Biochemistry, 1994 Jun 14, 33(23), 7278 - 87 Pseudosubstrate inhibition of CDPK, a protein kinase with a calmodulin-like domain; Harmon AC et al.; Between the catalytic and regulatory domains of calmodulin-like domain protein kinase, CDPK, is a junction domain which has some identity to the autoinhibitory domain of calmodulin-dependent protein kinase type II (Harper, J . F., Sussman, M . R., Schaller, G . E., Putnam-Evans, C., Charbonneau, H., & Harmon, A . C . (1991) Science 252, 951-954) . To investigate whether CDPK's junction domain also functions as an autoinhibitory domain, we determined the effect of synthetic peptides, corresponding to sequences within the junction domain, on the activity of native soybean CDPK . Three peptides, corresponding to residues 310-332, 318-332, 302-317, were competitive inhibitors with respect to syntide-2 and had Ki values of 5, 25, and 85 microM, respectively . These peptides were uncompetitive inhibitors with respect to ATP and had Ki values of 24, 220, and 510 microM, respectively . A fourth peptide, CDPK alpha 302-332, inhibited activity by a mixed mechanism with respect to both syntide-2 (Ki = 1.9 microM; K'i = 5.0 microM) and ATP (Ki = 15 microM; K'i = 4.5 microM) . Three of the peptides, CDPK alpha 302-332, 310-332, and 318-332, formed complexes with soybean calmodulin during electrophoresis in native polyacrylamide gels and were able to inhibit calmodulin-dependent protein kinases . Given the similarity between CDPK's calmodulin-like domain and calmodulin (40% sequence identity), it was possible that these peptides could inhibit activity through interaction with the calmodulin-like domain rather than the catalytic domain . To address this possibility, a cDNA encoding the first 312 residues of soybean CDPK alpha was constructed and expressed in Escherichia coli . This enzyme, which is missing most of the junction domain and all of the calmodulin-like domain, was active in the presence and absence of calcium . Peptide CDPK alpha 310-332 inhibited this truncated enzyme competitively with respect to syntide-2 (Ki = 4 microM) . These results show that the junction domain is capable of functioning as an autoinhibitory domain, possibly through a pseudosubstrate site located between residues 310 and 332. Biochemistry, 1994 Jun 14, 33(23), 7267 - 77 Genetic identification of an autoinhibitor in CDPK, a protein kinase with a calmodulin-like domain; Harper JF et al.; CDPKs are a family of calcium (Ca2+)-dependent protein kinases which are defined by a carboxyl-terminal calmodulin-like domain . Mutational analysis indicates that the junction domain, which joins the kinase and calmodulin-like domains, contains an autoinhibitor . CDPK isoform AK1 from Arabidopsis was expressed in Escherichia coli as a fusion protein sandwiched between glutathione S-transferase and six consecutive histidines at the N- and C-terminal ends, respectively . This fusion, called AK1-6H, was purified and displayed kinase activity which was stimulated up to 127-fold by Ca2+, with a typical specific activity of 2000 nmol min-1 mg-1, using syntide-2 as peptide substrate . A truncation which deletes the calmodulin-like domain, as in mutant delta C-6H, disrupts Ca2+ activation and leaves the enzyme with a basal level of activity . Delta C-6H could be activated 87-fold by preincubation with a purified polyclonal IgG which was raised against a junction domain fusion . A further deletion of the junction domain, as in mutant delta JC, results in a constitutively active enzyme . This indicates that the junction domain in delta C-6H can function as an autoinhibitor . Its function as an autoinhibitor in a full-length enzyme was confirmed by site-specific mutagenesis, as shown by mutant KJM23-6H, which had a six-residue substitution in the junction domain between A422 and A432 . Both delta JC and KJM23-6H encoded Ca(2+)-independent enzymes which had specific activities greater than 70% that of a fully active AK1-6H and displayed equivalent Km values for ATP and syntide-2 . Inhibition studies on delta JC, using peptides based on the autoinhibitory domains of Ca2+/calmodulin-dependent protein kinases, are consistent with a model where the junction domain contains a similar pseudosubstrate-type autoinhibitor. Biochemistry, 1994 Jun 14, 33(23), 7174 - 83 Mannose transporter of Escherichia coli . Backbone assignments and secondary structure of the IIA domain of the IIABMan subunit; Seip S et al.; The mannose transporter of Escherichia coli consists of two transmembrane and one peripheral protein subunit . The complex acts by a mechanism which couples translocation of the substrate with substrate phosphorylation . The peripheral IIABMan is a homodimer . The IIABMan monomer itself contains two domains which are linked by an Ala-Pro-rich hinge and which are both transiently phosphorylated at histidyl residues . The IIA and IIB domains can be separated by limited proteolysis . The IIA domain has a dimer molecular mass of 2 x 14 kDa . Almost complete 1H, 13C, and 15N NMR assignments of the backbone resonances of IIAMan have been achieved using 3D and 4D double-and triple-resonance techniques . Secondary structure elements were derived from NOE data . The IIA domain consists of a central beta-sheet of four parallel and one antiparallel strand (strand order 5 4 3 1 2) with helices on both sides of the sheet . The active-site His-10 is located in a loop at the C-terminus of beta-strand 1 . This loop and the loop after strand 3 are at the topological switch point of the sheet. Biochemistry, 1994 Jun 14, 33(23), 7127 - 33 Replication of DNA templates containing the alpha-anomer of deoxyadenosine, a major adenine lesion produced by hydroxyl radicals; Ide H et al.; The alpha-anomer of deoxyadenosine (alpha-dA) is a major adenine lesion produced by hydroxyl radicals in DNA . To assess its biochemical effects on DNA replication, alpha-dA was site-specifically incorporated into oligodeoxyribonucleotide templates using phosphoramidite chemistry . alpha-dA in the template constituted a transient block to DNA synthesis catalyzed by Escherichia coli DNA polymerase I Klenow fragment (polI), but translesional synthesis occurred after prolonged incubation . Primer extension assays and Maxam-Gilbert sequencing of newly synthesized products revealed that alpha-dA directed not only incorporation of the correct nucleotide, dTMP, opposite the lesion but also misincorporation of dAMP and dCMP . dGMP was barely incorporated under these conditions . The order of the incorporation frequency at the alpha-dA site was affected by the nearest neighbor base pair 3' to the lesion . T7 and Taq DNA polymerases, as well as RAV-2 reverse transcriptase, showed a selectivity similar to that of PolI with respect to the nucleotide incorporation opposite alpha-dA, suggesting that the discrimination of nucleotides associated with alpha-dA is independent of the origin of DNA polymerases and is an intrinsic feature of the lesion . The mutational spectrum predicted for alpha-dA (i.e., A-->G transitions and A-->T transversions) is significantly different from those reported for other hydroxyl radical induced DNA lesions such as abasic sites or 7,8-dihydro-8-oxoguanine, both primarily directing misincorporation of A . Possible biological consequences and the mechanism of dNTP discrimination associated with alpha-dA are discussed. Biochemistry, 1994 Jun 14, 33(23), 7120 - 6 Sequence-specific DNA displaces 6-p-toluidino-2-naphthalenesulfonate bound to a hydrophobic site on the DNA-binding domain of Drosophila c-myb; Madan A et al.; The N-terminal DNA-binding domain of c-myb oncoprotein binds to DNA in a sequence-specific manner . The domain, consisting of three imperfect tandem repeats, has tryptophan residues at very regular intervals and this is believed to be of some significance in the DNA-binding activity of the protein . We have found that the hydrophobic-site-specific probe 6-p-toluidino-2-naphthalenesulfonate (TNS) binds to the bacterially expressed DNA-binding domain of Drosophila c-myb protein (R123) . TNS has a single binding site on this protein with an apparent dissociation constant in the range of (5-8) x 10(-7) M . When the TNS-protein complex was treated with an oligomeric DNA duplex having a cognate myb-binding site, the TNS was displaced from the complex . Nonspecific DNA duplex oligomers were ineffective, indicating that TNS displacement was a sequence-specific process . We examined further some features of the TNS-binding site on the protein, taking advantage of the fluorescence properties of the protein and the bound TNS . Our data indicate that the TNS binding occurs in a peripheral site on the protein in a manner that allows the bound TNS to be solvent accessible . Furthermore, there are indications that tyrosine(s) and tryptophans of the protein mediate resonance energy transfer to the bound TNS . From fluorescence-quenching data of the protein and protein-TNS complex, we could assess that both solvent-accessible and internal tryptophans are in the vicinity of the bound TNS . (ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 Jun 14, 33(23), 7107 - 12 Substrate specificity is determined by amino acid binding pocket size in Escherichia coli phenylalanyl-tRNA synthetase; Ibba M et al.; Alanine at position 294 (Ala294) within the motif 3 consensus of Escherichia coli phenylalanyl-tRNA synthetase alpha subunit has previously been implicated as a determinant of amino acid specificity . To characterize the role of Ala294, the catalytic effects of amino acid replacements at this position were tested with purified wild-type and mutant phenylalanyl-tRNA synthetases . We show that Ala294 is involved in amino acid binding and that it influences specificity as a determinant of binding pocket size . Replacement of Ala294 by either glycine or serine, thereby increasing or decreasing the size of the binding pocket, respectively, reduces affinity for phenylalanine . The Gly294 mutant shows a relaxed specificity toward synthetic para-halogenated phenylalanine analogues, the apparent dissociation constant Km increasing in direct relation to an increase of the van der Waals radius of the para group, thus confirming the role of position 294 in determining amino acid binding pocket size . For the substrate analogue p-chlorophenylalanine, attachment to tRNA and in vivo incorporation into cellular protein by the Gly294 mutant were demonstrated . Tyrosine activation was also improved with this mutant, but the resulting enzyme-Tyr-adenylate complex was rapidly hydrolyzed, indicating the presence of a proofreading mechanism in E . coli phenylalanyl-tRNA synthetase. Biochemistry, 1994 Jun 14, 33(23), 7062 - 8 Site-directed mutagenesis and NMR studies of histidine-385 mutants of 5-enolpyruvylshikimate-3-phosphate synthase; Shuttleworth WA et al.; The site-directed mutagenesis of His-385 of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase is reported . The steady-state kinetics for two mutants, H385Q and H385A, are compared with that of the wild-type enzyme . H385Q EPSP synthase was found to have 25% wild-type enzyme activity, whereas H385A EPSP synthase retained 1% activity . The KM values for Pi and shikimate 3-phosphate were unaffected, whereas the KM for phosphoenolpyruvate (PEP) was increased 10 times for H385Q EPSP synthase . The KM for EPSP was unaffected in H385Q but raised by a factor of 10 in H385A EPSP synthase . The binding of glyphosate was studied by fluorescence spectroscopy and by 31P NMR spectroscopy . Direct observation of the enzyme-intermediate complexes by 13C NMR spectroscopy with {2,3-13C}phosphoenolpyruvate was studied for the mutant enzymes and compared with the wild type . Under equilibrium conditions, H385A EPSP synthase does not accumulate enzyme-bound EPSP . These results suggest that, while critically located in the PEP binding site, His-385 is not the residue responsible for initiating catalysis through the protonation of PEP. Biochemistry, 1994 Jun 14, 33(23), 7041 - 6 Serine 90 is required for enzymic activity by tRNA-guanine transglycosylase from Escherichia coli; Reuter K et al.; An Escherichia coli mutant described by Noguchi et al . {Noguchi, S., et al . (1982) J . Biol . Chem . 275, 6544-6550} contains tRNA lacking the hypermodified wobble nucleoside queuosine (Q) due to an inactive tRNA-guanine transglycosylase (TGT) . TGT catalyzes the posttranscriptional base exchange of the Q precursor preQ1 with the genetically encoded guanine in tRNA(Asp,Asn,His,Tyr) . The mutant tgt gene was cloned and sequenced; it contained a single point mutation resulting in the change of serine 90 to phenylalanine . Overexpression of the mutant gene yielded TGT(S90F) that showed a reduced solubility and did not purify in the same fashion as the wild-type enzyme . TGT(S90F) has no detectable enzymic activity . To determine whether serine 90 performs a catalytic role in the TGT reaction or whether the loss of activity was caused solely by a conformational change of the enzyme, we used site-specific mutagenesis to construct serine-to-alanine (S90A) and serine-to-cysteine (S90C) mutants . Both S90A and S90C mutants were purified in a manner identical to that used for the wild-type enzyme . SDS-PAGE of dimethyl suberimidate-cross-linked mutants showed a pattern identical to that of the wild-type TGT, indicative of a trimeric quaternary structure . Native PAGE of wild-type and mutant TGTs in the absence and presence of substrate tRNA exhibited band shifts indicating that both mutants retain the ability to bind tRNA.