Nucleic Acids Res, 1993 Jun 25, 21(12), 2899 - 905 Cleavage and binding of a DNA fragment containing a single 8-oxoguanine by wild type and mutant FPG proteins; Castaing B et al.; A 34-mer oligonucleotide containing a single 7,8-dihydro-8-oxoguanine (8-OxoG) residue was used to study the enzymatic and DNA binding properties of the Fpg protein from E . coli . The highest rates of incision of the 8-OxoG containing strand by the Fpg protein were observed for duplexes where 8-OxoG was opposite C (*G/C) or T (*G/T) . In contrast, the rates of incision of duplexes containing 8-OxoG opposite G (*G/G) and A (*G/A) were 5-fold and 200-fold slower . Gel retardation studies showed that the Fpg protein had a strong affinity for duplexes where the 8-OxoG was opposite pyrimidines and less affinity for duplexes where the 8-OxoG was opposite purines . KDapp values were 0.6 nM (*G/C), 1.0 nM (*G/T), 6.0 nM (*G/G) and 16.0 nM (*G/A) . The Fpg protein also binds to unmodified (G/C) duplex and a KDapp of 90 nM was measured . The cleavage and binding of the (*G/C) duplex were also studied using bacterial crude lysates . Wild type E . coli crude extract incised the 8-OxoG containing strand and formed a specific retardation complex with the (*G/C) duplex . These two reactions were mediated by the Fpg protein, since they were not observed with a crude extract from a bacterial strain whose fpg gene was inactivated . Furthermore, we have studied the properties of 6 mutant Fpg proteins with Cys-->Gly mutations . The results showed that the 2 Fpg proteins with Cys-->Gly mutations outside the zinc finger sequence cleaved the 8-OxoG containing strand, formed complexes with the (*G/C) duplex and suppressed the mutator phenotype of the fpg-1 mutant . In contrast, the 4 Fpg proteins with Cys-->Gly mutations within the zinc finger motif neither cleave nor bind the (*G/C) duplex, nor do these proteins suppress the fpg-1 mutator phenotype. Biochim Biophys Acta, 1993 Jun 25, 1173(3), 289 - 93 Synthesis of mammalian prolylendopeptidase in Escherichia coli and analysis of the recombinant protein; Sommer J; Prolylendopeptidase is a cytoplasmic serine proteinase which hydrolyses peptide bonds at the C-terminal side of prolines . Here, the expression in Escherichia coli of the cDNA coding for the enzyme from porcine brain is reported . Furthermore, the purification of active prolylendopeptidase from the extract of recombinant bacterial cells is described . The large amounts of protein obtained were used to gain insight in the substrate specificity of recombinant prolylendopeptidase, by using internally quenched fluorescent substrates. J Biol Chem, 1993 Jun 25, 268(18), 13253 - 60 Selective decay of Escherichia coli dnaG messenger RNA is initiated by RNase E; Yajnik V et al.; The rpsU-dnaG-rpoD operon messenger RNA that encodes S21, primase, and sigma-70 is cleaved under normal physiological conditions . The dnaG coding portion of the mRNA is then rapidly degraded . An endonuclease activity has been isolated from wild type Escherichia coli cells that cleaves dnaG mRNA . This activity has been identified as RNase E, and the identity confirmed by the accumulation of the unprocessed operon polycistronic mRNA in RNase E mutants, rne-3071 and ams-1, when incubated at nonpermissive temperatures . Extracts prepared from RNase E mutant strains failed to cleave dnaG mRNA in vitro . The dnaG mRNA RNase E cleavage site (5'-AAGUGAUUUA-3') is only 50% homologous to the ribosomal RNA RNase E cleavage site . By using computer programs predicting secondary structure, the dnaG RNase E cleavage site appears to be in a single stranded RNA loop. Nature, 1993 Jun 24, 363(6431), 744 - 7 Functional dissection of TFIIB domains required for TFIIB-TFIID-promoter complex formation and basal transcription activity; Hisatake K et al.; The protein TFIIB is a general transcription initiation factor that interacts with a promoter complex (D.DNA) containing the TATA-binding subunit (TFIID tau, or TBP) of TFIID to facilitate subsequent interaction with RNA polymerase II (ref . 2) through the associated TFIIF (ref . 3) . The potential bridging function of TFIIB raises the possibility of two structural domains and emphasizes the importance of TFIIB structure-function studies for a further understanding of preinitiation complex assembly and function . Here we show that human TFIIB (refs 5,6) is comprised of functionally distinct N- and C-terminal domains . The C-terminal domain, containing the direct repeats and associated basic regions, is necessary and sufficient for interaction with the D.DNA complex . By contrast, the N-terminal domain that is dispensable for formation of the TFIID tau-TFIIB-promoter (D.B.DNA) complex is required for subsequent events leading to basal transcription initiation . On the basis of these results, we discuss structural and functional similarities between TFIIB and TFIID tau, which have similar structural organization and motifs. Nature, 1993 Jun 24, 363(6431), 722 - 4 Characterization and localization of the FMR-1 gene product associated with fragile X syndrome; Verheij C et al.; The fragile X syndrome is the most frequent form of inherited mental retardation after Down's syndrome, having an incidence of one in 1,250 males . The fragile X syndrome results from amplification of the CGG repeat found in the FMR-1 gene . This CGG repeat shows length variation in normal individuals and is increased significantly in both carriers and patients; it is located 250 base pairs distal to a CpG island which is hypermethylated in fragile X patients . The methylation probably results in downregulation of FMR-1 gene expression . No information can be deduced about the function of the FMR-1 protein from its predicted sequence . Here we investigate the nature and function of the protein encoded by the FMR-1 gene using polyclonal antibodies raised against the predicted amino-acid sequences . Four different protein products, possibly resulting from alternative splicing, have been identified by immunoblotting in lymphoblastoid cell lines of healthy individuals . All these proteins were missing in cell lines from patients not expressing FMR-1 messenger RNA . The intracellular localization of the FMR-1 gene products was investigated by transient expression in COS-1 cells and found to be cytoplasmic . Localization was also predominantly cytoplasmic in the epithelium of the oesophagus, but in some cells was obviously nuclear. Biochim Biophys Acta, 1993 Jun 24, 1164(1), 36 - 46 Improved synthetic methods for the selective deuteration of aromatic amino acids: applications of selective protonation towards the identification of protein folding intermediates through nuclear magnetic resonance; Wishart DS et al.; In this report we describe several novel methods for the preparation of selectively deuterated aromatic amino acids . New syntheses for {2,3,5,6-2H4}phenylalanine and {2,4,6,7-2H4}tryptophan, as well as improved catalytic exchange methods for {2,3,5,6-2H4}tyrosine and {2,3,4,5,6-2H5}phenylalanine are presented . Isotopic substitution levels for all compounds are generally found to be greater than 95% . Biosynthetic incorporation of these amino acids is also shown to be possible with little or no evidence of isotopic scrambling . The products from these new syntheses, in combination with other selectively deuterated aromatic amino acids, are found to permit group-specific 'single-proton' labelling of proteins . This highly-efficient and very cost-effective method of selective protonation is shown to produce greatly simplified 1H-NMR spectra of the aromatic region of proteins . The utility of this approach to isotopic editing is demonstrated with the identification of a transient folding intermediate of Escherichia coli thioredoxin which is undetectable by standard 2-D NMR techniques. Biochemistry, 1993 Jun 22, 32(24), 6206 - 13 Environmental influences on the in vivo level of intramolecular triplex DNA in Escherichia coli; Ussery DW et al.; We describe an assay for detecting intramolecular triple-stranded DNA in living cells . The assay involves quantitative analysis of the differential photobinding of 4,5',8-trimethylpsoralen to 5'TA and 5'AT dinucleotides in a region of plasmid DNA containing the triplex-forming sequence (GA)7TA(GA)7 . Psoralen photobinds to the central 5'TA within the (GA)7TA(GA)7 sequence in duplex DNA but not when the sequence exists as an intramolecular triplex structure . The reactivity of photobinding sites in regions flanking the intramolecular triplex-forming sequence, those that comprise the duplex-triplex junctions, either increase or decrease upon formation of the intramolecular triplex structure from duplex DNA . The pattern of trimethylpsoralen reactivity provides an indication of the conformation of the (GA)7TA(GA)7 sequence in plasmid DNA . The formation of the intramolecular triplex structure is dependent on both pH and negative superhelical density in vitro . The fraction of the (GA)7TA(GA)7 sequence that existed as an intramolecular triplex structure in vivo was dependent on the level of DNA supercoiling in vivo and changes in growth conditions which influence the intracellular pH . The Hy3 isomer was detected in living Escherichia coli cells. Biochemistry, 1993 Jun 22, 32(24), 6134 - 40 Promoter recognition by Escherichia coli RNA polymerase . Effects of single base pair deletions and insertions in the spacer DNA separating the -10 and -35 regions are dependent on spacer DNA sequence; Warne SE et al.; Escherichia coli RNA polymerase contacts promoter DNA at two upstream regions separated by a spacer DNA . We had previously studied the effects of substitutions of simple DNA sequences in a stretch of the spacer DNA devoid of any known specific contacts with RNA polymerase . It was found that substitution of nine consecutive nonalternating dG-dC base pairs, but not nine alternating dG-dC base pairs, impaired promoter function . We proposed that this effect was due to the fact that the oligo(dG)-oligo(dC) sequence adopted a conformation (possibly A-helical) resulting in a reduction in its length and twist as compared with the B-form DNA of the alternating sequence . Here we test this hypothesis by combining the substitutions with single base pair insertions and deletions in the spacer DNA, which affect the length and the twist in known ways . Deletion and substitutions equally affect the activities of promoters with the presumed B-DNA substitutions . However, for promoters bearing the oligo(dG)-oligo(dC) substitution, a deletion in the spacer DNA impairs promoter activity to a much greater extent than the insertion of a base pair . This asymmetry is consistent with our hypothesis that the deleterious effects of the substitution are due to its having the reduced twist and/or length characteristic of A-DNA . Additionally, we present data that concern the sequence requirements for adoption of this structure that leads to reduced promoter function. Biochemistry, 1993 Jun 22, 32(24), 6171 - 8 Stabilization of Escherichia coli ribonuclease HI by cavity-filling mutations within a hydrophobic core; Ishikawa K et al.; The crystal structure of Escherichia coli ribonuclease HI has a cavity near Val-74 within the protein core . In order to fill the cavity space, we constructed two mutant proteins, V74L and V74I, in which Val-74 was replaced with either Leu or