Szyperski T et al.; DnaJ from Escherichia coli is a 376-amino acid protein that functions in conjunction with DnaK and GrpE as a chaperone machine . The N-terminal fragment of residues 2-108, DnaJ-(2-108), retains many of the activities of the full-length protein and contains a structural motif, the J domain of residues 2-72, which is highly conserved in a superfamily of proteins . In this paper, NMR spectroscopy was used to determine the secondary structure and the three-dimensional polypeptide backbone fold of DnaJ-(2-108) . By using 13C/15N doubly labeled DnaJ-(2-108), nearly complete sequence-specific assignments were obtained for 1H, 15N, 13C alpha, and 13C beta, and about 40% of the peripheral aliphatic carbon resonances were also assigned . Four alpha-helices in polypeptide segments of residues 6-11, 18-31, 41-55, and 61-68 in the J domain were identified by sequential and medium-range nuclear Overhauser effects . For the J domain, the three-dimensional structure was calculated with the program DIANA from an input of 536 nuclear Overhauser effect upper-distance constraints and 52 spin-spin coupling constants . The polypeptide backbone fold is characterized by the formation of an antiparallel bundle of two long helices, residues 18-31 and 41-55, which is stabilized by a hydrophobic core of side chains that are highly conserved in homologous J domain sequences . The Gly/Phe-rich region from residues 77 to 108 is flexibly disordered in solution. Biochemistry, 1994 Nov 22, 33(46), 13888 - 95 Reductive and oxidative half-reactions of glutathione reductase from Escherichia coli; Rietveld P et al.; Glutathione reductase catalyzes the reduction of glutathione disulfide by NADPH and has a redox active disulfide and an FAD cofactor in each monomer . In the reductive half-reaction, FAD is reduced by NADPH and electrons pass from the reduced flavin to the redox active disulfide . The oxidative half-reaction is dithiol-disulfide interchange between the enzyme dithiol and glutathione disulfide . We have investigated the reductive and oxidative half-reactions using wild-type glutathione reductase from Escherichia coli and in an altered form of the enzyme in which the active site acid-base catalyst, His439, has been changed to an alanine residue (H439A) . H439A has 0.3% activity in the NADPH/GSSG assay . The replacement affects both the oxidative half-reaction, as expected, and the reductive half-reaction--specifically, the passage of electrons from reduced flavin to the disulfide . Reduction of H439A by NADPH allows direct observation of flavin reduction . The NADPH-FAD charge transfer complex is formed in the dead time . Reduction of FAD, at a limiting rate of 250 s-1, is observed as a decrease at 460 nm and an increase at 670 nm (FADH(-)-NADP+ charge transfer) . Subsequent passage of electrons from FADH- to the disulfide (increase at 460 nm and a decrease at 670 nm) is very slow (6-7 s-1) and concentration independent in H439A . The monophasic oxidative half-reaction is very slow, as expected for reduced H439A.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 Nov 22, 33(46), 13693 - 9 Covalently bound pH-indicator dyes at selected extracellular or cytoplasmic sites in bacteriorhodopsin . 2 . Rotational orientation of helices D and E and kinetic correlation between M formation and proton release in bacteriorhodopsin micelles; Alexiev U et al.; The kinetics of the light-induced proton release in bacteriorhodopsin/lipid/detergent micelles was monitored with the optical pH-indicator fluorescein bound covalently to positions 127-134 (helices D and E and the DE loop) on the extracellular side of the protein (the proton release side) . Single cysteine residues were introduced in these positions by site-directed mutagenesis, and fluorescein was attached to the sulfhydryl group by reaction with (iodoacetamido)fluorescein . Two characteristic proton release times (approximately 20 and 70 microseconds) were observed . The faster time constant was recorded when fluorescein was attached to positions 127, 130, 131, 132, and 134, while the slower time was observed with the indicator bound to positions 128, 129, and 133 . The results are rationalized by assuming specific helical wheel orientations for helics D and E and by making a choice for the residues in the DE loop: (i) The fast time constants occur with fluorescein either attached to residues 130, 131, and 132 that form the DE loop or when pointing toward the interior of the protein with its aqueous proton channel {residues 127 (helix D) and 134 (helix E)} . (ii) The slower time constants are detected with fluorescein exposed to the exterior lipid/detergent phase when bound to residues 128, 129 (both helix D), and 133 (helix E) . This interpretation is supported by measurements of the polarity of the label environment which indicate for fluorescein in group i a more hydrophilic environment and for group ii a more hydrophobic environment . The fastest proton release time (10 microseconds) was observed with fluorescein bound to position 127.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 Nov 22, 33(46), 13684 - 92 Covalently bound pH-indicator dyes at selected extracellular or cytoplasmic sites in bacteriorhodopsin . 1 . Proton migration along the surface of bacteriorhodopsin micelles and its delayed transfer from surface to bulk; Scherrer P et al.; The kinetics of the light-induced release and uptake of protons was monitored with the optical pH-indicator fluorescein covalently bound to various sites on the extracellular and cytoplasmic surfaces of bacteriorhodopsin . Selective labeling was achieved by reacting (iodoacetamido)fluorescein with the single cysteine residues in bacteriorhodopsin introduced at the desired positions by site-directed mutagenesis . All measurements were performed with bacteriorhodopsin micelles in phospholipid/detergent mixtures in 150 mM KCl at 22 degrees C, pH 7.3 . Neither the replacements by cysteine nor the subsequent labeling affected the absorption spectrum of bacteriorhodopsin and the rise times of the M intermediate . Only the decay of M was altered for some bacteriorhodopsin mutants with cysteine residues on the cytoplasmic side . The proton release time detected with fluorescein attached to the extracellular surface (the proton release side) at position 72 (in the loop connecting helices B and C) or 130 (DE loop) was 22 +/- 4 microseconds, clearly faster than that measured with pyranine in the aqueous bulk phase (125 +/- 10 microseconds for wild-type and all mutants studied) . For bacteriorhodopsin mutants labeled at positions 35, 101, 160, 229, and 231 in the cytoplasmic loop region (the proton uptake side), the released proton was observed with a time of 61 +/- 4 microseconds . This was about 3-fold slower than the release time on the extracellular side, but still significantly faster than that measured with pyranine in the bulk phase . These results suggest that the released protons are retained on the micellar surface and move more rapidly along this surface to the cytoplasmic side than from the surface to the bulk medium.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 Nov 22, 33(46), 13662 - 7 Formation of functional cross-species heterodimers of ornithine decarboxylase; Osterman A et al.; The two active sites in ornithine decarboxylase (ODC) are formed at the dimer interface with Lys-69 and Cys-360 contributing to each active site from opposite monomers {Tobias, K . E., & Kahana, C . (1993) Biochemistry 32, 5842-5847} . To gain insight into the organization of the substrate binding site and the nature of the dimer interface, analysis of ornithine decarboxylase from two parasitic protozoa, Trypanosoma brucei and Leishmania donovani, and from mouse was undertaken . Though T . brucei and mouse ornithine decarboxylase share only 60% sequence identity, the cross-species heterodimers form spontaneously, as measured by the restoration of enzyme activity upon mixing inactive K69A and C360A mutant enzymes . Thus, the amino acid composition of the dimer interface is apparently highly conserved between the T . brucei and mouse enzymes . Cross-species heterodimers were not formed between either T . brucei or mouse ODC and L . donovani ODC . Unlike the mouse and T . brucei ODC, the subunits of L . donovani ODC are not in rapid equilibrium, and incubation with a denaturant is required to induce reassociation . Kinetic analysis of the wild-type mouse and parasite ODCs revealed differences in the substrate binding sites between the three enzymes . The substrate binding properties of the restored active site in the T . brucei:mouse cross-species heterodimer mimic the characteristics of the wild-type enzyme from the species which contributes the subunit with a functional Lys-69. Biochemistry, 1994 Nov 22, 33(46), 13581 - 92 Determination of the solution structure of the peptide hormone guanylin: observation of a novel form of topological stereoisomerism; Skelton NJ et al.; Guanylin is a 15 amino acid mammalian hormone containing two disulfide bonds . Guanylin shares sequence similarity with the bacterial heat-stable enterotoxin (STa) and is capable of binding to and stimulating the STa guanylyl cyclase receptor . Biologically active peptides have been prepared by two methods: (1) enzymatic treatment of a 99 residue proprotein (denoted proguanylin) expressed in Escherichia coli and (2) solid-phase chemical synthesis . Although both sources yield material that is pure by high-performance liquid chromatography and mass spectrometry, analysis by nuclear magnetic resonance (NMR) indicates that peptides from both sources contain two conformationally distinct species present in a 1:1 ratio . The chemical shift differences between the two species are large, allowing unambiguous sequential NMR assignments to be made for both sets of resonances . Exchange between the two forms was not observed even at 70 degrees C . Structural restraints have been generated from nuclear Overhauser effects and scalar coupling constants and used to calculate structures for both forms using distance geometry and restrained energy minimization . The resulting structures for the first isoform are well defined (root-mean-square deviation from the average structure for backbone atoms of 0.47 A) and adopt a right-handed spiral conformation, similar to that observed for heat stable enterotoxin . The second isoform is less well defined (root-mean-square deviation from the average structure