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 Jun 14, 33(23), 7021 - 6 Determination by Raman spectroscopy of the pKa of N5 of dihydrofolate bound to dihydrofolate reductase: mechanistic implications; Chen YQ et al.; Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate (H2folate) to tetrahydrofolate by NADPH, and this requires that the pteridine ring be protonated at N5 . A long-standing puzzle has been how, at physiological pH, the enzyme can protonate N5 in view of its solution pKa of 2.6 and the fact that the only proton-donating group in the pterdine binding site, Asp-27, hydrogen bonds not to N5 but to the 2-amino group and N3 of the pterin ring . We have determined the pKa of N5 of dihydrofolate in the Escherichia coli DHFR/NADP+/H2folate ternary complex by Raman difference spectroscopy and found that the value is 6.5 . In contrast, the pKa of N5 is less than 4.0 in either the binary complex, the ternary complex with an analogue of NADPH (H2NADPH), or the Asp27 to serine mutant DHFR (D27S) ternary complex with NADP+ . Thus, one need not invoke proton donation from Asp-27 to N5 via a series of bound water molecules and/or pteridine-ring substituents . We propose instead that the N5 protonated form of H2folate is stabilized directly at the active site in the DHFR/NADPH/H2folate complex by specific interactions that form only in the ternary complex, involving perhaps a bound water molecule, the carboxamide moiety of the coenzyme, and/or the local electrostatic field of the enzyme molecule, to which an important contribution may be made by Asp-27. FEBS Lett, 1994 Jun 13, 346(2-3), 263 - 7 Distinct kinetics of subunit autolysis in mammalian m-calpain activation; Saido TC et al.; Subunit autolysis of mammalian m-calpain upon activation was examined in kinetic terms using a set of antibodies recognizing different portions of the protease . Activation of m-calpain by calcium resulted in no apparent autolysis in the large catalytic subunit, whereas the small regulatory subunit underwent immediate autolysis followed by substrate proteolysis . This profile of subunit autolysis is distinct from that of the other ubiquitous isozyme, mu-calpain, in which autolysis of the large subunit and then of the small subunit precedes substrate proteolysis under the normal conditions . The activation state of m-calpain thus is not reflected by the large subunit autolysis . The mode and role of autolysis may vary among calpain isozymes. FEBS Lett, 1994 Jun 13, 346(2-3), 217 - 20 The immobilized movement proteins of two tobamoviruses form stable ribonucleoprotein complexes with full-length viral genomic RNA; Ivanov KI et al.; The movement proteins of two tobamoviruses (tobacco mosaic virus, TMV, common strain U1 and cruciferous TMV strain) containing amino-terminal hexahistidine affinity tags were overexpressed in Escherichia coli and purified by metal chelate affinity chromatography . Purified recombinant proteins were immobilized to a Ni(2+)-chelate adsorbent and their ability to interact with full-length genomic TMV RNA was tested . Here we report that binding of viral RNA to hexahistidine fusion movement proteins results in the formation of stable ribonucleoprotein complexes. FEBS Lett, 1994 Jun 13, 346(2-3), 175 - 80 In vitro mutation analysis of Arabidopsis thaliana small GTP-binding proteins and detection of GAP-like activities in plant cells; Anai T et al.; Previously, we have reported the molecular cloning of ara genes encoding a small GTP-binding protein from Arabidopsis thaliana . The criterion based on amino acid sequences suggest that such an ara gene family can be classified to be of the YPT/rab type . To examine the biochemical properties of ARA proteins, several deletions and point mutations were introduced into ara cDNAs . Mutant proteins were expressed in E . coli as GST-chimeric molecules and analyzed in terms of their GTP-binding or GTP-hydrolysing ability in vitro . The results indicate that four conserved amino acid sequence regions of ARA proteins are necessary for GTP-binding . A point mutation of Asn at position 72 for ARA-2, or 71 for ARA-4, to Ile decreased GTP-binding and a point mutation of Gln at position 126 for ARA-2, or 125 for ARA-4, to Leu suppressed GTP-hydrolysis activity . Furthermore, certain factors associated with the membrane fraction accelerated GTPase activities of ARA proteins, suggesting the presence of GTPase activating protein(s) (GAP(s)) in the vesicular transport system of higher plant cells. FEBS Lett, 1994 Jun 13, 346(2-3), 151 - 5 Cloning and expression of a human pro(tea)some beta-subunit cDNA: a homologue of the yeast PRE4-subunit essential for peptidylglutamyl-peptide hydrolase activity; Gerards WL et al.; The cDNA encoding a human prosome beta-subunit (HSBpros26) was isolated from a lymphoma library using the cDNA of the Xenopus homologue as a probe . The cDNA contains an open reading frame encoding a protein of 233 amino acids and a calculated molecular weight of 25,909 . Comparison with interspecies homologues of HSBpros26 from Xenopus (XLB), rat (RN3) and yeast (PRE4) reveals a high degree of identity between the beta-subunits except for the N-terminal end, which is probably cleaved post-translationally . The complete coding sequence of HSBpros26 has been expressed in E . coli . The produced protein of about 27 kDa reacts with the prosomal monoclonal antibody MCP205, kindly provided by Dr . K . Hendil . The molecular weight of the native protein is about 28 kDa indicating that the protein is present as monomers . Finally partially purified HSBpros26 preparations do not contain any proteolytical activity. Brain Res, 1994 Jun 13, 648(1), 171 - 5 Adenoviral vectors as functional retrograde neuronal tracers; Ridoux V et al.; Adenoviruses have been recently recognized as a highly efficient system for gene delivery to various tissues . The ability of replication-defective recombinant adenovirus to transfer the lacZ reporter gene encoding beta-galactosidase to nerve cells in various brain structures has been demonstrated . Here, on the continuation of these studies, we present evidence that the adenovirus can be transported in a retrograde manner to nerve cell bodies from axonal terminals . This method may be of great value for infecting selected subsets of specific neurons for either anatomo-functional studies or even therapeutic purposes. Biochim Biophys Acta, 1994 Jun 12, 1206(2), 197 - 202 Characterization and use of biotinylated Escherichia coli K99 lectin; Berger S et al.; K99 lectin from Escherichia coli was purified and biotinylated via the amino groups of lysine residues using N-biotinyl-6-amino-caproic acid N-hydroxysuccinimide ester (BcapNHS) . Biotin was detected on Lys-47 and Lys-87 . It was previously demonstrated (Jacobs, A.A.C., Van den Berg, P.A., Bak, H.J . and De Graaf, F.K . (1986) Biochim . Biophys . Acta 872, 92-97) that modification of lysine residues 132 and 133 with 4-chloro-3,5-dinitrobenzoate (CDNB) resulted in the loss of the binding capacity of K99 fimbriae . Due to the higher size of the biotin derivative compared to CDNB, Lys-132 or Lys-133, essential for the biological activity, were not modified . The biotinylation did not cause the loss of the haemagglutinating activity but was sufficient to permit detection of the lectin by streptavidin . A flow cytometric analysis was used for the detection of the receptors on the surface of erythrocytes. Nucleic Acids Res, 1994 Jun 11, 22(11), 2065 - 70 Phage P4 DNA replication in vitro; Diaz Orejas R et al.; Phage P4 DNA is replicated in cell-free extracts of Escherichia coli in the presence of partially purified P4 alpha protein {Krevolin and Calendar (1985), J . Mol . Biol . 182, 507-517} . Using a modified in vitro replication assay, we have further characterized this process . Analysis by agarose gel electrophoresis and autoradiography of in vitro replicated molecules demonstrates that the system yields supercoiled monomeric DNA as the main product . Electron microscopic analysis of in vitro generated intermediates indicates that DNA synthesis initiates in vitro mainly at ori, the origin of replication used in vivo . Replication proceeds from this origin bidirectionally, resulting in theta-type molecules . In contrast to the in vivo situation, no extensive single-stranded regions were found in these intermediates . The initiation proteins of the host, DnaB and DnaG, and the chaperones DnaJ and DnaK are not required for P4 replication, because polyclonal antibodies against those polypeptides do not inhibit the process . The reaction is inhibited by antibodies against the SSB protein, and by ara-CTP, a specific inhibitor of DNA polymerase III holoenzyme . Consistent with previous reports, P4 in vitro replication is independent of transcription by host RNA polymerase . Novobiocin, a DNA gyrase inhibitor, strongly inhibits P4 DNA synthesis, indicating that form I DNA is the required substrate. Nucleic Acids Res, 1994 Jun 11, 22(11), 2028 - 35 The heterodimeric subunit SRP9/14 of the signal recognition particle functions as permuted single polypeptide chain; Bovia F et al.; The targeting of nascent polypeptide chains to the endoplasmic reticulum is mediated by a cytoplasmic ribonucleoprotein, the signal recognition particle (SRP) . The 9 kD (SRP9) and the 14 kD (SRP14) subunits of SRP are required to confer elongation arrest activity to the particle . SRP9 and SRP14 form a heterodimer which specifically binds to SRP RNA . We have constructed cDNAs that encode single polypeptide chains comprising SRP9 and SRP14 sequences in the two possible permutations linked by a 17 amino acid peptide . We found that both fusion proteins specifically bound to SRP RNA as monomeric molecules folded into a heterodimer-like structure . Our results corroborate the previous hypothesis that the authentic heterodimer binds to SRP RNA in equimolar ratio . In addition, both fusion proteins conferred elongation arrest activity to SRP(-9/14), which lacks this function, and one fusion protein could functionally replace the heterodimer in the translocation assay . Thus, the normal N-and C-termini of both proteins have no essential role in folding, RNA-binding and in mediating the biological activities . The possibility to express the heterodimeric complex as a single polypeptide chain facilitates the analysis of its functions and its structure in vivo and in vitro. Nucleic Acids Res, 1994 Jun 11, 22(11), 1933 - 47 Molecular evolution of SRP cycle components: functional implications; Althoff S et al.; Signal recognition particle (SRP) is a cytoplasmic ribonucleoprotein that targets a subset of nascent presecretory proteins to the endoplasmic reticulum membrane . We have considered the SRP cycle from the perspective of molecular evolution, using recently determined sequences of genes or cDNAs encoding homologs of SRP (7SL) RNA, the Srp54 protein (Srp54p), and the alpha subunit of the SRP receptor (SR alpha) from a broad spectrum of organisms, together with the remaining five polypeptides of mammalian SRP . Our analysis provides insight into the significance of structural variation in SRP RNA and identifies novel conserved motifs in protein components of this pathway . The lack of congruence between an established phylogenetic tree and size variation in 7SL homologs implies the occurrence of several independent events that eliminated more than half the sequence content of this RNA during bacterial evolution . The apparently non-essential structures are domain I, a tRNA-like element that is constant in archaea, varies in size among eucaryotes, and is generally missing in bacteria, and domain III, a tightly base-paired hairpin that is present in all eucaryotic and archeal SRP RNAs but is invariably absent in bacteria . Based on both structural and functional considerations, we propose that the conserved core of SRP consists minimally of the 54 kDa signal sequence-binding protein complexed with the loosely base-paired domain IV helix of SRP RNA, and is also likely to contain a homolog of the Srp68 protein . Comparative sequence analysis of the methionine-rich M domains from a diverse array of Srp54p homologs reveals an extended region of amino acid identity that resembles a recently identified RNA recognition motif . Multiple sequence alignment of the G domains of Srp54p and SR alpha homologs indicates that these two polypeptides exhibit significant similarity even outside the four GTPase consensus motifs, including a block of nine contiguous amino acids in a location analogous to the binding site of the guanine nucleotide dissociation stimulator (GDS) for E . coli EF-Tu . The conservation of this sequence, in combination with the results of earlier genetic and biochemical studies of the SRP cycle, leads us to hypothesize that a component of the Srp68/72p heterodimer serves as the GDS for both Srp54p and SR alpha . Using an iterative alignment procedure, we demonstrate similarity between Srp68p and sequence motifs conserved among GDS proteins for small Ras-related GTPases . The conservation of SRP cycle components in organisms from all three major branches of the phylogenetic tree suggests that this pathway for protein export is of ancient evolutionary origin. J Biol Chem, 1994 Jun 10, 269(23), 16502 - 7 Drosophila kinesin motor domain extending to amino acid position 392 is dimeric when expressed in Escherichia coli; Huang TG et al.; A truncated domain of the alpha-subunit of Drosophila kinesin was obtained by expression in Escherichia coli and purified to homogeneity in the presence of MgATP . This domain (designated DKH392) extends to amino acid 392 and contains the complete N-terminal region of kinesin which is highly conserved between species . The DKH392 construct includes an additional 52 amino acids beyond the minimal motor domain of 340 amino acid residues which has been previously characterized as DKH340 (Huang, T.-G., and Hackney, D . D . (1994) J . Biol . Chem . 