Ile, respectively . The mutant proteins are stabilized, as revealed by a 2.1-3.7 degrees C increase in the Tm values, as compared to the wild-type protein at pH values of 3.0 and 5.5 . The mutant protein V74A, in which Val-74 is replaced with Ala, was also constructed to analyze the reverse effect . The stability of V74A decreases by 7.6 degrees C at pH 3.0 and 12.7 degrees C at pH 5.5 in Tm as compared to those values for the wild-type protein . None of the three mutations significantly affect the enzymatic activity . The crystal structures of V74L and V74I, determined at 1.8-A resolution, are almost identical to that of the wild-type protein, except for the mutation site . In the two mutant proteins, calculation by the Voronoi procedure shows that the cavity volumes around the individual mutation sites are remarkably reduced as compared to that in the wild-type protein . These results indicate that the introduction of a methylene group into the cavity, without causing steric clash, contributes to an increase in the hydrophobic interaction within the protein core and thereby enhances protein stability . We also discuss the role of the Leu side chain, which can assume many different local conformations on a helix without sacrificing thermostability. FEBS Lett, 1993 Jun 21, 324(3), 361 - 6 The KH domain occurs in a diverse set of RNA-binding proteins that include the antiterminator NusA and is probably involved in binding to nucleic acid; Gibson TJ et al.; New findings are presented for the approximately 50 residue KH motif, a domain recently discovered in RNA-binding proteins . The conserved sequence is approximately 10 residues larger than previously reported . Profile searches have revealed new members of this family, including two, E . coli NusA and human GAP-associated p62 phosphoprotein, for which RNA-binding data exists . A nusA homolog was detected in the RNA polymerase gene complex of six archaebacterial species and may encode an antiterminator . All KH-containing proteins are linked with RNA and the KH motif most probably functions as a nucleic acid binding domain. FEBS Lett, 1993 Jun 21, 324(3), 247 - 52 cDNA cloning and expression of cysteine synthase B localized in chloroplasts of Spinacia oleracea; Saito K et al.; The cDNA clones for cysteine synthase B, which is localized in chloroplasts of Spinacia oleracea L., were isolated by screening a library with synthetic oligonucleotides encoding a partial peptide sequence of the purified protein . Nucleotide sequence analysis revealed an open reading frame encoding a polypeptide of 383 amino acids containing a putative transit peptide of 52 amino acids . A bacterial expression vector of the cDNA clone could genetically complement an Escherichia coli auxotroph lacking cysteine synthase and could produce the functionally active and immuno-reactive cysteine synthase in E . coli . RNA blot hybridization suggested that the transcripts were primarily accumulated in leaves of spinach. Ann N Y Acad Sci, 1993 Jun 21, 681, 219 - 37 Approaches for studying the pathogenic T cells in autoimmune patients; Willcox N et al.; Our provisional conclusions from this work are as follows . (1) For screening responses of established lines, native human AChR is not prohibitively scarce, especially if it is concentrated onto beads, and class II-transfected TE671 cells may be useful too; both may give vital evidence of AChR-specificity, but it is still crucial to confirm that with synthetic peptides . (2) For mapping epitopes, panels of full-length and shorter recombinant human polypeptides, and of synthetic peptides, are invaluable complementary material: longer peptides tend to stimulate particularly strongly . (3) Initial selection with pooled synthetic peptides can easily generate interesting lines from both patients and controls, but they may depend on the artificial processing sites that are an inevitable consequence of arbitrarily chosen start and stop points . Of course, these might conceivably be employed in unusual antigen-presenting cells (such as thymic myoid cells), so we cannot totally dismiss such "cryptic" epitopes . This system can sometimes select T cells responding to "natural" epitopes too, as now reported for tetanus toxin . Nevertheless, for these and other reasons, at present, we strongly favor using the longest human recombinant material possible, because it is apparently processed more naturally . This must be combined with rigorous screening for reactivity to E . coli-derived contaminants plus concomitant mapping of epitopes as above . Use of intact AChR for initiating lines may yet become feasible . (4) The T cells thus isolated and characterized so far are proving to be heterogeneous in the epitopes and presenting class II molecules they recognize, and in their T-cell receptor gene usage . It is premature to claim key myasthenogenic epitopes or clonotypes, but HLA-DR3 and the linked -DQw2 do not appear to monopolize presentation . (5) Assessing the disease-relevance of these T cells is a separate problem, highlighted by their apparent similarity in healthy controls . In the meantime, to test their potential pathogenicity, we are assaying their cytokine profiles and ability to help specific antibody production in vitro . In the hope that they do prove to be relevant, we are also using some of them to test possible therapeutic strategies that might prove applicable in the patients. J Mol Biol, 1993 Jun 20, 231(4), 950 - 9 Factors influencing the repair of the mutagenic lesion O6-methylguanine in DNA by human O6-methylguanine-DNA methyltransferase; Liem LK et al.; Oligodeoxynucleotides of various chain lengths (p(Bp)nB, n < or = 9) and the eight possible dinucleotide phosphates (pm6GpB and pBpm6G), each containing a single O6-methylguanine residue (m6G), were used to study the repair kinetics of this lesion by the cloned DNA repair proteins; human 21 kDa O6-methylguanine-DNA methyltransferase (MGMT), human 43 kDa glutathione-S-transferase fused MGMT (GSTMGMT) and the Escherichia coli 39 kDa ada protein . The observed second-order repair rate constants are dependent upon both the chain length of the oligonucleotide substrates for all three proteins and in the case assuming O6-methylguanine is similar to B) . The differences observed in the ratios of the rate constants for the substrates with five and four base residues; 125 for the E . coli 39 kDa ada protein, 640 for the human MGMT and 27,800 for the human fusion protein GSTMGMT, suggest that the pentanucleotide phosphate containing this lesion is the "optimal" substrate for the proteins . Surprisingly, the human GSTMGMT is shown to be more effective in the repair of longer substrates with the second-order repair rate constants for TATA-Cm6GTATA being 6.16 x 10(6) for GSTMGMT, 2.00 x 10(6) for MGMT and 0.27 x 10(6) M-1 s-1 for the E . coli 39 kDa ada protein . Thus, the presence of an additional protein domain at the N terminus of human MGMT can alter its selectivity towards certain substrates . Although a number of peptide domains are conserved between the E . coli 39 kDa ada protein and phosphates can also be used to explain the observed sequence specific repair of this lesion within certain DNA sequences. J Mol Biol, 1993 Jun 20, 231(4), 1122 - 5 Crystallization and preliminary crystallographic analysis of Escherichia coli DNA photolyase; Park HW et al.; DNA photolyase from Escherichia coli (M(r) 54,000) consists of a polypeptide chain of 471 amino acids and the non-covalently bound cofactors methenyltetrahydrofolate (MTHF) and flavin adenine dinucleotide (FADH2) . Two crystal forms of the enzyme were obtained; both have symmetry of space group P1 . Form I has the unit cell dimensions a = 89.4 A, b = 97.3 A, c = 62.1 A, alpha = 108.3 degrees, beta = 97.4 degrees and gamma = 90.0 degrees . Diffraction from this form extends beyond 3 A resolution, but the crystals are radiation-sensitive and difficult to reproduce . Form II has the unit cell dimensions a = 62.6 A, b = 72.2 A, c = 58.5 A, alpha = 99.1 degrees, beta = 101.5 degrees and gamma = 72.0 degrees; most likely, the unit cell contains two molecules . High diffraction quality and reproducibility make form II suitable for structure analysis. J Mol Biol, 1993 Jun 20, 231(4), 1078 - 89 Methionyl-tRNA synthetase zinc binding domain . Three-dimensional structure and homology with rubredoxin and gag retroviral proteins; Fourmy D et al.; Methionyl-tRNA synthetase from Escherichia coli contains one tightly bound zinc atom per subunit . The region encompassing residues 138 to 163 of this enzyme is responsible for the metal binding . A 28-mer peptide corresponding to these residues was expressed in vivo and shown to contain approximately 1 mol of tightly bound Zn/mol of peptide . In this study, the three-dimensional solution structure of this peptide was solved by means of two-dimensional proton NMR spectroscopy . A total of 133 nuclear Overhauser effect distance constraints and 22 dihedral angle restraints were used for the calculations, using a hybrid distance-geometry-simulated annealing strategy . Excluding the first four residues, the resulting structure is well-defined (r.m.s.d . 0.71 A for backbone atoms) and composed of a series of four tight turns . The second and the fourth turns are composed of CXXC sequences which are structurally homologous to the NH-S turns found in the metal binding sites of gag retroviral proteins and rubredoxin . The solution structure of the zinc binding peptide shows significant discrepancies with the crystal structure of methionyl-tRNA synthetase. J Mol Biol, 1993 Jun 20, 231(4), 1068 - 77 Mapping of the zinc binding domain of Escherichia coli methionyl-tRNA synthetase; Fourmy D et al.; Cys/His motifs, found in several nucleic acid binding proteins, generally correspond to sites for the binding of metal atoms . Such a motif, comprising four Cys residues, occurs in the subunits of Escherichia coli methionyl-tRNA synthetase, a dimeric enzyme known to bind two zinc atoms . In this study, each of the four cysteines in the cysteine cluster (region 145 to 161) of E . coli methionyl-tRNA synthetase were successively changed into an alanine . Either substitution is sufficient to destabilize the tight binding of the zinc ion . Moreover, a peptide having a sequence corresponding to that of the 138 to 163 region of methionyl-tRNA synthetase has been prepared . It strongly binds one zinc atom, even in the presence of ethylene diamine tetraacetate . These data establish that, in methionyl-tRNA synthetase, the Cys motif of region 145 to 161 is actually the binding site for zinc . In addition, the mutation of each cysteine modifies the parameters of the methionine activation reaction, and appears to change the structure of the enzyme, as probed by an increased sensitivity of the mutant enzymes to trypsin attack . The possible role of the zinc atom and of its chelating residues in the folding of the active centre of methionyl-tRNA synthetase is discussed. Cas Lek Cesk, 1993 Jun 20, 132(14), 434 - 6 {Thrombosis of the inferior vena cava--an unusual cause of a chronic septic condition in a female patient with type 1 diabetes mellitus}; Klein D et al.; The authors describe the accidental detection of a clinically not very marked thrombosis of the vena cava inferior in a 29-year-old female diabetic which was the cause of permanent bacteriaemia and