Carbohydr Res, 1995 Jul 21, 272(1), 73 - 90 NMR reinvestigation of two N-acetylneuraminic acid-containing O-specific polysaccharides (O56 and O24) of Escherichia coli; Torgov VI et al.; Structures for the N-acetylneuraminic acid (Neu5Ac)-containing O56 and O24 polysaccharides of Escherichia coli have been reported previously . During these studies unusual chemical shifts had been observed for the NMR signals for H-3eq and C-3 of the Neu5Ac residues of both polysaccharides . In further pursuing this phenomenon, we have reinvestigated the O56 and O24 polysaccharides as well as derived oligosaccharides by one- and two-dimensional NMR spectroscopy . The results showed that structures of both polysaccharides (PSs) had to be modified and formulated as {formula: see text} 2D ROESY spectra revealed a strong NOE between H-3eq of Neu5Ac and the protons of the side-chain sugar (H-3 and H-5 of alpha-D-Gal p in the O56 PS and H-3 of alpha-D-Glc p in the O24 PS) and also between H-3ax of Neu5Ac and H-3 of beta-D-Glc p in the main chain . This indicated a close spatial association of the seven-linked alpha-Neu5Ac and the side-chain residues alpha-D-Gal p (O56 PS) and alpha-D-Glc p (O25 PS), respectively . The strong long-range spatial contacts caused the unusual chemical shifts of H-3eq and C-3 of Neu5Ac. J Mol Biol, 1995 Jul 21, 250(4), 407 - 19 Structural changes in base-paired region 28 in 16 S rRNA close to the decoding region of the 30 S ribosomal subunit are correlated to changes in tRNA binding; Ericson G et al.; Escherichia coli 30 S ribosomal subunits undergo a reversible change under low monovalent or divalent cation concentration and become inactive in tRNA binding and 50 S subunit association . In the inactive form, 16 S rRNA base-pairs (921-922).(1395-1396) and (923-925).(1391-1393), which are part of region 28, are unstable and an alternate arrangement, (921-923).(1532-1534), is detected by psoralen photochemical crosslinking . Site-directed mutagenesis has been used to investigate whether changes in base-paired region 28 or the alternate secondary structure is responsible for the inactivity of the subunit . 30 S subunits with the substitution C1533A or with deletion of nucleotides 1534 to 1542 can still be inactivated like the wild-type 30 S subunit . On the other hand, 30 S subunits that contain sequence changes in the 920 to 926 region show moderate to severe decreases in tRNA binding even under activating conditions . When 30 S subunits containing these mutations were subjected to chemical probing, they failed to show the normal hyper-reactivity of nucleotide G926 and, instead, reactivity was shifted to G925 or to G928, and G929 . Two mutations in the 920 region result in structures in which A1394 is base-paired rather than being unpaired as normal; deletion but not substitution of A1394 resulted in loss of tRNA binding activity and depression of the reactivity of G926 . Mutations were made to insert or delete a nucleotide at position 920 . The deletion mutant but not the insertion mutant has decreased tRNA binding activity and also low reactivity of G926 . We conclude that structural changes in region 28 account for the active/inactive difference in tRNA binding . Molecular models of region 28 were made using the program MC-SYM . Models that include a hydrogen bond interaction between A1394 and G1392 account for the G926 reactivity in the wild-type sequence and account for the effects of most of the mutations in changing the G926 reactivity. J Biol Chem, 1995 Jul 21, 270(29), 17394 - 9 A stable carbocyclic analog of 5-phosphoribosyl-1-pyrophosphate to probe the mechanism of catalysis and regulation of glutamine phosphoribosylpyrophosphate amidotransferase; Kim JH et al.; Glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase catalysis and regulation were studied using a new stable carbocyclic analog of PRPP, 1-alpha-pyrophosphoryl-2-alpha, 3-alpha-dihydroxy-4-beta-cyclopentane-methanol-5-phosphate (cPRPP) . Although cPRPP competes with PRPP for binding to the catalytic C site of the Escherichia coli enzyme, two lines of evidence demonstrate that cPRPP, unlike PRPP, does not promote an active enzyme conformation . First, cPRPP was not able to "activate" Cys1 for reaction with glutamine or a glutamine affinity analog . The ring oxygen of PRPP may thus be necessary for the conformation change that activates Cys1 for catalysis . Second, binding of cPRPP to the C site blocks binding of AMP and GMP, nucleotide end product inhibitors, to this site . However, the binding of nucleotide to the allosteric site was essentially unaffected by cPRPP in the C site . Since it is expected that nucleotide inhibitors would bind with low affinity to the active enzyme conformation, the nucleotide binding data support the conclusion that cPRPP does not activate the enzyme. Anal Biochem, 1995 Jul 20, 229(1), 99 - 105 Selection of high-affinity binding sites for sequence-specific, DNA binding proteins from random sequence oligonucleotides; Pierrou S et al.; We describe a rapid and sensitive method to isolate sets of high-affinity binding sites for sequence-specific DNA binding proteins . The DNA binding domain of the protein is expressed in Escherichia coli as a fusion with glutathione S-transferase (GST) . Binding reactions are set up with total soluble extract from induced bacteria and a double-stranded oligonucleotide for which the central 32 bp have been randomized . To ensure stringent conditions, binding is done in the presence of high levels of poly(dI:C) . The GST fusion protein is recovered by the addition of glutathione-Sepharose . Following extensive washing of the Sepharose beads, the bound oligonucleotides are rescued by polymerase chain reaction amplification . The amplified material is used in the next cycle of selection and amplification . Approximately five cycles are needed to obtain a pure population of high-affinity sites, which are then cloned and sequenced . This procedure should be applicable to any sequence-specific DNA binding protein for which the cDNA is available and which can be expressed in bacteria in a functional form. Biochim Biophys Acta, 1995 Jul 20, 1268(1), 81 - 7 Rotational asymmetry of Escherichia coli flagellar motor in the presence of arsenate; Welch M et al.; The flagellar motor of Escherichia coli (E . coli) is driven by a proton-motive force (PMF), hence it was of interest to determine whether the motor is symmetrical in the sense that it can be rotated by any polarity of PMF . For this purpose the cells had to be deenergized first . Conventional deenergization procedures caused irreversible loss of motility, presumably due to ATP-dependent degradative processes . However, E . coli cells deenergized by incubation with arsenate manifested a slow, reversible depletion of PMF . In this procedure there was a sufficiently long time window, during which a considerable proportion of the cells lost their motility and could be made to rotate again by an artificially-imposed PMF . The motors of these cells rotated in response to any PMF polarity, but positive and negative polarities rotated different sub-populations of cells and the direction was almost exclusively counterclockwise . The reason for the unidirectionality of the rotation was not the intervention of the chemotaxis system . A number of potential reasons are suggested . One is the arsenate effect on the motor function found previously {Margolin, Y., Barak, R . and Eisenbach, M . (1994) J . Bacteriol . 176, 5547-5549} . A possible interaction between arsenate and the motor is discussed. Nature, 1995 Jul 20, 376(6537), 274 - 7 Three-dimensional structure of the kinesin head-microtubule complex; Kikkawa M et al.; Kinesin is a microtubule (MT)-associated 'motor' molecule fundamental to organelle transport . Recently, various kinesin superfamily members (KIFs) have also been identified and suggested as being responsible for the transport of specific organelles . Kinesin is a heterotetramer composed of two heavy chains and two light chains . The heavy chains form two globular heads, a rod and a fan-like tail completed by the light chains . The globular head, which is composed of approximately 340 amino-terminal residues of the heavy chain, includes both ATP-binding and MT-binding domains, and its recombinant protein also has these properties . To improve the understanding of the mechanism of force generation by an MT-based molecular motor, kinesin, we report here the three-dimensional structure of the complex of a recombinant kinesin head and MTs, as revealed by helical reconstruction from cryo-electron micrographs . A kinesin head is a globular teardrop-like structure binding to the ridge of one protofilament of MTs . We have determined the polarity of the structure of the complex of MTs and the kinesin head in relation to MT polarity. Nature, 1995 Jul 20, 376(6537), 260 - 3 A novel receptor involved in T-cell activation; Cocks BG et al.; Optimal T-cell activation and T-cell expansion require triggering by T-cell antigen receptors and co-stimulatory signals provided by accessory cells . A major co-stimulatory pathway involves crosslinking the CD28 molecule on T cells by its ligands CD80 or CD86 expressed on antigen-presenting cells . But recent studies on CD28-deficient mice have indicated that CD28 is not required for all T-cell responses and that additional T-cell co-stimulatory pathways exist . Here we describe a novel glycoprotein, of relative molecular mass 70,000 (M(r) 70K), designated SLAM, that belongs to the immunoglobulin gene superfamily, which is involved in T-cell stimulation . SLAM is constitutively expressed on peripheral-blood CD45ROhigh memory T cells, T-cell clones, immature thymocytes, and a proportion of B cells, and is rapidly induced on naive T cells after activation . Engagement of SLAM enhances antigen-specific proliferation and cytokine production by T cells carrying the CD4 antigen (CD4+) . Particularly, the production of interferon-gamma (IFN-gamma) is strongly upregulated, even in T helper type 2 (Th2) CD4+ T-cell clones, whereas no induction of interleukin (IL)-4 or IL-5 production was observed in Th1 clones . In addition, the engagement of SLAM induces directly the proliferation of CD4+ T-cell clones and preactivated T cells, in the absence of any other stimuli, and without CD28 involvement . Thus SLAM is a novel receptor on T cells that, when engaged, potentiates T-cell expansion in a CD28-independent manner and induces a Th0/Th1 cytokine production profile. Nature, 1995 Jul 20, 376(6537), 230 - 5 Crystal structure of a complex between interferon-gamma and its soluble high-affinity receptor; Walter MR et al.; The crystal structure of interferon-gamma bound to the extracellular fragment of its high-affinity cell-surface receptor reveals the first view of a class-2 cytokine receptor-ligand complex . In the complex, one interferon-gamma homodimer binds two receptor molecules . Unlike the class-1 growth hormone receptor complex, the two interferon-gamma receptors do not interact with one another and are separated by 27 A . Upon receptor binding, the flexible AB loop of interferon-gamma undergoes a conformational change that includes the formation of a 3(10) helix. Mol Cell Biochem, 1995 Jul 19, 148(2), 105 - 13 Partial characterization of the RNA from LPS-stimulated macrophages that induces the release of chemotactic cytokines by resident macrophages; Ribeiro RA et al.; It is well established that exogenous RNA is incorporated into eukaryotic cells and is able to exert various biological responses . Little, however, is known about the effects of such RNA on macrophages . In this study, we demonstrate that RNA extracted from macrophages stimulated with Escherichia coli lipopolysaccharide (LPS), referred to as L-RNA, in contrast to RNA from non-stimulated macrophages (N-RNA), induces the release of a macrophage-derived neutrophil chemotactic factor (MNCF) and interleukin-8 (IL-8) from macrophage monolayers . The effect of L-RNA was dependent of the integrity of the polynucleotide chain and was not due to LPS contamination since its ability to induce MNCF and IL-8 release was strongly reduced by RNase but was not affected by DNase or polymyxin B . The poly A(+)L-RNA and poly A(-)L-RNA fractions were able to induce the release of MNCF and IL-8, indicating that the L-RNA could be acting at transcriptional and translational levels . The demonstration that actinomycin-D and cycloheximide inhibited the release of MNCF and IL-8 by L-RNA-stimulated macrophages confirms this assumption . Fractionation of the total L-RNA by centrifugation on a 5-20% sucrose gradient showed that the L-RNA which sediments in the 4-5S region of the gradient is the only fraction capable of inducing the release of MNCF from naive macrophages . We have previously shown that macrophage monolayers stimulated with interleukin-1 beta or LPS release a low molecular RNA which also sediments in the same 4-5S region . Taken together, these results support our proposal that resident macrophages, when activated by injurious stimuli, in addition to secreting cytokines, also release a low molecular weight (4-5S) RNA which may act on the surrounding macrophages to further stimulate the release of cytokines . This process would amplify the inflammatory response and would increase the mechanisms involved in the defense response or tissue injury. Biochim Biophys Acta, 1995 Jul 19, 1250(2), 197 - 203 Identification of two specific lysines responsible for the inhibition of phospholipase A2 by manoalide; Bianco ID et al.; Manoalide, a natural product of sponge, irreversibly inhibits phospholipase A2 (PLA2) by reacting with lysine residues . Cobra venom PLA2 mutants were constructed in which four of the six lysine residues were independently replaced by arginine or methionine, which cannot react with manoalide . The mutants were overexpressed in Escherichia coli, renatured, and purified . The enzyme mutants lacking Lys-6 (K6R and K6M) or Lys-79 (K79R) were inhibited only 40% by manoalide while the native cobra venom PLA2 was inhibited 80% under the same conditions . This means that the manoalide modification of either Lys-6 