for backbone atoms of 1.07 A) but clearly adopts a very different fold consisting of a left-hand spiral . The differences in structure suggest that the two forms may have very different affinities toward the STa receptor . The observation of such isomerism has important implications for the common practice of introducing multiple disulfide bonds into small peptides to limit conformational flexibility and enhance bioactivity. Biochemistry, 1994 Nov 22, 33(46), 13509 - 16 Secondary structure of the ETS domain places murine Ets-1 in the superfamily of winged helix-turn-helix DNA-binding proteins; Donaldson LW et al.; The members of the ets gene family of transcription factors are characterized by a conserved 85-residue DNA-binding region, termed the ETS domain, that lacks sequence homology to structurally characterized DNA-binding motifs . The secondary structure of the ETS domain of murine Ets-1 was determined on the basis of NMR chemical shifts, NOE and J-coupling constraints, amide hydrogen exchange, circular dichroism, and FT-IR spectroscopy . The ETS domain is composed of three alpha-helices (H) and four beta-strands (S) arranged in the order H1-S1-S2-H2-H3-S3-S4 . The four-stranded antiparallel beta-sheet is the scaffold for a putative helix-turn-helix DNA recognition motif formed by helices 2 and 3 . The 25 residues extending beyond the ETS domain to the native C-terminus of the truncated Ets-1 also contain a helical segment . On the basis of the similarity of this topology with that of catabolite activator protein (CAP), heat shock factor (HSF), and hepatocyte nuclear factor (HNF-3 gamma), we propose that ets proteins are members of the superfamily of winged helix-turn-helix DNA-binding proteins. Proc Natl Acad Sci U S A, 1994 Nov 22, 91(24), 11462 - 6 Domains of Escherichia coli primase: functional activity of a 47-kDa N-terminal proteolytic fragment; Sun W et al.; Endoproteinase Asp-N cleaves the 581-amino acid Escherichia coli primase (65,564 Da) into several major fragments . One of these, a 47-kDa fragment containing the complete N terminus and the first 422 amino acids of primase, is capable of primer RNA (pRNA) synthesis in the G4oric/single-stranded DNA binding protein/primase pRNA synthesis system . A cloned 398-amino acid N-terminal fragment of primase can also synthesize pRNA . The sizes of the pRNA synthesized by these N-terminal fragments, however, are smaller than those synthesized by intact primase, suggesting that the C-terminal region of primase plays a role in processivity or regulation of pRNA synthesis . Primase mutants with the last 10 and 40 C-terminal amino acids deleted synthesize pRNA as wild-type primase, indicating that any regulatory sequences must be internal to the C terminus of primase. Biochemistry, 1994 Nov 22, 33(46), 13836 - 47 Evolution of host cell RNA into efficient template RNA by Q beta replicase: the origin of RNA in untemplated reactions; Moody MD et al.; Q beta replicase can replicate a single molecule of certain species of RNA to 10(14) copies in minutes . This replication ability has been used for in vitro studies of molecular evolution and is currently being utilized as a method of amplifying RNAs that contain probe sequences . It has been observed that Q beta replicase can produce replicatable RNA even in the absence of exogenously added template RNA . The origin of this RNA has been ascribed either to contamination with replicatable RNA or to an ability of Q beta replicase to synthesize RNA de novo from the nucleotides present in the reaction . Technologies that employ Q beta replicase require a thorough understanding of the conditions that lead to this so-called spontaneous RNA production . We have created an expression system and purification method with which we produce gram quantities of highly purified Q beta replicase, and we have identified reaction conditions that prevent the amplification of RNA in assays that do not contain added RNA . However, when these reaction conditions are relaxed, spontaneous RNA replication is seen in up to 100% of the assays . To understand the origin of this RNA, we have cloned several spontaneously produced RNAs . Sequence analysis of one of these RNAs shows that it arose by the evolution of Escherichia coli tRNA into a replicatable template and not by de novo synthesis from nucleoside triphosphates in the reaction. Biochemistry, 1994 Nov 22, 33(46), 13775 - 86 Resonance assignments and solution structure of the second RNA-binding domain of sex-lethal determined by multidimensional heteronuclear magnetic resonance; Lee AL et al.; The RNA-binding protein Sex-lethal (Sxl) is a critical regulator of sexual differentiation and dosage compensation in Drosophila . This regulatory activity is a consequence of the ability of Sxl to bind uridine-rich RNA tracts involved in pre-mRNA splicing . Sxl contains two RNP consensus-type RNA-binding domains (RBDs) . A structural study of a portion of Sxl (amino acids 199-294) containing the second RNA-binding domain (RBD-2) using multidimensional heteronuclear NMR is presented here . Nearly complete 1H, 13C, and 15N resonance assignments have been obtained from 15N- and 13C/15N-uniformly labeled protein . These assignments were used to analyze 3D 15N-separated NOESY and 13C/13C-separated 4D NOESY spectra which produced 494 total and 169 long-range NOE-derived distance restraints . Along with 41 backbone dihedral restraints, these distance restraints were employed to generate an intermediate-resolution family of calculated structures, which exhibits the beta alpha beta-beta alpha beta tertiary fold found in other RBD-containing proteins . The RMSD to the average structure for the backbone atoms of residues 11-93 is 1.55 +/- 0.30 A, while the RMSD for backbone atoms involved in secondary structure is 0.76 +/- 0.14 A . A capping box {Harper, E.T., & Rose, G.D . (1993) Biochemistry 32, 7605-7609} was identified at the N-terminus of the first helix and has been characterized by short- and medium-range NOEs . Finally, significant structural similarities and differences between Sxl RBD-2 and other RBD-containing proteins are discussed. FEBS Lett, 1994 Nov 21, 355(1), 27 - 9 Heterologous expression of an active rat regulatory protein of glucokinase; Detheux M et al.; The cDNA presumed to encode the rat liver regulatory protein of glucokinase has been expressed in Escherichia coli and a partially soluble protein has been obtained . This recombinant protein was partially purified and found to have the same apparent molecular mass as the regulatory protein purified from rat liver . Like the latter, it inhibited rat liver glucokinase competitively with respect to glucose and its effect was sensitive to fructose 6-phosphate and fructose 1-phosphate. J Theor Biol, 1994 Nov 21, 171(2), 179 - 95 Evolution of genetic information flow from the viewpoint of protein sequence similarity; Fukuchi S et al.; As a course of inquiry into the evolution of genetic information flow, similarity relations of amino acid sequences between the proteins involved in translation, transcription and replication are investigated . The sequence data of these proteins are mostly accumulated from Escherichia coli, and the present investigation is carried out mainly on this organism by the FASTP program . This result reveals an interesting similarity linkage extending from ribosomal proteins to the proteins participating in translational elongation process and to the proteins in transcription and replication . Although the ribosomal proteins are of relatively short polypeptide chains, our systematic comparison between these proteins finds many similarity relations, being more than 100 in terms of "overlap", reducing them to about 14 elementary ribosomal proteins from which other ribosomal proteins would have diverged . Moreover, the proteins involved in translation, transcription and replication contain the regions similar to the elementary ribosomal proteins . In particular, some initiation and elongation factors in translation process are assigned to be similar to the elementary ribosomal proteins almost over the whole regions . To such an elongation factor Tu, the alpha and sigma 70 subunits of RNA polymerase and primase also show similarity in the wider regions than the individual ribosomal proteins, and they are shown to be fundamental for the similarity linkage extending to the other polypeptide chains involved in transcription and replication processes, although the latter polypeptide chains contain regions not similar to any ribosomal protein . This divergence pattern of similarity relations strongly suggests that the proteins involved in the contemporary genetic information flow DNA-->RNA-->protein have evolved from some elementary ribosomal proteins, first by gene fusion, in a primitive organism of the RNA-protein world, and then by the addition of the mechanism of domain shuffling from other genes in the DNA-RNA-protein world. J Mol Biol, 1994 Nov 18, 244(1), 36 - 51 Stability of Escherichia coli transcription complexes near an intrinsic terminator; Wilson KS et al.; Transcription complexes containing Escherichia coli RNA polymerase terminate RNA synthesis in response to intrinsic (rho-independent) terminators encoded in the DNA template . This type of termination involves the formation of a hairpin structure in the nascent RNA, leading to release of the RNA and polymerase from the DNA template . In this paper we show that RNA release occurs only within a zone extending over six template positions of the tR2 terminator of phage lambda . The upstream end of this zone is defined by a large and abrupt decrease in the stability of the transcription complex, and this decrease is reversed at the downstream end . Except within this termination zone, RNA chain elongation is completely processive at all template positions, over a wide range of temperatures (20 to 70 degrees) . The stability of ternary complexes halted at each template position by the incorporation of 3'-deoxynucleotide substrate analogues was determined by means of an ultrafiltration RNA release assay . This assay shows that the nascent RNA is released from halted ternary complexes within the same zone in which termination occurs during active transcription . The termination efficiency at each position within the tR2 termination zone is a sensitive function of the concentration of the next nucleotide substrate to be incorporated into the nascent RNA . These results suggest that