the source of embolization of the lungs . The patient was delivered of an infant six years previously by Caesarean section, subsequently complicated by a paranephritic abscess on the right which called for surgical treatment . Since then the patient suffered from frequent dermal infections and was dyspnoic . Because of deterioration of polyneuropathic complaints of the lower extremities resulting from diabetes the patient was admitted to the authors' clinic . Sonographic examination of the abdominal cavity aroused urgent suspicion of a thrombus in the vena cava inferior, as confirmed by subsequent cavography and computed tomography . The patient was treated for prolonged periods with anticoagulants and antibiotics . During the subsequent check-up examination after four months marked diminution of the thrombus was recorded and improvement of the patient's general condition. Cell, 1993 Jun 18, 73(6), 1165 - 73 LexA and lambda Cl repressors as enzymes: specific cleavage in an intermolecular reaction; Kim B et al.; During the SOS response, LexA repressor is inactivated by specific cleavage . Although cleavage requires RecA protein in vivo, RecA acts indirectly as a coprotease by stimulating an inherent self-cleavage activity of LexA . In lambda lysogens, cleavage of lambda Cl repressor in a similar but far slower reaction results in prophage induction . We describe an intermolecular cleavage reaction in which the C-terminal fragment of LexA acted as an enzyme to cleave other molecules of LexA . The C-terminal fragment of lambda repressor cleaved the LexA substrates about as efficiently as did the LexA enzyme, suggesting that the slow rate of Cl self-cleavage results from a weak interaction between its cleavage site and the active site. Science, 1993 Jun 18, 260(5115), 1773 - 7 From RNA to DNA, why so many ribonucleotide reductases? Reichard P. It is generally accepted that DNA appeared after RNA during the chemical evolution of life . To synthesize DNA, deoxyribonucleotides are required as building blocks . At present, these are formed from the corresponding ribonucleotides through the enzymatic action of ribonucleotide reductases . Three classes of enzymes are present in various organisms . There is little sequence similarity among the three classes of reductases . However, enzymic mechanisms and the allosteric behavior of the enzymes from various organisms are strongly conserved, suggesting that the enzymes might have evolved from a common ancestor, with the class III anaerobic Escherichia coli reductase as its closest relative. Biochim Biophys Acta, 1993 Jun 18, 1149(1), 29 - 39 Interaction of immunologically-active lipopeptides with membranes; Metzger JW et al.; Synthetic tripalmitoyl-S-glycerylcysteinyl (Pam3Cys) peptides are derived from the N-terminal part of bacterial lipoprotein and constitute polyclonal B-lymphocyte and macrophage activators . In order to elucidate the primary events of leukocyte activation, we investigated the biophysical interaction of lipopeptides containing spin labels or fluorescent markers with phosphatidylcholine vesicles or immune cells . Utilizing fluorescence microscopy and FACS analysis we found, that the surface of cells, after incubation with a fluorescein-labelled lipopeptide, was highly fluorescent . In addition, capping and patching was observed . Furthermore, fluorescence quenching experiments and electron paramagnetic resonance studies using vesicles incubated with lipopeptides suggested, that the peptide moiety and other more polar molecules linked to the lipo-amino acid are exposed to the hydrophilic compartment . These results show that in lipopeptide conjugates the Pam3Cys moiety acts as an efficient membrane anchor for molecules covalently coupled to it . The sequestering of the fatty-acid chains of the lipopeptide within the membrane is an early step of interaction, which might induce the uptake of the lipopeptide into the cell and the stimulation of immunocompetent cells. Biochim Biophys Acta, 1993 Jun 18, 1149(1), 151 - 65 Antibody epitope mapping of the gastric H+/K(+)-ATPase; Mercier F et al.; Several antibodies against the gastric H+/K(+)-ATPase were analysed for the topological and sequence location of their epitopes . Topological mapping was done by comparing indirect immunofluorescent staining in intact and permeabilised rat parietal cells . Epitope definition was by Western analysis of intact and of trypsin or V8-proteinase-fragmented hog gastric ATPase combined with N-terminal sequencing of the fragments; by Western analysis of fragments of rabbit alpha subunit expressed in Escherichia coli; by analysis of rabbit alpha and beta subunits expressed in baculovirus-transfected SF 9 cells and by ELISA assay of synthetic octamers of one region of the hog alpha subunit . It was confirmed that the monoclonal antibody, mAb 95-111, recognised a cytoplasmic region between M4 and M5, close to the ATP-binding domain . The major epitope for monoclonal antibody mAb 12-18 was also in this region, but a second epitope was confirmed to be present in the M7/M8 region . The monoclonal antibody, mAb 146-14, was shown to recognise an extracytoplasmic epitope dependent on intact disulfide bonds, present in the rat and the rabbit, but absent in the hog beta subunit, due to non-conservative amino-acid substitutions . This antibody also recognised an epitope present in the alpha subunit of the H+/K(+)-ATPase at the M7 extracytoplasmic interface, perhaps indicating structural association of these two regions . The polyclonal antibody, pAb39, raised against the C-terminal portion of the enzyme, reacted only with the cytoplasmic surface of the H+/K(+)-ATPase, showing that the alpha subunit of the enzyme has an even number of membrane spanning segments. Nature, 1993 Jun 17, 363(6430), 648 - 52 New eukaryotic transcriptional repressors; Saha S et al.; Transcriptional activating sequences have been described that are encoded by parts of the genome of Escherichia coli . These acidic peptides, fused to a DNA-binding fragment of the yeast transcriptional activator GAL4, activate transcription of a gene in a wide array of eukaryotes, provided that gene bears GAL4-binding sites nearby . Here we describe an E . coli-encoded sequence that, when attached to the same DNA-binding fragment (GAL4(1-147)), converts that fragment into a repressor . Thus, as assayed in yeast or in vitro in yeast extracts, this molecule represses transcription when bound upstream of a variety of different activators . Two additional repressing regions that work when tethered upstream, a multiple mutant derivative of the original isolate and a synthetic peptide are, like the original isolate, highly basic . At least one activator can be inhibited by the mutant but not by the parental repressing region . These and other findings suggest that these repressing regions interact with and inhibit the activity of activating regions bound nearby on DNA. Nature, 1993 Jun 17, 363(6430), 640 - 4 Fusion of CHOP to a novel RNA-binding protein in human myxoid liposarcoma; Crozat A et al.; Human myxoid liposarcomas contain a characteristic chromosomal translocation, t(12;16)(q13;p11), that is associated with a structural rearrangement of the gene encoding CHOP, a growth arrest and DNA-damage inducible member of the C/EBP family of transcription factors residing on 12q13.1 . Using a CHOP-specific complementary probe and antiserum we report here the presence of an abnormal CHOP transcript and protein in these tumours . Cloning of the translocation-associated CHOP gene product revealed a fusion between CHOP and a gene provisionally named TLS (translocated in liposarcoma) . TLS is a novel nuclear RNA-binding protein with extensive sequence similarity to EWS, the product of a gene commonly translocated in Ewing's sarcoma . In TLS-CHOP the RNA-binding domain of TLS is replaced by the DNA-binding and leucine zipper dimerization domain of CHOP . Targeting of a conserved effector domain of RNA-binding proteins to DNA may play a role in tumour formation. Proc Natl Acad Sci U S A, 1993 Jun 15, 90(12), 5863 - 7 Spectroscopic evidence for a heme-heme binuclear center in the cytochrome bd ubiquinol oxidase from Escherichia coli; Hill JJ et al.; The cytochrome bd complex is a ubiquinol oxidase, which is part of the aerobic respiratory chain of Escherichia coli . This enzyme is structurally unrelated to the heme-Cu oxidases such as cytochrome c oxidase . While the cytochrome bd complex contains no copper, it does have three heme prosthetic groups: heme b558, heme b595, and heme d (a chlorin) . Heme b558 appears to be involved in the oxidation of quinol, and heme d is known to be the site where oxygen binds and is reduced to water . The role of heme b595, which is high spin, is not known . In this paper, CO is used to probe the oxygen-binding site by use of Fourier transform infrared spectroscopy to monitor the stretching frequency of CO bound to the enzyme . Photodissociation at low temperature (e.g., 20 K) of the CO-heme d adduct results in CO associated with the protein within the heme binding pocket . This photodissociated CO can subsequently relax to form a kinetically trapped CO-heme b595 adduct . The data clearly show that heme d and heme b595 must reside within a common binding pocket in the enzyme . The catalytic active site where oxygen is reduced to water is, thus, properly considered to be a heme d-heme b595 binuclear center . This is analogous to the heme alpha 3-Cu(B) binuclear center in the heme-Cu oxidases . Heme b595 may play roles analogous to those proposed for the Cu(B) component of cytochrome c oxidase. Proc Natl Acad Sci U S A, 1993 Jun 15, 90(12), 5796 - 800 A stationary-phase protein of Escherichia coli that affects the mode of association between the trp repressor protein and operator-bearing DNA; Yang W et al.; Highly purified preparations of trp repressor (TrpR) protein derived from Escherichia coli strains that were engineered to overexpress this material were found to contain another protein, of 21 kDa . The second protein, designated WrbA {for tryptophan (W) repressor-binding protein} remained associated with its namesake through several sequential protein fractionation steps . The N-terminal amino acid sequence of the WrbA protein guided the design of two degenerate oligonucleotides that were used as probes in the cloning of the wrbA gene (198 codons) . The WrbA protein, in purified form, was found by several criteria to enhance the formation and/or stability of noncovalent complexes between TrpR holorepressor and its primary operator targets . The formation of an operator-holorepressor-WrbA ternary complex was demonstrated by gel mobility-shift analysis . The WrbA protein alone does not interact with the trp operator . During the stationary phase, cells deficient in the WrbA protein were less efficient than wild type in their ability to repress the trp promoter . It is proposed that the WrbA protein functions as an accessory element in blocking TrpR-specific transcriptional processes that might be physiologically disadvantageous in the stationary phase of the bacterial life cycle. Proc Natl Acad Sci U S A, 1993 Jun 15, 90(12), 5791 - 5 Molecular characterization of the 128-kDa immunodominant antigen of Helicobacter pylori associated with cytotoxicity and duodenal ulcer; Covacci A et al.; Helicobacter pylori has been associated