269, 16493-16501) . The s20,w values for DKH340 and DKH392 are 3.3 and 5.2 S and the D20,w values are 7.7 x 10(-7) and 4.9 x 10(-7) cm3 s-1, respectively . These results indicate that DKH340 is a monomer in solution, but DKH392 is a dimer . In the presence of adenosine 5-(beta,gamma-imido)triphosphate, DKH392 binds to microtubules with a stoichiometry of two head domains (one DKH392 dimer) per tubulin heterodimer in contrast to the tight binding of one DKH340 per tubulin heterodimer . Electron microscopy indicates that both DKH340 monomers and DKH392 dimers decorate microtubules with a periodicity of 8 nm. J Biol Chem, 1994 Jun 10, 269(23), 16493 - 501 Drosophila kinesin minimal motor domain expressed in Escherichia coli . Purification and kinetic characterization; Huang TG et al.; A truncated motor domain of the alpha subunit of Drosophila kinesin was obtained by expression in Escherichia coli and purified to homogeneity in the presence of MgATP . This domain (designated DKH340) extends from the N terminus to amino acid 340 . The isolated protein contains a stoichiometric level of tightly bound ADP and has a low basal rate of ATP hydrolysis of 0.029 +/- 0.002 s-1 in the absence of microtubules . The rate of release of bound ADP is 0.026 +/- 0.003 s-1 . The approximate equality of the ADP release rate and the steady state ATPase rate indicates that ADP release is the rate-limiting step in ATP hydrolysis in the absence of microtubules . The rate of ATP hydrolysis is stimulated 3000 fold-by addition of microtubules (MT) (kcat = 80 s-1; KMT0.5,ATPase = 160 nM for half-saturation of the ATPase rate by microtubules at saturating ATP levels; KMT0.5ATPase = 43 microns for half-saturation of the ATPase rate by ATP at saturating microtubule levels) . Binding of DKH340 to MTs is biphasic in the presence of adenosine 5-(beta-gamma-imido)t-riphosphate . One DKH340 binds tightly per tubulin heterodimer, but greater than one DKH340/tubulin heterodimer can be bound at higher ratios of DKH340/microtubules . In the presence of MgATP, KMT0.5,Binding for physical binding of DKH340 to microtubules is weaker than KMT0.5,ATPase for stimulation of hydrolysis . These results are consistent with a model in which DKH340 cycles on and off the microtubule during hydrolysis of each ATP molecule . For this model, the kcat/KMT0.5,ATPase ratio of 5 x 10(8) M-1 s-1 is at least as large as the bimolecular rate constant for association with microtubules, and this value approaches the diffusion controlled limit . Nucleotide-free DKH340 can be produced by gel filtration in the absence of Mg2+, but it reforms tightly bound ADP slowly in the presence of MgATP (t1/2 > or = 10 min), and thus it is likely to be in a conformational state which is not produced during steady state ATP hydrolysis. J Biol Chem, 1994 Jun 10, 269(23), 16449 - 54 Identification of volatile forms of methyl groups released by Halobacterium salinarium; Nordmann B et al.; halobacterium salinarium (formerly H . halobium) is a chemotactic and phototactic archaeon from which volatile methyl groups are released continually, a phenomenon related to its sensory system . We found that released methyl groups comprised two different chemical species, methanol and methanethiol, the sulfur analog of methanol . Radiolabeling experiments showed that the methyl groups of both compounds, as well as the sulfur of methanethiol, were derived from methionine but were donated to cellular components and subsequently cleaved to produce the respective volatile compounds . Previous work had shown that chemostimuli and photostimuli result in transient increases in the rate of release of volatile methyl groups . We found that these increases reflected increased release of methanol but not of methanethiol . Thus, the methyl group chemistry of the H . salinarium sensory system is analogous to the well-studied chemotactic system of Escherichia coli . The reactions that result in methanethiol release are of unknown function and have unusual features . They may involve a methionine-gamma-lyase activity we detected in H . salinarium . Sulfur derived from methionine was found attached to specific proteins in reduction-sensitive disulfide linkages. J Biol Chem, 1994 Jun 10, 269(23), 16371 - 5 Topoisomerase IV can support oriC DNA replication in vitro; Hiasa H et al.; Escherichia coli has two type II topoisomerases, DNA gyrase and topoisomerase IV (Topo IV) . Topo IV is required for the decatenation of the linked daughter chromosomes at the terminal stages of DNA replication, whereas gyrase, because of its ability to convert to negative supercoils the positive supercoils generated by replication fork progression in a circular chromosome, is required to support nascent chain elongation . Using an oriC DNA replication system in vitro, we show that Topo IV, which can relax positive supercoils, can also support replication fork progression . This activity is only observed at substoichiometric ratios of Topo IV to template, at higher ratios, the template becomes relaxed and initiation of DNA replication cannot occur . Topo IV was capable of supporting bidirectional DNA replication from oriC, although, unlike the case with gyrase, some templates apparently replicated unidirectionally . This suggests that either gyrase itself or a certain minimum superhelical density is required for proper initiation of DNA replication from oriC. J Biol Chem, 1994 Jun 10, 269(23), 16364 - 70 Plasmodium falciparum S-adenosylhomocysteine hydrolase . cDNA identification, predicted protein sequence, and expression in Escherichia coli; Creedon KA et al.; Compounds that specifically inhibit S-adenosylhomocysteine hydrolase (SAHH; EC 3.3.1.1) interfere with the proliferation of Plasmodium malarial parasites, but efforts to identify the enzyme directly in parasite extracts have been unsuccessful . Here we report genetic and biochemical evidence for the presence of a gene encoding P . falciparum SAHH . The gene is transcribed as a 2.8-kilobase mRNA in erythrocytic stage parasites . Analysis of the open reading frame predicts a 53.9-kDa protein having conserved regions thought to be involved in NAD binding . The cDNA sequence has been incorporated into an Escherichia coli expression construct to confirm the function of the sahh product . Transformed E . coli cells produce a protein with a relative molecular weight of 56,000 which possesses SAHH activity as evidenced by the conversion of 3-deazaadenosine to S-3-deazaadenosylhomocysteine . Several amino acid residues that have been suggested to be at the SAHH active site in other organisms show nonconserved replacements in P . falciparum, suggesting that some current proposals for the enzyme mechanism may need to be revised . The structural differences between the P . falciparum and mammalian SAHH enzymes may foster innovative strategies for drug development against malaria. J Biol Chem, 1994 Jun 10, 269(23), 16333 - 9 rheb, a growth factor- and synaptic activity-regulated gene, encodes a novel Ras-related protein; Yamagata K et al.; Neuronal activity results in long term cellular changes that underlie normal brain development and synaptic plasticity . To examine the molecular basis of activity-dependent plasticity, we have used differential cloning techniques to identify genes that are rapidly induced in brain neurons by synaptic activity . Here we describe an inducible novel member of the Ras family of small GTP-binding proteins we have termed Rheb . rheb mRNA is rapidly and transiently induced in hippocampal granule cells by seizures and by NMDA-dependent synaptic activity in the long term potentiation paradigm . The predicted amino acid sequence of Rheb is most closely homologous to yeast Ras1 and human Rap2 . The putative GTP binding regions are highly conserved . A bacterial fusion protein of Rheb binds GTP and exhibits intrinsic GTPase activity . Like Ha-Ras, the carboxylterminal sequence encodes a CAAX box that is predicted to signal post-translational farnesylation and to target Rheb to specific membranes . rheb mRNA is expressed at comparatively high levels in normal adult cortex as well as a number of peripheral tissues, including lung and intestine . In the developing brain, rheb mRNA is expressed at relatively high levels in embryonic day 19 cortical plate, and expression remains at stable levels throughout the remainder of prenatal and postnatal development . Its close homology with ras and its rapid inducibility by receptor-dependent synaptic activity suggest that rheb may play an important role in long term activity-dependent neuronal responses. J Biol Chem, 1994 Jun 10, 269(23), 16305 - 10 Signal peptide cleavage regions . Functional limits on length and topological implications; Jain RG et al.; As a first step toward understanding the topology of the signal peptide with respect to the membrane during the protein export process, we have examined the constraints on the length of the cleavage region needed to achieve signal peptidase recognition and cleavage . Using the signal peptide of Escherichia coli alkaline phosphatase, a series of cleavage region mutants has been constructed . Variations in length were brought about by replacing the wild type cleavage region of the signal peptide with polymers of increasingly more residues . In each case, alanine residues are used exclusively in the -1 and -3 positions to provide only one viable cleavage site . Glutamine residues are used in all other positions in order to vary the length from 3 to 13 total residues . Analysis of these mutants revealed that cleavage regions ranging from 3 to 9 residues are completely and efficiently processed . The extent of processing drops substantially thereafter, with no processing observed for signal peptides with 13-residue long cleavage regions . A second mutant with a 13-residue long cleavage region was designed and analyzed to ensure that the lack of processing reflected a cleavage problem and not a translocation defect . The results are consistent with the notion that the signal peptidase active site is in close proximity to the periplasmic surface of the inner membrane and that interaction of the cleavage region with the signal peptidase probably depends on, and is constrained by, other interactions involving the signal peptide. J Biol Chem, 1994 Jun 10, 269(23), 16260 - 8 Purification and characterization of a novel deoxyinosine-specific enzyme, deoxyinosine 3' endonuclease, from Escherichia coli; Yao M et al.; We have purified a novel endonuclease from Escherichia coli that recognizes deoxyinosine, a deamination product of deoxyadenosine in DNA . This activity, which we named deoxyinosine 3' endonuclease, is different from the known hypoxanthine DNA N-glycosylases which have been partially characterized in E . coli and other organisms . The enzyme was purified 24,800-fold to apparent homogeneity . SDS- and activity PAGE analyses indicate that the enzyme has an apparent molecular mass of 25 kDa . Deoxyinosine 3' endonuclease recognized deoxyinosine in both single- and double-stranded DNA but exhibited a 4-fold preference for double stranded over single-stranded DNA . In addition to deoxyinosine, the enzyme recognized urea residues and AP sites . Deoxyinosine 3' endonuclease has an obligatory requirement for Mg2+, but other cations such as Co2+ and Mn2+ could partially replace Mg2+ . The optimal pH for deoxyinosine 3' endonuclease was around 7.5 . In contrast to most of the known repair enzymes, deoxyinosine 3' endonuclease makes an incision at the second phosphodiester bond 3' to a deoxyinosine or AP site, leaving behind the intact lesion on the nicked DNA . Therefore, deoxyinosine 3' endonuclease recognizes, but does not remove, the lesion from the DNA molecule . The biological significance of this novel activity is discussed with reference to other repair activities in E . coli. J Biol Chem, 1994 Jun 10, 269(23), 16236 - 41 Effects of guanosine 3',5'-bisdiphosphate (ppGpp) on rate of transcription elongation in isoleucine-starved Escherichia coli; Vogel U et al.; We measured the transcription elongation rate on two mRNA genes, i.e . infB and lacZ, and on a part of the rrnB gene under conditions when wild type (rel+) Escherichia coli and relaxed (relA) mutants were exposed to isoleucine starvation . The RNA chain growth rates were calculated from the time lag between induction of transcription and the appearance of specific hybridization to probes complementary to the 3' ends of the genes, i.e . from the transcription time . The rate of mRNA chain elongation responded differently in rel+ and relA strains exposed to isoleucine starvation as it decreased (approximately 50%) in rel+ strains that accumulated high concentrations of guanosine 3',5'-bisdiphosphate (ppGpp) and increased (approximately 15%) the relA mutant whose ppGpp pool decayed during starvation . These results show that ppGpp inhibits mRNA chain elongation in vivo . However, stable RNA chain elongation appeared unaffected by ppGpp pool size and was twice as fast as mRNA chain elongation in exponentially growing cells. J Biol Chem, 1994 Jun 10, 269(23), 16223 - 8 Effect of selenium deficiency on type I 5'-deiodinase; DePalo D et al.; The type I iodothyronine 5'-deiodinase (5'-DI) present in rat liver and kidney has recently been demonstrated to be a selenoprotein . The goal of the present study was to examine in detail the effect of selenium (Se) deficiency on 5'-DI at the protein and mRNA levels . In weanling rats fed a selenium-deficient (Se(-)) diet for 6 weeks, 5'-DI activity was decreased 91 and 69% relative to control activities in liver and kidney, respectively . Administration of 3,5,3'-triiodothyronine resulted in a 2-fold increase in 5'-DI activity in control animals, but had little or no effect on 5'-DI activity in Se(-) animals . Western analysis using a specific antiserum directed against a bacterial fusion protein containing the carboxyl-terminal half of the 5'-DI protein demonstrated that this decrease in 5'-DI activity in Se(-) animals was explained by a marked decrease in 5'-DI protein . Administration of Se to Se(-) animals resulted in parallel increases in 5'-DI protein and activity over a 72-h time period . It was also shown that selenium deficiency was accompanied by a 40% decrease in 5'-DI mRNA levels in the kidney, but not in the liver . In both tissues, the administration of 3,5,3'-triiodothyronine resulted in increased 5'-DI mRNA levels which were not altered by selenium status . These studies indicate that selenium deficiency decreases 5'-DI activity by decreasing the amount of 5'-DI protein . The mechanism of this impairment in enzyme synthesis appears to be a defect in translation, presumably due to a block in the UGA-directed selenocysteine incorporation in selenium deficiency. J Biol Chem, 1994 Jun 10, 269(23), 16163 - 9 The importance of conserved nucleotides of 23 S ribosomal RNA and transfer RNA in ribosome catalyzed peptide bond