termination positions are defined by the thermodynamic stability of the polymerase complex, while the efficiencies of termination are determined by a kinetic competition between RNA elongation and release at each position . Based on a kinetic competition model, the temperature dependence of the efficiency indicates that the elongation pathway is favored enthalpically, while the termination pathway is favored entropically. J Mol Biol, 1994 Nov 18, 244(1), 13 - 22 Molecular modeling of RNA polymerase II mutations onto DNA polymerase I; Kim WJ et al.; Genetic and molecular analysis in Drosophila melanogaster identifies eight suppressor mutations in the second largest subunit of RNA polymerase II . The suppressor mutations fall into two classes: five are strong, result from the same serine to cysteine amino acid residue substitution and rescue one conditional lethal allele in the largest subunit of RNA polymerase II; three are mild, result from a change in the same methionine residue to either isoleucine or valine, are located seven amino acid residues away from the strong suppressors and rescue two conditional lethal alleles in the largest subunit . Sequence analysis of the three regions around these mutations demonstrates that they are located within highly conserved domains but fails to explain the observed genetic interactions . One of the conditional lethal alleles maps within a region previously reported to share sequence similarity to Escherichia coli DNA polymerase I . As the gross structure of RNA polymerase II and DNA polymerase I is similar, even though their primary sequence is not, we predict that more similarities exist but may be too highly divergent to be detected by normal homology searches . We identify the most similar regions between each of the three conserved domains of RNA polymerase II, identified as functionally important because of the mutations we isolated, and DNA polymerase I . Molecular modeling these regions of RNA polymerase II onto the tertiary structure of DNA polymerase I predicts that all lie adjacent to the DNA binding cleft in positions such that they could interact with the phosphate backbone of DNA . This juxtaposition of mutations in the two largest subunits of RNA polymerase II suggest a mechanism for their genetic interactions. J Biol Chem, 1994 Nov 18, 269(46), 29247 - 51 On the relationship between the metabolic and thermodynamic stabilities of T4 lysozymes . Measurements in eukaryotic cells; Inoue I et al.; We have measured the metabolic stabilities of wild-type and 17 temperature-sensitive mutants of T4 lysozyme in HeLa cells, in Xenopus egg extract, and in reticulocyte lysate . {35S}Methionine-labeled T4 lysozymes were expressed in Escherichia coli, purified, injected into HeLa cells, and their degradation rates were determined . Wild-type T4 lysozyme has a half-life of 4 h; the half-lives of 16 lysozyme variants ranged from 2 to 10 h . Surprisingly, the most temperature-sensitive enzyme in the set, R96H, was significantly more stable (half-life = 10 h) . Different T4 lysozyme variants yield conflicting answers to the proposed relationship between thermal and metabolic stabilities . For mutations at Thr157 there is no correlation between melting temperature and half-life . By contrast, T4 lysozymes mutated at various positions show a definite correlation between the two parameters . Treatment of injected HeLa cells with the lysosomotropic agents chloroquine or ammonium chloride did not alter the stability of T4 lysozyme . However, the enzyme's half-life increased 10-fold in HeLa cells depleted of ATP . Although T4 lysozyme is degraded rapidly within HeLa cells, the molecule is stable in reticulocyte lysate and Xenopus egg extract . Presumably, there is a specific proteolytic event(s) in HeLa cells which is not manifest in the in vitro extracts. J Biol Chem, 1994 Nov 18, 269(46), 29241 - 6 On the relationship between the metabolic and thermodynamic stabilities of T4 lysozymes . Measurements in Escherichia coli; Inoue I et al.; Escherichia coli cells were transformed with plasmids encoding 12 site-specific variants of T4 lysozyme, and pulse-chase protocols were used to measure the metabolic stability of each protein . The resulting half-lives ranged from 1 h to more than 50 h . Although the metabolic half-lives of T4 lysozymes correlated roughly with their thermal stabilities, three mutant enzymes were clear exceptions . A reasonably temperature-resistant variant, G156D, exhibited a half-life of 1 h . By contrast, two temperature-sensitive variants, T157I and I3G, were as metabolically stable as wild-type T4 lysozyme . Degradation of two short lived variants, L91P and G156P, required ATP both in vivo and in vitro . Degradation of variant and wild-type enzymes was unimpaired in cells lacking the Lon protease, Clp A or Clp P . However, degradation of L91P and G156D was inhibited in Clp B-cells . Decreased proteolysis of L91P was accompanied by its accumulation in inclusion bodies, indicating that Clp B prevents accumulation of aggregated protein either by preventing aggregation of misfolded polypeptides or solubilizing aggregates. J Biol Chem, 1994 Nov 18, 269(46), 29077 - 84 Participation of lysine 516 and phenylalanine 530 of diphtheria toxin in receptor recognition; Shen WH et al.; To identify amino acid residues within diphtheria toxin that participate in receptor recognition, we made alanine replacements for each of 12 solvent-accessible residues within a loop (residues 516-530) of the toxin's R domain, which prominently extends from the main surface of the domain . Amino acid replacements were generated in an enzymatically attenuated form of the toxin (in compliance with regulations for cloning in Escherichia coli), and the mutant toxins were purified and assayed for toxicity on Vero cells . The largest effects were seen with K516A and F530A, which caused approximately 22- and approximately 10-fold increases, respectively, in the toxin concentration required for half-maximal inhibition of protein synthesis (IC50) . Smaller effects were seen at certain other sites and no effect at still others . K516A caused approximately 500-fold reduction in ability to compete with radiolabeled wild-type toxin for receptors, and F530A gave approximately 100-fold reduction . The small differences in IC50 with the mutants, compared with differences in receptor binding, are attributable to nonlinearity in the cytotoxicity assay with the enzymatically attenuated toxin . K516A and F530A also inhibited the receptor-blocking activity of the isolated R domain . Neither mutation caused a change in the circular dichroism spectrum of the R domain . These results indicate important roles for Lys-516 and Phe-530 in receptor recognition . In addition, on the basis of cytotoxicity data, four other residues (Tyr-514, Val-523, Asn-524, and Lys-526) are proposed play roles in receptor binding . These findings support the notion that the toxin's receptor binds to the solvent-exposed face of the R domain opposite the catalytic domain. J Biol Chem, 1994 Nov 18, 269(46), 28988 - 96 Nonenzymatic bilin addition to the alpha subunit of an apophycoerythrin; Fairchild CD et al.; C-Phycoerythrin is a light-harvesting protein whose alpha and beta subunits carry thioether-linked phycoerythrobilin (PEB) at cysteine residues alpha-82, alpha-139, beta-48,59 (doubly-linked), beta-80, and beta-165 . The two subunits of Calothrix sp . PCC 7601 C-phycoerythrin, overexpressed together as apopolypeptides in Escherichia coli, formed inclusion bodies . Purified apo-alpha was soluble in the absence of urea, whereas the apo-beta subunit was only soluble at high urea concentrations . Products of nonenzymatic addition of PEB and phycocyanobilin (PCB) to apo-alpha were characterized by isolation of bilin peptides and spectroscopy . Reaction of PEB with the apo-alpha subunit led primarily to 15,16-dihydrobiliverdin (Cys-82) or urobilin (Cys-139) adducts, and small amounts of the natural PEB adducts at both Cys-82 and Cys-139 . PCB reacted primarily with Cys-82 to form phycocyanobilin and mesobiliverdin adducts . Both PEB and PCB also formed relatively small amounts of adducts with Cys-59, which is not a bilin attachment residue in natural phycoerythrin . Sodium azide was found to promote the addition of PEB to simple thiols but not to apo-alpha phycoerythrin. J Biol Chem, 1994 Nov 18, 269(46), 28908 - 12 Identification of a novel quinone-binding site in the cytochrome bo complex from Escherichia coli; Sato-Watanabe M et al.; The cytochrome bo complex is a heme BO-type heme-copper quinol oxidase in the aerobic respiratory chain of Escherichia coli and functions as an electron transfer-linked proton pump . To study the protein-mediated electron transfer from substrates to metal centers, we carried out quantitative and qualitative analyses of a bound quinone in the purified oxidase and found that it has a novel high affinity ubiquinone-binding site distinct from the quinol oxidation site . Enzymatic and spectroscopic studies suggest that the quinone-binding site is located close to both the quinol oxidation site in subunit II and low-spin heme B in subunit I . The quinone-binding site of a bound ubiquinone-free oxidase was reconstituted with the potent quinol oxidation site inhibitor 2,6-dichloro-4-nitrophenol, which decreased the Vmax value of the ubiquinol-1 oxidase activity to one-fourth of the control activity . These results indicate that the quinone-binding site is essential for the catalytic functions of the cytochrome bo complex and mediates electron transfer from the quinol oxidation site to the low-spin heme. J Biol Chem, 1994 Nov 18, 269(46), 28899 - 907 Structure-function studies on the ubiquinol oxidation site of the cytochrome bo complex from Escherichia coli using p-benzoquinones and substituted phenols; Sato-Watanabe M et al.; To characterize the structural features of the quinol oxidation site (the QL site) of the cytochrome bo complex, a heme-copper respiratory oxidase in Escherichia coli, we carried out structure-inhibitory potency analyses using 7 p-benzoquinones and 33 substituted phenols . Their effects on its ubiquinol-1 oxidase activity were compared with those on the cytochrome bd complex in E . coli and on cytochromes o and alpha 1 in Acetobacter aceti . They showed similar structural properties of the QL site, although cytochrome o was more sensitive to 4-cyanophenols, suggesting a specific interaction of the hydrogen