with gastritis, peptic ulcer, and gastric adenocarcinoma . We report the nucleotide sequence and expression of an immunodominant antigen of H . pylori and the immune response to the antigen during disease . The antigen, named CagA (cytotoxin-associated gene A), is a hydrophilic, surface-exposed protein of 128 kDa produced by most clinical isolates . The size of the cagA gene and its protein varies in different strains by a mechanism that involves duplication of regions within the gene . Clinical isolates that do not produce the antigen do not have the gene and are unable to produce an active vacuolating cytotoxin . An ELISA to detect the immune response against a recombinant fragment of this protein detects 75.3% of patients with gastroduodenal diseases and 100% of patients with duodenal ulcer (P < 0.0005), suggesting that only bacteria harboring this protein are associated with disease. Proc Natl Acad Sci U S A, 1993 Jun 15, 90(12), 5638 - 42 Functional domains of the AraC protein; Bustos SA et al.; The AraC protein, which regulates the L-arabinose operons in Escherichia coli, was dissected into two domains that function in chimeric proteins . One provides a dimerization capability and binds the ligand arabinose, and the other provides a site-specific DNA-binding capability and activates transcription . In vivo and in vitro experiments showed that a fusion protein consisting of the N-terminal half of the AraC protein and the DNA-binding domain of the LexA repressor dimerizes, binds well to a LexA operator, and represses expression of a LexA operator-beta-galactosidase fusion gene in an arabinose-responsive manner . In vivo and in vitro experiments also showed that a fusion protein consisting of the C-terminal half of the AraC protein and the leucine zipper dimerization domain from the C/EBP transcriptional activator binds to araI and activates transcription from a PBAD promoter-beta-galactosidase fusion gene . Dimerization was necessary for occupancy and activation of the wild-type AraC binding site. Proc Natl Acad Sci U S A, 1993 Jun 15, 90(12), 5598 - 602 Essential role of the HMG domain in the function of yeast mitochondrial histone HM: functional complementation of HM by the nuclear nonhistone protein NHP6A; Kao LR et al.; The yeast mitochondrial histone protein HM is required for maintenance of the mitochondrial genome, and disruption of the gene encoding HM (HIM1/ABF2) results in formation of a respiration-deficient petite mutant phenotype . HM contains two homologous regions, which share sequence similarity with the eukaryotic nuclear nonhistone protein, HMG-1 . Experiments with various deletion mutants of HM show that a single HMG domain of HM is functional and can restore respiration competency to cells that lack HM protein (him1 mutant cells) . The gene encoding the putative yeast nuclear HMG-1 homolog, the NHP6A protein, can functionally complement the him1 mutation . These results suggest that the HMG domain is the basic unit for the function of HM in mitochondria and that the function of HMG-1 proteins in the nucleus and HM in the mitochondrion may be equivalent. Proc Natl Acad Sci U S A, 1993 Jun 15, 90(12), 5469 - 73 On the directional specificity of ribosome frameshifting at a "hungry" codon; Lindsley D et al.; Limitation for aminoacyl-tRNA promotes ribosome frameshifting at certain sites . We have previously demonstrated ribosome frameshifting to the right (3') at an AAG site in one context, and to the left (5') at an AAG site in a different context . Here, we demonstrate that the "rightwing" context is largely specific for frameshifting to the right, and the "leftwing" context is largely specific for frameshifting to the left . Analysis of these context rules, and the conversion of a sequence that promotes leftward frameshifting to one that promotes rightward frameshifting, demonstrated here, permits us to define a minimal heptanucleotide sequence sufficient for shiftiness in each direction at an AAG codon whose lysyl-tRNA is in short supply. Proc Natl Acad Sci U S A, 1993 Jun 15, 90(12), 5433 - 7 Yeast DNA-repair gene RAD14 encodes a zinc metalloprotein with affinity for ultraviolet-damaged DNA; Guzder SN et al.; Xeroderma pigmentosum (XP) patients suffer from a high incidence of skin cancers due to a defect in excision repair of UV light-damaged DNA . Of the seven XP complementation groups, A-G, group A represents a severe and frequent form of the disease . The Saccharomyces cerevisiae RAD14 gene is a homolog of the XP-A correcting (XPAC) gene . Like XP-A cells, rad14-null mutants are defective in the incision step of excision repair of UV-damaged DNA . We have purified RAD14 protein to homogeneity from extract of a yeast strain genetically tailored to overexpress RAD14 . As determined by atomic emission spectroscopy, RAD14 contains one zinc atom . We also show in vitro that RAD14 binds zinc but does not bind other divalent metal ions . In DNA mobility-shift assays, RAD14 binds specifically to UV-damaged DNA . Removal of cyclobutane pyrimidine dimers from damaged DNA by enzymatic photoreactivation has no effect on binding, strongly suggesting that RAD14 recognizes pyrimidine(6-4)pyrimidone photoproduct sites . These findings indicate that RAD14 functions in damage recognition during excision repair. Proc Natl Acad Sci U S A, 1993 Jun 15, 90(12), 5428 - 32 A double-filter method for nitrocellulose-filter binding: application to protein-nucleic acid interactions; Wong I et al.; Nitrocellulose-filter binding is a powerful technique commonly used to study protein-nucleic acid interactions; however, its utility in quantitative studies is often compromised by its lack of precision . To improve precision and accuracy, we have introduced two modifications to the traditional technique: the use of a 96-well dot-blot apparatus and the addition of a DEAE membrane beneath the nitrocellulose membrane . Using the dot-blot apparatus, an entire triplicate set of data spanning 20-24 titrant concentrations can be collected on a single 4.5 x 5 inch sheet of nitrocellulose, obviating the need to manipulate separate filters for each titration point . The entire titration can then be quantitated simultaneously with direct two-dimensional beta-emission imaging technology . The DEAE second membrane traps all DNA that does not bind to the nitrocellulose, enabling a direct determination of the total amount of DNA filtered . This measurement improves precision by allowing the amount of DNA retained by the nitrocellulose to be normalized against the total amount of DNA filtered . The DEAE membrane also permits a more accurate quantitation of filter-retention efficiency and nonspecific background retention based on free DNA rather than total DNA filtered . The general approach and methods of analysis to obtain equilibrium binding isotherms are discussed, using as examples our studies of the Escherichia coli Rep protein, a helicase, and its interactions with short oligodeoxynucleotides. Proc Natl Acad Sci U S A, 1993 Jun 15, 90(12), 5418 - 22 Phosphorylation of the regulatory subunit of type II beta cAMP-dependent protein kinase by cyclin B/p34cdc2 kinase impairs its binding to microtubule-associated protein 2; Keryer G et al.; Subcellular localization of type II cAMP-dependent protein kinase is determined by the interactions of the regulatory subunit (RII) with specific RII-anchoring proteins . By using truncated NH2-terminal RII beta fusion proteins expressed in Escherichia coli and the mitotic protein kinase p34cdc2 isolated from HeLa cells or starfish oocytes, we investigated the in vitro phosphorylation of RII beta by these kinases . The putative site for phosphorylation by the mitotic kinases is Thr-69 in the NH2-terminal domain of RII beta . This phosphorylation site matches the consensus sequence X(T/S)PX(K/R) for p34cdc2 recognition and belongs to a well-conserved sequence found in all RII beta sequences known to date . In contrast to phosphorylation by casein kinase II or the cAMP-dependent protein kinase catalytic subunit, phosphorylation of RII beta by mitotic kinases impaired its interaction with a well-known RII-anchoring protein, the neuronal microtubule-associated protein 2 . The potential regulatory significance of the phosphorylation of this site on the interaction with microtubule-associated protein 2 and other RII-anchoring proteins and the physiological relevance of this cyclin B/p34cdc2 kinase-catalyzed modification of RII beta (or phosphorylation by other proline-directed protein kinases) are discussed. Biochem Biophys Res Commun, 1993 Jun 15, 193(2), 779 - 86 Cloning and expression of a novel human DNA binding protein, PO-GA; Lu Y et al.; We have cloned a novel human DNA-binding protein from a HeLa cDNA expression library using the cognate DNA binding site of a transcription factor, PO-B . Further hybridization screening with the initial clone produced contiguous cDNA sequence of 4508 bp and a complete open reading frame encoding a 128 kDa protein, PO-GA . Northern analysis revealed a wide distribution of PO-GA mRNA in most human tissue . However, PO-GA mRNA levels were lower in lung and kidney and undetectable in placental tissue . A DNA-binding fragment of PO-GA expressed in E . Coli bound selectively to the PO-B element and other GA-rich double-stranded DNA sequences and to certain single-stranded DNA sequences . PO-GA has regions of homology to E . coli and yeast DNA ligases, and to proteins involved in DNA repair . Thus, in addition to a potential role in transcription, PO-GA may also be involved in DNA repair or replication. J Biol Chem, 1993 Jun 15, 268(17), 12958 - 63 NADPH inhibits transcription of the Escherichia coli manganese superoxide dismutase gene (sodA) in vitro; Gardner PR et al.; We have previously reported that the thiols glutathione, dithiothreitol, and beta-mercaptoethanol suppress transcription of the Escherichia coli manganese-containing superoxide dismutase gene (sodA) in an in vitro coupled transcription plus translation system (Gardner, P . R., and Fridovich, I . (1987) J . Biol . Chem . 