or Lys-79 accounted for only half of the manoalide inhibition . The double mutant (K6R79R) was not inhibited by manoalide at all . Lys-56 (K56R) and Lys-65 (K65R) mutants were inhibited to the same extent as the native enzyme which indicates that these residues are not responsible for any of the inhibitory effects produced by manoalide . These results demonstrate that the reaction of manoalide with both Lys-6 and Lys-79 can account for all of its inhibition of cobra venom PLA2 . The inhibition of PLA2 and its mutants with manoalide did not affect the activity of the enzyme toward monomeric substrate, which suggests that manoalide does not modify the catalytic site residues, that it does not block access to this site, and that its inhibition requires an interface . Furthermore, as with native PLA2, the activation of phosphatidylethanolamine hydrolysis by phosphorylcholine-containing compounds was exhibited by all of the mutants suggesting that none of the lysines examined are essential for this activation. Proc Natl Acad Sci U S A, 1995 Jul 18, 92(15), 7021 - 5 A small bispecific antibody construct expressed as a functional single-chain molecule with high tumor cell cytotoxicity; Mack M et al.; Construction of a bispecific single-chain antibody derivative is described that consists of two different single-chain Fv fragments joined through a Gly-Ser linker . One specificity of the two Fv fragments is directed against the CD3 antigen of human T cells and the other is directed against the epithelial 17-1A antigen; the latter had been found in a clinical trial to be a suitable target for antibody therapy of minimal residual colorectal cancer . The construct could be expressed in CHO cells as a fully functional protein, while its periplasmic expression in Escherichia coli resulted in a nonfunctional protein only . The antigen-binding properties of the bispecific single-chain antibody are indistinguishable from those of the corresponding univalent single-chain Fv fragments . By redirecting human peripheral T lymphocytes against 17-1A-positive tumor cells, the bispecific antibody proved to be highly cytotoxic at nanomolar concentrations as demonstrated by 51Cr release assay on various cell lines . The described bispecific construct has a molecular mass of 60 kDa and can be easily purified by its C-terminal histidine tail on a Ni-NTA chromatography column . As bispecific antibodies have already been shown to be effective in vivo in experimental tumor systems as well as in phase-one clinical trials, the small CD3/17-1A-bispecific antibody may be more efficacious than intact antibodies against minimal residual cancer cells. Biochemistry, 1995 Jul 18, 34(28), 9227 - 34 Role of Asp274 in lac repressor: diminished sugar binding and altered conformational effects in mutants; Chang WI et al.; The role of Asp274 in inducer binding of lac repressor has been explored by spectroscopic measurements, fluorescence quenching, in vitro induction assays, and chemical modification of mutants with conservative substitutions at this site . Although no fluorescence emission shift or characteristic UV difference spectrum was observed at high inducer concentration, fluorescence quenching, effects on operator binding, and chemical modification results indicate indirectly that the mutants Asp274-->Asn and Asp274-->Glu bind sugar, albeit with very low affinity (> 0.1 M) . Consistent with very weak inducer binding indicated by protection from fluorescence quenching by iodide, operator binding activity of these two mutant proteins is altered at very high IPTG concentration, although in opposite directions . The distinct effects of inducer on operator binding in these two mutant proteins as well as substantial differences in the effect of sugar ligand on chemical modification of Cys107 and Cys140 by 2-(bromoacetamido)-4-nitrophenol suggest that the conformation of the protein before and after association with sugar may differ in these mutant proteins . Fluorescence quenching assays of lac mutant proteins at Asp274 indicate the proximity of Trp220 to the side chain at position 274, consistent with the location of this residue in the structural model of lac repressor and in the crystallographic structure of the homologous purine repressor . From these results, we conclude that Asp274 is in the inducer binding site, that the character of this residue is crucial to inducer binding, and that interaction of sugar with the side chain at this position may be associated with the conformational change necessary for generating high affinity ligand binding. Biochemistry, 1995 Jul 18, 34(28), 9172 - 6 The binding site of the nicotinic acetylcholine receptor in animal species resistant to alpha-bungarotoxin; Barchan D et al.; The ligand binding site of the nicotinic acetylcholine receptor (AChR) is located in the alpha-subunit, within a small fragment containing the tandem cysteines at positions 192 and 193 . We have been analyzing the binding site domain of AChRs from several animal species exhibiting various degrees of resistance to alpha-bungarotoxin (alpha-BTX) . Our earlier work on the snake and mongoose AChR, both of which do not bind alpha-BTX, suggested that amino acid substitutions at positions 187, 189, and 194 of the AChR alpha-subunit are important in determining the resistance of these AChRs to alpha-BTX . In the present study, we have examined the correlation between alpha-BTX binding and the structure of the binding site domain of AChR from the hedgehog, shrew, cat, and human . Fragments of the AChR alpha-subunit corresponding to residues 122-205 from these species were cloned, sequenced, and expressed in Escherichia coli . The hedgehog fragment does not bind alpha-BTX, in common with the snake and mongoose AChR, and the human fragment is a partial binder . The shrew and cat fragments bind alpha-BTX to a similar extent as the mouse fragment . The hedgehog and human AChRs have nonaromatic amino acid residues at positions 187 and 189 of the alpha-subunit, as is seen with the "toxin resistant" snake and mongoose, and in contrast with the "toxin binders", which have aromatic residues at these two positions.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1995 Jul 18, 34(28), 9166 - 71 Tryptophan-scanning mutagenesis of MotB, an integral membrane protein essential for flagellar rotation in Escherichia coli; Sharp LL et al.; The MotB protein of Escherichia coli is an essential component of the flagella that functions together with the MotA protein in transmembrane proton conduction . MotB has a single hydrophobic segment that spans the membrane . In order to determine which parts of the membrane-spanning segment can tolerate the introduction of a large, hydrophobic side chain, single Trp residues were substituted into many consecutive positions in the segment and the effects on function were measured . Trp residues were tolerated at positions near the periplasmic end of the MotB segment but not at positions near the cytoplasmic end . These results are different from what was seen in a similar mutational study of MotA, in that protein Trp residues were tolerated at positions that would be clustered together on one face of each hydrophobic segment if they are alpha-helices {Sharp, L . L., Zhou, J., & Blair, D . F . (1995) Proc . Natl . Acad . Sci . U.S.A . (in press)} . Those results suggested that the membrane-spanning segments of MotA are alpha-helices arranged in a bundle so that each has a face directed toward the lipid . The contrasting results seen with MotB indicate that its relationship to neighboring protein segments is different . Double-Trp substitutions, one each in MotA and MotB, also were studied . Many double substitutions had strongly synergistic effects which imply that the membrane segments of these proteins interact . (ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1995 Jul 18, 34(28), 9103 - 10 Cloning, Zn2+ binding, and structural characterization of the guanine nucleotide exchange factor human Mss4; Yu H et al.; The Sec4/Ypt1/Rab family of small GTP-binding proteins are involved in the regulation of intracellular vesicular transport . A rat gene, mss4, that encodes a guanine nucleotide exchange factor (GEF) for Sec4 was recently cloned by its ability to rescue defects in protein transport of a yeast temperature-sensitive (ts) mutant, sec4-8 . We describe herein the cloning, bacterial expression, and biochemical characterization of human Mss4 . As expected, both the cDNA and its encoded amino acid sequences are highly conserved between the human and rat mss4 . Soluble and functional Mss4 protein was obtained by expressing the gene as a glutathione-S-transferase fusion protein in Escherichia coli . Subsequent biochemical analysis revealed that Mss4 binds 1 equiv of Zn2+, and zinc is essential for the stability of the protein . Utilizing multidimensional heteronuclear NMR techniques, we assigned most of the 1H, 15N, and 13C resonances of this 14-kDa protein . Its secondary structure was also deduced from slowly exchanging amide protons, characteristic NOEs, and 3JNH-C alpha H coupling constants . The protein contains a central seven-stranded antiparallel beta sheet, flanked by two small beta sheets . Many resonances pertaining to a loop region of the molecule cannot be identified, suggesting that it might be involved in local movements . These studies provide the first structural insights into a protein possessing GEF activity. Biochemistry, 1995 Jul 18, 34(28), 9095 - 102 Rat guanylyl cyclase C expressed in COS-7 cells exhibits multiple affinities for Escherichia coli heat-stable enterotoxin; Deshmane SP et al.; Intestinal cells exhibit binding sites with different affinities for Escherichia coli heat-stable enterotoxin (ST) and guanylin, suggesting the existence of different receptors for these peptides . Guanylyl cyclase C from intestinal cells has been identified as one receptor for these peptides . Equilibrium and kinetic binding characteristics of rat guanylyl cyclase C expressed in COS-7 cells were examined, employing ST, to determine if this receptor exhibited multiple affinities . Scatchard analysis of equilibrium binding yielded curvilinear isotherms consistent with the presence of high (pM) and low (nM) affinity sites . Kinetic analysis of binding demonstrated that these sites exhibited similar dissociation but different association kinetics . In addition, two distinct affinity states of low affinity sites were identified with dissociation constants of 0.15 and 5.85 nM . Association of ST and low affinity sites was biphasic, while dissociation from these sites was unimodal . Close agreement of equilibrium and kinetic dissociation constants suggested that low affinity sites were in the lowest affinity state at equilibrium . Comparison of the ligand dependence of guanylyl cyclase activity (EC50 = 110 nM) with receptor occupancy revealed that binding of ST to the lowest affinity state of low affinity sites (EC50 = 80 nM) is directly coupled to catalytic activation . These studies suggest that binding sites with different affinities for ST exhibited by intestinal cells reflect the expression of a single gene product, guanylyl cyclase C, rather than different receptors for the ligand . The shift in affinity state of low affinity sites and its correlation with catalytic activation suggest a central role for this phenomenon in mechanisms mediating receptor-effector coupling of membrane guanylyl cyclases. Biochemistry, 1995 Jul 18, 34(28), 9000 - 8 Amino acid differences at positions 10, 11, and 104 explain the profound catalytic differences between two murine pi-class glutathione S-transferases; Bammler TK et al.; The glutathione S-transferases play a pivotal role in the detoxification of toxic and carcinogenic electrophiles . We have previously reported the isolation of two actively transcribed murine pi-class glutathione S-transferase genes . In this study the two proteins encoded by these genes, Gst p-1 and Gst p-2, were expressed in Escherichia coli and found to exhibit profoundly different catalytic activities, the activity of Gst p-2 toward a panel of electrophilic substrates being 1-3 orders of magnitude lower than that of Gst p-1 . In order to establish the basis for the difference between these highly homologous proteins, mutants were generated where specific amino acids had been exchanged . Kinetic analysis of the wild-type and mutant enzymes revealed that the amino acid differences occurring at positions 10 (Val/Ser), 11 (Arg/Pro), and 104 (Val/Gly) are responsible for the reduced enzymatic activity of Gst p-2 . This analysis together with computer graphics modeling for Gst p-2 indicated that these changes affected both substrate and glutathione binding to the enzyme. Biochemistry, 1995 Jul 18, 34(28), 8931 - 9 Aspartic acid 26 in reduced Escherichia coli thioredoxin has a pKa > 9; Wilson NA et al.; Apparent pKa values of active site residues Asp26, Cys32, and Cys35 in reduced thioredoxin have been characterized . Both wild-type thioredoxin and mutant D26A thioredoxin were selectively 13C-enriched on cysteine beta-carbons . In both proteins, the variation with pH of 1HB1, 1HB2, and 13CB NMR chemical shifts has been measured . In wild-type reduced thioredoxin, for both cysteines, the pH versus chemical shift plots of HB1 protons can be fit to one titration with pKa values of 7.0-7.1 . In contrast, the HB2 protons and beta-carbons give pH--chemical shift plots that clearly reflect more than one titration; fits to the data give apparent pKa values of 7.0-7.3 and 9.5 for HB2 protons and 7.5-7.9 and 9.2-10.2 for CB carbons . In reduced D26A, all three probe chemical shifts have a pH dependence that is fit by one titration with pKa of 7.4-7.9 . The absence of a titration with pKa > 9 in D26A, taken together with cysteine thiol pKa values of 7.1 and 7.9 determined by Raman spectroscopy {Li et al . (1993) Biochemistry 32, 5800-5808}, indicates that the pKa > 9 in reduced thioredoxin is that of Asp26 . This is highly significant in view of the previous observation that, in oxidized thioredoxin, Asp26 pKa is 7.5 {Langsetmo et al . (1991) Biochemistry 30, 7603-7609} . The very high pKa values of these carboxyls is consistent with their local environment in the three-dimensional structure; the Asp26 side chain in oxidized thioredoxin is almost but not completely buried, and in reduced thioredoxin it may be even more buried.