bond-accepting cyano group with the binding pocket . Replacing one of the methyl groups of 2,6-dimethyl-p-benzoquinone, which is the most potent competitive inhibitor, with an ethyl group markedly decreased the inhibitory activity, indicating that the QL site specifically recognizes one C = O group with two methyl groups as the ortho-substituents . In substituted phenols, ortho-chlorine substituents were the most effective in recognition, and the electron-withdrawing ability of the para-substituent determined an inhibitory potency, probably by stabilizing an anionic form . Based on these observations, we postulate that the QL site of the cytochrome bo complex asymmetrically recognizes exogenous ligands and that this property accounts for the sequential electron transfer from ubiquinols to the low-spin heme. J Biol Chem, 1994 Nov 18, 269(46), 28871 - 7 Nucleotide-binding sites on Escherichia coli F1-ATPase . Specificity of noncatalytic sites and inhibition at catalytic sites by MgADP; Hyndman DJ et al.; Nucleotide-depleted EcF1 binds a maximum of two GTP, ATP, or ADP at noncatalytic sites, whereas all three sites can only be filled by a combination of nucleoside di- and triphosphates . MgPPi prevents binding of GTP and significantly slows ATP binding, suggesting that non-catalytic sites also bind PPi . No binding of GDP at non-catalytic sites could be detected . The slow rate of GTP dissociation from noncatalytic sites (t1/2 = 175 min) is increased 2-8-fold by EDTA, MgPPi, MgADP, or EDTA/ATP, but 23-fold by conditions for ATP hydrolysis . ATP hydrolysis by EcF1, depleted of both its inhibitory epsilon-subunit and endogenous nucleotides, shows a burst of activity . However, it shows a lag if preincubated with MgADP but not when preincubated with Mg2+ alone . For epsilon-depleted EcF1 containing endogenous inhibitory ADP, preincubation with an ATP-regenerating system results in a burst of activity, whereas the control shows a lag . This same enzyme form shows significant inhibition with increasing concentrations of Mg2+ during ATP hydrolysis but lesser levels of inhibition when other NTP substrates are used . With the five-subunit enzyme, increasing amounts of azide cause an increase in the level of inhibition with a corresponding increase in amount of bound nucleotide resistant to rapid chase . Azide-trappable nucleotide is bound at catalytic sites as shown by covalent incorporation of 2-azido-ADP . The results suggest that ligand specificity may not be a reliable means of distinguishing between catalytic and noncatalytic sites and that MgADP inhibition should be taken into account in the kinetic analysis of EcF1 mutants. J Biol Chem, 1994 Nov 18, 269(46), 28865 - 70 Thioredoxin increases the proliferation of human B-cell lines through a protein kinase C-dependent mechanism; Biguet C et al.; Thioredoxin (Trx) catalyzes thiol-disulfide oxidoreductions . We and others recently showed that human Trx could function as an autocrine growth factor for human lymphoid cells immortalized by the human T-lymphotrophic virus type I or the Epstein-Barr virus . Here we report that reduced Trx from Escherichia coli generated by NADPH and thioredoxin reductase increases the proliferation of an Epstein-barr virus(+)-B cell line 1G8, which constitutively produces low amounts of human Trx . This proliferative effect involved the activation of protein kinase C through its translocation to the membrane . Staurosporin and calphostin C, two inhibitors of protein kinase C, but not of H8, a protein kinase A inhibitor, were able to block Trx-dependent proliferation . The addition of Trx to 1G8 cells resulted in the formation of inositol 1,4,5-triphosphate and sn-1,2-diacylglycerol by a phosphoinositide-specific phospholipase C, as well as increased free calcium concentration . Diacylglycerol showed a biphasic increase; the first phase, corresponding to an early peak (30 s) of inositol 1,4,5-triphosphate and a second larger, prolonged phase . The second phase was inhibited by propranolol, a specific inhibitor of phosphohydrolase, indicating that it is most likely derived from phosphatidylcholine hydrolysis by the sequential action of phospholipase D and phosphatidic acid phosphohydrolase . Our data suggest that enhanced phosphoinositide-specific phospholipase C activity induced by the dithiol form of Trx in 1G8 cells is associated to protein kinase C activation, and thus plays a role in the permanent growth of Epstein-Barr virus-infected B cells. J Biol Chem, 1994 Nov 18, 269(46), 28844 - 50 Site-specific mutagenesis by a propanodeoxyguanosine adduct carried on an M13 genome; Burcham PC et al.; The spectrum of mutations induced upon in vivo replication of an M13 genome containing a site-specifically located propanodeoxyguanosine (PdG) adduct was determined . PdG was used as a model for the major deoxyguanosine adduct produced on reaction of DNA with the endogenous genotoxin malondialdehyde . PdG was introduced at position 6256 of M13MB102 by ligating the oligodeoxynucleotide 5'-GGT(PdG)TCCG-3' into an 8-base gap in the (-)-strand of duplex M13MB102 . Replication of the adducted strand was maximized by incorporation of uracil into the unadducted (+)-strand . Following replication of dG-containing and PdG-containing M13MB102 genomes in Escherichia coli JM105, frameshift mutations were detected as phenotypic changes in the lacZ alpha marker gene . Base pair substitutions were detected by differential hybridization using 32P-labeled 13-mers bearing different bases opposite position 6256 . Neither frameshift nor base pair substitution mutations were detected following replication of PdG-adducted genomes in non-SOS-induced JM105 . However, PdG-->T transversions and PdG-->A transitions were detected following transformation of PdG-adducted M13MB102 into SOS-induced JM105 . Both types of mutations were detected at comparable frequencies, and the total mutation frequency was approximately 2% . The results indicate that PdG is an efficient premutagenic lesion in E . coli strains in which the SOS response is induced. J Biol Chem, 1994 Nov 18, 269(46), 28834 - 8 Identification of the ubiquinol-binding site in the cytochrome bo3-ubiquinol oxidase of Escherichia coli; Welter R et al.; The cytochrome bo3-ubiquinol oxidase, one of two ubiquinol oxidases in Escherichia coli, is a member of the heme-copper oxidase superfamily . The enzyme contains four protein subunits (I-IV) with apparent molecular masses of 58, 33, 22, and 17 kDa, respectively . Cytochrome bo3 catalyzes the 2-electron oxidation of ubiquinol and the reduction of molecular oxygen to water . Although the primary structures of all four subunits have been determined, the ubiquinol-binding site has not been investigated . The photoreactive radiolabeled azidoubiquinone derivative 3-{3H}azido-2-methyl-5-methoxy-6-geranyl-1,4-benzoquinone (azido-Q), which has been widely used in locating the ubiquinone-binding sites of other enzymes, was used to identify the subunit(s) involved in the binding of quinol to cytochrome bo3 . When reduced by dithioerythritol, the azido-Q derivative functioned as a substrate with partial effectiveness, suggesting that azido-Q interacts with a legitimate quinol-binding site . When cytochrome bo3 was incubated with an 8-fold molar excess of azido-Q, illumination by UV light for 10 min resulted in a 50% loss of activity . The uptake of radiolabeled azido-Q by the oxidase complex upon illumination correlated with the photoinactivation . In the presence of the competitive inhibitor 2-heptyl-4-hydroxyquinoline or ubiquinol, the rate of azido-Q uptake and the loss of enzyme activity upon illumination decreased . Analysis of the distribution of radioactivity among the subunits after separation by SDS-polyacrylamide gel electrophoresis showed that subunit II was heavily labeled by azido-Q, but that the other subunits were not . This suggests that the ubiquinol-binding site of the cytochrome bo3 complex is located at least partially on subunit II. J Biol Chem, 1994 Nov 18, 269(46), 28822 - 8 Mutations eliminating the protein export function of a membrane-spanning sequence; Lee E et al.; Individual membrane protein spanning sequences can promote protein export . To help define the sequence features necessary for this action, we identified mutations disrupting export mediated by the first spanning sequence (TM1) of the Escherichia coli serine chemoreceptor . Mutant spanning sequences were generated and characterized using beta-galactosidase and alkaline phosphatase gene fusions . The protein export function of TM1 was remarkably tolerant of single charged residues, and the introduction of pairs of charged amino acids was necessary to eliminate export . The results are accommodated by a model in which export requires a stretch of uncharged residues whose summed hydrophobicity exceeds a particular threshold value . This threshold approximates the minimum hydrophobicity required for cleavable signal sequence function . In addition, the threshold was near the minimum hydrophobicity observed for wild-type spanning sequences in a collection of topologically characterized membrane proteins. J Biol Chem, 1994 Nov 18, 269(46), 28809 - 14 Two N-acetylglucosaminyltransferases catalyze the biosynthesis of heparan sulfate; Fritz TA et al.; We report that two N-acetylglucosaminyltransferases catalyze the biosynthesis of heparan sulfate in Chinese hamster ovary cells . The first enzyme initiates heparan sulfate biosynthesis and can be measured by the transfer of GlcNAc from UDP-GlcNAc to GlcUA beta 1-3Gal beta 1-O-naphthalenemethanol . The second enzyme catalyzes the polymerization of heparan sulfate and can be measured by the transfer of GlcNAc from UDP-GlcNAc to the nonreducing terminal GlcUA present in oligosaccharide fragments prepared from the Escherichia coli K5 capsular polysaccharide, N-acetylheparosan . Kinetic characterization of the initiating GlcNAc-transferase (alpha-GlcNAc-TI) indicates an apparent Km for UDP-GlcNAc of 36 +/- 4 microM . The apparent Km for UDP-GlcNAc of the polymerizing GlcNAc-transferase (alpha-GlcNAc-TII) is 230 +/- 30 microM . Both enzymes have broad pH optima and require a divalent cation for activity . alpha-GlcNAc-TI can use both Mn2+ and Ca2+, while alpha-GlcNAc-TII will use only Mn2+ . Chinese hamster ovary cells deficient in the synthesis of heparan sulfate and lacking alpha-GlcNAc-TII activity and S49 Thy 1-a lymphoma cells deficient in alpha GlcNAc addition to phosphatidylinositol