262, 17591-17595) . We now report that NADPH, but not NADH, selectively decreases transcription of sodA in vitro and that an NADPH generating system utilizing glucose 6-phosphate and the corresponding dehydrogenase markedly augments this suppressive effect . A redox buffer containing various ratios of oxidized and reduced glutathione also modulated transcription of sodA thus demonstrating the existence of a redox-sensitive mechanism controlling sodA transcription . Fusion of a 120-base pair fragment, containing 90 base pairs of DNA upstream of the sodA transcription initiation site, to a promoterless galactokinase gene (galK) conferred redox-sensitivity to GalK synthesis . We propose that these redox effects act through a redox-sensitive regulator of sodA and that the anabolic reduction charge, {NADPH}/({NADPH}+{NADP+}), is one cellular signal controlling sodA transcription. J Biol Chem, 1993 Jun 15, 268(17), 12901 - 11 Messenger RNAs expressed in intestine of adult but not baby rabbits . Isolation of cognate cDNAs and characterization of a novel brush border protein with esterase and phospholipase activity; Boll W et al.; Using a subtractive hybridization method, we have cloned cDNAs corresponding to 10 different mRNAs which share the property of being expressed in the intestine of adult but not baby rabbits . Four could be identified as coding for previously known gene products (sucrase-isomaltase, a glutathione S-transferase, a cytochrome P450, and a long form of ferritin mRNA), while six code for previously unknown proteins . One clone, AdRab-B, codes for a protein of 1458 amino acids, including (i) a putative signal sequence at the NH2 terminus, (ii) four internal repeats, 308-346 amino acids in length, (iii) a hydrophobic stretch near the COOH terminus, which represents a potential membrane anchor, and (iv) a short hydrophilic stretch at the very COOH terminus . The corresponding protein was studied with the aid of antibodies prepared against polypeptides expressed from segments of the cDNA in Escherichia coli . The protein was shown to be proteolytically processed in the intestine (but not when expressed in COS cells) and to be targeted to the brush border membrane of the enterocytes . Finally, the protein was found to have esterase and phospholipase A/lysophospholipase activity. J Biol Chem, 1993 Jun 15, 268(17), 12812 - 7 Topological analysis of quinoprotein glucose dehydrogenase in Escherichia coli and its ubiquinone-binding site; Yamada M et al.; Topological structure of quinoprotein glucose dehydrogenase in the inner membrane of Escherichia coli was determined by constructing protein fusions with alkaline phosphatase or beta-galactosidase . Analysis of the fusions revealed that the dehydrogenase possesses five membrane-spanning segments, and the N-terminal and C-terminal portions resided at the cytoplasmic and periplasmic side of the membrane, respectively . These results agreed with the hydropathy profile based on its primary structure . The topological structure suggests that the predicted binding site of the prosthetic group pyrroloquinoline quinone is located at the periplasmic side and that the amino acid residues corresponding to those that were presumed to interact with ubiquinone in one subunit of mitochondrial NADH dehydrogenase also occur at the periplasmic side . When the purified glucose dehydrogenase and cytochrome o ubiquinol oxidase were reconstituted together with ubiquinone into liposomes, a membrane potential could be generated by the electron transfer at the site of the ubiquinol oxidase but not of the dehydrogenase . These results suggest that glucose dehydrogenase has a ubiquinone reacting site close to the periplasmic side of the membrane, and thus its electron transfer to ubiquinone appears to be incapable of forming a proton electrochemical gradient across the inner membrane of E . coli. J Biol Chem, 1993 Jun 15, 268(17), 12787 - 95 Carbohydrate regulation of the rat L-type pyruvate kinase gene requires two nuclear factors: LF-A1 and a member of the c-myc family; Liu Z et al.; Transcription of the L-type pyruvate kinase (L-PK) gene is induced in response to increased carbohydrate metabolism in the liver . We have demonstrated previously that a segment of the 5'-flanking region of the L-PK gene between -183 and -96 is necessary and sufficient for the glucose response in primary hepatocytes . To explore the protein factors that are involved in carbohydrate regulation, we have performed mutational analyses and in vitro binding studies of this segment . Sequences critical for the glucose response were mapped from -171 to -124 . This segment contains the consensus binding sites for two nuclear transcription factors: LF-A1 and MLTF . Both factors are capable of binding to the corresponding L-PK sites in vitro . Mutational and functional analyses indicated that LF-A1 is indeed involved in glucose induction of the L-PK gene . The PK MLTF-like site consists of two imperfect CACGTG motifs, the core binding site for a family of transcription factors related to c-myc . Unexpectedly, mutations in either motif that resulted in defective glucose stimulation retained in vitro binding to MLTF . Furthermore, an authentic MLTF binding site from the adenovirus major late promoter was not functionally interchangeable with the natural sequence . These data indicate that binding of MLTF, in presence of LF-A1, is not capable of supporting the glucose response . Conversion of either imperfect motif to CACGTG within the context of the mutations disrupting the opposite site restored the response to elevated glucose . Thus, the factor that recognizes the PK MLTF-like site and participates in mediating the carbohydrate response of the L-PK gene appears to be a member of the c-myc family distinct from MLTF. J Biol Chem, 1993 Jun 15, 268(17), 12744 - 8 Production and characterization of recombinant heteropolymers of human ferritin H and L chains; Santambrogio P et al.; Vertebrate ferritins are iron storage proteins composed by 24 subunits of one or more types . The recombinant homopolymers of human ferritin H- and L-type chains differ in iron uptake and in physical stability, but the properties of heteropolymers with various proportions of H- and L-type chains cannot be predicted . Present study shows that unfolded human ferritin H- and L- type chains renature under similar conditions to form homopolymers indistinguishable from the native ones and that, when mixed, the unfolded H and L chains renature to form heteropolymers with restricted heterogeneity and with the expected H:L ratios . Seven of these ferritins with different H:L ratios were analyzed; electrophoretic mobility, immunological reactivity, and stability to guanidine denaturation varied as predicted, based on the homopolymers . In contrast, the rate of iron uptake, monitored by the variation of absorbance at 310 nm, increased in the ferritins that ranged in H chain content from 0 to 35%; further increments in H chains had no additional effect . This finding indicates that, under the present conditions, only a limited number of H chains are needed for the maximum rate of ferritin iron uptake . Variations of L- and H-type chains in vivo may thus have biological relevance. J Biol Chem, 1993 Jun 15, 268(17), 12730 - 5 Peptide-dependent stimulation of the ATPase activity of the molecular chaperone BiP is the result of conversion of oligomers to active monomers; Blond-Elguindi S et al.; The molecular chaperone BiP purified from bovine liver (bBiP) exhibits a low basal level of ATPase activity that can be stimulated 3-6-fold by synthetic peptides (Flynn, G . C., Chappell, T . G., and Rothman, J . E . (1989) Science 245, 385-390) . By contrast, recombinant murine BiP (rBiP) purified to homogeneity following expression in Escherichia coli exhibits a higher basal level of ATPase activity and is much less stimulated by synthetic peptides . Nondenaturing gel electrophoresis showed that rBiP is predominantly monomeric, while bBiP exists in multiple forms probably corresponding to differentially modified monomeric, dimeric, and higher oligomeric species . Some, but not all, synthetic peptides cause conversion of the oligomeric and modified species of bBiP to a monomeric form . We propose that the peptide-dependent ATPase stimulation observed for BiP reflects the conversion of inactive oligomeric and/or modified species into active monomers. J Biol Chem, 1993 Jun 15, 268(17), 12504 - 11 The influence of effectors and subunit interactions on Escherichia coli carbamoyl-phosphate synthetase studied by differential scanning calorimetry; Cervera J et al.; Differential scanning calorimetry of Escherichia coli carbamoyl-phosphate synthetase and its isolated large and small subunits reveals in each case an irreversible, kinetically controlled transition, at a temperature 14 degrees C higher for the holoenzyme than for the subunits, indicating dramatic stabilization of the subunits in the heterodimer . The deletion of the COOH-terminal 171 (mutant CarB'2373) or 385 (mutant CarB2177) residues of the large subunit results in more asymmetric transitions at a temperature 7 degrees C lower than for the wild type . The allosteric effectors IMP, UMP, and ornithine induce small reversible transitions at low temperature in the endotherm for the wild-type enzyme, but not for CarB'2373, as expected if the effectors bind in the 171-residue, COOH-terminal region . In contrast, two ligands that bind outside the deleted region, Ap5A (a ligand of both ATP sites) and glycine (an analog of glutamine) decrease and increase, respectively, the stability of the two mutants and of the wild type . The stabilization by glycine requires that the subunits are associated . The results support the implication of the 20-kDa COOH-terminal domain of the large subunit in the allosteric modulation by all the effectors and are consistent with the folding of the large subunit as a pseudohomodimer of its two homologous halves. J Biol Chem, 1993 Jun 15, 268(17), 12498 - 503 Rabbit liver microsomal endopeptidase with substrate specificity for processing proproteins is structurally related to rat testes metalloendopeptidase 24.15; Kawabata S et al.; The detergent extract of rabbit liver microsomes contains an endopeptidase (MEP) with substrate specificity for peptides containing Arg residues at the P1 and P4 positions in the cleavage site (Kawabata, S., and Davie, E . W . (1992) J . Biol . Chem . 267, 10331-10336) . These sequences occur in many proproteins such as the vitamin K-dependent proproteins and prohormones . A cDNA coding for MEP has been obtained from three overlapping clones isolated from two rabbit liver lambda gt10 cDNA libraries . The longest open reading frame of the 3507-base pair cDNA codes for a protein of 704 amino acids, of which 406 residues were confirmed by amino acid sequence analysis . MEP contains a putative active site of -His-Glu-X-X-His-, which is typical of mammalian zinc metallopeptidases . Based on a hydropathy plot, MEP is a hydrophilic protein with no transmembrane domain and no NH2-terminal signal sequence . Amino acid sequence analysis identified Asn at the three potential N-glycosylation sites in the enzyme, indicating that MEP contains no N-linked sugar . MEP is homologous with rat testes metalloendopeptidase 24.15 (60% identity), rat mitochondrial intermediate peptidase (24% identity), Escherichia coli dipeptidyl carboxypeptidase (25% identity), and the open reading frame YCL57w present in yeast chromosome III (35% identity). J Biol Chem, 1993 Jun 15, 268(17), 12325 - 33 The involvement of the arginine 17 residue in the active site of the histidine-containing protein, HPr, of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli; Anderson JW et al.; Histidine-containing protein, HPr, of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system has an active site His-15 that is phosphorylated to form N delta 1-P-histidine . The nearby conserved residue, Arg-17, has been replaced by: lysine, histidine, glutamate, glycine, serine, and cysteine . All mutations resulted in impairment of the phosphoacceptor function of HPr with enzyme I: kcat/Km values between 6% (Ser-17) and 0.1% (Glu-17), relative to wild type . Several sugar-specific enzymes II had different responses . Both the Vmax and Km of enzyme IIN-acetylglucosamine were altered, while for enzyme IImannose only Km was affected, except for R17E . For both enzymes, kcat/Km values were between 0.5 and 3%, with R17E being 10-fold lower . Except for R17E, minimal effects were observed for enzyme IImannitol . These results suggest that there are different rate-limiting steps in the enzymes II . Phosphohydrolysis