(ABSTRACT TRUNCATED AT 250 WORDS) Proc Natl Acad Sci U S A, 1995 Jul 18, 92(15), 6883 - 6 The tRNA processing enzyme RNase T is essential for maturation of 5S RNA; Li Z et al.; The maturation of 5S RNA in Escherichia coli is poorly understood . Although it is known that large precursors of 5S RNA accumulate in mutant cells lacking the endoribonuclease-RNase E, almost nothing is known about how the mature 5' and 3' termini of these molecules are generated . We have examined 5S RNA maturation in wild-type and single- or multiple-exoribonuclease-deficient cells by Northern blot and primer-extension analysis . Our results indicate that no mature 5S RNA is made in RNase T-deficient strains . Rather, 5S RNA precursors containing predominantly 2 extra nucleotides at the 3' end accumulate . Apparently, these 5S RNAs are functional inasmuch as mutant cells are viable, growing only slightly slower than wild type . Purified RNase T can remove the extra 3' residues, showing that it is directly involved in the trimming reaction . In contrast, mutations affecting other 3' exoribonucleases have no effect on 5S RNA maturation . Approximately 90% of the 5S RNAs in both wild-type and RNase T- cells contain mature 5' termini, indicating that 5' processing is independent of RNase T action . These data identify the enzyme responsible for generating the mature 3' terminus of 5S RNA molecules and also demonstrate that a completely processed 5S RNA molecule is not essential for cell survival. Biochem Pharmacol, 1995 Jul 17, 50(2), 163 - 8 Differences in kinetic properties of pure recombinant human and mouse deoxycytidine kinase; Johansson M et al.; Human and mouse deoxycytidine kinase (dCK) (EC 2.7.1.74) were cloned and expressed in Escherichia coli . Michaelis-Menten kinetics were determined for the purified enzymes with 2'-deoxycytidine (dCyd), 2'-deoxyadenosine (dAdo), 2-chloro-2'-deoxyadenosine (CdA), 2',3'-dideoxycytidine (ddCyd) and 9-beta-D-arabinofuranosylguanine (araG) as substrates and ATP and UTP as phosphate donors . Both human and mouse dCK showed highest affinity to dCyd with Km values of 0.05-0.2 microM . The anti-leukaemic compound CdA was the superior substrate of the nucleoside analogues tested . Both enzymes were able to efficiently utilize ATP and UTP as phosphate donors . However, the use of UTP instead of ATP as phosphate donor decreased Km values for all substrates investigated . The kinetic properties of mouse and human dCK differed in that the human enzyme showed higher affinity for the substrates dAdo, CdA, ddCyd and araG . The human enzyme also showed higher affinity for ATP and UTP . The ability to phosphorylate dCyd was, however, similar for both human and mouse dCK . At physiological concentration of the feedback inhibitor dCTP, mouse dCK showed lower activity than human dCK for all substrates investigated. FEBS Lett, 1995 Jul 17, 368(2), 315 - 20 Recognition of 2'-hydroxyl groups by Escherichia coli ribonuclease HI; Iwai S et al.; In order to investigate the hydrogen-bonding interactions between Escherichia coli ribonuclease HI and the 2'-hydroxyl functions of the substrate, oligonucleotide duplexes containing 2'-amino-2'-deoxyuridine or 2'-fluoro-2'-deoxyuridine at a specific site were used, and their affinities for the enzyme were determined by kinetic analyses . The results indicate that the hydroxyl groups of the nucleoside 3'-adjacent to the cleaved phosphodiester linkage and the second nucleoside 5' to the cleaved phosphodiester act as both a proton donor and an acceptor and as a proton acceptor, respectively, in the enzyme-substrate complex . A molecular model was constructed using the interactions derived from the results. FEBS Lett, 1995 Jul 17, 368(2), 263 - 6 Evidence for SecA- and delta pH-independent insertion of D1 into thylakoids; van Wijk KJ et al.; Many nuclear-encoded proteins are targeted into chloroplast thylakoids by an azide sensitive Sec-related mechanism or by a delta pH-driven mechanism . In this report, the requirements for the integration of chloroplast-encoded thylakoid proteins have been analysed in pulse-labeled intact chloroplasts . We show that the integration of the photosystem II reaction centre protein, D1, continues in the absence of a delta pH and in the presence of azide . A range of other proteins are similarly targeted to thylakoids in the presence of azide, suggesting that the SecA-related mechanism is not widely used for the targeting of chloroplast-encoded proteins. FEBS Lett, 1995 Jul 17, 368(2), 235 - 8 The gamma subunit in the Escherichia coli ATP synthase complex (ECF1F0) extends through the stalk and contacts the c subunits of the F0 part; Watts SD et al.; A mutant, in which a cysteine has been site-directed into the polar loop region of the c subunit at residue 44, has been studied . Cross-linking of the c subunit to both the gamma and epsilon subunits was observed with cupric 1,10-phenanthrolinate treatment . The linkage between the c and gamma subunits was localized to that part of the gamma subunit between residues 202-286, based on peptide analysis . Reference to the high resolution structure of F1 {Abrahams et al . (1994) Nature 370, 621-628} appears to limit this contact site to the region including residues 202-230 . This segment contains 4 tyrosines and 1 tryptophan as possible reactive residues for cross-linking with the c subunit cysteine. FEBS Lett, 1995 Jul 17, 368(2), 220 - 4 Direct electrochemistry and EPR spectroscopy of spinach ferredoxin mutants with modified electron transfer properties; Aliverti A et al.; Mutations of the conserved residue Glu-92 to lysine, glutamine, and alanine have been performed in the recombinant ferredoxin I of spinach leaves . The purified ferredoxin mutants were found twice as active with respect to wild-type protein in the NADPH-cytochrome c reductase reaction catalyzed by ferredoxin-NADP+ reductase in the presence of ferredoxin . Cyclic voltammetry and EPR measurements showed that the mutations cause a change in the {2Fe-2S} cluster geometry, whose redox potential becomes approximately 80 mV less negative . These data point to a role of the Glu-92 side-chain in determining the low redox potential typical of the {2Fe-2S} cluster of chloroplast and cyanobacterial ferredoxins . Also a ferredoxin/ferredoxin-NADP+ reductase chimeric protein obtained by gene fusion was overproduced in Escherichia coli and purified . Fusion of the ferredoxin with its reductase causes only minor effects to the iron-sulfur cluster, as judged by cyclic voltammetry and EPR measurements. EMBO J, 1995 Jul 17, 14(14), 3415 - 24 Identification and characterization of a new disulfide isomerase-like protein (DsbD) in Escherichia coli; Missiakas D et al.; Previous studies have established that DsbA and DsbC, periplasmic proteins of Escherichia coli, are two key players involved in disulfide bond formation . A search for extragenic mutations able to compensate for the lack of dsbA function in vivo led us to the identification of a new gene, designated dsbD . Lack of DsbD protein leads to some, but not all, of the phenotypic defects observed with other dsb mutations, such as hypersensitivity to dithiothreitol and to benzylpenicillin . In addition, unlike the rest of the dsb genes, dsbD is essential for bacterial growth at temperatures above 42 degrees C . Cloning of the wild-type gene and sequencing and overexpression of the protein show that dsbD is part of an operon and encodes an inner membrane protein . A 138 amino acid subdomain of the protein was purified and shown to possess an oxido-reductase activity in vitro . Expressing this subdomain in the periplasmic space helped restore the phenotypic defects associated with a dsbD null mutation . Interestingly, this domain shares 45% identity with the portion of the eukaryotic protein disulfide isomerase carrying the active site . We further show that in dsbD mutant bacteria the dithiol active sites of DsbA and DsbC proteins are mostly oxidized, as compared with wild-type bacteria . Our results argue that DsbD generates a reducing source in the periplasm, which is required for maintaining proper redox conditions . The finding that overexpression of DsbD leads to a Dsb- phenotype, very similar to that exhibited by dsbA null mutants, is in good agreement with such a model. EMBO J, 1995 Jul 17, 14(14), 3365 - 72 A novel periplasmic carrier protein involved in the sorting and transport of Escherichia coli lipoproteins destined for the outer membrane; Matsuyama S et al.; Lipoproteins are localized in the outer or inner membrane of Escherichia coli, depending on the species of amino acid located next to the N-terminal fatty acylated Cys . The major outer membrane lipoprotein (Lpp) expressed in spheroplasts was, however, retained in the inner membrane as a mature form . A novel protein that is essential for the release of Lpp from the inner membrane was discovered in the periplasm and purified . The partial amino acid sequence of this 20 kDa protein (p20) was determined and used to clone a gene for p20 . Sequencing of the gene revealed that p20 is synthesized as a precursor with a signal sequence . p20 formed a soluble complex only with outer membrane-directed lipoproteins such as Lpp, indicating that p20 plays a critical role in the sorting of lipoproteins . Lpp released from the inner membrane in the presence of p20 was specifically assembled into the outer membrane in vitro . These results indicate that p20 is a periplasmic carrier protein involved in the translocation of lipoproteins from the inner to the outer membrane. EMBO J, 1995 Jul 17, 14(14), 3302 - 10 Branch migration of three-strand recombination intermediates by RecG, a possible pathway for securing exchanges initiated by 3'-tailed duplex DNA; Whitby MC et al.; RecG protein is required for normal levels of recombination and DNA repair in Escherichia coli . This 76 kDa polypeptide is a junction-specific DNA helicase that acts post-synaptically to drive branch migration of Holliday junction intermediates made by RecA during the strand exchange stage of recombination . To gain further insight into the role of RecG, we studied its activity on three-strand intermediates formed by RecA between circular single-stranded and linear duplex DNAs . Once RecA is removed, RecG drives branch migration of these intermediates by a junction-targeted activity that depends on hydrolysis of ATP . RuvAB has a similar activity . However, when RecG is added to a RecA strand exchange reaction it severely reduces the accumulation of joint molecule intermediates by driving branch migration of junctions in the reverse direction to that catalysed by RecA strand exchange . In comparison, RuvAB has little effect on the reaction . We discuss how reverse branch migration by RecG, which acts counter of the 5'-->3' polarity of RecA binding and strand exchange, could serve to promote or abort the early stages of recombination, depending on the orientation of the single DNA strand initiating the exchange relative to the adjacent duplex region. Biochem Biophys Res Commun, 1995 Jul 17, 212(2), 317 - 25 Characterisation of a gain-of-function mutant of poly(ADP-ribose) polymerase; Miranda EA et al.; In order to examine the structure-function relationship of the poly (ADP-ribose) polymerase (PARP) catalytic domain, potential active-site residues in the catalytic domain have previously been described . Here, we have used mutagenesis with hydroxylamine to generate a random library of PARP mutants . The identification, overproduction in insect cells, purification and characterization of a gain-of-function mutant (L713F) is described . We show that the kcat of this mutant is increased over nine times compared to the wild-type enzyme; the Km for NAD+ is unchanged . The size and the branching structure of the ADP-ribose polymers are similar in both the wild-type and the mutant enzyme . This mutation may have an allosteric effect on the catalytic site and could be useful in analyzing the consequences of poly ADP-ribose overproduction in vivo on cell survival following DNA damage. J Immunol Methods, 1995 Jul 17, 184(1), 101 - 12 Biotinylation of interleukin-2 (IL-2) for flow cytometric analysis of IL-2 receptor expression . Comparison of different methods; De Jong MO et al.; The main prerequisites for the use of biotinylated ligands to study the expression of growth factor receptors on heterogeneous cell populations, such as peripheral blood or bone marrow, by flow cytometric methods, are that the biotinylated ligand retains its binding ability and that binding of the biotinylated ligand to the receptor does not inhibit the subsequent interaction of biotin with fluorescently tagged avidin or streptavidin . Using interleukin-2 (IL-2), we compared the usefulness of various biotinylation reagents, NHS-biotin, S-NHS-biotin, S-NHS-LC-biotin, DBB and photobiotin, and developed optimal biotinylation conditions for the preparation of biologically active biotin-labeled IL-2 and the detection of IL-2 receptor expressing cells by flow cytometry . As determined by spot blot analysis, biotinylation of IL-2 was most efficient at the highest biotin-to-protein (B:P) ratio used . At a B:P ratio of 100, most of the biological activity of IL-2 was retained when S-NHS-LC-biotin was used . In contrast, most of the biological activity of IL-2 samples that were labeled with NHS-biotin or photobiotin was lost under these conditions . Biotin-labeled IL-2 preparations were tested in order to detect IL-2 receptors on IL-2 dependent CTLL-2 cells by flow cytometry after sequential staining with the biotinylated IL-2 and fluorescence tagged streptavidin . A high B:P ratio generally resulted in a high specific fluorescence intensity of the cells, particularly when S-NHS-LC-biotin was used as the biotinylation reagent . Biotin-IL-2 could also be used to detect IL-2 receptors expressed by lymphocytes in peripheral blood and bone marrow . Comparison