have wild-type alpha-GlcNAc-TI activity . Thus, distinct alpha-GlcNAc-transferases catalyze the initiation and polymerization of heparan sulfate. J Biol Chem, 1994 Nov 18, 269(46), 28790 - 7 Primary structure and functional expression of human Glycyl-tRNA synthetase, an autoantigen in myositis; Ge Q et al.; Two identical cDNAs encoding human glycyl-tRNA synthetase were isolated from K562 and HEL cell lambda gt11 libraries by screening with "anti-EJ" autoantibodies from a patient with dermatomyositis, previously shown to specifically inhibit this enzyme . The sequenced cDNA had 2,385 base pairs, with a predicted protein of 685 amino acids and M(r) 77,500 . The antigen reactive with anti-EJ was immunoaffinity-purified from HeLa cells, and two proteolytic peptides matched areas of the predicted amino acid sequence . The sequence was 61.6% identical with that of silk moth glycyl-tRNA synthetase . Several very highly conserved regions were identified, including the single class II synthetase motif region and an N-terminal region similar to one found in at least three other synthetases . Near identity in other regions suggested that they also had important functions . Expression of the cDNA in Escherichia coli yielded a protein of expected size that reacted with the autoantibodies and was active as glycyl-tRNA synthetase. J Biol Chem, 1994 Nov 18, 269(46), 28777 - 82 Contribution to antibody-antigen interaction of structurally perturbed antigenic residues upon antibody binding; Tsumoto K et al.; For elucidating the contribution of structurally perturbed antigenic residues upon antibody binding to antigen-antibody interaction, the interaction between hen egg white lysozyme (HEL) and HyHEL10 Fv fragment, which is one of several monoclonal antibodies against HEL and structurally well defined (Padlan, E.A., Silverton, E . W., Sheriff, S., Cohen, G . H., Smith-Gill, S . J., and Davies, D . R . (1989) Proc . Natl . Acad . Sci . U . S . A . 86, 5938-5942), was investigated . Asp-101 and Trp-62 of HEL, whose conformations are perturbed by the binding of antibody HyHEL10 in this interaction, were replaced with Gly, and the resulting interactions were studied by assay of the inhibition of the lysozyme activity with the Fv fragment and by titration calorimetry . The results can be summarized as follows . 1) It was possible to prepare the fully functional Fv fragment of HyHEL10 using a secretory expression system in Escherichia coli . Its inhibition profile for HEL activity was almost indistinguishable from that of HyHEL10 IgG, and the contribution of enthalpy to driving the interaction was shown to be significant . 2) A thermodynamic study of the interaction between the D101G mutant HEL and the Fv fragment revealed that, although the negative enthalpy change was smaller than that for the wild type, the Gibbs energy was almost identical to that of the wild type, which resulted from the smaller entropy loss . 3) Study of the interaction between the W62G mutant HEL and this Fv fragment indicated that the rotation of the Trp-62 indole ring upon binding of the antibody made an enthalpic contribution to antibody-antigen interaction, although Trp-62 of HEL was proposed not to be the direct contact residue in the HyHEL10.HEL complex . 4) From these results, it was confirmed experimentally that structural perturbations of antigenic residues upon antibody binding of antigen would contribute to the gain of enthalpic energy, in spite of partial offset by entropic loss, and to driving the interaction. J Biol Chem, 1994 Nov 18, 269(46), 28724 - 31 Random chimeragenesis of G-protein-coupled receptors . Mapping the affinity of the cAMP chemoattractant receptors in Dictyostelium; Kim JY et al.; G-protein-coupled receptors mediate a wide variety of responses to extracellular stimuli in eucaryotic cells . Binding of the ligand to these receptors is thought to involve contacts within a pocket from the sequences of these genes . A family of four surface cAMP receptors that mediate responses to secreted cAMP coordinates the developmental program of Dictyostelium . A large difference in affinity for cAMP exists between cAR1 (25 and 230 nM) and cAR2 (> 5 microns) . To understand the basis for this affinity difference, we generated an extensive series of cAR1/cAR2 and cAR2/cAR1 chimeras using a technique designated "random chimeragenesis." When a linearized plasmid was transformed into Escherichia coli, tandemly positioned cAR1 and cAR2 genes crossed over at homologous regions . The cAMP binding properties and EC50 values for agonist-induced phosphorylation of each of the chimeras were characterized in order to map the domains that determine the affinity . These studies implicated a domain in the second extracellular loop in which only 5 residues differ between the two receptors as the major determinant of affinity . A secondary domain including residues 110-147 (11 residue differences) was identified as a minor determinant of affinity. J Biol Chem, 1994 Nov 18, 269(46), 28670 - 5 Regulation of lipid polymorphism is essential for the viability of phosphatidylethanolamine-deficient Escherichia coli cells; Rietveld AG et al.; Escherichia coli strain AD93 is unable to synthesize the nonbilayer lipid phosphatidylethanolamine and requires high concentrations of specific divalent cations for growth . Previous studies suggested that in this strain, cardiolipin in combination with divalent cations functionally replaces phosphatidylethanolamine, reflecting polymorphic regulation of membrane lipid composition . However, it is also possible that divalent cations are required for regulation of lipid packing or membrane surface potential . 2H NMR was employed to measure the effect of different divalent cations on lipid packing in aqueous dispersions of lipid extracts isolated from AD93 and the wild type parental strain W3899, which were grown with {11,11-2H2}oleic acid . The results indicate that a range of acyl chain order is compatible with growth and that Ba2+, which cannot support growth of AD93, can increase chain packing to the wild type level . By means of microelectrophoresis, it was shown that the growth-promoting cations and Ba2+ have a strong and comparable ability to screen the surface charge of large unilamellar vesicles prepared from AD93 lipid extracts . Therefore, it is unlikely that the growth-promoting capacity of divalent cations is primarily due to their effect on lipid packing or their potency to decrease the surface potential . Furthermore, the addition of small amounts of Ba2+ to a AD93 lipid dispersion with excess Mg2+ diminished HII phase formation . This observation can explain the growth arrest in AD93 cultures upon the addition of Ba2+ and further supports the conclusion that the cation requirement of this strain arises mainly from polymorphic regulation of lipid composition. J Biol Chem, 1994 Nov 18, 269(46), 28629 - 34 The manganese superoxide dismutase of Escherichia coli K-12 associates with DNA; Steinman HM et al.; Superoxide dismutases (SODs) are vital components in the resistance of aerobic organisms to the toxicity of oxygen . Escherichia coli contains two highly homologous cytoplasmic SODs, a manganese- and an iron-containing enzyme (MnSOD, FeSOD) . We previously demonstrated that MnSOD and FeSOD have different physiological functions and that MnSOD is more effective in preventing oxidative damage to DNA . In this report, purified E . coli MnSOD was shown to bind nonspecifically to DNA by electrophoretic mobility shift assay and nitrocellulose-filter binding methodologies . From electrophoretic mobility shift assay, the equilibrium dissociation constants for interaction with a variety of double-stranded and single-stranded oligonucleotides ranged from 1.5 +/- 0.2 to 8.4 +/- 1.3 microM at 20 degrees C . This range of concentrations corresponds to MnSOD concentrations in aerobically grown E . coli . In vivo binding of MnSOD to DNA was supported by colocalization of MnSOD and the E . coli nucleoid in immunoelectron microscopy . Both MnSOD and DNA were inhomogeneously distributed in the cytosol, the concentration of each being higher in the center of the cell and relatively low near the inner membrane . In contrast, there was no evidence for physiologically relevant interaction of FeSOD with DNA . Binding to DNA in vitro was weak, Kd > 40-220 microM, concentrations 7-40 times higher than found in vivo . In addition, the cytoplasmic distribution of FeSOD did not correlate with DNA . FeSOD concentration was higher near the inner membrane and lower in the center of the cytosol . These results demonstrate that E . coli MnSOD associates with DNA in vitro and in vivo . Combined with prior data demonstrating that MnSOD preferentially protects DNA in vivo while an equal enzymatic activity of FeSOD does not (Hopkin, K . A., Papazian, M . A., and Steinman, H . M . (1992) J . Biol . Chem . 267, 24253-24258), our data suggest that E . coli MnSOD acts as a "tethered antioxidant"; association of MnSOD with DNA localizes dismutase activity near a target of oxidative stress and increases protection of DNA from oxidative damage . This model has implications for the therapeutic use of SODs as antioxidants. J Biol Chem, 1994 Nov 18, 269(46), 28584 - 90 Binding of a new Ca2+ sensitizer, levosimendan, to recombinant human cardiac troponin C . A molecular modelling, fluorescence probe, and proton nuclear magnetic resonance study; Pollesello P et al.; The binding of a new calcium sensitizer, levosimendan, to human cardiac troponin C (cTnC) is described . Fluorescence studies done on dansylated recombinant human cTnC and a site-directed mutant showed that levosimendan modulated the calcium-induced conformational change in cTnC, and revealed the role of Asp-88 in the binding of the drug to the NH2-terminal domain of cTnC . Furthermore, NMR studies performed on the NH2-terminal fragment of cTnC showed a spatial proximity between levosimendan and Met81, Met85, and Phe77 in the drug-protein complex . These data were used to build an optimized model of the drug-protein complex, in which levosimendan binds cTnC at the hydrophobic pocket of the NH2-terminal domain . The role of the binding of levosimendan to cTnC in the pharmacological action of this drug in vivo is discussed. Gene, 1994 Nov 18, 149(2), 363 - 6 Cloning and sequence analysis of the gene encoding human lymphocyte prolyl endopeptidase; Vanhoof G et al.; The human cDNA encoding prolyl endopeptidase, a cytoplasmic