formation; Lieberman KR et al.; We have constructed the double mutant G2252C/G2253C in Escherichia coli 23 S rRNA by site-directed mutagenesis . These phylogenetically conserved residues are protected from chemical modification by the 3' CCA terminus of the peptidyl-tRNA site (P site)-bound tRNA . Expression of C2252/C2253 23 S rRNA in E . coli severely compromises cell growth . Mutant rRNA is assembled into 50 S subunits and 70 S ribosomes but is discriminated against in polysomes . Mutant ribosomes function at lower rates in peptidyltransferase assays than wild type ribosomes . To test whether this defect derives from disruption of base pairing with the 2 cytidines of the invariant 3' CCA terminus of tRNA, a mutant E . coli tRNAPhe gene was constructed, with the CCA sequence changed to GGA . As deacylated species, mutant and wild type tRNAPhe inhibit peptidyl transfer identically . Mutant tRNAPhe was aminoacylated in vitro but failed to react as a P site substrate, with either mutant or wild type ribosomes . These results support a role for G2252 and G2253 of 23 S rRNA in peptidyltransferase function and a role for the 3' residues of peptidyl-tRNA in catalytically productive P site interaction; but they fail to provide evidence supporting canonical base pairing between these 23 S residues and the 3' end of peptidyl-tRNA. J Biol Chem, 1994 Jun 10, 269(23), 16091 - 100 Identification of the gene encoding lipoate-protein ligase A of Escherichia coli . Molecular cloning and characterization of the lplA gene and gene product; Morris TW et al.; R(+)-Lipoic acid is a cofactor required for function of the alpha-keto acid dehydrogenase and glycine cleavage enzyme complexes . The naturally occurring form of lipoate is attached by amide linkage to the epsilon-amino group of a specific lysine residue within conserved lipoate-accepting protein domains . Lipoate-protein ligase(s) catalyze the formation of this amide bond between lipoyl groups and specific apoproteins . We report the isolation of the lplA gene which encodes an Escherichia coli lipoate-protein ligase . Strains with lplA null mutations transport lipoic acid normally but have severe defects in the incorporation and utilization of exogenously supplied lipoic acid and lipoic acid analogs . These strains are also highly resistant to selenolipoate (a growth-inhibiting lipoate analog) and contain no detectable lipoate-protein ligase activity in cell extracts . The lplA gene has been cloned, sequenced, and physically mapped to min 99.6 (4657 kilobases) of the E . coli chromosome . Upon overexpression, the 38-kDa lplA gene product was purified to homogeneity and shown to have a mass, N-terminal sequence and amino acid composition consistent with the deduced 337 residue primary sequence . Enzyme assays show that purified LplA catalyzes the ATP-dependent attachment of {35S}lipoic acid to apoprotein, thus confirming that lplA encodes lipoate-protein ligase A . Analysis of lplA null mutants also indicates the existence of a second (lplA-independent) lipoyl-ligase enzyme in E . coli . This is the first identification of a lipoate ligase gene and the first analysis of a purified lipoate ligase enzyme. J Biol Chem, 1994 Jun 10, 269(23), 16082 - 90 Direct binding of myosin II to phospholipid vesicles via tail regions and phosphorylation of the heavy chains by protein kinase C; Murakami N et al.; Recent cloning and sequencing studies suggest that heavy chains of all non-muscle myosins II have a protein kinase C (PKC) phosphorylation site within their tail regions . A fragment of human macrophage myosin heavy chain, encompassing its COOH-terminal 396 amino acids (MIIAF46), was expressed in Escherichia coli to provide a model system for study of PKC-mediated phosphorylation . PKC phosphorylated this fragment when phosphatidylserine (PS) liposomes were present, but not when liposomes made from PS/phosphatidylcholine (PC) were used . The reaction required Ca2+, but not other activators such as diacylglycerol (DG) or phosphatidylinositol 4,5-bisphosphate . Phosphorylation of MIIAF46 was not observed in the presence of micelles of PS or PS/DG . Similar results were obtained using native myosin II purified from bovine brain and chicken intestine brush border . Phosphorylation of light chains, in contrast, occurred even with PS/PC liposomes if DG was present . Addition of the PS and PS/DG liposomes significantly increased the turbidities at 340 nm of MIIAF46 and native myosin II, and the extent of increase depended upon the type of myosin used . Also, PS and PS/DG liposomes shifted the gel filtration elution positions of MIIAF46 and myosin II . In contrast, liposomes of PS/PC and PS/PC/DG gave only a slight increase in turbidity with all myosins and fragments and did not noticeably shift their gel filtration elution positions . These results suggest that myosins II bind to PS liposomes via the COOH-terminal regions of their heavy chains with affinities specific to each myosin isoform, that the binding is dependent upon the PS composition, and that PKC phosphorylates the PS-bound heavy chains. J Biol Chem, 1994 Jun 10, 269(23), 16020 - 8 Model of subunit composition of gamma-aminobutyric acid A receptor subtypes expressed in rat cerebellum with respect to their alpha and gamma/delta subunits; Quirk K et al.; Antibodies specific for subunits of the gamma-aminobutyric acid A (GABAA) receptor have been used to immunoprecipitate {3H}muscimol, {3H}Ro 15-4513, and {3H}Ro 15-1788 binding sites from deoxycholate-solubilized preparations of rat cerebellum . Of the antisera raised against alpha subunits, those specific for alpha 6 immunoprecipitated the largest proportion of receptors . Two populations of alpha 6-containing GABAA receptors were identified . The first was labeled with {3H}Ro 15-4513 and exhibited a pharmacological profile consistent with that observed for alpha 6 beta 2 gamma 2 in transfected cells (Luddens, H., Pritchett, D . B., Kohler, M., Killisch, I., Keinanen, K., Monyer, H., Sprengel, R., and Seeberg, P . H . (1990) Nature 346, 648-651) . The second population was labeled only with {3H}muscimol and was deduced, from quantitative immunoprecipitation studies using combinations of antibodies, to contain both alpha 6 and delta subunits . The alpha 6 subunit was not observed to be present in combination with other alpha subunits or the gamma 1 subunit . Each of the other alpha subunits was found to be present in only one population of receptors in the cerebellum . Some subunits (alpha 4, alpha 5, and gamma 3) were not detectable . By combining information from quantitative immunoprecipitation experiments and Western blot analysis, a model describing the composition of all GABAA receptors in the cerebellum was constructed that defined the following alpha and gamma/delta combinations (percentage of cerebellar GABAA receptors): alpha 6 gamma 2 (36%), alpha 6 delta (23%), alpha 1 gamma 2 (28%), alpha 2 gamma 1 (8%), and alpha 3 gamma 2 (5%). Gene, 1994 Jun 10, 143(2), 239 - 43 Secretory production of chicken ovomucoid domain 3 by Escherichia coli and alteration of inhibitory specificity toward proteases by substitution of the P1 site residue; Kojima S et al.; Ovomucoids are commonly present in bird egg white and exhibit inhibitory activity toward various serine proteases . To investigate the structure-function relationship of ovomucoid domain 3, we established a secretory expression system for the chicken ovomucoid domain 3 (OMCHI3)-encoding gene in Escherichia coli by ligating it downstream from the tac promoter and signal peptide of E . coli alkaline phosphatase . E . coli JM105 was transformed with the resulting plasmid and induced with 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG) . The mature OMCHI3 was detected in the culture supernatant, and was purified to homogeneity by three-step chromatography . Amino-acid sequence analysis showed that processing by the signal peptidase was carried out exactly at the expected site . Measurements of circular dichroism spectra and inhibitory activity indicated that OMCHI3 was produced in the properly folded form . Furthermore, site-specific replacement of the Ala residue at the P1 site with Met or Lys resulted in acquisition of inhibitory activity toward chymotrypsin or trypsin, respectively, indicating that the P1 site is the predominant determinant for inhibitory specificity. Gene, 1994 Jun 10, 143(2), 201 - 9 Translational regulation of a recombinant operon containing human platelet-derived growth factor (PDGF)-encoding genes in Escherichia coli: genetic titration of the peptide chains of the heterodimer AB; Schneppe B et al.; A new strategy is described for the production of recombinant heteromultimeric proteins using Escherichia coli as host . A recombinant operon was constructed containing modified cDNA sequences encoding the mature A and B chains of human platelet-derived growth factor (PDGF) . The relative expression rates of the PDGF genes were varied over a range equivalent to A:B ratios from 0.8 to 3.7 by means of translational regulation . This was achieved using two different translational initiation sequences (TIS) upstream from the respective coding regions, one derived from the E . coli atpE translational initiation region, and the other containing a sequence with extended complementarity to the 3' end of the 16S rRNA . The generation of mature PDGF A and B chains in different relative amounts in E . coli provided the basis for developing a novel procedure for the production of the biologically active PDGF heterodimer AB in large quantities . The general strategy is applicable to the preparation of a wide range of heteromultimeric complexes . Moreover, the described PDGF operon constitutes a compact and versatile model system for studies of the posttranscriptional regulation of gene expression. J Mol Biol, 1994 Jun 10, 239(3), 433 - 5 Nucleotide sequence of the Escherichia coli K12 histidine operon revisited; Jovanovic G et al.; We report on a significant difference between the published nucleotide sequence of the Escherichia coli K12 histidine operon and our sequencing results repeatedly obtained from a number of different E . coli K12 strains . The discrepancies include 39 base-pair changes and one addition located predominantly in the proximal portion of the operon . Our data also suggest that neutral and near-neutral mutations do not accumulate to a significant extent in the histidine operon of E . coli strains harbouring strong mutator alleles. Biochemistry, 1994 Jun 7, 33(22), 6998 - 7004 Effects of autophosphorylation on casein kinase II activity: evidence from mutations in the beta subunit; Lin WJ et al.; Casein kinase II is a heterotetramer composed of two catalytic (alpha) and two regulatory (beta) subunits . To examine the effects of autophosphorylation of the beta subunit on enzyme activity, two mutants of the beta subunit from Drosophila were constructed in which either Ser4 or Ser2-4 was changed to alanine residues by oligonucleotide-directed mutagenesis and the proteins were expressed in Escherichia coli . The wild-type alpha and individual beta subunits present in inclusion bodies were renatured, and the biochemical properties of the reconstituted holoenzymes were examined . Analysis of autophosphorylation revealed that phosphate incorporation was about 0.8 mol/mol of beta subunit for the wild type and Ala4 mutant; Ser2 and Ser3 were the major sites of autophosphorylation with some phosphate in Ser4 as shown by Edman degradation . No autophosphorylation was observed with the Ala2-4 mutant . Substitution of alanine for serine residues at positions 4 or 2-4 of the beta subunits did not influence the reassociation of the alpha and beta subunits to form holoenzyme, or the function of the beta subunit in stimulating catalytic activity or in responding to basic compounds . To measure the effects of autophosphorylation on casein kinase II activity, the wild-type and mutant holoenzymes were preincubated in the presence and absence of ATP, and the rate of phosphorylation was measured with various substrates . In the absence of autophosphorylation, the wild-type, Ala4, and Ala2-4 forms of the holoenzyme displayed similar rates of phosphorylation of glycogen synthase . After preincubation with ATP, the rate of phosphorylation of glycogen synthase by the wild-type and Ala4 enzymes was inhibited by 30%.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 Jun 7, 33(22), 6812 - 21 Time-resolved solid-state NMR spectroscopy of 5-enolpyruvylshikimate-3-phosphate synthase; Appleyard RJ et al.; The novel technique of time-resolved solid-state NMR spectroscopy has been used to characterize the enzyme, 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, in both the forward and reverse directions over time periods ranging from 5 to 300 ms . The wealth of data currently available for EPSP synthase, in particular the pre-steady-state kinetics performed using chemical quench-flow experiments {Anderson, K . S., Sikorski, J . A., & Johnson, K . A . (1988) Biochemistry 27, 7395-7406}, has made the enzyme an obvious choice as a proving ground for this new technique . Pre-steady-state 13C TOSS CP-MAS spectra have been obtained with a much improved signal-to-noise ratio, and corrections have been made to some previously reported assignments {Evans, J.N.S., Appleyard, R.J., & Shuttleworth, W.A . (1993) J . Am . Chem . Soc . 