of staining of lymphocytes with biotinylated IL-2 and an antibody against the IL-2 receptor alpha chain demonstrated that only a subset of the cells that showed a strong fluorescence signal after staining with biotinylated IL-2 expressed high numbers of the IL-2 receptor alpha chain . This is in agreement with the expression of functional IL-2 receptors on resting T cells and NK cells which do not express the alpha chain . After stimulation with PHA, virtually all lymphocytes expressed the alpha chain, whereas only part of these cells showed a strong fluorescence signal after staining with biotin-IL-2, while the rest of the cells had very low numbers of IL-2 binding sites . Our results demonstrate that, in addition to staining individual receptor subunits with antibodies, staining with biotinylated IL-2 is a useful indicator of functional IL-2 receptor expression. Biochem Biophys Res Commun, 1995 Jul 17, 212(2), 705 - 11 Homology cloning of GTP-cyclohydrolase I from various unrelated eukaryotes by reverse-transcription polymerase chain reaction using a general set of degenerate primers; Maier J et al.; GTP-cyclohydrolase I is the primary enzyme of tetrahydrobiopterin and folic acid biosynthesis . cDNA fragments of GTP-cyclohydrolase I were obtained from rainbow trout, chicken, the fungi Neurospora crassa, Phycomyces blakesleeanus and Saccharomyces cerevisiae, the cellular slime mold Dictyostelium discoideum, the phytoflagellate Euglena gracilis and the higher plant Mucuna hassjo using primers specific for conserved regions of the open reading frame and the reverse transcription polymerase chain reaction (RT-PCR) technique . A number of regions were found to be strictly conserved between unrelated eukaryotes . These regions may be essential for the function of GTP-cyclohydrolase I and are discussed with respect to the recently resolved crystal structure of the Escherichia coli enzyme. Anal Chem, 1995 Jul 15, 67(14), 2493 - 7 Ion/molecule reactions for improved effective mass resolution in electrospray mass spectrometry; McLuckey SA et al.; Tandem mass spectrometry is shown to improve the effective mass resolution in electrospray mass spectrometry . The technique involves selecting a population of ions within a narrow range of mass-to-charge values and allowing the ions to undergo proton transfer reactions . The shifts in mass-to-charge ratios associated with product ions formed by proton transfer allow for mass and charge assignment . The success of the technique relies on the relative enrichment of ions of a particular charge state that occurs in the mass-to-charge selection step . This approach can be used to extend the polymer mass range amenable to measurement, analyze mixtures that might otherwise be too complex for reliable mass measurements, and improve mass measurement precision when a mixture of cations is present within a given charge state . The technique is illustrated with a quadrupole ion trap using multiply-charged ions of cytochrome c, transfer ribonucleic acid from E . coli, strain W, and a synthetic deoxyribonucleic acid 30-mer. Structure, 1995 Jul 15, 3(7), 729 - 41 Crystal structure of Escherichia coli pyruvate kinase type I: molecular basis of the allosteric transition; Mattevi A et al.; BACKGROUND: Pyruvate kinase (PK) plays a major role in the regulation of glycolysis . Its catalytic activity is controlled by the substrate phosphoenolpyruvate and by one or more allosteric effectors . The crystal structures of the non-allosteric PKs from cat and rabbit muscle are known . We have determined the three-dimensional structure of the allosteric type I PK from Escherichia coli, in order to study the mechanism of allosteric regulation . RESULTS: The 2.5 A resolution crystal structure of the unligated type I PK in the inactive T-state shows that each subunit of the homotetrameric enzyme comprises a (beta/alpha)8-barrel domain, a flexible beta-barrel domain and a C-terminal domain . The allosteric and active sites are located at the domain interfaces . Comparison of the T-state E . coli PK with the non-allosteric muscle enzyme, which is thought to adopt a conformation similar to the active R-state, reveals differences in the orientations of the beta-barrel and C-terminal domains of each subunit, which are rotated by 17 degrees and 15 degrees, respectively . Moreover, the relative orientation of the four subunits differs by about 16 degrees in the two enzymes . Highly conserved residues at the subunit interfaces couple these movements to conformational changes in the substrate and allosteric effector binding sites . The subunit rotations observed in the T-state PK induce a shift in loop 6 of the (beta/alpha)8-barrel domain, leading to a distortion of the phosphoenolpyruvate-binding site accounting for the low substrate affinity of the T-state enzyme . CONCLUSIONS: Our results suggest that allosteric control of PK is accomplished through remarkable domain and subunit rotations . On transition from the T- to the R-state all 12 domains of the functional tetramer modify their relative orientations . These concerted motions are the molecular basis of the coupling between the active centre and the allosteric site. Structure, 1995 Jul 15, 3(7), 669 - 79 Three new crystal structures of point mutation variants of monoTIM: conformational flexibility of loop-1, loop-4 and loop-8; Borchert TV et al.; BACKGROUND: Wild-type triosephosphate isomerase (TIM) is a very stable dimeric enzyme . This dimer can be converted into a stable monomeric protein (monoTIM) by replacing the 15-residue interface loop (loop-3) by a shorter, 8-residue, loop . The crystal structure of monoTIM shows that two active-site loops (loop-1 and loop-4), which are at the dimer interface in wild-type TIM, have acquired rather different structural properties . Nevertheless, monoTIM has residual catalytic activity . RESULTS: Three new structures of variants of monoTIM are presented, a double-point mutant crystallized in the presence and absence of bound inhibitor, and a single-point mutant in the presence of a different inhibitor . These new structures show large structural variability for the active-site loops, loop-1, loop-4 and loop-8 . In the structures with inhibitor bound, the catalytic lysine (Lys13 in loop-1) and the catalytic histidine (His95 in loop-4) adopt conformations similar to those observed in wild-type TIM, but very different from the monoTIM structure . CONCLUSIONS: The residual catalytic activity of monoTIM can now be rationalized . In the presence of substrate analogues the active-site loops, loop-1, loop-4 and loop-8, as well as the catalytic residues, adopt conformations similar to those seen in the wild-type protein . These loops lack conformational flexibility in wild-type TIM . The data suggest that the rigidity of these loops in wild-type TIM, resulting from subunit-subunit contacts at the dimer interface, is important for optimal catalysis. Structure, 1995 Jul 15, 3(7), 635 - 9 Counting the calories to stay in the groove; Ladbury JE; High-sensitivity microcalorimetry is beginning to make an impact on the determination of thermodynamic parameters associated with protein-DNA interactions and the understanding of the relationship of these data to structural details of complex formation. Eur J Biochem, 1995 Jul 15, 231(2), 491 - 7 Human topoisomerase II alpha is phosphorylated in a cell-cycle phase-dependent manner by a proline-directed kinase; Wells NJ et al.; Topoisomerase II is essential for chromosome condensation and segregation at mitosis in eukaryotic cells, but the mechanism of its regulation is not clearly understood . We have investigated whether or not the alpha isozyme of human topoisomerase II is phosphorylated in a cell-cycle phase-dependent manner . Two-dimensional tryptic phosphopeptide mapping revealed that several sites on HeLa topoisomerase II alpha protein were phosphorylated predominantly or exclusively during the G2 and M phases . To identify the protein kinases involved in this cell-cycle phase-specific phosphorylation, oligohistidine-tagged recombinant domains of the topoisomerase II alpha protein were expressed in Escherichia coli, purified by affinity chromatography and phosphorylated in vitro by different protein kinases . Phosphorylation of the C-terminal domain of the topoisomerase II alpha protein by the universal mitotic controller, p34cdc2, generated multiple tryptic phosphopeptides, many of which corresponded to the G2/M-phase-specific phosphorylation sites observed in vivo . The same phosphopeptides were obtained following phosphorylation of the C-terminal domain in vitro by the mitogen-activated protein kinase . Site-directed mutagenesis studies identified five of these sites of phosphorylation, each of which comprised a serine-proline motif . Our data implicate one or more proline-directed kinases in the cell-cycle-dependent regulation of topoisomerase II alpha enzyme activity in human cells. Eur J Biochem, 1995 Jul 15, 231(2), 396 - 404 Characterization of a 2{4Fe-4S} ferredoxin obtained by chemical insertion of the Fe-S clusters into the apoferredoxin II from Rhodobacter capsulatus; Armengaud J et al.; The Rhodobacter capsulatus ferredoxin II (FdII) belongs to a family of 7Fe ferredoxins containing one {3Fe-4S} cluster and one {4Fe-4S} cluster . This protein, encoded by the fdxA gene, has been overproduced in Escherichia coli as a soluble apoferredoxin . The purified recombinant protein was subjected to reconstitution experiments by chemical incorporation of the Fe-S clusters under anaerobic conditions . A brown protein was obtained, the formation of which was dependent upon the complete unfolding of the polypeptide prior to incorporation of iron and sulfur atoms . The yield of the reconstituted product was higher when the reaction was carried out at slightly basic pH . The reconstituted ferredoxin was purified and shown to be distinct from the native {7Fe-8S} ferredoxin, based on several biochemical and spectroscopic criteria . In the oxidized state, EPR revealed the quasi-absence of {3Fe-4S} cluster . 1H-NMR spectroscopic analyses provided evidence that the protein was reconstituted as a 2{4Fe-4S} ferredoxin . This conclusion was further supported by the determination by electrospray mass spectrometry of the molecular mass of the reconstituted protein, which matched within 2 Da to the mass of the FdII polypeptide incremented of eight atoms each of iron and sulfur . Exposure of the reconstituted protein to air resulted in a fast and irreversible oxidative denaturation of the Fe-S clusters, without formation of {7Fe-8S} form . Unlike the natural 7Fe ferredoxin, the reconstituted ferredoxin appeared incompetent in an electron-transfer assay coupled to nitrogenase activity . The fact that the apoFdII was reconstituted as a highly unstable 8Fe ferredoxin instead of the 7Fe naturally occurring FdII is discussed in relation to the results obtained with other types of ferredoxins. Eur J Biochem, 1995 Jul 15, 231(2), 306 - 11 Recombinant production and biological properties of rat cytokine-induced neutrophil chemoattractants, GRO/CINC-2 alpha, CINC-2 beta and CINC-3; Shibata F et al.; Recently we found four cytokine-induced neutrophil chemoattractants, CINC-1, CINC-2 alpha, CINC-2 beta and CINC-3/macrophage inflammatory protein 2 (MIP-2), in conditioned medium of granulation tissue obtained from carrageenin-induced inflammation in rats {Nakagawa, H., Komorita, N., Shibata, F., Ikesue, A., Konishi, K., Fujioka, M . & Kato, H . (1994) Biochem . J . 301, 545-550} . In the present report, we describe recombinant production of CINC-2 alpha, CINC-2 beta and CINC-3 in Escherichia coli, and biological properties of these chemokines . Neutrophil chemotactic activities of CINC-2 alpha and 2 beta in vitro were the same as the activity of CINC-1 . CINC-3 had an activity comparable to other CINCs, but showed a decrease at high concentrations . Stimulation of neutrophils with CINCs induced an increase in intracellular {Ca2+} dose-dependently . CINC-3 was more potent than the other CINCs and still induced an increase in intracellular {Ca2+} in rat neutrophils stimulated first with other CINCs . CINC-2 alpha, CINC-2 beta and CINC-3 induced a comparable response to CINC-1 in the release of cathepsin G from rat neutrophils . Injection of CINC-2 alpha, 2 beta and 3 into preformed air-pouch on the back of rat induced infiltration of neutrophils to an extent similar to that caused by the injection of CINC-1 . These data indicate CINC-2 alpha, 2 beta and 3 as well as CINC-1 are chemoattractants specific for neutrophil in vivo. Biochem J, 1995 Jul 15, 309 ( Pt 2), 689 - 93 Over-expression and characterization of active recombinant rat liver carnitine palmitoyltransferase II using baculovirus; Johnson TM et al.; The cDNA encoding rat liver carnitine palmitoyltransferase II (CPT-II) was heterologously expressed using a recombinant baculovirus/insect cell system . Unlike Escherichia coli, the baculovirus-infected insect cells expressed mostly soluble active recombinant CPT-II (rCPT-II) . CPT activity from crude lysates of recombinant baculovirus-infected insect cells was maximal between 50 and 72 h post-infection, with a peak specific activity of 100-200 times that found in the mock- or wild-type-infected control lysates . Milligram quantities (up to 1.8 mg/l of culture) of active rCPT-II were chromatographically purified from large-scale cultures of insect cells infected with the recombinant baculovirus . The rCPT-II was found to be: (1) similar in size to the native rat liver enzyme (approximately 70 kDa) as judged by SDS/PAGE; (2) immunoreactive with a polyclonal serum raised against rat liver CPT-II; and (3) not glycosylated . Kinetic analysis of soluble rCPT-II revealed Km values for carnitine and palmitoyl-CoA of 950 +/- 27 microM and 34 +/- 5.6 microM respectively. Biochem J, 1995 Jul 15, 309 ( Pt 2), 601 - 5 Isolation and characterization of the flavin-binding domain of flavocytochrome b2 expressed independently in Escherichia coli; Balme A et al.; Flavocytochrome b2 consists of two distinct domains . The N-terminal domain contains protohaem IX and the larger, C-terminal domain contains flavin mononucleotide (FMN) . We describe here the isolation of the flavin-binding domain expressed in Escherichia coli independent of the cytochrome domain . The isolated domain is an efficient lactate dehydrogenase with ferricyanide as electron acceptor but reduces cytochrome c, the physiological oxidant for flavocytochrome b2, extremely poorly; electron transfer from the flavin-binding domain to the separately expressed cytochrome domain is undetectable . FMN reduction by lactate occurs as a single exponential process in the isolated flavin-binding domain, in contrast to the biphasic kinetics observed with native flavocytochrome b2. Biochem J, 1995 Jul 15, 309 ( Pt 2), 465 - 72 Structural and functional properties of heparin analogues obtained by chemical sulphation of Escherichia coli K5 capsular polysaccharide; Razi N et al.; Capsular polysaccharide from Escherichia coli K5, with the basic structure (GlcA beta 1-4GlcNAc alpha 1-4)n, was chemically modified through N-deacetylation, N-sulphation and O-sulphation {Casu, Grazioli, Razi, Guerrini, Naggi, Torri, Oreste, Tursi, Zoppetti and Lindahl (1994) Carbohydr . Res . 