properties and the pKa values for His-15 and phosphorylated His-15 determined by NMR for both wild type and mutant HPrs suggest that Arg-17 is partly responsible for the instability of P-His-15 and the depressed pKa values in wild type HPr . Other feature(s) of the tertiary structure influence the protonation of His-15 and the phosphohydrolysis properties of phosphorylated His-15. J Biol Chem, 1993 Jun 15, 268(17), 12282 - 8 Structural analysis of the purine repressor, an Escherichia coli DNA-binding protein; Schumacher MA et al.; The purine repressor protein, PurR, is a member of the lac repressor, LacI, family of Escherichia coli DNA-binding proteins that bind DNA via a highly conserved N-terminal helix-turn-helix motif . Additionally, the members of this family display strong sequence homologies between their larger C-terminal effector binding/oligomerization domains . Analysis of the PurR primary and secondary structures reveals that its C-terminal corepressor binding domain is highly homologous to another group of E . coli-binding proteins, the periplasmic binding proteins, particularly to the ribose-binding protein (RBP) . The high resolution x-ray structure of RBP allows this protein to serve as a template with which to model the predicted secondary structure of the corepressor binding domain of PurR . Similarly, the N-terminal DNA binding domain of PurR can be modeled using the NMR-determined structure of the corresponding region (residues 1-56) from LacI as a template . Combining the two, results in a description of the likely secondary structure topology of PurR and implicates residues important for corepressor binding and dimerization . CD spectroscopic studies on PurR, its corepressor binding domain and RBP result in secondary structure estimates nearly identical with those obtained by sequence analyses, thereby providing further corroborating physical evidence for this topological assignment. J Biol Chem, 1993 Jun 15, 268(17), 12250 - 2 The aleu207-->arg mutation in F1F0-ATP synthase from Escherichia coli . A model for human mitochondrial disease; Hartzog PE et al.; The mitochondrial ATPase 6 gene encodes a subunit of F1F0 adenosine triphosphate (ATP) synthase . A mutation in the ATPase 6 gene has been genetically linked to two maternally inherited genetic diseases: neurological muscle weakness, ataxia, and retinitis pigmentosa (NARP) and certain cases of subacute necrotizing encephalopathy (SNE) . Although the severity of both NARP and SNE disease were correlated with the quantity of the ATPase 6leu156-->arg mutation in each patient, the mutation could not be shown to alter F1F0-ATP synthase activity . To investigate the biochemical effects of the ATPase 6leu156-->arg mutation on F1F0-ATP synthase, the aleu207-->arg mutation was constructed in the F1F0-ATP synthase from Escherichia coli to serve as a model for the disease mutation . Characterization of the model bacterial enzyme revealed that the mutation abolishes detectable ATP synthesis via oxidative phosphorylation . The aleu207-->arg mutation results in a structural perturbation blocking proton translocation through F1F0-ATP synthase . The results suggest that a structural defect in human F1F0-ATP synthase is the biochemical basis for NARP and SNE. Gene, 1993 Jun 15, 128(1), 135 - 40 Functional display of human plasminogen-activator inhibitor 1 (PAI-1) on phages: novel perspectives for structure-function analysis by error-prone DNA synthesis; Pannekoek H et al.; The synthesis of the human plasminogen-activator inhibitor 1 (PAI-1) protein in the cytoplasm of transformed Escherichia coli cells results in inactive protein preparations that can be activated by denaturation and renaturation . We have used the phagemid pComb3, designed for combinatorial immunoglobulin repertoire cloning, for routing of PAI-1 to the periplasm and subsequent exposure on the surface of filamentous phages . Phage-displayed PAI-1 specifically binds to immobilized polyclonal and monoclonal anti-human PAI-1 antibodies . In addition, PAI-1 retains its capacity to form equimolar complexes with its target serine protease tissue-type plasminogen activator (t-PA), as well as its ability to inhibit t-PA activity . Finally, we have explored and manipulated the error-prone property of TaqI DNA polymerase during PCR amplification of the full-length PAI-1 cDNA to generate a large library of predominantly single, random PAI-1 mutants . In addition, a computer simulation program has been devised that converts the number of mutations per codogenic region (in this case PAI-1) into actual mutant proteins . The PAI-1-phage mutant library is composed of 46% single and 34% double mutants and 20% wild-type PAI-1 and can be employed to isolate mutants defective in interactions of PAI-1 with other components . The method described here is applicable to other studies on the structure-function analysis of eukaryotic proteins. Gene, 1993 Jun 15, 128(1), 13 - 7 Biochemical diversity in a phage display library of random decapeptides; DeGraaf ME et al.; Detailed knowledge and understanding of the structure and function of biologically important macromolecules is frequently insufficient to permit rational, de novo design of recognition molecules and therapeutics . Traditional drug discovery has, thus, focused on screening and identifying native molecules as drugs or as templates for genetic engineering or organic synthesis . The number and novelty of lead compounds for drug discovery might be expanded significantly, however, by the ability to express and screen large libraries of peptide structures with phage display technologies {Scott and Smith, Science 249 (1990) 386-390} . The significance of such libraries as sources of novel biological ligands will depend in part on the depth and degree of biochemical diversity they comprise . We have prepared a phage display library of greater than 2 x 10(6) individual decapeptides produced as N-terminal fusions to the pIII surface protein of fd filamentous phage . The decapeptides were expressed from a degenerate DNA insert sequence that was chemically synthesized with an equal mixture of all four nucleotide bases at the three positions in each of the ten codons . Fifty-two clones were picked randomly and without prior selection from the population and the sequences of their peptide inserts were determined . Our results confirm and document the broad representation at the primary amino acid sequence level that is expected in a library expressed from random DNA inserts . More significantly, biochemical characterization shows these insert sequences correspond to structures comprising a wide range and combination of isoelectric, hydropathic, and biochemical properties necessary in drug discovery to access a significant percentage of the repertoire of possible peptide structures by affinity or activity screening. Biochemistry, 1993 Jun 15, 32(23), 6011 - 8 Molecular characterization of a conserved, guanine nucleotide-dependent ADP-ribosylation factor in Drosophila melanogaster; Murtagh JJ Jr et al.; ADP-Ribosylation factors (ARFs) are ubiquitous approximately 20-kDa guanine nucleotide-binding proteins that stimulate cholera toxin-catalyzed ADP-ribosylation in vitro . Because the functional role(s) of ARF in mammalian systems is (are) elusive, we looked for ARF in Drosophila melanogaster, and report the partial purification and molecular cloning of an ARF from Drosophila . We cloned the Drosophila ARF 1 gene without library screening by a combination of 5 polymerase chain reactions (PCRs), yielding a 546-base open reading frame encoding 182 amino acids, which are > 93% identical to those of mammalian class I ARFs . This ARF gene maps to 79F3-6 in the proximal region of the left arm of Drosophila chromosome 3 . The Drosophila ARF1 gene structure, including placement of introns, is highly conserved relative to mammalian class 1 ARF genes . A single ARF mRNA species of 1.8 kb was abundant in all Drosophila body segments . Recombinant Drosophila ARF 1 synthesized in Escherichia coli had biochemical and immunochemical activities similar to those of mammalian ARF . The similarities of sequence and biochemical properties between Drosophila and mammalian ARFs contrast with their differences from Drosophila arl (ARF-like protein), which does not stimulate cholera toxin-catalyzed ADP-ribosylation, and is only approximately 52-56% identical in amino acid sequence to mammalian ARFs. FEMS Microbiol Lett, 1993 Jun 15, 110(2), 185 - 9 Evidence for protein kinase C stimulation in rat enterocytes pretreated with heat stable enterotoxin of Escherichia coli; Chaudhuri AG et al.; Rat intestinal epithelial cells were isolated and the activity of the calcium- and phospholipid-dependent protein kinase C (PKC) was investigated . The stimulation of activity by Escherichia coli heat stable enterotoxin (STa) was about 5-fold compared to control activity (16.91 +/- 1.69 vs 93.56 +/- 10.40 nmol/mg protein/min) and was dose dependent . Maximum enzyme activity was observed after incubation for 1 min with 6 ng of purified STa . The synergistic effects of calcium, phosphatidylserine and diolein on the enzyme activity were noted both in control and STa-treated cells . Staurosporine, a potent PKC inhibitor, significantly reduced the enzyme activity . Autoradiographic analysis of polyacrylamide gel electrophoresis revealed that pretreatment of the cells with STa also resulted in the phosphorylation of specific membrane proteins each with a molecular mass of 37 kDa, 100 kDa and 140 kDa . However, STa had no direct role on the enzyme activity . Our results, therefore, provide evidence for the involvement of PKC in STa-induced signal transduction in rat enterocytes. Proc Natl Acad Sci U S A, 1993 Jun 15, 90(12), 5833 - 7 Cloning, heterologous expression, and chromosomal localization of human inositol polyphosphate 1-phosphatase; York JD et al.; Inositol polyphosphate 1-phosphatase, an enzyme in the phosphatidylinositol signaling pathway, catalyzes the hydrolysis of the 1 position phosphate from inositol 1,3,4-trisphosphate and inositol 1,4-bisphosphate . We used a cDNA that encodes bovine inositol polyphosphate 1-phosphatase as a probe to isolate the human counterpart by low-stringency hybridization . The 1.74-kb human cDNA has 341 bp of 5' untranslated region, 180 bp of 3' untranslated region, poly(A)32, and predicts a protein of 399 amino acids . Human and bovine inositol polyphosphate 1-phosphatases show 84% amino acid sequence identity . Northern blot analysis from a variety of human tissues demonstrates that a 1.9-kb mRNA is ubiquitously expressed with highest levels in pancreas and kidney . Several higher molecular weight mRNAs also are expressed in brain, muscle, heart, and liver . We have confirmed the functional identity of the human cDNA by heterologous expression in NIH 3T3 fibroblasts, COS-7 cells and Escherichia coli . Polymerase chain reaction assay of a panel of human-rodent somatic cell hybrid DNA using human inositol polyphosphate 1-phosphatase-specific DNA primers resulted in amplification of a specific product using chromosome 2 DNA as template . Fluorescence in situ hybridization of metaphase chromosomes localizes the gene to chromosome 2 band q32 . The identification of the human inositol polyphosphate 1-phosphatase gene locus provides a target for linkage analysis to identify