115, 1588-1590} . Peak fitting has allowed the extrapolation of NMR integral intensities of species involved in the reaction . These show a good correlation with concentrations calculated by simulations using the kinetic parameters obtained from the chemical quench-flow experiments . It is proposed that careful optimization of the contact time used will be necessary to obtain accurate, relative concentrations that will enable an independent kinetic simulation by time-resolved solid-state NMR . The technique shows much promise due to its nondestructive quenching procedure, which allows the direct observation of enzyme intermediates on a reaction pathway . However, its requirement of significantly larger amounts of enzyme does limit the technique to those proteins which naturally occur in high abundance or have been hyperexpressed. Proc Natl Acad Sci U S A, 1994 Jun 7, 91(12), 5508 - 12 Stable DNA transformation in the obligate intracellular parasite Toxoplasma gondii by complementation of tryptophan auxotrophy; Sibley LD et al.; The protozoan parasite Toxoplasma gondii infects a wide range of vertebrate hosts and is an important opportunistic pathogen in immunocompromised humans . Although Toxoplasma is amenable to both biochemical and cellular experimental approaches, the molecular basis of its success as an intracellular parasite is poorly understood . To provide a system for molecular genetic analyses, we have developed a stable DNA transformation system for Toxoplasma based on complementation of its naturally occurring tryptophan auxotrophy . Complementation was accomplished by expressing the Escherichia coli trpB gene, encoding the beta subunit of tryptophan synthase (EC 4.2.1.20), the enzyme that catalyzes the formation of tryptophan from indole plus serine . Transformants were obtained by electroporation of a plasmid, called SAG1/trpB, containing the trpB gene flanked by Toxoplasma surface antigen 1 (SAG1) gene sequences and selection for growth on indole . Transformants were obtained with circular forms of the SAG1/trpB plasmid with efficiencies of 10(-4) per cell . Transformation with either circular or linear SAG1/trpB resulted in integration into the genome at distinct, nonhomologous sites . Trp+ transformants typically contained tandemly repeated copies of the SAG1/trpB plasmid and were stable in the absence of continued selection . The Trp phenotype provides a dominant selectable marker that should allow expression of foreign or altered genes in Toxoplasma and facilitate molecular analyses of genes important for intracellular survival. Proc Natl Acad Sci U S A, 1994 Jun 7, 91(12), 5446 - 50 A role for destabilizing amino acid replacements in light-chain amyloidosis; Hurle MR et al.; Light-chain (L-chain) amyloidosis is characterized by deposition of fibrillar aggregates composed of the N-terminal L-chain variable region (VL) domain of an immunoglobulin, generally in individuals overproducing a monoclonal L chain . In addition to proteolytic fragmentation and high protein concentration, particular amino acid substitutions may also contribute to the tendency of an L chain to aggregate in L-chain amyloidosis, although evidence in support of this has been limited and difficult to interpret . In this paper we identify particular amino acid replacements at specific positions in the VL domain that are occupied at frequencies significantly higher in those L chains associated with amyloidosis . Analysis of the structural model for the VL domain of the Bence-Jones protein REI suggests that these positions play important roles in maintaining domain structure and stability . Using an Escherichia coli expression system, we prepared single-point mutants of REI VL incorporating amyloid-associated amino acid replacements that are both rare and located at structurally important positions . These mutants support ordered aggregate formation in an in vitro L-chain fibril formation model in which wild-type REI VL remains soluble . Moreover, the ability of these sequences to aggregate in vitro correlates well with the extent to which domain stability is decreased in denaturant-induced unfolding . The results are consistent with a mechanism for the disease process in which the VL domain, either before or after proteolytic cleavage from the L-chain constant region domain, unfolds by virtue of one or more destabilizing amino acid replacements to generate an aggregation-prone nonnative state. Proc Natl Acad Sci U S A, 1994 Jun 7, 91(12), 5421 - 5 Properties of permease dimer, a fusion protein containing two lactose permease molecules from Escherichia coli; Sahin-Toth M et al.; An engineered fusion protein containing two tandem lactose permease molecules (permease dimer) exhibits high transport activity and is used to test the phenomenon of negative dominance . Introduction of the mutation Glu-325-->Cys into either the first or the second half of the dimer results in a 50% decrease in activity, whereas introduction of the mutation into both halves of the dimer abolishes transport . Lactose transport by permease dimer is completely inactivated by N-ethylmaleimide; however, 40-45% activity is retained after N-ethylmaleimide treatment when either the first or the second half of the dimer is replaced with a mutant devoid of cysteine residues . The observations demonstrate that both halves of the fusion protein are equally active and suggest that each half may function independently . To test the possibility that oligomerization between dimers might account for the findings, a permease dimer was constructed that contains two different deletion mutants that complement functionally when expressed as untethered molecules . Because this construct does not catalyze lactose transport to any extent whatsoever, it is unlikely that the two halves of the dimer interact or that there is an oligomeric interaction between dimers . The approach is consistent with the contention that the functional unit of lactose permease is a monomer. Proc Natl Acad Sci U S A, 1994 Jun 7, 91(12), 5242 - 6 The cooperativity and allosteric inhibition of Escherichia coli phosphofructokinase depend on the interaction between threonine-125 and ATP; Auzat I et al.; During the reaction catalyzed by the phosphofructokinase (EC 2.7.1.11) from Escherichia coli, the phosphoryl group transferred from ATP interacts with Thr-125 {Shirakihara, Y . & Evans, P . R . (1988) J . Mol . Biol . 204, 973-994} . The replacement of Thr-125 by serine changes the saturation by fructose 6-phosphate from cooperative to hyperbolic and abolishes the allosteric inhibition by phosphoenolpyruvate . The same changes, a saturation by fructose 6-phosphate that is no longer cooperative and an activity that is no longer inhibited by phosphoenolpyruvate, are observed with wild-type phosphofructokinase when adenosine 5'-{gamma-thio}triphosphate is used instead of ATP as the phosphoryl donor . These two perturbations of the ATP-Thr-125 interaction lead to the suppression of both the allosteric inhibition by phosphoenolpyruvate and the cooperativity of fructose-6-phosphate saturation, as if replacing the neutral oxygen of ATP by sulfur or removing the methyl group of Thr-125 had "locked" phosphofructokinase in its active conformation . The geometry of this ATP-Thr-125 interaction and/or the presence of the methyl group on the beta-carbon of Thr-125 are crucial for the regulatory properties of phosphofructokinase . This interaction could be a hydrogen bond between the neutral oxygen of the gamma-phosphate of ATP and the hydroxyl group of Thr-125. Biochemistry, 1994 Jun 7, 33(22), 7014 - 20 A single mutation in the recombinant light chain of tetanus toxin abolishes its proteolytic activity and removes the toxicity seen after reconstitution with native heavy chain; Li Y et al.; Specific proteolysis by the tetanus toxin light chain of a vesicle-associated membrane protein (VAMP) involved in exocytosis is thought to underlie its intracellular blockade of neurotransmitter release . To substantiate this mechanism, recombinant light chain was expressed as a maltose binding protein-light chain fusion product in Escherichia coli . After purification of affinity chromatography and cleavage with factor Xa, the resultant light chain was isolated and its identity confirmed by Western blotting and N-terminal sequencing . It exhibited activity similar to that of the native light chain in proteolyzing its target in isolated bovine small synaptic vesicles and in hydrolyzing a 62-residue synthetic polypeptide spanning the cleavage site of the substrate . The importance of Glu234 in the catalytic activity of the light chain, possibly analogous to Glu143 of thermolysin, was examined using site-directed mutagenesis . Changing Glu234 to Ala abolished the protease activity of the light chain, but its ability to bind the polypeptide substrate was retained . Each recombinant light chain could be reconstituted with the heavy chain of tetanus toxin, yielding the same level of disulfide-linked species as the two native chains . Whereas the toxin formed with wild-type light chain exhibited appreciable neuromuscular paralysis activity and mouse lethality, the equivalent dichain material containing the Ala234 mutant lacked neurotoxicity in both the in vitro and in vivo assays . Thus, these results demonstrate directly, for the first time, that the lethality of tetanus toxin and its inhibition of exocytosis in intact neurons are attributable largely, if not exclusively, to endoprotease activity. FEBS Lett, 1994 Jun 6, 346(1), 65 - 8 Biochemical characterization of the presecretory protein translocation machinery of Escherichia coli; Tokuda H; The protein translocation apparatus in Escherichia coli has been studied both genetically and biochemically . In vitro protein translocation systems involving everted membrane vesicles or reconstituted proteoliposomes have significantly contributed to biochemical clarification of the structure, mechanism and energetics of the apparatus . It is established that SecA, SecY and SecE are essential components, and play fundamental roles in the translocation reaction, and that both ATP and a proton motive force are required for the translocation . A new membrane factor, SecG, was found to participate in the formation of the apparatus, causing significant enhancement of the activity . SecD was found to play a role in the release of translocated proteins from the outer surface of the cytoplasmic membrane. FEBS Lett, 1994 Jun 6, 346(1), 55 - 8 Maltose transport system of Escherichia coli: an ABC-type transporter; Nikaido H; The maltose transport system of E . coli is composed of a periplasmic maltose-binding protein (MBP), the presumed transmembrane channel made up of MalF and MalG proteins, and two copies of the ATPase subunit, MalK . The membrane-associated transporter complex was purified in a functional form both from the wild-type strain and from mutants that do not require MBP for transport, and was reconstituted into proteoliposomes . A major function of MBP is to send a transmembrane signal, in the presence of ligands, to the ATPase subunits on the inner side of the membrane . In addition, MBP performs a special function in the translocation of the larger ligands, maltodextrins, perhaps by aligning them for entry into the channel. FEBS Lett, 1994 Jun 6, 346(1), 48 - 54 Mitochondrial carrier proteins; Palmieri F; Ten mitochondrial carriers have been purified from animal mitochondria . They are small proteins with a molecular mass ranging from 28 to 34 kDa on SDS-PAGE . So far, five of these proteins have been sequenced . Their polypeptide chain consists of three tandemly related sequences of about 100 amino acids . The repeats of the different proteins are related and probably fold into two transmembrane alpha-helices linked by an extra-membrane loop . The features of this family are also present in several proteins of unknown function characterized by DNA sequencing . Isoforms of some carriers have been found . All mitochondrial carriers investigated in proteoliposomes function according to a simultaneous (sequential) mechanism of transport . The only exception is the carnitine carrier that proceeds via a ping-pong mechanism . Three mitochondrial carriers have been expressed in yeast and two overexpressed in E . coli and refolded in active form. Mol Gen Genet, 1994 Jun 3, 243(5), 584 - 92 Different UmuC requirements for generation of different kinds of UV-induced mutations in Escherichia coli; Nowicka A et al.; An Escherichia coli strain bearing the dnaQ49 mutation, which results in a defective epsilon subunit of DNA polymerase III, and carrying the lexA71 mutation, which causes derepression of the SOS regulon, is totally unable to maintain high-copy-number plasmids containing the umuDC operon . The strain is also unable to maintain the pAN4 plasmid containing a partial deletion of the umuD gene but retaining the wild-type umuC gene . These results suggest that a high cellular level of UmuC is exceptionally harmful to the defective DNA polymerase III of the dnaQ49 mutant . We have used this finding as a basis for selection of new plasmid umuC mutants . The properties of two such mutants, bearing the umuC61 or umuC95 mutation, are described in detail . In the umuC122::Tn5 strain harbouring the mutant plasmids, UV-induced mutagenesis is severely decreased compared to that observed with the parental umuDC+ plasmid . Interestingly, while the frequency of UV-induced GC-->AT transitions is greatly reduced, the frequency of AT-->TA transversions is not affected . Both mutant plasmids bear frameshift mutations within the same run of seven A residues present in umuC+; in umuC61 the run is shortened to six A whereas in umuC95 is lengthened to eight A . We have found in both umuC61 and umuC95 that translation is partially restored to the proper reading frame . We propose that under conditions of limiting amounts of UmuC, the protein preferentially facilitates processing of only some kinds of UV-induced lesions. Mol Gen Genet, 1994 Jun 3, 243(5), 525 - 31 Structure of the 5' upstream region and the regulation of the rpoS gene of Escherichia coli; Takayanagi Y et al.; The nucleotide sequence of the 5' upstream region of the Escherichia coli rpoS gene was determined and analyzed . At least four promoters responsible for rpoS transcription were identified, and designated P1, P2, P3 and P4, P1 being furthest from the upstream . Using lacZ fusion genes, the P2 promoter was found to be the strongest of the four . All of these promoters are regulated similarly, and their activity is enhanced 2 to 3-fold in stationary phase . P1 and P2 transcription start sites were determined by primer extension analyses . The P2 promoter region shows similarity to the consensus sigma 70-type promoter sequence, and was recognized by both E sigma 70 and E sigma 38 holoenzymes in vitro . The mRNA transcribed from the most