263, 271-284} . Depending on the reaction conditions, the products showed different proportions of components with high affinity for antithrombin (AT) . A high-affinity subfraction, M(r) approx . 36,000, was shown by near-UV CD, UV-absorption difference spectroscopy and fluorescence to cause conformational changes in the AT molecule very similar to those induced by high-affinity heparin . Fluorescence titrations demonstrated about two AT-binding sites per polysaccharide chain, each with a Kd of approx . 200 nM . The anti-(Factor Xa) activity was 170 units/mg, similar to that of the IIId international heparin standard and markedly higher than activities of previously described heparin analogues . Another preparation, M(r) approx . 13,000, of higher overall O-sulphate content, exhibited a single binding site per chain, with Kd approx . 1 microM, and an anti-(Factor Xa) activity of 70 units/mg . Compositional analysis of polysaccharide fractions revealed a correlation between the contents of -GlcA-GlcNSO3(3,6-di-OSO3)- disaccharide units and affinity for AT; the 3-O-sulphated GlcN unit has previously been identified as a marker component of the AT-binding pentasaccharide sequence in heparin . The abundance of the implicated disaccharide unit approximately equalled that of AT-binding sites in the 36,000-M(r) polysaccharide fraction, and approached one per high-affinity oligosaccharide (predominantly 10-12 monosaccharide units) isolated after partial depolymerization of AT-binding polysaccharide . These findings suggest that the modified bacterial polysaccharide interacts with AT and promotes its anticoagulant action in a manner similar to that of heparin. Biochem J, 1995 Jul 15, 309 ( Pt 2), 425 - 30 Processing of iduronate 2-sulphatase in human fibroblasts; Froissart R et al.; Iduronate 2-sulphatase (IDS) is a lysosomal enzyme involved in degradation of dermatan sulphate and heparan sulphate . Antigenic material was obtained either by purification of placental IDS (A and B forms) or by expression of three different fusion peptides in Escherichia coli allowing the production of five specific antibodies . Pulse-chase-labelling experiments in over-expressing fibroblasts showed poor IDS processing but large amounts of precursors were secreted into the medium . The endocytosis of the 35S- or 33P-labelled precursors by deleted fibroblasts together with glycosylation studies and proteolysis inhibition by leupeptin allowed better elucidation of IDS maturation . The initial 73-78 kDa form is converted into a phosphorylated 90 kDa precursor after modification of its oligosaccharide chains in the Golgi apparatus . This precursor is processed by proteolytic cleavage through various intermediates to a major 55 kDa intermediate, with the release of an 18 kDa polypeptide . Further proteolytic cleavage by a thiol protease gives the 45 kDa mature form containing hybrid and complex-type oligosaccharide chains. Biochem J, 1995 Jul 15, 309 ( Pt 2), 411 - 7 Mistranslation of a TGA termination codon as tryptophan in recombinant platelet-derived growth factor expressed in Escherichia coli; Lu KV et al.; The mature 109-amino-acid human platelet-derived growth factor B (PDGF-B) peptide is derived by intracellular processing from a 241-amino-acid precursor synthesized in mammalian cells, with removal of 81 N-terminal and 51 C-terminal amino acids . In order to produce directly the mature 109-amino acid PDGF-B peptide as a recombinant protein in Escherichia coli, a CGA codon at position 110 of a DNA sequence encoding the full-length precursor form of PDGF-B was converted into the translation termination codon TGA by in vitro mutagenesis . Expression of this DNA via a plasmid vector in E . coli resulted in production of two distinct PDGF-B proteins having apparent molecular masses of 15 and 19 kDa, with the latter species predominating . Structural characterization employing N- and C-terminal amino acid sequencing and MS analyses indicated that the 15 kDa protein is the expected 109-amino-acid PDGF-B, and that the 19 kDa protein represents a C-terminal extended PDGF-B containing 160 amino acids . Characterization of a unique tryptic peptide derived from the 19 kDa protein revealed that this longer form of PDGF-B results from mistranslation of the introduced TGA termination codon at position 110 as tryptophan, with translation subsequently proceeding to the naturally occurring TAG termination codon at position 161 . Owing to the high rate of translation readthrough of TGA codons in this and occasionally other proteins, it appears that the use of TGA as a translation termination codon for proteins to be expressed in E . coli should be avoided when possible. FEMS Microbiol Lett, 1995 Jul 15, 130(1), 13 - 7 Dr fimbriae coding region associated hemolytic activity of Escherichia coli; Goluszko P et al.; We investigated the hemolytic activity of Escherichia coli strain EC901 carrying plasmid pBJN406 containing genes draA-E involved in expression of the mannose-resistant Dr hemagglutinin, and in its isogenic insertion mutants devised with Tn5, Tn3, and TnphoA . While E . coli BN406 displayed rapid hemolytic activity against equine erythrocytes, insertion mutations in draD and draE, but not in draA, draB, and draC, abolished all hemolytic activity . These data suggest a role for draD and draE in the expression of hemolysis. FEMS Microbiol Lett, 1995 Jul 15, 130(1), 1 - 6 Tyrosine protein phosphorylation in murine B lymphocytes by stimulation with lipopolysaccharide from Porphyromonas gingivalis; Kimura S et al.; The molecular effect of lipopolysaccharides (LPS) from porphyromonas gingivalis as well as Escherichia coli on the tyrosine protein phosphorylation in the splenic B lymphocytes from LPS-responsive C3H/HeN and LPS-hyporesponsive C3H/HeJ mice was examined . P . gingivalis LPS induced tyrosine phosphorylation of selected membrane proteins that included the phosphoproteins with apparent molecular masses of 24.8 kDa and 26.0 kDa (p24.8 and p26.) in the B lymphocytes from both strains of mice, while E . coli LPS induced p24.8 and p26.0 in C3H/HeN B Lymphocytes only . These findings suggest that through the same tyrosine phosphorylation pathway as observed in C3H/HeN B lymphocytes, P . gingivalis LPS induced the activation of C3H/HeJ B lymphocytes in which a trigger signal by E . coli LPS could not be transduced to initiate tyrosine protein phosphorylation. Eur J Pharmacol, 1995 Jul 14, 280(3), 339 - 42 Nitric oxide and sensory afferent neurones modulate the protective effects of low-dose endotoxin on rat gastric mucosal damage; Barrachina D et al.; Pretreatment (1 h) with low doses (5-40 micrograms/kg i.p.) of Escherichia coli endotoxin dose dependently reduced the gastric mucosal damage induced by a 10 min challenge with 1 ml ethanol (50% and 100%) in conscious rats . Treatment with the nitric oxide synthesis inhibitor, NG-nitro-L-arginine methyl ester (L-NAME, 5 and 10 mg/kg i.p.), significantly inhibited the protective effects of endotoxin (40 micrograms/kg i.p.) . The actions of L-NAME were reversed by the prior administration of L-arginine (100 mg/kg i.p.) . The protective effects of endotoxin were not influenced by pretreatment with dexamethasone (5 mg/kg s.c . twice) or indomethacin (5 mg/kg s.c.) . However, ablation of sensory afferent neurons by capsaicin pretreatment (20, 30 and 50 mg/kg s.c.) abolished the mucosa protective effects of endotoxin (40 micrograms/kg) . These findings suggest that the protection elicited by low doses of endotoxin against ethanol-induced mucosal damage involves synthesis of nitric oxide and activation of sensory neurones. J Biol Chem, 1995 Jul 14, 270(28), 16903 - 10 Nucleotide-induced conformational changes in the ATPase and substrate binding domains of the DnaK chaperone provide evidence for interdomain communication; Buchberger A et al.; Interactions of the DnaK (Hsp70) chaperone from Escherichia coli with substrates are controlled by ATP . Nucleotide-induced changes in DnaK conformation were investigated by monitoring changes in tryptic digestion pattern and tryptophan fluorescence . Using nucleotide-free DnaK preparations, not only the known ATP-induced major changes in kinetics and pattern of proteolysis but also minor ADP-induced changes were detected . Similar ATP-induced conformational changes occurred in the DnaK-T199A mutant protein defective in ATPase activity, demonstrating that they result from binding, not hydrolysis, of ATP . N-terminal sequencing and immunological mapping of tryptic fragments of DnaK identified cleavage sites that, upon ATP addition, appeared within the proposed C-terminal substrate binding region and disappeared in the N-terminal ATPase domain . They hence reflect structural alterations in DnaK correlated to substrate release and indicate ATP-dependent domain interactions . Domain interactions are a prerequisite for efficient tryptic degradation as fragments of DnaK comprising the ATPase and C-terminal domains were highly protease-resistant . Fluorescence analysis of the N-terminally located single tryptophan residue of DnaK revealed that the known ATP-induced alteration of the emission spectrum, proposed to result directly from conformational changes in the ATPase domain, requires the presence of the C-terminal domain and therefore mainly results from altered domain interaction . Analyses of the C-terminally truncated DnaK163 mutant protein revealed that nucleotide-dependent interdomain communication requires a 15-kDa segment assumed to constitute the substrate binding site. J Biol Chem, 1995 Jul 14, 270(28), 16653 - 9 Proton-translocating nicotinamide nucleotide transhydrogenase of Escherichia coli . Involvement of aspartate 213 in the membrane-intercalating domain of the beta subunit in energy transduction; Yamaguchi M et al.; Mutations in the beta subunit of Escherichia coli proton-translocating nicotinamide nucleotide transhydrogenase of the conserved residue beta Asp-213 to Asn (beta D213N) and Ile (beta D213I) resulted in the loss, respectively, of about 70% and 90% NADPH-->3-acetylpyridine adenine dinucleotide (AcPyAD) transhydrogenation and coupled proton translocation activities . However, the cyclic NADP(H)-dependent NADH-->AcPyAD transhydrogenase activities of the mutants were only approximately 35% inhibited . The latter transhydrogenation, which is not coupled to proton translocation, occurs apparently via NADP under conditions that enzyme-NADP(H) complex is stabilized . Mutations beta D213N and beta D213I also resulted in decreases in apparent KmNADPH for the NADPH-->AcPyAD and S0.5NADPH (NADPH concentration needed for half-maximal activity) for the cyclic NADH-->AcPyAD transhydrogenation reactions, and in KdNADPH, as determined by equilibrium binding studies on the purified wild-type and the beta D213I mutant enzymes . These results point to a structural role of beta Asp-213 in energy transduction and are discussed in relation to our previous suggestion that proton translocation coupled to NADPH-->NAD (or AcPyAD) transhydrogenation is driven mainly by the difference in the binding energies of NADPH and NADP. J Biol Chem, 1995 Jul 14, 270(28), 16610 - 4 Factor for inversion stimulation-dependent growth rate regulation of serine and threonine tRNA species; Emilsson V et al.; We have previously shown that the accumulation of 20 tRNA species in Escherichia coli is individually regulated as a function of cellular growth rate . We have also reported that the growth rate regulation of some but not all tRNA species is dependent on the activity of the factor for inversion stimulation (FIS) . In present work, we studied the growth rate regulation of the serine- and threonine-accepting tRNA families . We show that the levels of tRNA(3Thr), tRNA(3Ser), tRNA(2Thr), tRNA(3Thr), and tRNA(4Thr) are reduced in fis cells as the growth rate increases . The accumulation of these tRNA species is reduced 2-5-fold at the fastest bacterial growth rate . The strongest effect is observed for the two minor tRNA species; tRNA(2Ser) and tRNA(2Thr) . In contrast, we find that the accumulation of tRNA(1Ser), tRNA(5Ser), and tRNA(1Thr) is similar in wild type and fis bacteria . The data presented provide further evidence for the suggestion that FIS is a stimulating factor that is involved, directly or indirectly, in the high expression level of some tRNA genes at fast bacterial growth rates. J Biol Chem, 1995 Jul 14, 270(28), 16499 - 502 Identification of a yeast karyopherin heterodimer that targets import substrate to mammalian nuclear pore complexes; Enenkel C et al.; Targeting of import substrate to nuclear pore complexes of permeabilized vertebrate cells was previously shown to require a protein complex composed of two subunits, termed karyopherin . Yeast contain a homologue of karyopherin alpha named Srp1p, which was initially identified as a genetic suppressor of mutations in a subunit of RNA polymerase I . To determine whether yeast contain a karyopherin complex that includes Srp1p as the karyopherin alpha