endoprotease which hydrolyses the peptide bond at the C-terminal side of proline, was sequenced . After the isolation of the 3' terminal fragment of the pep cDNA sequence from a human lymphocyte cDNA library, an approach based on the polymerase chain reaction (PCR) was undertaken to obtain the complete pep cDNA . Overlapping DNA fragments were generated by PCR from cDNA synthesized from human lymphocyte mRNA . The DNA fragments were subcloned and sequenced . The complete cDNA is 2562 nucleotides (nt) in length and contains an open reading frame coding for a protein of 710 amino acids (aa) . Comparison of the primary PEP sequences from human lymphocyte and pig brain shows 97% identify . The aa sequence analysis shows homology with bacterial PEPs and with protease II from Escherichia coli . Asp641 probably participates in the active site of PEP. Gene, 1994 Nov 18, 149(2), 261 - 5 Characterization of the gene encoding the iron-sulfur protein subunit of succinate dehydrogenase from Drosophila melanogaster; Au HC et al.; The iron-sulfur protein (Ip) subunit of succinate dehydrogenase (and complex II of the electron transport chain) is highly conserved in evolution {Gould et al., Proc . Natl . Acad . Sci . USA 86 (1989) 1934-1938} . We have cloned the Drosophila melanogaster Ip-encoding gene (SdhB) by genomic library screening using the human Ip-encoding cDNA as a probe at low stringency . A 2.7-kb fragment containing SdhB has been sequenced and shown to comprise the entire transcribed region and more than 900 bp of promoter region . The gene contains three exons and two small introns of 272 and 56 nt, respectively, and is transcribed into an mRNA of 1205 nt (plus poly(A) tail) . The deduced amino-acid (aa) sequence shows strong similarities with Ip peptides from Escherichia coli, yeasts, plants and mammals, with 100% aa identity around the three Cys clusters which form the non-heme iron-sulfur centers . In situ hybridization on polytene chromosomes maps SdhB to band 42D 1-5 on the right arm of the second chromosome next to the centromere . Developmental and tissue-specific Northern blots show a single transcript of 1.3 kb in all tissues . However, its abundance varies during development and in the major body segments of the adult fly . Pupae have very low levels of transcript, in contrast to larvae . It is most abundant in the adult thorax and low in abdominal tissues. Gene, 1994 Nov 18, 149(2), 193 - 201 Generation of long flavivirus expression cassettes by in vivo recombination and transient dominant selection; Yamshchikov VF et al.; Assembly of expression cassettes coding for large segments of viral polyproteins is often complicated or impossible due to the instability of the resulting recombinant (re-) plasmids during propagation in Escherichia coli . Using the transient dominant selection approach described for the construction of vaccinia virus recombinants (re-VV), we have constructed several intermediate vectors and developed a procedure which enables direct assembly of long expression cassettes in the VV genome by in vivo recombination and does not require preliminary assembly of long cassettes in intermediate plasmids, thus eliminating the instability problems . The procedure was used to construct re-VV carrying fragments of the West Nile (WN), Murray Valley encephalitis (MVE), tick-borne encephalitis (TBE) and dengue type-2 (DEN2) viral genomes . Using this procedure, we have assembled a WN expression cassette which represents 86% of the WN genome and codes for 91% of its polyprotein and constitutes the longest flavivirus (FV) expression cassette inserted so far into the VV genome . Analysis of FV protein expression from the obtained recombinants indicates that recombination occurs with a high degree of specificity and the ORF remains intact . The procedure described offers a possible approach for the assembly of infectious cDNA clones. Cell, 1994 Nov 18, 79(4), 629 - 38 Starvation in vivo for aminoacyl-tRNA increases the spatial separation between the two ribosomal subunits; Ofverstedt LG et al.; Structures in situ of individual ribosomes in E . coli have been determined by computer-aided electron microscope tomography using a tilt series of positively stained embedded cellular sections . Amino acid starvation of a bacterial culture, causing a deficiency for aminoacyl-tRNA, induces a spatial separation between the ribosomal subunits compared with ribosomes in exponentially growing cells . Eight ribosomes from each growth condition were aligned to each other, and the two average structures were determined . Comparison of these suggests that the distance between the two subunits increases by approximately 3 nm upon starvation for aminoacyl-tRNA during protein synthesis . Ribosomes in most other states of the translational elongation cycle in exponentially growing cells show a more compact structure than previously realized. J Mol Biol, 1994 Nov 18, 244(1), 74 - 85 Mutational and structural analysis of the RNA binding site for Escherichia coli ribosomal protein S7; Dragon F et al.; Ribosomal protein S7 binds to a small RNA fragment of about 100 nucleotides within the lower half of the 3' major domain of E . coli 16 S rRNA . This fragment (D3M) comprises two large internal loops, A and B, connected by helix 29, a six-base-pair helix containing a G.U pair . Two hairpins with non-canonical base-pairs, 42' and 43, protrude from loops A and B, respectively . We used site-directed mutagenesis and molecular probing to further define which parts of D3M are important for S7 binding . Changing the stem of hairpin 42' into a Watson-Crick helix did not affect S7 binding, indicating that the non-canonical pairs of 42' do not provide recognition features for S7 . However, deletion of this hairpin decreased S7 binding affinity by about threefold and altered the conformation of loop A . Deletion of the upper part of hairpin 43 (the loop and the adjacent four base-pairs) did not affect S7 binding, whereas the lower part of this hairpin (three base-pairs) was found to be required for proper S7 binding . Moreover, replacing the U.G pair with a C.G pair in this lower part decreased S7 binding affinity by twofold, suggesting that the U.G pair is a recognition signal for S7 . S7 binding was also affected by mutations in helix 29 . Insertion of one nucleotide 5' to the G or 3' to the U of the G.U pair decreased S7 binding affinity by about threefold and twofold, respectively, whereas replacement of the G.U pair by a G.C pair enhanced the affinity about twofold, and lengthening the helix by inserting a C.G pair upstream from the G.U pair had no effect . Taken together, these results are consistent with a bipartite binding site for S7 on 16 S rRNA, involving two regions of interaction: one centered around helix 29 and extending on the adjacent part of loop A, and the other one centered around the lower part of hairpin 43 and probably extending on the adjoining part of loop B. J Biol Chem, 1994 Nov 18, 269(46), 29112 - 20 Human mitochondrial transcription termination exhibits RNA polymerase independence and biased bipolarity in vitro; Shang J et al.; Human mitochondrial 16 S rRNA 3'-end formation requires a tridecamer template sequence and a trans-acting protein of approximately 34 kDa . This protein binds tightly to its target sequence and further analysis of the protein-DNA complex revealed that the DNA is bent . Either T3, T7, Escherichia coli, or yeast mitochondrial RNA polymerase produced transcripts mapping at this termination site . With these heterologous RNA polymerase, RNA 3'-end formation was detected only in the transcription polarity opposite that of mitochondrial rRNA synthesis; the efficiency of termination in the homologous human RNA polymerase system is approximately 2-fold greater in this same opposite polarity . These results suggested the possible importance of biased bipolar transcription termination in vivo . For wild-type mtDNA, the apparent relative efficiency of termination in vivo reflected the values determined in vitro . Examination of a pathogenic human mtDNA mutation known to result in impaired termination in vitro showed no significant differences in relative transcript abundances in vivo, despite a loss of in vitro termination efficiency in both directions . Recently, six additional mitochondrial disease-associated point mutations have been reported that cluster at the human mitochondrial transcription termination site . None of these resulted in significantly impaired transcription termination in vitro. Biochim Biophys Acta, 1994 Nov 17, 1215(1-2), 115 - 20 Cloning, expression and partial characterization of a novel rat phospholipase A2; Chen J et al.; We report the cloning of a novel rat cDNA encoding a Ca(2+)-dependent, low molecular weight phospholipase A2 (PLA2) . A rat RNA blot hybridized with the cDNA exhibited a putative 2.4 kb transcript in heart . When the cDNA was expressed in human 293s cells, PLA2 activity accumulated in the culture medium . This conditioned medium optimally hydrolyzed the phospholipids of {1-14C}oleate-labeled Escherichia coli at neutral to alkaline pH with 10 mM or greater Ca2+ . When single substrates were tested, L-alpha-palmitoyl-2-oleoyl phosphatidylcholine was more efficiently hydrolyzed than L-alpha-1-palmitoyl-2-arachidonyl phosphatidylcholine, L-alpha-1-palmitoyl-2-arachidonyl phosphatidylethanolamine or L-alpha-1-stearoyl-2-arachidonyl phosphatidylinositol. Biochim Biophys Acta, 1994 Nov 16, 1209(1), 95 - 100 Site-directed mutagenesis of human ferrochelatase: identification of histidine-263 as a binding site for metal ions; Kohno H et al.; In nature, ferrochelatase catalyzes the insertion of ferrous ion into the porphyrin macrocycle of protoporphyrin IX to exclude two protons to form protoheme IX: other porphyrin substrates, including mesoporphyrin IX may be used in vitro . Based on the deduced amino-acid sequences, one histidine residue (H263 of human enzyme) is conserved among all ferrochelatases cloned from human to bacterial cells, and three histidine residues (H157, H341 and H388 of human enzyme) are conserved among eukaryotic ferrochelatases; no cysteine residue is conserved . To attempt to clarify the binding site of ferrous ion, we converted four highly conserved histidine residues in human ferrochelatase to alanine, using site-directed mutagenesis . The mutant enzymes were expressed in Escherichia coli, and iron- and zinc-chelating activities were examined . Mutants