distal promoter, P1, appears to include another open reading frame (orf-281), indicating that the two open reading frames comprise an operon . The rpoS gene product (sigma 38) was rapidly degraded after addition of chloramphenicol to cultures in the exponential, but not the stationary phase . This strongly suggests that posttranslational regulation is involved in the control of rpoS expression. Mol Gen Genet, 1994 Jun 3, 243(5), 500 - 5 Hydrogen peroxide induces G:C to T:A and G:C to C:G transversions in the supF gene of Escherichia coli; Akasaka S et al.; A vector plasmid, pZ189, carrying an Escherichia coli supF gene as a target for mutations, was treated with a combination of hydrogen peroxide and Fe3+/EDTA complex and propagated in E . coli host cells that had been induced for SOS functions by ultraviolet irradiation . The mutations frequency increased by up to 30-fold over spontaneous background levels with increasing concentrations of hydrogen peroxide . The increase in mutation frequency correlated with an increase in the formation of 8-hydroxydeoxyguanosine in the pZ189 DNA . Sequence analysis of 82 independent supF mutant plasmids revealed that 70 mutants contained base substitutions, with 63 of the 70 involving a G:C base pair, and with G:C-->C:G (28 cases) and G:C-->T:A (26 cases) transversions predominating . Investigation of the influence of the local DNA sequence on the transversions revealed that the guanine at the center of the triplet 5'-PuGA-3' was five times more likely to mutate after treatment with hydrogen peroxide than that at the center of 5'PyGN3' . G:C-->T:A transversions presumably resulted from mispairing of an altered G (probably 8-hydroxydeoxyguanosine) with deoxyadenosine . The origin of the G:C-->C:G transversions may be an as yet unidentified lesion generated by hydrogen peroxide . Mutagenic hotspots for base substitutions were found at positions 133, 160 and 168 . Mutation spectra and the positions of mutagenic hotspots, when compared with a previously determined spontaneous mutagenesis spectrum, also provide information on the mechanism of spontaneous mutagenesis. J Mol Biol, 1994 Jun 3, 239(2), 285 - 305 Crystal structures of Escherichia coli aspartate aminotransferase in two conformations . Comparison of an unliganded open and two liganded closed forms; Jager J et al.; Three crystal structures of wild type E . coli aspartate aminotransferase (E.C.2.6.1.1) in space group P2(1) have been determined at resolution limits between 2.6 and 2.35 A . The unliganded enzyme and its complexes with the substrate analogues maleate and 2-methylaspartate resulted in different conformations . The unit cell parameters of the unliganded and the inhibited enzyme are a = 87.2, b = 79.9, c = 89.8 A and beta = 119.1 degrees, and a = 85.4, b = 79.8, c = 89.5 A and beta = 118.6 degrees, respectively . The crystallographic symmetry is pseudo-C222(1) . The liganded enzyme structures were solved by difference Fourier techniques from that of a Val39-->Leu mutant partially refined to an R-factor of 0.22 at 2.85 A . They have a "closed" conformation like the chicken mAATase:maleate complex . The models were refined to R-factors of 0.19 (maleate complex) and 0.18 (2-methylaspartate complex) by molecular dynamics and restrained least squares methods . The unliganded crystal form was solved by molecular replacement and refined to an R-factor of 0.19 at 2.5 A resolution . The structure is in a "half-open" conformation, with the small domain rotated about 6 degrees from the closed conformation . The cofactor pyridoxal phosphate has a more relaxed conformation than in mAATase . Both maleate and 2-methylaspartate are hydrogen-bonded to the active site as in mAATase . The C alpha-CH3 bond of 2-methylaspartate is oriented at right angles to the cofactor pyridine ring, the most productive orientation for alpha-deprotonation of the substrate L-aspartate . Comparisons with earlier determined eAATase structures in space group C222(1) revealed differences that can probably be attributed to the somewhat lower resolution of the orthorhombic structures and/or mutations in the eAATases used in those studies . The present P2(1) structures confirm the justification of extrapolating properties of active site point mutants to the vertebrate isozymes . They will serve as reference in the interpretation of the properties of further site-directed mutants in continued studies of structure-function relationships of this enzyme. J Biol Chem, 1994 Jun 3, 269(22), 15838 - 45 Molecular cloning of a chiral-specific 3 alpha-hydroxysteroid sulfotransferase; Lee YC et al.; A novel stereoselective hydroxysteroid sulfotransferase (HST) that acts on neutral steroids having the 3-hydroxyl group in the alpha orientation but not on steroids where the 3-hydroxyl group is oriented in the beta position has been cloned and expressed . Primary screening of a guinea pig adrenal cDNA library was performed by colony hybridization using an oligonucleotide probe based on a highly homologous region among known steroid sulfotransferases . Selected clones consisted of overlapping sequences but were incomplete . The rapid amplification of cDNA ends procedure was used to construct a full-length guinea pig HST cDNA . The guinea pig HST cDNA transiently transfected into Chinese hamster ovary K1 cells expressed a protein identical in size to that of purified guinea pig HST specific for 3 alpha-hydroxylated neutral steroids that was recently reported (Driscoll, W . J., Martin, B . M., Chen, H.-C., and Strott, C . A . (1993) J . Biol . Chem . 268, 23496-23503) . The expressed HST likewise exhibited sulfotransferase activity that was directed specifically toward steroid substrates containing a 3-hydroxyl group in the alpha orientation; on the other hand, steroids with a 3 beta-hydroxyl group were not sulfonated by the expressed HST . Thus, the cloned HST cDNA clearly coded for a steroid sulfotransferase with chiral specificity for 3 alpha-hydroxylated neutral steroids and was, therefore, given the designation of guinea pig 3 alpha-hydroxysteroid sulfotransferase (gp3 alpha-HST) . The full-length gp3 alpha-HST cDNA consisted of 1182 base pairs and coded for a protein containing 287 amino acids . The deduced amino acid sequence of the protein shares 65/79, 65/80, and 62/76% identity/similarity with rat, mouse, and human HST, respectively . Northern blot analysis of guinea pig tissues revealed no apparent gender differences in either mRNA species or distribution; a single 1.4-kilobase HST mRNA species was present in adrenal and liver tissue . Interestingly, the adrenal mRNA content was considerably more abundant than that found in the liver . Evidence for 3 alpha-HST mRNA was not detected in kidney, heart, lung, muscle, spleen, or uterus. J Biol Chem, 1994 Jun 3, 269(22), 15808 - 13 Recombinant pulmonary surfactant protein D . Post-translational modification and molecular assembly; Crouch E et al.; Pulmonary surfactant protein D (SP-D) is a member of a family of collagenous C-type lectins that includes the serum mannose binding proteins and surfactant protein A . Recent studies have shown that rat SP-D (rSP-D) molecules are assembled as tetramers of trimeric subunits (12 mers) and that dodecamers can participate in higher orders of molecular assembly involving interactions of the amino-terminal peptide domains . In order to further study the assembly of SP-D in vitro, Chinese hamster ovary K1 cells were transfected with a full-length rat SP-D cDNA, and stable transfectants with high levels of SP-D production (approximately 6 x 10(6) dodecamers/cell/24 h) were obtained using a glutamine synthetase selection system . The secreted molecules (RrSP-D), which were purified by affinity chromatography on maltosyl-agarose, comigrated with rSP-D on SDS-polyacrylamide gel electrophoresis in the presence and absence of reduction, and coeluted with rSP-D dodecamers from 4% agarose . The major bacterial collagenase-resistant peptide showed a decreased mobility on reduction consistent with the formation of intrachain disulfide bonds . A 17-kDa pepsin-resistant fragment was isolated following overnight digestion with pepsin at 27 degrees C, confirming the formation of a triple helical domain comparable in size and thermal stability to that of natural SP-D . The expressed protein contained sialylated endoglycosidase F-sensitive carbohydrate; amino acid analysis of acid and alkaline hydrolysates demonstrated essentially normal levels of hydroxyproline, hydroxylysine, and hydroxylysine-glycosides . Electron microscopic studies showed a molecular structure indistinguishable from lung SP-D, with a similar small subpopulation of molecules showing higher orders of multimerization . Solid-phase neoglycoprotein binding assays gave the same saccharide inhibition profile as natural rat SP-D, and both proteins showed efficient saccharide-dependent agglutination of Escherichia coli . These studies demonstrate that a single genetically distinct chain type can account for the various and complex molecular assemblies of SP-D, and further verify the potential physiologic significance of the disulfide-bonded multimers and higher aggregates isolated from rat, bovine, and human lung lavage. J Biol Chem, 1994 Jun 3, 269(22), 15795 - 802 High level expression in Escherichia coli and characterization of the EF-hand calcium-binding protein caltractin; Weber C et al.; Caltractin is a member of the calmodulin superfamily of Ca(2+)-binding proteins that was originally cloned at the DNA level from the unicellular green alga Chlamydomonas reinhardtii . Human and mouse homologs to algal caltractin have been recently characterized . In the studies reported here, recombinant Chlamydomonas caltractin was expressed at high levels in Escherichia coli and purified to homogeneity . The use of the ompT-host BL21 proved critical for obtaining high yields of homogeneous full-length protein . Growth and purification protocols were optimized to allow reproducible and efficient production of tens of milligrams of pure protein from 1-liter cultures . Caltractin has a distinct UV spectrum which is largely dominated by the fine structure due to the 9 Phe residues . Unlike other members of the same protein family, the UV and the CD spectra do not change upon addition of Ca2+ to the apoprotein . However, the 1H NMR spectrum shows distinct changes upon Ca2+ binding, which are indicative of structural and/or dynamic changes largely reminiscent of other members of the calmodulin superfamily . Ca2+ binding measurements demonstrated the binding of four Ca2+ ions to caltractin with two higher affinity (Kd = 1.2 x 10(-6) M) and two lower affinity (Kd = 1.6 x 10(-4) M) sites . Caltractin is highly stable in both the apo- and the Ca(2+)-loaded states . The unusual stability of apocaltractin makes this protein highly suited for structural studies by multidimensional NMR aimed at understanding the structural and dynamic consequences of Ca2+ binding, and the molecular basis of Ca2+ signal transduction. J Biol Chem, 1994 Jun 3, 269(22), 15697 - 702 Purification and characterization of a novel organometallic receptor protein regulating the expression of the broad spectrum mercury-resistant operon of plasmid pDU1358; Yu H et al.; The narrow spectrum mercury-resistant (mer) operons of transposons Tn21 and Tn501 are inducible by inorganic mercury salts . The major regulatory gene merR is transcribed divergently from the other mer genes, which are cotranscribed . The MerR protein represses its own expression, as well as the expression of the other mer genes in the absence of the inducers . The synthesis of the polycistronic mer message is stimulated by MerR in the presence of the inducers . The MerRBS protein encoded by the broad spectrum mer operon of plasmid pDU1358 was characterized as a novel organomercurial receptor, distinguishing it from the narrow spectrum MerRNS proteins, described above . Several organomercurial compounds directly effected cellular activation of the mer operon transcription via the receptor protein MerRBS, but not by MerRNS . The merR gene from pDU1358 was cloned under the tac promoter, and the overexpressed MerRBS protein was soluble in buffer solutions containing 0.5 M NaCl at pH 7.5, but precipitated when NaCl concentration was reduced to 0.1 M (MerRBS concentrations at or above 0.1 mg/ml) . MerRBS was purified to near homogeneity by selective precipitation and solubilization by varying the salt concentration in buffer solutions, followed by Sephadex G-75 column chromatography . Both MerRBS and Tn21-encoded MerRNS bound with DNA fragments containing the pDU1358 mer operator sequence with comparable affinities . In vitro run-off transcription studies revealed that MerRBS activated mer operon expression in the presence of Hg2+ or phenylmercuric acetate . Phenylmercuric acetate did not induce mer operon expression when the MerRNS was used in the assay. J Biol Chem, 1994 Jun 3, 269(22), 15583 - 7 Isolation of recombinant ADP-ribosylation factor 6, an approximately 20-kDa guanine nucleotide-binding protein, in an activated GTP-bound state; Welsh CF et al.; ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins, which, like other members of the ras superfamily, are activated by exchanging bound GDP for GTP and inactivated through hydrolysis of the gamma-phosphate of bound GTP to form GDP in a highly regulated cycle . ARF 6, a class III ARF, was expressed in Escherichia coli with its amino terminus fused to maltose-binding protein . Following release from maltose-binding protein, recombinant ARF 6 (rARF 6) exhibited maximal activity with or without GTP . Such constitutive activation was due to the predominance of ARF-GTP over ARF-GDP, as demonstrated by nucleotide analysis . rARF 6 expressed in E . coli without amino-terminal extension was bound primarily to GDP and exhibited typical GTP-dependent activity . After release from maltose-binding protein, rARF 6-GTP was stable; only a fraction of the nucleotide was removed using EDTA, whereas urea denaturation restored complete GTP dependence . {alpha-32P}GTP bound to rARF 6 was in part protected from hydrolysis by alkaline