homologue, we genetically replaced Srp1p with a Srp1-Protein A chimera . Cytosol from this strain contained a complex, composed of the chimera and a protein of 95 kDa, that was purified using affinity chromatography on IgG Sepharose . Microsequence analysis showed that the 95-kDa protein was identical with a yeast protein encoded by gene L8300.15 on chromosome XII . Sequence comparison revealed that the L8300.15 gene product is the closest structural homologue of vertebrate karyopherin beta . The yeast alpha and beta karyopherin subunits were expressed in Escherichia coli and were purified . When combined, they formed a heterodimeric complex and were active in targeting import substrate to nuclear envelopes of mammalian cells . We propose that all karyopherins function as alpha/beta heterodimers. J Mol Biol, 1995 Jul 14, 250(3), 309 - 14 Monomer-dimer equilibrium of the pSC101 RepA protein; Ingmer H et al.; The pSC101 RepA protein, which is required for plasmid DNA replication, but is inhibitory to replication at high concentration, has been found in both monomeric and dimeric forms . While RepA monomers bind to direct repeat iterons near the pSC101 replication origin, dimers bind to sequences that autoregulate RepA synthesis . We investigated the solution properties of purified RepA protein by analytical ultracentrifugation analysis, and found that RepA exists in Escherichia coli cells in a monomer-dimer equilibrium (Kd = 4 microM), and, moreover, that RepA is primarily in the monomeric form at the concentration (500 molecules per cell; 2 microM) we found by Western blot analysis to occur in cells carrying replicating wild-type pSC101 plasmids . However, at concentrations inhibitory to pSC101 DNA replication, the majority of RepA molecules exist as dimers . Our findings provide experimental support for the proposal that the equilibrium between monomer and dimer forms of RepA has a key role in determining its effect on the replication of pSC101. Nature, 1995 Jul 13, 376(6536), 191 - 4 Crystal structure of the zeta isoform of the 14-3-3 protein; Liu D et al.; The 14-3-3 family of proteins have recently been identified as regulatory elements in intracellular signalling pathways: 14-3-3 proteins bind to oncogene and proto-oncogene products, including c-Raf-1 (refs 2-5), c-Bcr (ref . 6) and polyomavirus middle-T antigen; overexpression of 14-3-3 activates Raf kinase in yeast and induces meiotic maturation in Xenopus oocytes . Here we report the crystal structure of the major isoform of mammalian 14-3-3 proteins at 2.9 A resolution . Each subunit of the dimeric protein consists of a bundle of nine antiparallel helices that form a palisade around an amphipathic groove . The groove is large enough to accommodate a tenth helix, and we propose that binding to an amphipathic helix represents a general mechanism for the interaction of 14-3-3 with diverse cellular proteins . The residues in the dimer interface and the putative ligand-binding surface are invariant among vertebrates, yeast and plants, suggesting a conservation of structure and function throughout the 14-3-3 family. Nature, 1995 Jul 13, 376(6536), 188 - 91 Structure of a 14-3-3 protein and implications for coordination of multiple signalling pathways; Xiao B et al.; A broad range of organisms and tissues contain 14-3-3 proteins, which have been associated with many diverse functions including critical roles in signal transduction pathways, exocytosis and cell cycle regulation . We report here the crystal structure of the human T-cell 14-3-3 isoform (tau) dimer at 2.6 A resolution . Each monomer (Mr 28K) is composed of an unusual arrangement of nine antiparallel alpha-helices organized as two structural domains . The dimer creates a large, negatively charged channel approximately 35 A broad, 35 A wide and 20 A deep . Overall, invariant residues line the interior of this channel whereas the more variable residues are distributed on the outer surface . At the base of this channel is a 16-residue segment of 14-3-3 which has been implicated in the binding of 14-3-3 to protein kinase C. Nature, 1995 Jul 13, 376(6536), 184 - 8 A giant nucleopore protein that binds Ran/TC4; Yokoyama N et al.; Ran/TC4 is a small nuclear G protein that forms a complex with the chromatin-bound guanine nucleotide release factor RCC1 (ref . 2) . Loss of RCC1 causes defects in cell cycle progression, RNA export and nuclear protein import . Some of these can be suppressed by overexpression of Ran/TC4 (ref . 1), suggesting that Ran/TC4 functions downstream of RCC1 . We have searched for proteins that bind Ran/TC4 by using a two-hybrid screen, and here we report the identification of RanBP2, a novel protein of 3,224 residues . This giant protein comprises an amino-terminal 700-residue leucine-rich region, four RanBP1-homologous (refs 9, 10) domains, eight zinc-finger motifs similar to those of NUP153 (refs 11, 12), and a carboxy terminus with high homology to cyclophilin . The molecule contains the XFXFG pentapeptide motif characteristic of nuclear pore complex (NPC) proteins, and immunolocalization suggests that RanBP2 is a constituent of the NPC . The fact that NLS-mediated nuclear import can be inhibited by an antibody directed against RanBP2 supports a functional role in protein import through the NPC. Nucleic Acids Res, 1995 Jul 11, 23(13), 2519 - 25 The site-specific DNA endonuclease encoded by a group I intron in the Chlamydomonas pallidostigmatica chloroplast small subunit rRNA gene introduces a single-strand break at low concentrations of Mg2+; Turmel M et al.; Two group I introns (CpSSU.1 and CpSSU.2) that each potentially encode a protein with two copies of the LAGLI-DADG motif were identified in the Chlamydomonas pallidostigmatica chloroplast small subunit rRNA gene . They both belong to subgroup IA3 and represent novel insertion positions in this gene (sites 508 and 793 in the Escherichia coli 16S rRNA) . The proteins encoded by the two introns were synthesized in vitro and tested for their ability to cleave the homing site of their respective introns . Only the CpSSU.1-encoded protein (I-CpaII) was found to display specific DNA endonuclease activity . At 0.1 mM MgCl2, I-CpaII nicks only the bottom (transcribed) DNA strand, but at concentrations ranging from 0.5 to 5.0 mM, it cleaves both DNA strands (leaving a 4 nucleotide single-stranded extension with 3'-OH overhangs) while preferentially nicking the bottom strand . The rate of cleavage of the top strand increases with increasing concentration of MgCl2 . The preliminary data derived from these endonuclease assays suggest that the mode of DNA cleavage by I-CpaII is directed by the availability of Mg2+ and the affinity of different binding sites for this cation. Nucleic Acids Res, 1995 Jul 11, 23(13), 2506 - 11 Phosphorylation of a chloroplast RNA-binding protein changes its affinity to RNA; Lisitsky I et al.; An RNA-binding protein of 28 kDa (28RNP) was previously isolated from spinach chloroplasts and found to be required for 3' end-processing of chloroplast mRNAs . The amino acid sequence of 28RNP revealed two approximately 80 amino-acid RNA-binding domains, as well as an acidic- and glycine-rich amino terminal domain . Upon analysis of the RNA-binding properties of the 'native' 28RNP in comparison to the recombinant bacterial expressed protein, differences were detected in the affinity to some chloroplastic 3' end RNAs . It was suggested that post-translational modification can modulate the affinity of the 28RNP in the chloroplast to different RNAs . In order to determine if phosphorylation accounts for this post-translational modification, we examined if the 28RNP is a phosphoprotein and if it can serve as a substrate for protein kinases . It was found that the 28RNP was phosphorylated when intact chloroplasts were metabolically labeled with {32P} orthophosphate, and that recombinant 28RNP served as an excellent substrate in vitro for protein kinase isolated from spinach chloroplasts or recombinant alpha subunit of maize casein kinase II . The 28RNP was apparently phosphorylated at one site located in the acidic domain at the N-terminus of the protein . Site-directed mutagenesis of the serines in that region revealed that the phosphorylation of the protein was eliminated when serine number 22 from the N-terminus was changed to tryptophan . RNA-binding analysis of the phosphorylated 28RNP revealed that the affinity of the phosphorylated protein was reduced approximately 3-4-fold in comparison to the non-phosphorylated protein . Therefore, phosphorylation of the 28RNP modulates its affinity to RNA and may play a significant role in its biological function in the chloroplast. Nucleic Acids Res, 1995 Jul 11, 23(13), 2472 - 8 Metalloregulation of the cyanobacterial smt locus: identification of SmtB binding sites and direct interaction with metals; Erbe JL et al.; The smtB gene of Synechococcus PCC 7942 encodes a trans-acting repressor of the metal-regulated smtA gene that encodes a class II metallothionein . Recombinant SmtB has been expressed in Escherichia coli and purified . Electrophoretic mobility shift assays using recombinant SmtB or a protein extract from Synechococcus PCC 6301 reveal the concentration-dependent formation of three specific complexes with the smt operator/promoter . SmtB is also capable of direct interaction with metals as evidenced by 65Zn binding to the SmtB protein as well as the inhibition of repressor-DNA complex formation in the presence of various metal ions . Methylation interference analysis of such complexes identifies four protein contact points within the smt operator/promoter DNA . The points of contact appear to represent two pairs of binding sites, one pair in each of two inverted repeats (nt 548-563, 589-602) . The contact points within each pair lie on opposing DNA strands and are separated by 10 bp, placing the repressor binding sites on opposite sides of the DNA helix . Based on electrophoretic mobility shift assays, methylation interference and molecular size calculations we propose that recombinant SmtB binds to the smt operator/promoter in multimeric fashion. Nucleic Acids Res, 1995 Jul 11, 23(13), 2396 - 403 Cooperative assembly of proteins in the ribosomal GTPase centre demonstrated by their interactions with mutant 23S rRNAs; Rosendahl G et al.; The ribosomal protein L11 binds to the region of 23S rRNA associated with the GTPase-dependent steps of protein synthesis . Nucleotides 1054-1107 within this region of the Escherichia coli 23S rRNA gene were mutagenized with bisulphite . Twenty point mutations (G-->A and C-->T transitions) and numerous multiple mutations were generated . Expression of mutant 23S rRNAs in vivo shows that all the mutations detectably alter the phenotype, with effects ranging from a slight growth rate reduction to lack of viability . Temperature sensitivity is conferred by 1071G-->A and 1092C-->U substitutions . These effects are relieved by point mutations at other sites, indicating functional interconnections within the higher order structure of this 23S rRNA region . Several mutations prevent direct binding of r-protein L11 to 23S rRNA in vitro . These mutations are mainly in a short irregular stem (1087-1102) and within a hairpin loop (1068-1072), where the protein probably makes nucleotide contacts . Some of these mutations also interfere with binding of the r-protein complex L10.(L12)4 to an adjacent site on the rRNA . When added together to rRNA, proteins L10.(L12)4 and L11 bind cooperatively to overcome the effects of mutations at 1091 and 1099 . The proteins also stimulate each others binding to rRNA mutated at 1087 or 1092, although in these cases binding remains clearly substoichiometric . Surprisingly, none of the mutations prevents incorporation of L11 into ribosomes in vivo, indicating that other, as yet unidentified, factors are involved in the cooperative assembly process. Nucleic Acids Res, 1995 Jul 11, 23(13), 2389 - 95 Cloning and characterisation of a nuclear, site specific ssDNA binding protein; Smidt MP et al.; Estradiol inducible, liver-specific expression of the apoVLDL II gene is mediated through the estrogen receptor and a variety of other DNA-binding proteins . In the present study we report the cloning and characterisation of a single-strand DNA binding protein that interacts with the lower strand of a complex regulatory site, which includes the major estrogen responsive element and a site that resembles the rat albumin site D (apoVLDL II site D) . Based on its binding specificity determined with electro-mobility shift assays, the protein is named single-strand D-box binding factor (ssDBF) . Analysis of the deduced 302 amino acid sequence revealed that the protein belongs to the heteronuclear ribonucleoprotein A/B family (hnRNP A/B) and resembles other known eukaryotic single-strand DNA binding proteins . Transient transfection experiments in a chicken liver cell-line showed that the protein represses estrogen-induced transcription . A protein with similar binding characteristics is present in liver nuclear extract . The relevance of the occurrence of this protein to the expression of the apoVLDL II gene is discussed. Nucleic Acids Res, 1995 Jul 11, 23(13), 2371 - 80 Mapping the path of the nascent peptide chain through the 23S RNA in the 50S ribosomal subunit; Stade K et al.; Peptides of different lengths encoded by suitable mRNA fragments were biosynthesized in situ on Escherichia coli ribosomes . The peptides carried a diazirine derivative bound to their N-terminal methionine residue, which was photoactivated whilst the peptides were still attached to the ribosome . Subsequently, the sites of photo-cross-linking to 23S RNA were analyzed by our standard procedures . The N-termini of peptides of increasing length became progressively cross-linked to nucleotide 750 (peptides of 6, 9 or 13-15 amino acids), to nucleotide 1614 and concomitantly to a second site between nucleotides 1305 and 1350 (a peptide of 25-26 amino acids), and to nucleotide 91 (a peptide of 29-33 amino acids) . Previously we had shown that peptides of 1 or 2 amino acids were cross-linked to nucleotides 2062, 2506 and 2585 within