defects in patients with inherited psychiatric disorders that respond to lithium ions, an inhibitor of the enzyme. Biochemistry, 1993 Jun 15, 32(23), 6065 - 72 Structure of the heme-copper binuclear center of the cytochrome bo complex of Escherichia coli: EPR and Fourier transform infrared spectroscopic studies; Tsubaki M et al.; The cytochrome bo complex is a terminal quinol oxidase in the aerobic respiratory chain of Escherichia coli and functions as a redox-coupled proton pump . To clarify the structural differences of the binuclear reaction center between the cytochrome bo complex and the mitochondrial cytochrome c oxidase, a combined study using EPR and Fourier transform infrared spectroscopies was carried out . The EPR spectrum of the highly purified cytochrome bo complex in the air-oxidized state showed a broad EPR signal (peak g* = 3.7) from an integer spin system . This confirms the existence of the spin-spin exchange-coupled binuclear site, in which the Feo3+ and CuB2+ centers were bridged by an unknown ligand (X) . Binding of azide at the binuclear site as an ionic modulator weakened the strength of the spin-spin exchange coupling and thus caused a narrowing of the broad EPR signal . Binding of another modulator, formate, at the binuclear site caused the formation of EPR signals at g' = 12 and 2.7, which are very similar to those observed for cytochrome c oxidase . Cyanide replaced the bridging ligand (X) to form an Feo(3+)-C-N-CuB2+ structure in which strong spin-spin exchange coupling is expected, leading to a complete EPR-invisible state . Infrared evidence (a 2146 cm-1 C-N stretching band for the cyanide complex and a 2041 cm-1 azide antisymmetric stretching band for the azide complex) supported the theory that these ligands form bridging structures at the binuclear center, as previously observed for cytochrome c oxidase.(ABSTRACT TRUNCATED AT 250 WORDS) FEMS Microbiol Lett, 1993 Jun 15, 110(2), 243 - 8 Distribution in the genus Streptomyces of a homolog to nusG, a gene encoding a transcriptional antiterminator; Puttikhunt C et al.; The presence of the vbrA gene encoding the transcriptional antiterminator NusG equivalent protein of Streptomyces virginiae was tested for in 73 Streptomyces species by Southern hybridization . Fifty-five strains (75%) including S . griseus, S . lividans TK-21 and S . coelicolor A3(2) showed clear hybridization signals, indicating wide distribution of vbrA or vbrA homologs in Streptomyces species . With hybridization patterns against 3 different probes, i.e., probes covering vbrA alone, the downstream gene rplK alone, and both vbrA-rplK, the 55 strains were classified into 4 groups . In the groups I, II and III (total 50 strains) vbrA was found to be adjacent to rplK, indicating that the gene arrangement vbrA-rplK is common in Streptomyces and that these genes may constitute a part of gene cluster encoding several components of the transcription and translation apparatus, as in Escherichia coli. FEMS Microbiol Lett, 1993 Jun 15, 110(2), 223 - 9 Eimeria refractile body proteins contain two potentially functional characteristics: transhydrogenase and carbohydrate transport; Vermeulen AN et al.; cDNA encoding an immunogenic protein from partially sporulated oocysts of Eimeria acervulina was cloned and used to search for the homologous counterpart in Eimeria tenella . Monospecific antibodies were raised against the recombinant expression product . Using these antibodies, the parasite proteins were found to be localised in the refractile bodies . The derived amino-acid sequences were compared by computer using the SWISSPROT protein database . In addition to high homology between the Eimeria species, extensive similarity was found with pyridine nucleotide transhydrogenase from Escherichia coli . Comparison with the sugar signature database also resulted in a possible sugar binding domain present only in the Eimeria proteins . It is possible that the corresponding parasite proteins play a role in the recently discovered mannitol cycle of Eimeria. Eur J Biochem, 1993 Jun 15, 214(3), 787 - 94 Expression and mutation of soybean beta-amylase in Escherichia coli; Totsuka A et al.; The cDNA clones corresponding to soybean beta-amylase mRNA were isolated and sequenced . The cDNA contained an open-reading frame composed of 496 amino acids . The comparison of the amino acid sequence deduced from the cDNA with the N-terminal peptide sequence from mature enzyme proved that beta-amylase had no leader sequence . Employing the cDNA, the beta-amylase was directly synthesized in Escherichia coli by the expression vector pKK233-2 controlled by the tac promoter . The enzyme activity detected in E . coli lysate drastically increased with a lower cultivation temperature, and the total activity and specific activity of the enzyme in E . coli lysate cultured at 13 degrees C was 130-fold and 280-fold, respectively, the value at 37 degrees C . The enzyme produced in E . coli was purified by the affinity column chromatography of cyclomaltohexaose-immobilized Sepharose 6B . Employing the established expression and purification system of the enzyme, the functional ionizable groups in the active site were searched . His93, involving an imidazole, and Asp348, involving a carboxylate, in the highly conserved regions within the beta-amylases were replaced by Arg (H93R) and Ash (D348N) by site-directed mutagenesis, respectively . All beta-amylases, including the non-mutant and mutant beta-amylases, produced in E . coli exhibited lower Vmax values than that of beta-amylase isolated conventionally from soybean seeds . Especially the Vmax value of {H93R}beta-amylase was reduced drastically compared to that of the non-mutant; however, none of them lost their enzyme activities completely . Therefore, neither His93 nor Asp348 may participate in the catalytic reaction directly. Eur J Biochem, 1993 Jun 15, 214(3), 695 - 701 Chemical structure of the lipid A of Escherichia coli J-5; Holst O et al.; The lipopolysaccharide, and particularly its lipid A moiety, of the J-5 mutant of Escherichia coli O111 plays a central role in studies on potential induction of cross-reactive and cross-protective antibodies, however, its chemical and antigenic structure was hitherto unknown . Here, the chemical structure of the J-5 lipid A is reported . It is composed of the bisphosphorylated disaccharide beta-D-GlcpN-4-P-(1-6)-alpha-D-GlcpN-1-P which carries four residues of 3-hydroxytetradecanoic acid, one each at positions 2, 3, 2', and 3' . The hydroxyl groups of the acyl residues at 2' and 3' are esterified with dodecanoic and tetradecanoic acid, respectively . The hydroxyl group at C-6' functions in the lipopolysaccharide as the attachment site of the core oligosaccharide . Furthermore, a new method to isolate the hydrophilic backbone, i.e . the 1,4'-bisphosphorylated glucosamine disaccharide, and its structural analysis by 1H-, 13C-, and 31P-NMR spectroscopy, are described, leading to a new and easier strategy in structural analysis of lipid A from bacterial lipopolysaccharides. Eur J Biochem, 1993 Jun 15, 214(3), 635 - 9 Characterization of the receptor and translocator domains of colicin N; el Kouhen R et al.; Intact colicin N and various colicin derivatives, including a natural fragment lacking the first 36 amino-acid residues, a chymotryptic fragment lacking the first 66 amino acids and a thermolytic fragment comprising residues 183-387, were used to locate the regions involved in colicin-N uptake by sensitive Escherichia coli cells . Two separate domains of the molecule participate in colicin-N entry . Specific binding to OmpF receptor site requires a region located between residues 67-182 . A N-terminal domain, located between residues 17-66, is involved during the translocation step after binding to receptor . Two sub-regions, residues 17-36 and residues 37-36, can be defined in this domain . The location and interactions between these domains are discussed in comparison to other colicins which use similar cell components for their uptake. Biochem J, 1993 Jun 15, 292 ( Pt 3), 833 - 8 Characterization of phosphorylation sites in the cytoplasmic domain of the 300 kDa mannose-6-phosphate receptor; Rosorius O et al.; The human 300 kDa mannose-6-phosphate receptor (MPR 300) is phosphorylated in vivo at serine residues of its cytoplasmic domain . Two-dimensional separation can resolve tryptic phosphopeptides into four major species . To identify the kinases involved in MPR 300 phosphorylation and the phosphorylation sites the entire coding sequence of the cytoplasmic tail was expressed in Escherichia coli . The isolated cytoplasmic domain was used as a substrate for four purified serine/threonine kinases {casein kinase II (CK II), protein kinase A (PKA), protein kinase C and Ca2+/calmodulin kinase} . All kinases phosphorylate the cytoplasmic tail exclusively on serine residues . Inhibition studies using synthetic peptides, partial sequencing of isolated tryptic phosphopeptides and co-migration with tryptic phosphopeptides from MPR 300 labelled in vivo showed that (i) PKA phosphorylates the cytoplasmic MPR 300 domain at Ser20 and at a non-identified site, neither of which are phosphorylated in vivo, and that (ii) the two sites phosphorylated by CK II in vivo and in vitro are Ser82 and Ser157 . The results indicate that the human MPR 300 is a physiological substrate of either CK II or a related kinase which may play a role in the transport function of MPR 300 and/or interaction with other proteins. Biochem J, 1993 Jun 15, 292 ( Pt 3), 825 - 32 Expression and characterization of rat kallikrein-binding protein in Escherichia coli; Ma JX et al.; Rat kallikrein-binding protein is a novel serine-proteinase inhibitor that forms a covalent complex with tissue kallikrein . We have purified rat kallikrein-binding protein and cloned the cDNA and the gene encoding rat kallikrein-binding protein {Chao, Chai, Chen, Xiong, Chao, Woodley-Miller, Wang, Lu and Chao (1990) J . Biol . Chem . 265, 16394-16401; Chai, Ma, Murray, Chao and Chao (1991) J . Biol . Chem . 