phosphatase and resulted in the formation of {alpha-32P}GTP, -GDP, and -GMP, whereas unbound nucleotide was completely hydrolyzed to guanosine . Thus, amino-terminal extension of rARF 6, by maltose-binding protein, promoted the formation of a constitutively activated GTP-bound species . By analysis of this species, we confirmed that rARF 6 lacks the intrinsic ability to hydrolyze bound GTP and speculate that maltose-binding protein may inhibit hydrolysis by extrinsic factors. J Biol Chem, 1994 Jun 3, 269(22), 15577 - 82 Biochemical mechanism of HIV-I Vpr function . Specific interaction with a cellular protein; Zhao LJ et al.; vpr is an accessory gene of human immunodeficiency virus I (HIV-I) . Although unnecessary for viral replication in T cell lines, growing evidence suggests that it is essential for virus replication in monocytes/macrophages and for replication in vivo . We expressed HIV-I vpr in Escherichia coli and purified Vpr by affinity chromatography . In a coprecipitation assay, the purified Vpr interacted specifically with a cellular protein designated as Vpr-interacting protein, or RIP . Mutational analysis suggested that this interaction required a domain rich in leucine/isoleucine residues and highly conserved between HIV-I and SIVmac Vprs . During transient expression in mammalian cells, HIV-I Vpr was localized in the nucleus . However, mutational analysis failed to identify in Vpr a typical nuclear localization signal rich in basic amino acid residues . Instead, Vpr nuclear localization seemed to correlate with Vpr interaction with RIP . Mutations in the C-terminal 20-amino acid region containing a cryptic nuclear localization signal did not abolish Vpr nuclear localization or interaction with RIP, whereas point mutations in the leucine/isoleucine-rich domain abolished Vpr interaction with RIP and rendered Vpr unstable during transient expression . These results suggest that RIP may be involved in Vpr function. J Biol Chem, 1994 Jun 3, 269(22), 15546 - 52 The effect of Met-->Leu mutations on calmodulin's ability to activate cyclic nucleotide phosphodiesterase; Zhang M et al.; Calmodulin (CaM) has two hydrophobic surface patches that are particularly rich in Met residues, and these are the major contact areas where CaM interacts with its target enzymes . The amino acid Leu has been introduced by site-directed mutagenesis to replace all the Met residues in CaM . All nine individual Met-->Leu mutants of CaM as well as some double and quadruple mutants were expressed in Escherichia coli . All mutants could be purified by calcium-dependent hydrophobic affinity chromatography, indicating that they still expose their hydrophobic surfaces upon binding calcium . Each single Met-->Leu mutation in the C-terminal domain of the protein had little effect on its ability to activate phosphodiesterase (PDE), while a quadruple mutant with four C-terminal Leu residues instead of Met has a significantly lower affinity for PDE . The M36L mutant is a poor activator compared with the other three N-terminal single Met-->Leu mutants, which have a slightly lower affinity for PDE than wild-type CaM . The introduction of a positively charged Arg for Met-145 resulted in an almost complete loss of CaM's ability to activate PDE . Nuclear magnetic resonance spectroscopy was used to show that most CaM mutants retain their overall three-dimensional structure . Thus, the altered activation properties appear to arise from differences in the flexibility and polarizability of the Met and Leu sidechains, rather than from structural perturbations. J Biol Chem, 1994 Jun 3, 269(22), 15498 - 504 Mammalian topoisomerase I has base mismatch nicking activity; Yeh YC et al.; The all-type nicking enzyme (ATE) from human HeLa cells or calf thymus can nick DNA at the first phosphodiester bond 5' to all 8 possible mismatched bases . The strand disparity of this nicking is influenced by the neighboring nucleotide sequences . After nicking, the ATE covalently binds to the 3' end of the DNA product to form a cleavable complex, whose formation is insensitive to camptothecin, a specific inhibitor of eukaryotic topoisomerase I (Topo-I) . During the purification of ATE from calf thymus, a Mg(2+)-independent relaxation activity, characteristic of eukaryotic Topo-I, copurifies with the mismatch-nicking activity . The ATE from calf thymus may be a breakdown product of Topo-I . N-terminal amino acid analysis indicates that one of the polypeptides with ATE activity contains the C-terminal portion of Topo-I . Moreover, active human Topo-I, expressed as a fusion protein in Escherichia coli, is also capable of nicking all 8 base mispairs in the absence of Mg2+ . This mismatch-specific nicking activity may be a novel property of the mammalian Topo-I. J Biol Chem, 1994 Jun 3, 269(22), 15427 - 30 Rab-GDI presents functional Rab9 to the intracellular transport machinery and contributes selectivity to Rab9 membrane recruitment; Dirac-Svejstrup AB et al.; Rab proteins occur in the cytosol bound to Rab-GDP dissociation inhibitor (GDI) . We demonstrate here that cytosolic complexes of Rab9 bound to GDI represent a functional pool of Rab9 protein that can be utilized for transport from late endosomes to the trans Golgi network in vitro . Immunodepletion of GDI and Rab proteins bound to GDI led to the loss of cytosol activity; readdition of pure Rab9-GDI complexes fully restored cytosol activity . Delipidated serum albumin could solubilize prenylated Rab9 protein, but unlike Rab9-GDI complexes, Rab9-serum albumin complexes led to indiscriminate membrane association of Rab9 protein . Rab9 delivered to membranes by serum albumin was functional, but GDI increased the efficiency of Rab9 utilization, presumably because it suppressed Rab9 protein mistargeting . Finally, GDI inhibited transport of proteins from late endosomes to the trans Golgi network, likely because of its capacity to inhibit the membrane recruitment of cytosolic Rab9 . These experiments show that GDI contributes to the selectivity of Rab9 membrane recruitment and presents functional Rab9 to the endosome-trans Golgi network transport machinery. J Chromatogr B Biomed Appl, 1994 Jun 3, 656(1), 127 - 33 Production and simple purification of a protein encoded by part of the gag gene of HIV-1 in the Escherichia coli HB101F+ expression system inducible by lactose and isopropyl-beta-D-thiogalactopyranoside; Liska V et al.; The development of the Escherichia coli expression system, which was prepared by transferring the F' episome from strain 71/18 to a highly to a transformable F- strain HB101, is described . These new HB101 (F+) cells, which produced high levels of lac repressor, were capable of taking up lactose and grew under strict selection conditions . A relatively simple two-step purification of part of a protein (M(r) 27,000) encoded by the gag gene of HIV-1 in this expression system is described . The supernatant prepared by removal of cell debris was precipitated by 30% saturation of ammonium sulphate . The protein spectrum was characterized by gel electrophoresis, immunoblotting and ion-exchange titration curves . Optimum separation was achieved using a strong anion exchanger (Mono Q) at pH 8.0 . The purified protein did not cross-react with antibodies to E . coli. J Chromatogr B Biomed Appl, 1994 Jun 3, 656(1), 123 - 6 Procedure for refolding and purification of recombinant proteins from Escherichia coli inclusion bodies using a strong anion exchanger; Suttnar J et al.; Using Escherichia coli system expressing papilloma virus HPV16 E7MS2 fusion protein as a model system, a novel procedure was applied to solubilize, purify and refold recombinant proteins from E . coli inclusion bodies . The necessity to reactivate proteins at low protein concentrations (owing to their tendency to aggregate at high concentrations) was overcome by solubilization of inclusion bodies in alkaline solution and immobilization of proteins on a strong and resistant anion exchanger . This procedure has an inherent advantage of combining refolding and purification procedures in one step . The solubilization of the fusion protein in an alkaline reagent with the use of an anion exchanger resulted in considerable purification of the recombinant protein at a fairly high concentration . The protein was soluble under mild conditions and reacted with antibodies against the "native" papilloma virus. J Mol Biol, 1994 Jun 3, 239(2), 339 - 41 Crystallization and preliminary X-ray diffraction study of a bacterially produced T-cell antigen receptor V alpha domain; Fields BA et al.; A recombinant form of the variable domain of the alpha chain of a murine T-cell receptor specific for the N-terminal nonapeptide of myelin basin protein in association with the major histocompatibility complex class II I-Au molecule has been crystallized in a form suitable for X-ray diffraction analysis . This protein was secreted into the periplasmic space of Escherichia coli cells and affinity-purified using a nickel chelate adsorbent . The crystals are orthorhombic, space group P2(1)2(1)2, with unit cell dimensions a = 97.7 A, b = 79.6 A, c = 30.4 A and diffract to beyond 2.2 A resolution . The ability to crystallize a T-cell receptor domain produced in bacteria strongly suggests that the periplasmic space can provide a suitable environment for the correct in vivo folding of this class of antigen recognition molecules. J Neurosci, 1994 Jun, 14(6), 3548 - 64 Spinal cord neuroblasts proliferate in response to basic fibroblast growth factor; Ray J et al.; Trophic factors may function as one of the epigenic signals responsible for the proliferation, growth, migration, and differentiation of neurons and glia during embryogenesis . The present study reports that basic fibroblast growth factor (bFGF) at high concentrations (10-100 ng/ml) is a mitogen for embryonic spinal cord cells that have already committed to a neuronal pathway and are expressing neuronal phenotypes (neuroblasts) . Neuroblasts proliferate with a doubling time of 2.5 d . To characterize the nature of cells proliferating in response to bFGF, we have established long-term cultures of neuroblasts that can be passaged, freeze thawed, and recultured . In cultures the proportion of astrocytes remained the same, indicating limited survival and proliferation of these cells in response to bFGF . These results indicate that bFGF has mitogenic effects preferably on neuroblasts . The morphological and biochemical characterizations of the neuronal populations present in the long-term neuroblast cultures are presented here . The presence of cholinergic and GABAergic neurons in the cultures was established by immunocytochemical analysis . The cultures contain a small number of motoneurons as judged by their immunostaining with ChAT, low-affinity NGF receptor (LNGFR), and large size . Among all other growth factors tested for their mitogenic effects on embryonic spinal cord cells in culture, only epidermal growth factor (EGF) showed such effects, but to a lesser degree . The proliferative nature of neuroblasts has made it possible to transduce the Escherichia coli beta-galactosidase (LacZ) gene stably into these cells in vitro using a retroviral vector . The transfected cells expressing the foreign gene can be passaged, freeze thawed, and recultured without the loss of transgenes . The ability to transduce foreign genes stably into these cells permits implantation of these cells in the spinal cord to study cellular and biochemical behaviors and gene expression in defined neuronal populations in in vivo environments. J Bacteriol, 1994 Jun, 176(12), 3785 - 9 The RNA polymerase of Chlamydia trachomatis has a flexible sequence requirement at the -10 and -35 boxes of its promoters; Mathews SA et al.; Mutated variants of the predicted promoter of the countertranscript of the Chlamydia trachomatis plasmid were tested by in vitro transcription with chlamydial extract . A 3-bp deletion within the -10 region of the putative promoter caused the RNA polymerase to initiate transcription 3 bases downstream . Many single mutations in the -10 and -35 regions did not alter promoter function . However, some multiple mutations in both hexamers rendered the promoter inefficient or ineffective . Taken together, these results indicate that (i) the sequence requirement for chlamydial promoters differs from that for Escherichia coli and (ii) chlamydial RNA polymerase can tolerate considerably more variation at the -10 and -35 regions . These results are paradoxical considering the homology between C . trachomatis sigma 66 and E . coli sigma 70. J Bacteriol, 1994 Jun, 176(12), 3775 - 84 The gene coding for polynucleotide phosphorylase in Photorhabdus sp . strain K122 is induced at low temperatures; Clarke DJ et al.; Photorhabdus sp . strain K122 was found to produce higher levels of the protein CAP87K when cultured at 9 degrees C than when cultured at 28 degrees C . NH2-terminal sequencing of this protein revealed homology with the NH2 terminus of Escherichia coli polynucleotide phosphorylase . A 4.5-kb DNA fragment from strain K122 was cloned and sequenced and found to have 75% identity to the E . coli rpsO-pnp operon coding for ribosomal protein S15 and polynucleotide phosphorylase, respectively . Predicted proteins encoded by this sequence were found to have 86% identity with ribosomal protein S15 and polynucleotide phosphorylase from E . coli, and the genes were called rpsO and pnp, respectively . Quantitation of rpsO and pnp mRNA transcripts from K122 revealed that there was a 2.4-fold increase in the level of pnp mRNA and a 1.9-fold decrease in the level of rpsO mRNA at 9 degrees C relative to 28 degrees C . Primer extension analysis revealed the positions of possible promoters controlling the expression of rpsO and pnp in K122, suggesting that the genes are expressed independently . The increase in the level of pnp mRNA at 9 degrees C was not due to any relative increase in its stability compared with that of the rpsO transcript . However, there was evidence to suggest that it may be a result of a cold-inducible promoter, P2, in the intergenic region between rpsO and pnp . Several features of P2 support the suggestion that it may be cold inducible. J Bacteriol, 1994 Jun, 176(12), 3738 - 48 Growth phase variation of integration host factor level in Escherichia coli; Ditto MD et al.; We have measured the intracellular abundance of integration host factor (IHF), a site-specific, heterodimeric DNA-binding protein, in exponential- and stationary-phase cultures of Escherichia coli K-12 . Western immunoblot analysis showed that cultures that had been growing exponentially for several generations contained 0.5 to 1.0 ng of IHF subunits per microgram of total protein and that this increased to 5 to 6 ng/microgram in late-stationary-phase cultures . IHF is about one-third to one-half as abundant in exponentially growing cells as HU, a structurally related protein that binds DNA with little or no site specificity . Wild-type IHF is metabolically stable, but deletion mutations that eliminated one subunit reduced the abundance of the other when cells enter stationary phase . We attribute this reduction to the loss of stabilizing interactions between subunits . A mutation that inactivates IHF function but not subunit interaction increased IHF abundance, consistent with results of previous work showing that IHF synthesis is negatively autoregulated . We estimate that steady-state exponential-phase cultures contain about 8,500 to 17,000 IHF dimers per cell, a surprisingly large number for a site-specific DNA-binding protein with a limited number of specific sites . Nevertheless, small reductions in IHF abundance had significant effects on several IHF-dependent functions, suggesting that the wild-type exponential phase level is not in large excess of the minimum required for occupancy of physiologically important IHF-binding sites. J Bacteriol, 1994 Jun, 176(12), 3661 - 72 RecOR suppression of recF mutant phenotypes in Escherichia coli K-12; Sandler SJ et al.; The recF, recO, and recR genes form the recFOR epistasis group for DNA repair . recF mutants are sensitive to UV irradiation and fail to properly induce the SOS response . Using plasmid derivatives that overexpress combinations of the recO+ and recR+ genes, we tested the hypothesis that high-level expression of recO+ and recR+ (recOR) in vivo will indirectly suppress the recF mutant phenotypes mentioned above . We found that overexpression of just recR+ from the plasmid will partially suppress both phenotypes . Expression of the chromosomal recO+ gene is essential for the recR+ suppression . Hence we call this RecOR suppression of recF mutant phenotypes . RecOR suppression of SOS induction is more efficient with recO+ expression from a plasmid than with recO+ expression from the chromosome . This is not true for RecOR suppression of UV sensitivity (the two are equal) . Comparison of RecOR suppression with the suppression caused by recA801 and recA803 shows that RecOR suppression of UV sensitivity is more effective than recA803 suppression and that RecOR suppression of UV sensitivity, like recA801 suppression, requires recJ+ . We present a model that explains the data and proposes a function for the recFOR epistasis group in the induction of the SOS response and recombinational DNA repair. J Bacteriol, 1994 Jun, 176(12), 3638 - 45 The Escherichia coli proU promoter element and its contribution to osmotically signaled transcription activation; Mellies J et al.; The proU operon of Escherichia coli encodes a high-affinity glycine betaine transport system which is osmotically inducible and enables the organism to recover from the deleterious effects of hyperosmotic shock . Regulation occurs at the transcriptional level . KMnO4 footprinting showed that the preponderance of transcription initiated at a single primary promoter region and that proU transcription activation did not occur differentially at alternate promoters in response to various levels of salt shock . Mutational analysis confirmed the location of the primary promoter and identified an extended -10 region required for promoter activity . Specific nucleotides within the spacer, between position -10 and position -35, were important for maximal expression, but every mutant which retained transcriptional activity remained responsive to osmotic signals . A chromosomal 90-bp minimal promoter fragment fused to lacZ was not significantly osmotically inducible . However, transcription from this fragment was resistant to inhibition by salt shock . A mutation in osmZ, which encodes the DNA-binding protein H-NS, derepressed wild-type proU expression by sevenfold but did not alter expression from the minimal promoter . The current data support a model in which the role of the proU promoter is to function efficiently at high ionic strength while other cis-acting elements receive and respond to the osmotic signal. J Bacteriol, 1994 Jun, 176(12), 3606 - 13 Comparative analysis of functional and structural features in the primase-dependent priming signals, G sites, from phages and plasmids; Tanaka K et al.; The primase-dependent priming signals, G sites, are directly recognized by the Escherichia coli primase (dnaG gene product) and conduct the synthesis of primer RNAs . In nucleotide sequence and secondary structure, there is no striking resemblance between the phage- and plasmid-derived G sites, except for the limited sequence homology near the start position of primer RNA synthesis . In this study, we analyzed the structure and function of a G site of plasmid R100, G site (R100), and discovered the necessity of the coexistence of two domains (domains I and III), which contains blocks A, B, and C, which are nucleotide sequences highly conserved among the plasmid-derived G sites . However, neither the internal region, domain II, between domains I and III nor the potential secondary structure proposed by Bahk et al . (J . D . Bahk, N . Kioka, H . Sakai, and T . Komano, Plasmid 20:266-270, 1988) is essential for single-stranded DNA initiation activity . Furthermore, chimeric G sites constructed between a G site of phage G4, G site(G4), and G site(R100) maintained significant single-stranded DNA initiation activities . These results strongly suggest that phage- and plasmid-derived G sites have functionally equivalent domains . The primase-dependent priming mechanisms of phage- and plasmid-derived G sites are discussed. J Bacteriol, 1994 Jun, 176(12), 3559 - 67 Characterization of genetic determinants for R body synthesis and assembly in Caedibacter taeniospiralis 47 and 116; Heruth DP et al.; Caedibacter taeniospiralis, an obligate bacterial endosymbiont of Paramecium tetraurelia, confers a killing trait upon its host paramecium . Type 51 R bodies (refractile inclusion bodies) are synthesized by these endosymbionts and are required for expression of the killing trait . The nucleotide sequence of the genetic determinants for type 51 R body synthesis and assembly was determined for C . taeniospiralis 47 and 116 . Three independently transcribed genes (rebA, rebB, and rebC) were characterized . To date these are the only genes from C . taeniospiralis to be sequenced and characterized . DNA regulatory regions are recognized by Escherichia coli, and codon usage appears similar to that in E . coli . A fourth open reading frame with appropriate regulatory sequences was found within the reb locus, but no evidence was obtained to suggest that this putative gene is expressed in E . coli . The R body-encoding sequences from both strains are identical . Two-dimensional gel electrophoresis of deletion derivatives shows that two polymerization events are involved in R body assembly . One polymerization event requires only RebB and RebC; the other requires all three proteins . Expression of RebC is necessary for the posttranslational modification of RebA and RebB into species with three and two different molecular weights, respectively . In the presence of RebC, each species of RebB with a different molecular weight has six different isoelectric points. J Bacteriol, 1994 Jun, 176(12), 3466 - 73 Isolation and characterization of the Methylophilus sp . strain DM11 gene encoding dichloromethane dehalogenase/glutathione S-transferase; Bader R et al.; The restricted facultative methylotroph Methylophilus sp . strain DM11 utilizes dichloromethane as the sole carbon and energy source . It differs from other dichloromethane-utilizing methylotrophs by faster growth on this substrate and by possession of a group B dichloromethane dehalogenase catalyzing dechlorination at a fivefold-higher rate than the group A enzymes of slow-growing strains . We isolated dcmA, the structural gene of the strain DM11 dichloromethane dehalogenase, to elucidate its relationship to the previously characterized dcmA gene of Methylobacterium sp . strain DM4, which encodes a group A enzyme . Nucleotide sequence determination of dcmA from strain DM11 predicts a protein of 267 amino acids, corresponding to a molecular mass of 31,197 Da . The 5' terminus of in vivo dcmA transcripts was determined by primer extension to be 70 bp upstream of the translation initiation codon . It was preceded by a putative promoter sequence with high resemblance to the Escherichia coli sigma 70 consensus promoter sequence . dcmA and 130 bp of its upstream sequence were brought under control of the tac promoter and expressed in E . coli to approximately 20% of the total cellular protein by induction with isopropylthiogalactopyranoside (IPTG) and growth at 25 degrees C . Expression at 37 degrees C led to massive formation of inclusion bodies . Comparison of the strain DM11 and strain DM4 dichloromethane dehalogenase sequences revealed 59% identity at the DNA level and 56% identity at the protein level, thus indicating an ancient divergence of the two enzymes . Both dehalogenases are more closely related to eukaryotic class theta glutathione S-transferases than to a number of bacterial glutathione S-transferases. Arch Biochem Biophys, 1994 Jun, 311(2), 509 - 16 Initial kinetic and mechanistic characterization of Escherichia coli fumarase A; Flint DH; The protein encoded by the fumA gene in Escherichia coli is shown herein to be a highly efficient and specific catalyst of the fumarase reaction . In an investigation of 21 substrate analogs, this protein only had substantial activity as a hydro-lyase on fumarate, malate, acetylene dicarboxylate, fluorofumarate, and 2(S),3(S)-tartrate . The kcat and kcat/Km for the hydration of fumarate by this protein are 3100 s-1 and 5 x 10(6) mol-1 s-1, respectively . It is likely that one physiological role of this protein is a catalyst of the fumarase reaction; therefore, it is appropriate to name it fumarase A . Fumarase A specifically removes the 3-pro-R in the dehydration of (2S)-malate . The product of the action of fumarase A on acetylene dicarboxylate, fluorofumarate and 2(S),3(S)-tartrate is oxalacetate . The nitronate form of 2-hydroxy-3-nitro-propionate is a potent inhibitor of fumarase A, implying that the enzyme forms an intermediate with an anion at C-3 . No kinetic isotope effect was found with (2S,3R)-3-{2H}malate . The effects of pH on the kcat and kcat/Km for fumarate as a substrate show that the pKas of the groups involved in catalysis differ markedly from porcine fumarase . The possible roles of the proteins encoded by the three fumarase genes in E . coli are briefly discussed. Arch Biochem Biophys, 1994 Jun, 311(2), 487 - 95 A comparison of the enzymatic and physicochemical properties of human glutathione transferase M4-4 and three other human Mu class enzymes; Comstock KE et al.; The multigene family of cytosolic glutathione S-transferases (GSTs) consists of four classes (Alpha, Mu, Pi, and Theta), all involved in the detoxication of reactive electrophiles . The human Mu class GSTs consist of at least four expressed isozyme subunits, GST M1, GST M2, GST M3, and GST M4, which have 70-90% amino acid sequence identity . The gene and cDNA sequences for GST M4 have been determined recently (K . E . Comstock, K . J . Johnson, D . Rifenbery, and W . D . Henner, J . Biol . Chem . (1993) 268, 16958-16965) . Cloning of GST M4 cDNA into an Escherichia coli expression system permitted the production of the corresponding protein . The enzyme was purified and shown to have a relatively low specific activity with the standard GST substrate 1-chloro-2,4-dinitrobenzene (1.4 +/- 0.2 mumol min-1 mg-1 protein), but an activity equivalent to other Mu class enzymes with other tested substrates . The protein forms functional dimers composed of subunits with a M(r) of approximately 26,400 . A detailed comparison of the activity with various substrates and inhibitors was performed between GST M4-4 and other human Mu class GSTs, GST M1a-1a, GST M2-2, and GST M3-3, produced in bacterial expression systems . Despite the high level of amino acid sequence identity, the enzymatic properties of these enzymes were quite different . Comparisons with the crystallographic structure of a homologous rat GST, GST 3-3, indicate that a number of the nonconserved amino acid residues can be assigned to the putative active site of GST M4-4 . This suggests that diversification in the evolution of these genes has occurred primarily in the substrate binding regions to cope with an increasing variety of foreign compounds. Arch Biochem Biophys, 1994 Jun, 311(2), 418 - 24 Interaction of the catalytic and the membrane subunits of an oxyanion-translocating ATPase; Dey S et al.; Resistance to arsenical and antimonial compounds in Escherichia coli is due to active extrusion of these compounds from cells expressing the ars operon . The arsenical pump is an ion-translocating ATPase which consists of two polypeptide components, the ArsA and ArsB proteins . The ArsB protein, the inner membrane component of the pump, has been shown to function as the membrane anchor for the catalytic subunit, the ArsA protein . The properties and nature of interaction between these two components of the pump were investigated using an in vitro binding assay . Purified ArsA protein bound to the membrane in a saturable manner . In the absence of arsenite or antimonite an apparent positive cooperativity in the binding of the ArsA protein to me