the peptidyl transferase ring, whereas tri-and tetrapeptides were additionally cross-linked to nucleotides 2609 and 1781 . Taken together, the data demonstrate that the path of the nascent peptide chain moves from the peptidyl transferase ring in domain V of the 23S RNA to domain IV, then to domain II, then to domain III, and finally to domain I . These cross-linking results are correlated with other types of topographical data relating to the 50S subunit. Biochemistry, 1995 Jul 11, 34(27), 8924 - 30 Mutagenesis in Escherichia coli by three O6-substituted guanines in double-stranded or gapped plasmids; Pauly GT et al.; Plasmids were constructed with guanine (G) or O6-methyl- (m6G), O6-ethyl-(e6G), or O6-benzyl- (b6G) guanine in the initiation codon (ATG) of the lacZ' gene . Four deoxyuridine residues were incorporated near the modified guanine in the complementary strand . The deoxyuridine-containing plasmids exhibited similarly high transformation efficiencies in ung- Escherichia coli, although the frequency of mutations induced by m6G, e6G, and b6G residues was relatively low . Treatment of the plasmids with uracil-DNA glycosylase (UDG), to remove the uracil residues, or UDG and exonuclease III, to create a gap in the deoxyuridine-containing strand, reduced transformation efficiency for adduct-containing plasmids but did not affect transformation efficiency for control plasmids . However, the same treatments dramatically enhanced mutagenesis by m6G, e6G, and b6G . These results were consistent with blockage of replication by the modified guanines in double-stranded plasmids resulting in preferential replication of the complementary strand . Replication past the modified guanines was forced in the gapped plasmids . The frequency of modified guanine-induced mutations in gapped vectors was similar in strains of E . coli that were proficient in DNA polymerase III but deficient in either DNA polymerase I or II or both polymerase I and II suggesting either that polymerase III was primarily responsible for adduct bypass in all strains or that the probability of base misinsertion during bypass by either polymerase I or II was similar to that for polymerase III . Repair studies with gapped plasmids indicated that m6G was subject to repair by Ada methyltransferase and to postreplication processing by methylation-directed mismatch repair . Neither e6G nor b6G were similarly repaired.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1995 Jul 11, 34(27), 8914 - 23 Cytosine methyltransferase from Escherichia coli in which active site cysteine is replaced with serine is partially active; Gabbara S et al.; EcoRII methyltransferase (M.EcoRII) catalyzes the transfer of methyl groups from S-adenosyl-L-methionine (SAM) to C-5 position of second cytosine in the DNA sequence 5'-CCWGG (W = A or T) . The reaction is initiated by a nucleophilic attack of the C-6 of target cytosine by a cysteine that is conserved among all cytosine methyltransferases . We have replaced this cysteine in M.EcoRII with serine or alanine and purified the proteins to homogeneity . The catalytic efficiency (kcat/Km) of the mutant enzyme with serine (C186S) for methyl transfer is about 10,000 times less than that of WT but is substantially higher than the efficiency of the C186A mutant . We show that the WT enzyme and C186S mutant are proficient in exchange of proton at C-5 and that this activity is reduced in the mutant to the same extent as the methyl transfer activity . The C186S mutant is insensitive to a cysteine-specific inhibitor, and it transfers methyl groups to the same position of cytosine as the WT enzyme . The ability of serine to act as a nucleophile in the enzyme reaction suggests that it--and probably the cysteine in the WT enzyme--is activated by a nearby base . Like the WT enzyme, C186S forms stable SDS-resistant complexes with DNA containing 5-azacytosine; but unlike the WT enzyme, the mutant reacts faster with 5-azacytosine than with normal cytosine . Apparently, greater reactivity of 5-azacytosine assists the C186S mutant in catalysis. Biochemistry, 1995 Jul 11, 34(27), 8896 - 903 Isolated RNA binding domain of a class I tRNA synthetase; Gale AJ et al.; Class I tRNA synthetases typically have two major domains consisting of a class-defining N-terminal nucleotide binding fold, which contains the active site, and an idiosyncratic C-terminal domain, which in many instances provides for interactions with the tRNA anticodon . Whether the C-terminal domain can function in specific RNA binding when disconnected from the catalytic core is not known . We fused the anticodon binding domain of Escherichia coli methionyl tRNA synthetase to maltose binding protein . This fusion protein and the released, isolated domain are stable and have native-like structural characteristics, as shown by circular dichroism and thermal denaturation studies . Both the fusion protein and the isolated domain bind specifically to a small RNA hairpin oligonucleotide that recapitulates the anticodon stem-loop of tRNAfMet . Neither protein binds to an RNA hairpin with a point mutation in the anticodon trinucleotide . The binding specificity and affinity of these proteins duplicate those of the interaction between methionyl tRNA synthetase and the anticodon stem-loop oligonucleotide . Thus, the anticodon binding domain is functionally independent of the class-defining catalytic core and can be joined to another protein with little change in RNA binding characteristics. Biochemistry, 1995 Jul 11, 34(27), 8752 - 62 Self-association of Escherichia coli DNA-dependent RNA polymerase core enzyme; Harris SJ et al.; The extent of self-association of Escherichia coli DNA-dependent RNA polymerase core enzyme has been investigated by velocity sedimentation as a function of both NaCl and protein concentrations . The core enzyme, existing as essentially monomeric species having a sedimentation coefficient of 13.1 S at NaCl concentrations greater than 0.2 M, undergoes reversible self-association at lower salt concentrations . Estimates for the stoichiometry of association and equilibrium constants of reaction were determined from the effect of protein concentration on the weight-average sedimentation coefficient measured at different NaCl concentrations . Data analysis by a nonlinear curve-fitting procedure indicated that protein self-association is best described by a sequential model characterized by weaker association constants for each additional step of oligomerization, and any model that involves cooperative formation of oligomeric species can be excluded . These findings are at variance with the conclusion of a previous study {Shaner, S . L., Platt, D . M., Wensley, C . G., Yu, H., Burgess, R . R., & Record, M . T . (1982) Biochemistry 26, 5539-5551} which suggested that core RNA polymerase exists in equilibrium between monomeric and tetrameric forms of the enzyme and excluded the existence of intermediate species . Simulation of sedimentation velocity boundary and gradient profiles are used to assess the validity of both models of association of core protein . It was clear that had the core enzyme undergone a cooperative monomer<-->tetramer mode of association, then bimodality would have been observed in the derivative tracings of the sedimentation boundary under these experimental conditions . Nevertheless, no such observation was reported by Shaner et al . and this study . The sequential model favored by the results of this study is consistent with the proposed model resulted from a small-angle X-ray study {Heumann, H, Meisenberger O., & Pilz, I . (1982) FEBS Lett . 138, 273-276} . Further analysis of the data by the Wyman linked-function relationship {Wyman, J . (1964) Adv . Protein Chem . 19, 223-286} implies that core enzyme monomer loses approximately three counterions per contact upon association to higher oligomeric species. Biochemistry, 1995 Jul 11, 34(27), 8513 - 9 Interactions of Escherichia coli primary replicative helicase DnaB protein with single-stranded DNA . The nucleic acid does not wrap around the protein hexamer; Bujalowski W et al.; The interactions of the Escherichia coli primary replicative helicase DnaB protein with single-stranded (ss) DNA have been studied using the thermodynamically rigorous fluorescence titration technique, which allowed us to obtain absolute stoichiometries of the formed complexes and interaction parameters without any assumptions about the relationship between the observed signal change and the degree of binding . Binding of the DnaB protein to the ssDNA fluorescent derivative poly(d epsilon A) is accompanied by a strong increase of the nucleic acid fluorescence . We show that, in the presence of the ATP nonhydrolyzable analog AMP-PNP, the DnaB helicase binds polymer ssDNA with the site-size of 20 +/- 3 nucleotides per protein hexamer . This stoichiometry has been fully confirmed in the binding experiments with ssDNA oligomers of 40 and 20 residues in length . Two DnaB hexamers bind to 40-mer, and one DnaB hexamer binds to 20-mer . Thermodynamic studies of the 20-mer binding to the DnaB hexamer show that the hexamer has a single, strong binding site for ssDNA . Moreover, photo-cross-linking experiments indicate that only a single subunit is primarily in contact with ssDNA . This surprisingly very low site-size of the large hexameric helicase--ssDNA complex, the existence of only a single, strong ssDNA binding site on the hexamer, and the results of photo-cross-linking experiments preclude the possibility of extensive wrapping of the ssDNA around the hexamer and formation of the complex in which all six protomers are simultaneously bound to ss nucleic acid.(ABSTRACT TRUNCATED AT 250 WORDS) Arch Biochem Biophys, 1995 Jul 10, 320(2), 224 - 35 Structural characterization by nuclear magnetic resonance spectroscopy of a genetically engineered high-affinity calmodulin-binding peptide derived from Bordetella pertussis adenylate cyclase; Munier H et al.; This paper reports the solution conformation of a peptide (P196-267) derived from the calmodulin-binding domain of Bordetella pertussis adenylate cyclase . P196-267 corresponding to the protein fragment situated between amino acid residues 196-267 was overproduced by a recombinant Escherichia coli strain . Its affinity for calmodulin is only one order of magnitude lower (Kd = 2.4 nM) than that of the whole bacterial enzyme (Kd = 0.2 nM) . The proton resonances of the NMR spectra of P196-267 were assigned using homonuclear two-dimensional techniques (double-quantum-filtered J-correlated spectroscopy, total correlation spectroscopy, and nuclear Overhauser enhancement spectroscopy) and a standard assignment procedure . Analysis of the nuclear Overhauser effect connectivities and the secondary shift distribution of C alpha protons along the sequence allowed us to identify the elements of regular secondary structure . The peptide is flexible in solution, being in equilibrium between random coil and helical structures . Two segments of 11 amino acids (situated between V215 and A225) and 15 amino acids (situated between L233 and A247) populate in a significant proportion the helix conformational state . The two helices can be considerably stabilized in a mixed solvent, trifluoroethanol/water (30/70), suggesting that the corresponding fragment in the intact protein assumes a similar secondary conformation . No elements of tertiary structure organization were detected by the present experiments . The conformational properties of the isolated calmodulin target fragment are discussed in relation with the available NMR and X-ray data on various peptides complexed to calmodulin. FEBS Lett, 1995 Jul 10, 368(1), 77 - 80 ERK2/p42 MAP kinase stimulates both autonomous and SRF-dependent DNA binding by Elk-1; Sharrocks AD; A ternary complex comprised of SRF, ternary complex factor (TCF) and the c-fos SRE is the target of several extracellular signal regulated pathways . Phosphorylation of the TCF Elk-1 is a key event in the activation of this complex . We demonstrate that ERK2/p42 phosphorylation of Elk-1 stimulates its recruitment into ternary complexes with SRF . Moreover, phosphorylation of Elk-1 also stimulates its autonomous SRF-independent binding to high affinity binding sites . Thus part of the effect of ERK2/p42 phosphorylation is to stimulate DNA-binding by the ETS DNA-binding domain of Elk-1. FEBS Lett, 1995 Jul 10, 368(1), 55 - 8 Isoforms of 14-3-3 protein can form homo- and heterodimers in vivo and in vitro: implications for function as adapter proteins; Jones DH et al.; 14-3-3 proteins play a role in many cellular functions: they bind to and regulate several proteins which are critical for cell proliferation and differentiation . 