H157A and H388A lost most of their activities and concomitantly the enzyme became susceptible to proteolytic degradation . Kinetic studies with the residual activities showed no significant change of Km values for metal ions or for mesoporphyrin IX . Mutation at H341 did not alter the enzyme activities . Iron- and zinc-chelating activities of mutant H263A were reduced to 30% and 21% of the activities of the wild type, respectively . Moreover, this mutation resulted in 18- and 3.4-fold increases in Km values toward ferrous and zinc ions, respectively, while the Km value for mesoporphyrin remained unchanged . These results indicate that the binding site for metal ions in ferrochelatase is distinct from that for the porphyrin, and suggest that histidine-263 contributes significantly to the binding of metal ions . Maintenance of the structure of the protein molecule may involve functions related to histidine-157 and -388. Biochim Biophys Acta, 1994 Nov 16, 1209(1), 83 - 8 The guanidine-induced conformational changes of the chaperonin GroEL from Escherichia coli . Evidence for the existence of an unfolding intermediate state; Mizobata T et al.; Equilibrium unfolding experiments of the E . coli chaperonin GroEL were performed in guanidine hydrochloride . A reversible unfolding intermediate was observed in very low concentrations of denaturant (< 0.5 M guanidine hydrochloride) . This intermediate was characterized by a decreased light scattering intensity and an increased binding of the fluorescent probe 1-anilino-8-naphthalene sulfonate . No significant changes in circular dichroism spectra were observed for this unfolding intermediate . A second decrease in fluorescence intensity and light scattering was observed in higher concentrations of guanidine hydrochloride, with a transitional midpoint of 1.15 M . This transition was accompanied by the complete loss of secondary structure, as monitored by circular dichroism spectroscopy . This second transition agreed well with the results previously reported in this journal (Price et al . (1993) Biochim . Biophys . Acta 1161, 52-58). Biochim Biophys Acta, 1994 Nov 16, 1209(1), 40 - 50 Interactions of L-serine at the active site of serine hydroxymethyltransferases: induction of thermal stability; Bhaskar B et al.; Serine hydroxymethyltransferase (SHMT), EC 2.1.2.1, exhibits broad substrate and reaction specificity . In addition to cleaving many 3-hydroxyamino acids to glycine and an aldehyde, the enzyme also catalyzed the decarboxylation, transamination and racemization of several substrate analogues of amino acids . To elucidate the mechanism of interaction of substrates, especially L-serine with the enzyme, a comparative study of interaction of L-serine with the enzyme from sheep liver and Escherichia coli, was carried out . The heat stability of both the enzymes was enhanced in the presence of serine, although to different extents . Thermal denaturation monitored by spectral changes indicated an alteration in the apparent Tm of sheep liver and E . coli SHMTs from 55 +/- 1 degrees C to 72 +/- 3 degrees C at 40 mM serine and from 67 +/- 1 degrees C to 72 +/- 1 degrees C at 20 mM serine, respectively . Using stopped flow spectrophotometry k values of (49 +/- 5) x 10(-3) s-1 and (69 +/- 7) x 10(-3) s-1 for sheep liver and E . coli enzymes were determined at 50 mM serine . The binding of serine monitored by intrinsic fluorescence and sedimentation velocity measurements indicated that there was no generalized change in the structure of both proteins . However, visible CD measurements indicated a change in the asymmetric environment of pyridoxal 5'-phosphate at the active site upon binding of serine to both the enzymes . The formation of an external aldimine was accompanied by a change in the secondary structure of the enzymes monitored by far UV-CD spectra . Titration microcalorimetric studies in the presence of serine (8 mM) also demonstrated a single class of binding and the conformational changes accompanying the binding of serine to the enzyme resulted in a more compact structure leading to increased thermal stability of the enzyme. Biochim Biophys Acta, 1994 Nov 16, 1209(1), 10 - 8 Measurement of subnanomolar retinoic acid binding affinities for cellular retinoic acid binding proteins by fluorometric titration; Norris AW et al.; Cellular retinoic acid binding protein I (CRABP-I) and cellular retinoic acid binding protein II (CRABP-II) are small, cytoplasmic proteins which bind all-trans-retinoic acid with high affinity . Both of these proteins belong to a family of intracellular proteins which bind amphiphilic lipids, including fatty acids, bile salts, and retinoids . Because CRABP-I and -II exhibit different tissue distributions and differential transcriptional regulation, they are proposed to serve different functions . The binding properties of mouse CRABP-I and -II purified from Escherichia coli were examined to further understand their role in intracellular retinoic acid processing . Fluorescence titrations were performed using nanomolar protein concentrations, near the obtained dissociation constants, and analyzed by direct mathematical fitting to raw data, in order to extend the range and accuracy of binding constant determination . The apparent dissociation constants, K'd, of mouse CRABP-I and CRABP-II binding all-trans-retinoic acid were determined to be 0.4 +/- 0.3 nM and 2 +/- 1 nM respectively, stronger binding than previously reported . The K'd of mCRABP-I and mCRABP-II complexing with acitretin, a pharmacologically active synthetic retinoid used in the treatment of psoriasis, was 3 +/- 1 nM and 15 +/- 11 nM . Both CRABPs bound 9-cis-retinoic acid with a K'd of roughly 200 nM, and neither exhibited significant binding of 13-cis-retinoic acid. FEMS Microbiol Lett, 1994 Nov 15, 124(1), 93 - 8 Regulation of Escherichia coli K5 capsular polysaccharide expression: evidence for involvement of RfaH in the expression of group II capsules; Stevens MP et al.; Expression of the Escherichia coli K5 antigen was used as a model system to study the role of known regulators of gene expression on production of group II capsules in E . coli . Only mutations in the rfaH gene had an effect on production of the K5 antigen, abolishing the expression of any detectable capsule at 37 degrees C . None of the mutations studied induced capsule expression at 18 degrees C . A sequence, termed JUMPstart, found in group II capsule gene clusters and upstream of a number of polysaccharide biosynthesis genes in enteric bacteria is homologous to sequences found in RfaH regulated operons . This may indicate a common mode of regulation of these polysaccharide biosynthesis genes by RfaH. Biochem J, 1994 Nov 15, 304 ( Pt 1), 61 - 8 Interaction of procollagen I and other collagens with colligin; Jain N et al.; Colligin is a collagen-binding glycoprotein of molecular mass 46000 Da localized to the endoplasmic reticulum (ER) of diverse kinds of cells that produce collagen I . In order to help define its role in collagen biosynthesis and to study the interaction of colligin with procollagen I in detail, the binding characteristics of colligin purified from L6 myoblasts have been studied . A total of 3 mol were found to bind/mol of procollagen I, with a Kd of about 25 nM . Both pure and separated pro alpha 1(I) and procollagen alpha 2 (I) chains were able to compete with procollagen I for binding to colligin . However, colligin binds to pro alpha 2 (I) with higher affinity than to pro alpha 1 (I) . To find if the binding activity of colligin was altered during purification, an assay to measure colligin binding to procollagen in crude myoblast cell extracts was developed . This procedure gave the same binding parameters as did the highly purified colligin . Among different collagen types, colligin was found to bind to collagen I and collagen IV, but not to collagen III . In order to examine whether glycosylation or phosphorylation of colligin were required for the binding of colligin to procollagen I and to obtain enough colligin for further studies, recombinant protein was produced in Escherichia coli . An immunoaffinity purification scheme was used to get virtually pure protein in milligram yields . Comparison of the recombinant colligin with that isolated from L6 myoblasts showed that both types existed in solution as monomers and dimers . In addition, both types of colligins showed identical properties with regard to their binding to procollagen I and the isolated pro alpha 1(I) and pro alpha 2(I) chains . Post-translational modifications of colligin were thus not essential for binding to procollagen I. Biochem Biophys Res Commun, 1994 Nov 15, 204(3), 1119 - 24 Efficient unwinding of triplex DNA by a DNA helicase; Maine IP et al.; Sequence specific triple helix formation shows promise as a strategy for gene-specific inhibition of gene expression by blocking promoters or enhancers . Therefore, it is important to understand how this unusual structure affects DNA metabolic processes other than transcription . It has been shown that triplexes block in vitro DNA synthesis catalyzed by purified DNA polymerases . We report here that a purified DNA helicase unwinds a triple helical substrate with an efficiency similar to that observed with a comparable duplex species . These model studies suggest that triple helices will not seriously inhibit DNA replication or recombination in vivo, since DNA polymerases are preceded by helicases in the fully assembled replication holoenzyme. Arch Biochem Biophys, 1994 Nov 15, 315(1), 74 - 81 Preparation and characterization of Mn-salophen complex with superoxide scavenging activity; Liu ZX et al.; Mn(III)-salophen complex with superoxide scavenging activity was prepared from manganese(III) acetate dihydrate and salophen in ethanol . Visible absorption spectrum of the red-brown complex exhibits a shoulder at 430 nm which was absent with either salophen or manganic acetate alone . Titration of salophen with manganese(III) is consistent with a 1:1 Mn to salophen stoichiometry of the complex based on changes in the absorbance at 500 nm or of superoxide scavenging activity . The superoxide dismutase (SOD)-like activity of the complex in the xanthine-xanthine oxidase/cytochrome c assay was 1450 units/mg salophen . The SOD activity of the complex was suppressed 50% in the presence of EDTA (1 mM), but was not altered in the presence of bovine serum albumin (1 mg/ml) or crude protein extract of