14-3-3 proteins exist as dimers, and in this study we have shown that diverse 14-3-3 proteins can form both homo- and heterodimers in vitro (by cross-linking studies) and in vivo (by coimmunoprecipitation and Western blot analysis); this interaction is mediated solely through the N-terminal domain of the proteins . The composition of 14-3-3 dimers within a cell may play a key part in the role of this family of proteins as modulators or adapters which facilitate the interaction of distinct components of signalling pathways. FEBS Lett, 1995 Jul 10, 368(1), 49 - 54 Analysis and crystallization of a 25 kDa C-terminal fragment of cloned elongation factor Ts from Escherichia coli; Bogestrand S et al.; A 25 kDa C-terminal tryptic fragment of elongation factor Ts has been purified to homogeneity . Experimental evidence suggests that the 25 kDa C-terminal and the 5.3 kDa N-terminal fragments are structurally independent domains . The N-terminal fragment is shown to be essential for the nucleotide exchange activity . Crystals of the C-terminal fragment belong to space group P2 or P2(1) . The diffraction pattern shows a pronounced pseudo-C2 symmetry at low resolution . This pseudo symmetry increases when the crystals are irradiated with X-rays for a few hours. FEBS Lett, 1995 Jul 10, 368(1), 39 - 44 Biochemical characterization of recombinant yeast PPZ1, a protein phosphatase involved in salt tolerance; Posas F et al.; The Saccharomyces cerevisiae gene PPZ1 codes for a 692-residues protein that shows in its carboxyl-terminal half about 60% identity with the catalytic subunit of mammalian and yeast protein phosphatase-1 and that is involved in salt homeostasis . The complete PPZ1 protein has been successfully expressed as a soluble glutathione-S-transferase fusion protein . The recombinant protein, after purification by a single affinity chromatography step, displayed phosphatase activity towards a number of substrates, including myelin basic protein, histone 2A and casein, but was ineffective in dephosphorylating glycogen phosphorylase . It was also active towards p-nitrophenylphosphate . The activity was severalfold increased by the presence of Mn2+ ions and by limited trypsinolysis . The enzyme was inhibited by okadaic acid and microcystin-LR at concentrations comparable to what is found for type 1 protein phosphatase although it was much less sensitive to inhibitor-2 . The recombinant protein was phosphorylated in vitro by cAMP-dependent protein kinase, protein kinase C and casein kinase-2 . Phosphorylation affected preferentially sites located in the amino-terminal half of the protein and did not alter the activity of the phosphatase. FEBS Lett, 1995 Jul 10, 368(1), 15 - 8 Interaction between cardiolipin and the mitochondrial presequence of cytochrome c oxidase subunit IV favours lipid mixing without destabilizing the bilayer structure; Mandieau V et al.; We demonstrate the ability of a peptide corresponding to the presequence of the cytochrome c oxidase subunit IV to induce lipid mixing between large unilamellar liposomes . This lipid mixing requires the presence of CL or PE, lipids able to form non-bilayer structures, and is not observed with other negatively charged lipids . However, the fact that this mixing occurs without mixing of the liposome aqueous phases and without destabilizing the lipid organisation is unusual and has not been observed for other amphiphilic peptides . This observation supports the idea that the presequence could play a role in the formation of translocation contact sites between the two mitochondrial membranes and facilitate the structural rearrangements of the outer and inner membrane proteins involved in the two import machineries in a way to permit the formation of a continuous import channel through the two mitochondrial membranes without mixing the cytoplasmic and mitochondrial aqueous contents. FEBS Lett, 1995 Jul 10, 368(1), 105 - 9 Identification of additional homologues of subunits VII and VIII of the ubiquinol-cytochrome c oxidoreductase enables definition of consensus sequences; Boumans H et al.; The Candida utilis QCR7 gene encoding subunit VII of the ubiquinol-cytochrome c oxidoreductase was isolated by functional complementation of the Saccharomyces cerevisiae subunit VII-null mutant . Several other subunit VII homologues as well as homologues for subunit VIII were identified by screening the GenBank database . Some of these homologues for subunit VII could only be identified as such using a consensus sequence that was derived from the multiple sequence alignment . Definition of the consensus should facilitate further analysis of structure/function relationships in this protein. Virology, 1995 Jul 10, 210(2), 391 - 9 Identification of a linear neutralization domain in the protein VP2 of African horse sickness virus; Martinez-Torrecuadrada JL et al.; Overlapping fragments of the outermost capsid protein VP2 of African horse sickness virus serotype 4 (AHSV-4) have been expressed in Escherichia coli . Horse sera from infected and vaccinated animals, rabbit sera, and mice monoclonal antibodies specific for AHSV were used to screen these fragments for antigenic regions . The screening revealed that the major antigenic domain of the AHSV-4 VP2 is localized in a central region (amino acids 200 to 413) and that both the N-terminal region (aa 1-159) and the half C-terminal region (aa 414-1060) are not immunogenic . All the fragments containing a region between amino acids 253 and 413 (fragment H) were able to elicit consistently high titers of neutralizing antibodies . The ability of several subfragments of this region to evoke neutralizing antibodies indicates the presence of several sites inside this domain . However, neutralizing antibodies in sera of horse infected or vaccinated with attenuated viruses were not absorbed by fragment H, indicating that this domain is not immunodominant in AHSV . This information might be useful in designing a subunit vaccine against AHSV infection. J Biol Chem, 1995 Jul 7, 270(27), 16476 - 81 Disruption of base-paired U4.U6 small nuclear RNAs induced by mammalian heterogeneous nuclear ribonucleoprotein C protein; Forne T et al.; Due to 3' end modifications, mammalian U6 small nuclear RNA (snRNA) is heterogeneous in size . The major form terminates with five U residues and a 2',3'-cyclic phosphate, but multiple RNAs containing up to 12 U residues have a 3'-OH end . They are labeled in the presence of {alpha-32P}UTP by the terminal uridylyl transferase activity present in HeLa cell nuclear extracts . That these forms all enter the U6 snRNA-containing particles, U4.U6, U4.U5.U6, and the spliceosome, has been demonstrated previously . Here, we report an interaction between the heterogeneous nuclear ribonucleoprotein (hnRNP) C protein, an abundant nuclear pre-mRNA binding protein, and the U6 snRNAs that have the longest uridylate stretches . This U6 snRNA subset is free of any one of the other snRNPs, since anti-Sm antibodies failed to immunoprecipitate hnRNP C protein . Furthermore, isolated U4.U6 snRNPs containing U6 snRNAs with long oligouridylate stretches are disrupted upon binding of hnRNP C protein either purified from HeLa cells or produced as recombinant protein from Escherichia coli . In view of these data and our previous proposal that the U6 snRNA active in splicing has 3'-OH end, we discuss a model where the hnRNP C protein has a decisive function in the catalytic activation of the spliceosome by allowing the release of U4 snRNP. J Biol Chem, 1995 Jul 7, 270(27), 16360 - 70 Role of the Escherichia coli recombination hotspot, chi, in RecABCD-dependent homologous pairing; Dixon DA et al.; Genetic recombination occurring in wild type Escherichia coli is stimulated at DNA sequences known as chi sites, 5'-GCTGGTGG-3' . In vitro, homologous pairing between duplex DNA substrates dependent upon the RecA, RecBCD, and SSB proteins is stimulated by the presence of a chi sequence in the donor linear double-stranded DNA . We show that this stimulation is due to two factors: 1) the enhanced production of chi-specific single-stranded DNA fragments and 2) their preferential use in the RecA protein-promoted pairing step . Furthermore, under conditions of limiting Mg2+ concentration, joint molecule formation does not occur, even though DNA unwinding and chi-specific single-stranded DNA fragment production are observed . Also, under these conditions, chi-specific fragments derived from both the upstream and downstream regions of the DNA strand containing chi and from cleavage of the non-chi-containing DNA strand are detected . Finally, the behavior of mutant RecBCD enzymes (RecBC*D and RecBCD not equal to) in this in vitro reaction is shown to parallel their in vivo phenotypes with respect to chi stimulation of recombination . Thus we suggest that, in addition to its ability to regulate the degradative activities of RecBCD enzyme, chi itself may be a preferred site for initiation of homologous pairing in this concerted process. J Biol Chem, 1995 Jul 7, 270(27), 16321 - 6 Molecular cloning and characterization of a plant serine acetyltransferase playing a regulatory role in cysteine biosynthesis from watermelon; Saito K et al.; Serine acetyltransferase (SATase; EC 2.3.1.30), which catalyzes the reaction connecting serine and cysteine/methionine metabolism, plays a regulatory role in cysteine biosynthesis in plants . We have isolated a cDNA clone encoding SATase by direct genetic complementation of a Cys- mutation in Escherichia coli using an expression library of Citrullus vulgaris (watermelon) cDNA . The cDNA encodes a polypeptide of 294 amino acids (31,536 Da) exhibiting 51% homology with that of E . coli SATase . DNA-blot analysis indicated the presence of a single copy of the SATase gene (sat) in watermelon . RNA hybridization analysis suggested the relatively ubiquitous and preferential expression in the hypocotyls of etiolated seedlings . Immunoblot analysis indicated the accumulation of SATase predominantly in etiolated plants . L-Cysteine, an end product of the cysteine biosynthetic pathway, inhibited the SATase in an allosteric manner, indicating the regulatory function of SATase in this metabolic pathway, whereas beta-(pyrazole-1-yl)-L-alanine, a secondary metabolite formed partly through the cysteine biosynthetic pathway, showed no inhibitory effect . A multi-enzyme complex was formed from recombinant proteins of SATase and cysteine synthase (O-acetylserine(thiol)-lyase) from watermelon, suggesting efficient metabolic channeling from serine to cysteine, preventing the diffusion of intermediary O-acetyl-L-serine. J Biol Chem, 1995 Jul 7, 270(27), 16251 - 7 The conserved motif, GXXX(D/E)(R/K)XG{X}(R/K)(R/K), in hydrophilic loop 2/3 of the lactose permease; Jessen-Marshall AE et al.; A conserved motif, GXXX(D/E)(R/K)XG(R/K)(R/K), has been identified among a large group of evolutionarily related membrane proteins involved in the transport of small molecules across the membrane . To determine the importance of this motif within the lactose permease of Escherichia coli, a total of 28 site-directed mutations at the conserved first, fifth, sixth, eighth, ninth, and tenth positions were analyzed . A dramatic inhibition of activity was observed with all bulky mutations at the first-position glycine . Based on these results, together with sequence comparisons within the superfamily, it seems likely that small side chain volume (and possibly high beta-turn propensity) may be structurally important at this position . The acidic residue at the fifth position was also found to be very important for transport activity and even a conservative glutamate at this location exhibited marginal transport activity . In contrast, many substitutions at the eighth-position glycine, even those with a high side chain volume and/or low beta-turn propensity, still retained high levels of transport activity . Similarly, none of the basic residues within the motif were essential for transport activity when replaced individually by nonbasic residues . However, certain substitutions at the basic residue sites as well as the eighth-position glycine were observed to have moderately reduced levels of active transport of lactose . Taken together, the results of this study confirm the importance of the conserved loop 2/3 motif in transport function . It is suggested that this motif may be important in promoting global conformational changes within the permease. J Biol Chem, 1995 Jul 7, 270(27), 16230 - 5 Cytosolic mercaptopyruvate sulfurtransferase is evolutionarily related to mitochondrial rhodanese . Striking similarity in active site amino acid sequence and the increase in the mercaptopyruvate sulfurtransferase activity of rhodanese by site-directed mutagenesis; Nagahara N et al.; Rat liver mercaptopyruvate sulfurtransferase (MST) was purified to homogeneity . MST is very similar to rhodanese in physicochemical properties . Further, rhodanese cross-reacts with anti-MST antibody . Both purified authentic MST and expressed rhodanese possess MST and rhodanese activities, although the ratio of rhodanese to MST activity is low in MST and high in rhodanese . In order to compare the active site regions of MST and rhodanese, the primary structure of a possible active site region of MST was determined . The sequence showed 66% homology with that of rat liver rhodanese . An active site cysteine residue (Cys246; site of formation of persulfide in catalysis) and an arginine residue (Arg185; substrate binding site) in rhodanese were also conserved in MST . On the other hand, two other active site residues (Arg247 and Lys248) were replaced by Gly and Ser, respectively . Conversion of rhodanese to MST was tried by site-directed mutagenesis . After cloning of rat liver rhodanese, recombinant wild type and three mutants (Arg247-->Gly and/or Lys248-->Ser) were constructed . The enzymes were expressed in Escherichia coli strain BL21 (DE3) with a T7 promoter system . The mutation of these residues decreases rhodanese activity and increases MST activity. J Biol Chem, 1995 Jul 7, 270(27), 16213 - 20 Cytochrome bd oxidase from Azotobacter vinelandii . Purification and quantitation of ligand binding to the oxygen reduction site; Junemann S et al.; Cytochrome bd has been purified from Azotobacter vinelandii by a new simplified procedure . The heme and total iron content has been measured, as has the number of high affinity CO and NO binding sites . Spectral changes indicate high affinity binding of CO and NO to heme d only, with a stoichiometry of 1 molecule of gas per 2 molecules of heme b or per 3 atoms of iron . The results clearly define a stoichiometry of one heme d per complex . Low affinity binding of CO and NO to heme b595 also occurs at higher ligand concentrations . EPR heme-nitrosyl signals are seen with NO bound to both hemes b595 and d but with no indication of spin exchange coupling . Exposure of the air-oxidized complex to alkaline pH results in removal of molecular oxygen from heme d and a change in line shape of the high spin region of the EPR spectrum . Cyanide binds to both heme d and heme b595 in the air-oxidized complex, displacing molecular oxygen from heme d . The rate of cyanide binding to heme d as assessed by spectral changes at 650 nm does not correlate with the rate of binding to heme b595 as assessed by the loss of the high spin EPR signal . In addition, the cyanide binding rate in the presence of reductant is only 3 times that of the rate of binding to the air-oxidized enzyme, in contrast to the copper-containing oxidases where strong redox cooperativity makes these