Escherichia coli QC779 sodA-sodB- (1 mg/ml) . E . coli QC779 sodA-sodB- grew scantily after an 8-h lag phase in aerobic M63 glucose minimal medium . The aerobic growth of the E . coli SOD double mutant in glucose minimal medium was greatly enhanced in the presence of 5 or 10 microM Mn-salophen complex compared to that of control after 24 h incubation . Mn-desferal green complex (10 microM) and pink complex (5 microM) also increased growth rate of E . coli QC779 sodA-sodB- but to a lesser extent than Mn-salophen complex . However, the growth was completely inhibited by 50 microM Mn-salophen complex, 100 microM Mn-desferal green complex, or 10 microM Mn-desferal pink complex. Arch Biochem Biophys, 1994 Nov 15, 315(1), 104 - 10 Heterologous expression of gamma E-crystallin produces protein with an aberrant tertiary structure; Goode D et al.; Expression of complete rat gamma E-crystallin cDNA in Saccheromyces cerevisiae and in Escherichia coli at 25 degrees C produced soluble proteins similar to rat gamma E-crystallin but with an altered tertiary structure as judged by tryptophan fluorescence . Expression of rat gamma E-crystallin cDNA in E . coli at 37 degrees C produced insoluble inclusion bodies . Refolding of denatured rat gamma E-crystallin from these inclusion bodies produced a protein similar to rat gamma E-crystallin but with altered secondary and tertiary structure, judged by tryptophan fluorescence and circular dichroism . The secondary structure of the refolded gamma-crystallin was similar in beta-sheet content to the native gamma-crystallin structure but was somewhat shifted from the native spectrum, suggesting some alteration in the relative position of the beta-sheets in the refolded structure . These results have implications for crystallin folding, in particular the importance of lens-specific factors (alpha-crystallin, low water content, redox potential) and crystallin hydration. Virology, 1994 Nov 15, 205(1), 290 - 9 In vivo expression and mutational analysis of the barley yellow dwarf virus readthrough gene; Filichkin SA et al.; The barley yellow dwarf virus (BYDV) coat protein gene is separated from an adjacent downstream open reading frame (ORF) by a single termination codon . Immunological analysis of this downstream "readthrough" region reveals multiple coat protein-readthrough products . A full-length 72-kDa (P72) coat protein-readthrough fusion product is detected in total lysates from infected cells . However, purified aphid transmissible virions contain only a 50-kDa (P50) coat protein-readthrough product . Virion-associated P50 lacks the C-terminal domain predicted by its ORF sequence . A separate 33-kDa polypeptide (P33) corresponding to the readthrough C-terminus domain is detected in the crude cellular membrane fraction . Site-directed and deletion mutational analysis demonstrate that the readthrough ORF is dispensable for BYDV replication and virion accumulation in protoplasts . In contrast, a mutant which results in a continuous fusion product of coat and readthrough sequences is not viable . Point mutations were used to map regions required for P50 and P72 synthesis . A model explaining the relationships between the three forms of the readthrough polypeptides is proposed. Virology, 1994 Nov 15, 205(1), 141 - 50 Processing of E1 and E2 glycoproteins of hepatitis C virus expressed in mammalian and insect cells; Matsuura Y et al.; Processing of the envelope glycoproteins (E1 and E2) of hepatitis C virus (HCV) was investigated by using cDNA clones covering the structural and part of the nonstructural (NS) protein regions . The cDNA clones expressed in mammalian and insect cells were immunoprecipitated by serum of a hepatitis C patient and by monoclonal and polyclonal antibodies raised against the recombinant proteins expressed in insect cells or Escherichia coli . The E2 protein expressed in both insect and mammalian cells was a glycoprotein of 60 kDa (gp60) and removal of the sugar residues by N-glycanase yielded 38- and 40-kDa proteins . Pulse-chase experiments revealed that efficient expression and processing of the envelope proteins required coexpression with the flanking core and NS2 proteins . Not only E1 and E2 proteins but also NS2 and NS3 proteins were coprecipitated by anti-E1 or anti-E2 monoclonal antibody in the cells infected with the recombinant baculovirus expressing structural and NS proteins (NS2 and NS3), while only the NS3 protein was precipitated by anti-NS3 antibody . The association of E1 and E2 proteins was not influenced by the presence of a reducing agent and was still observed in the cells coinfected with the deletion mutants lacking both internal and C-terminal hydrophobic regions of each protein . Furthermore, the truncated forms of the E1 and E2 proteins were secreted into the culture supernatant and some of them were still associated with each other. Eur J Biochem, 1994 Nov 15, 226(1), 99 - 107 Regulation of 70-kDa heat-shock-protein ATPase activity and substrate binding by human DnaJ-like proteins, HSJ1a and HSJ1b; Cheetham ME et al.; The DnaJ family of molecular chaperones is characterized by the presence of a highly conserved 70-amino-acid J domain . Escherichia coli DnaJ interacts with the 70-kDa heat-shock protein (DnaK), in vitro, to stimulate the 70-kDa heat-shock protein ATPase activity and modify substrate binding . The conservation of the interaction of DnaJ-like proteins with the 70-kDa heat-shock proteins has been demonstrated for the yeast protein YDJ1, a protein that shows full domain conservation with E . coli DnaJ . Human neurone-specific DnaJ-like proteins, HSJ1a and HSJ1b, possess a J domain and a glycine/phenylalanine-rich region in common with E . coli DnaJ, although the overall amino acid identity is less than 23% . We have investigated, in vitro, the interaction of HSJ1a and HSJ1b with the mammalian brain constitutive 70-kDa heat-shock protein (hsc70) . The weak intrinsic ATPase activity of the constitutive 70-kDa heat-shock protein is enhanced more than fivefold by stoichiometric amounts of both HSJ1a and HSJ1b . This enhancement is mediated by an increase in the rate of bound ATP hydrolysis, whereas the rate of ADP release is unaffected . HSJ1 proteins appear to regulate the affinity of the 70-kDa constitutive heat-shock protein for the permanently unfolded substrate, carboxymethylated alpha-lactalbumin . A recent report {Palleros, D . R., Reid, K . L., Shi, L., Welch, W . J . & Fink, A . L . (1993) Nature 365, 664-666} has suggested that substrate release by 70-kDa heat-shock proteins requires a conformational change in these proteins induced by K+ in concert with ATP binding . In the presence of ATP, HSJ1 proteins reduce 70-kDa constitutive heat-shock protein/carboxymethylated alpha-lactalbumin complex formation both in the presence and absence of K+ . This suggests that HSJ1 proteins induce a conformational change in the 70-kDa constitutive heat-shock protein that can mimic the effect mediated by K+ and therefore modulate 70-kDa heat-shock protein substrate release by another mechanism rather than merely stimulating the 70-kDa heat-shock protein ATPase activity . As HSJ1 proteins have limited similarity to DnaJ, we suggest that this action is being mediated by the J domain alone, and that this modulation of 70-kDa heat-shock-protein substrate binding will be common to all proteins that contain a J domain. Eur J Biochem, 1994 Nov 15, 226(1), 71 - 80 Purification and characterization of the poly(hydroxyalkanoic acid) synthase from Chromatium vinosum and localization of the enzyme at the surface of poly(hydroxyalkanoic acid) granules; Liebergesell M et al.; A recombinant strain of Escherichia coli, which overexpressed phaC and phaE from Chromatium vinosum, was used to isolate poly(3-hydroxyalkanoic acid) synthase . The isolation was performed by a two-step procedure including chromatography on DEAE-Sephacel and Procion Blue H-ERD . The poly(3-hydroxyalkanoic acid) synthase consisted of two different kinds of subunit (PhaC, M(r) 39,500 and PhaE, M(r) 40.500) . PhaC was separated from the poly(3-hydroxyalkanoic acid) synthase complex by chromatography on phenyl-Sepharose: PhaE was enriched by solubilization of protein inclusion bodies . The stoichiometry of PhaC and PhaE in the enzyme complex was not determined . The poly(3-hydroxyalkanoic acid) synthase (PhaEC) exhibited a native relative molecular mass of M(r) 400,000 and most probably consists of ten subunits . The Km value of the enzyme for D(-)-3-hydroxybutyryl-CoA was 0.063 mM . The enzyme synthesized poly(3-hydroxybutyric acid) in vitro from D(-)-3-hydroxybutyryl-CoA or, together with propionyl-CoA transferase in a coupled enzyme reaction, synthesized the same product from acetyl-CoA plus D(-)-3-hydroxybutyric acid . Antibodies were raised against both subunits of the poly(3-hydroxyalkanoic acid) synthase . By immunoelectron microscopy, the poly(3-hydroxyalkanoic acid) synthase was localized within the cytoplasm in cells of C . vinosum grown under non-storage conditions . In cells grown under poly(3-hydroxybutyric acid) storage conditions, the enzyme was observed to be located at the surface of the poly(3-hydroxybutyric acid) granules . Immunoblots with anti-PhaC, anti-PhaE IgG and crude extract proteins indicated that poly(3-hydroxyalkanoic acid) synthases with partial sequence similarities are widespread among purple sulphur bacteria. Eur J Biochem, 1994 Nov 15, 226(1), 219 - 26 Site-directed mutagenesis clarifies the substrate position within the three-dimensional model of the active site of herpes simplex virus type-1 thymidine kinase; Michael M et al.; Site-directed mutagenesis was used to experimentally verify the 3D model of the active site of herpes simplex virus type-1 thymidine kinase (HSV 1 TK) obtained by homology modelling . For this purpose, D215 and K317 were replaced by R and G, respectively, at homologous positions in the aciclovir-insensitive bovine herpes virus type-1 thymidine kinase (BHV 1 TK) . Wild-type and mutated enzymes were expressed in Escherichia coli using a gene fusion vector and purified to homogeneity . While both mutants had the same Km value for thymidine as the recombinant wild-type enzyme (0.2 microM), Vmax was decreased to 20-25% of the original wild-type value . The recombinant wild-type enzyme was inhibited by the substrate analogue aciclovir with a Ki of 146 microM . Both mutants were able to phosphorylate aciclovir to about the same extent as the wild-