266, 16029-16036} . In the present study, we have expressed rat kallikrein-binding protein in Escherichia coli with a T7-polymerase/promoter expression system . A high level of expression was detected by an e.l.i.s.a . with an average of 24.2 mg of recombinant rat kallikrein-binding protein per 1 of culture . The recombinant protein appeared as a major protein in a crude extract of Escherichia coli on SDS/PAGE . It showed a molecular mass of 43 kDa and was recognized by polyclonal antibody to the native rat kallikrein-binding protein in Western-blot analysis . The recombinant rat kallikrein-binding protein has been purified to apparent homogeneity by DEAE-Sepharose CL-6B, hydroxyapatite Bio-Gel HPHT and Mono P 5/5 column chromatography . The purified recombinant rat kallikrein-binding protein showed immunological identity with the native rat kallikrein-binding protein purified from rat serum, in a specific e.l.i.s.a . To confirm the fidelity of the expression, the N-terminal ten amino acids of the recombinant rat kallikrein-binding protein were sequenced and were shown to match perfectly with those of the native rat kallikrein-binding protein . The purified recombinant rat kallikrein-binding protein formed SDS- and heat-stable complexes with rat tissue kallikrein (rK1) and T-kininogenase (rK10) in vitro, but not with other enzymes in the rat kallikrein gene family, such as tonin (rK2) and S3 protein (rK9), which indicates enzyme-specific binding . The properties of the recombinant rat kallikrein-binding protein including its size, charge, complex formation with target enzymes and immunological characteristics were compared with those of the native protein . This expression system provides a simple way to obtain a large amount of the biologically active recombinant protein, to study structure-function relationships of the rat kallikrein-binding protein and its interaction with its target enzymes. J Biol Chem, 1993 Jun 15, 268(17), 12837 - 42 Recombinant vaccinia virus K3L gene product prevents activation of double-stranded RNA-dependent, initiation factor 2 alpha-specific protein kinase; Carroll K et al.; Deletion of the vaccinia virus K3L gene, a homologue of the alpha subunit of protein synthesis initiation factor 2, has been reported to reduce the ability of the virus to grow in interferon-treated cells (Beattie, E., Tattaglia, J., and Paoletti, E . (1991) Virology 183, 419-422) . Purified recombinant K3L gene product, pK3r, has potent effects on activation of double-stranded (ds) RNA-dependent, initiation factor-2 alpha (eIF-2 alpha)-specific protein kinase (PKR) in in vitro reactions . Recombinant pK3 prevents the inhibition of protein synthesis by dsRNA in a cell-free translation system from rabbit reticulocytes at levels equal to, or lower than, the level of endogenous eIF-2 alpha . In the cell-free translation system, pK3r exerts its effects at all dsRNA concentrations tested, by preventing phosphorylation of eIF-2 alpha . In addition, pK3r reduces the autophosphorylation of immunopurified PKR, as well as its ability to phosphorylate the alpha subunit of purified eIF-2 . At 400 mM NaCl, in vitro translated {35S}methionine-radiolabeled pK3 can be co-immunoprecipitated with human PKR, using a monoclonal antibody to PKR . This tight binding is consistent with a role for pK3 as a pseudosubstrate for the kinase, and identifies the amino-terminal 30% of eIF-2 alpha as the domain recognized by the eIF-2 alpha-specific protein kinases . In addition, the tight binding opens up the possibility of using binding assays to identify functional domains within the kinase and pK3 . Recombinant pK3 also prevents activation of the heme-sensitive eIF-2 alpha-specific protein kinase, eIF-2 alpha-PKh, in both cell-free translation systems as well as in partially purified preparations . This suggests some similarity between the eIF-2 alpha binding domains of the two eIF-2 alpha specific protein kinases. J Biol Chem, 1993 Jun 15, 268(17), 12724 - 9 Cysteine to serine replacements in 6-hydroxy-D-nicotine oxidase . Consequences for enzyme activity, cofactor incorporation, and formation of high molecular weight protein complexes with molecular chaperones (GroEL); Brandsch R et al.; 6-Hydroxy-D-nicotine oxidase, an enzyme with FAD covalently attached to the protein, contains 6 cysteine residues in positions 45 (Cys1), 59 (Cys2), 136 (Cys3), 173 (Cys4), 260 (Cys5) and 433 (Cys6) . Cys2, 3, 5, and 6 were replaced with serine by site-directed mutagenesis . The effects of these exchanges on enzyme activity, the autocatalytic incorporation of the cofactor, and the interaction of the mutant proteins with molecular chaperones were analyzed . The flavinylation of 6-hydroxy-D-nicotine oxidase is dependent on the presence of allosteric effectors, e.g . glycerol 3-phosphate or other phosphorylated tricarbon compounds . Replacement of Cys2 or Cys5 abolished this dependence . Covalent incorporation of FAD was reduced to an undetectable level in the Cys3 and Cys5 mutants . Replacement of Cys6 by Ser had no significant effect on enzyme activity and cofactor attachment . Deletion of two amino acids, Phe and Arg, situated 12 and 11 amino acid residues, respectively, from the carboxyl terminus of the protein, resulted in an inactive enzyme with no covalently bound FAD . This result indicates that almost the entire protein chain has to be synthesized before the cofactor can be incorporated, making a cotranslational flavinylation step rather unlikely . The distribution of the 6-hydroxy-D-nicotine oxidase polypeptide between the high molecular weight complexes and the free soluble form was analyzed by gel filtration on Sephacryl S-200 . The wild-type holoenzyme as well as the wild-type apoenzyme were recovered in the eluent fraction of the column while the mutant proteins were retained in high molecular weight complexes, predominantly in those associated with GroEL, as revealed by immunoprecipitation . The extent of complex formation with this molecular chaperone depended on the position of the mutated Cys residue within the protein . Complex formation was highest with protein from the mutants Cys2 and Cys3, less with the Cys5, and absent with the Cys6 mutant protein . Thus, alterations in the amino-terminal part of the 6-hydroxy-D-nicotine oxidase appear more important for the interaction with molecular chaperones than alterations situated in the carboxyl-terminal part of the protein. Eur J Biochem, 1993 Jun 15, 214(3), 703 - 10 Structural analysis of two oligosaccharide bisphosphates isolated from the lipopolysaccharide of a recombinant strain of Escherichia coli F515 (Re chemotype) expressing the genus-specific epitope of Chlamydia lipopolysaccharide; Holst O et al.; The lipopolysaccharide of the recombinant strain Escherichia coli F515-207, expressing the genus-specific epitope of Chlamydia lipopolysaccharide, was sequentially de-O- and de-N-acylated by mild hydrazinolysis and treatment with 4 M KOH, respectively, yielding two oligosaccharide bisphosphates which were isolated by high-performance anion-exchange chromatography and gel-permeation chromatography . Their structures were determined by chemical analysis, NMR spectroscopy, and mass spectrometry as alpha-Kdo-(2-4)-alpha-Kdo-(2-6)-beta-D-GlcN-(1-6)-alpha-D-GlcN 1,4'-P2 (tetrasaccharide bisphosphate) and alpha-Kdo-(2-8)-alpha-Kdo-(2-4)-alpha-Kdo-(2-6)-beta-D-GlcN-(1-6)-alpha- D-GlcN 1,4'-P2 (pentasaccharide bisphosphate). J Immunol, 1993 Jun 15, 150(12), 5391 - 9 Cloning and expression of immunologically active recombinant Amb a V allergen of short ragweed (Ambrosia artemisiifolia) pollen; Ghosh B et al.; We have cloned and sequenced Amb a V, an Ambrosia artemisiifolia (short ragweed) pollen allergen that has proved to be particularly useful in the genetic analysis of human immune responsiveness . The amino acid sequence deduced from the cloned cDNA sequence corresponds to the published sequence of the protein, except that the cDNA sequence encodes an extra 10 amino acids at the C-terminus . The expressed 55-residue protein is then presumably cleaved enzymatically at the C-terminal lysine found in the 45-residue protein isolated from the pollen . The cloning and sequencing of Amb a V genomic DNA confirmed the cDNA sequence and showed that the Amb a V gene has no introns . Recombinant Amb a V allergen, expressed in Escherichia coli, bound to IgG and IgE antibodies in all Amb a V-allergic individuals tested and inhibition studies demonstrated that the recombinant protein contains a subset of the antigenic epitopes found on native Amb a V . In addition, recombinant Amb a V released histamine efficiently from basophils from Amb a V-allergic patients . The recombinant Amb a V allergen and mutants of Amb a V should, therefore, be useful in studies of allergen epitopes in humans, as well as providing a diagnostic tool. J Biol Chem, 1993 Jun 15, 268(17), 12983 - 9 Primary structure of human deoxycytidylate deaminase and overexpression of its functional protein in Escherichia coli; Weiner KX et al.; The cDNA encoding human dCMP deaminase was isolated from a lambda ZAPII expression library using an antibody generated against highly purified HeLa cell dCMP deaminase . The cloned cDNA consists of 1856 base pairs and encodes a protein of 178 amino acids with a calculated molecular mass of 19,985 daltons . The sequence of several cyanogen bromide-cleaved peptides derived from HeLa cell dCMP deaminase are all contained within the deduced amino acid sequence . A zinc binding region is present in the enzyme, similar to that reported for cytidine deaminase (Yang, E . C., Carlow, D., Wolfenden, R., and Short, S . A . (1992) Biochemistry 31, 4168-4174) . Northern blot analysis revealed a predominant messenger RNA species of 1.9 kilobases . Expression of the active protein to about 10% of Escherichia coli's total protein was achieved by subcloning the open reading frame into a high expression system using the polymerase chain reaction . Polyacrylamide gel electrophoresis revealed a prominent protein band which comigrated with affinity purified HeLa dCMP deaminase, while Western blot analysis yielded an immunoreactive band which comigrated with the single immunoreactive affinity column purified dCMP deaminase band . The enzyme which possesses a kcat of 1.02 x 10(3) s-1 was purified to homogeneity in over 60% yield . The overexpression of dCMP deaminase should permit more exacting studies on the regulation of this important allosteric enzyme which provides substrate for DNA synthesis. J Biol Chem, 1993 Jun 15, 268(17), 12655 - 62 The synapse-specific phosphoprotein F1-20 is identical to the clathrin assembly protein AP-3; Zhou S et al.; F1-20 and AP-3 are independently described, synapse-associated, developmentally regulated phosphoproteins with similar apparent molecular masses on SDS-polyacrylamide gel electrophoresis (PAGE) . F1-20 was cloned and characterized because of its synapse specificity . AP-3 was purified and studied biochemically because of its function as a clathrin assembly protein . Here we present evidence that establishes the identity of F1-20 and AP-3 . Monoclonal antibodies against F1-20 and AP-3 both specifically recognize a single protein from mouse brain with an apparent molecular mass of 190 kDa on SDS-PAGE . These monoclonal antibodies also specifically recognize the cloned F1-20 protein expressed in Escherichia coli . The anti-F1-20 monoclonal antibody (mAb) stains a bovine protein with an apparent molecular mass on SDS-PAGE of 190 kDa that copurifies with brain clathrin-coated vesicles (CCVs) and that can be extracted from the brain CCVs under conditions that extract AP-3 . The anti-F1-20 and anti-AP-3 mAbs specifically recognize the same spot on a two-dimensional gel run on a bovine brain clathrin-coated vesicle extract . AP-3 purified from bovine brain CCVs is recognized by both the anti-F1-20 and anti-AP-3 mAbs . Purified preparations of bovine AP-3 and bacterially expressed mouse F1-20 give identical patterns of protease digestion with bromelain and subtilisin . Sequence analyses reveal that F1-20 has an essentially neutral 30-kDa NH2-terminal domain with an amino acid composition typical of a globular structure and an acidic COOH-terminal domain rich in proline, serine, threonine, and alanine . This is consistent with proteolysis experiments that suggested that AP-3 could be divided into a 30-kDa globular uncharged clathrin-binding domain and an acidic, anomalously migrating domain. Gene, 1993 Jun 15, 128(1), 97 - 101 A family of vectors for surface display and production of antibodies; Dubel S et al.; Expression vectors for surface display and production of single-chain (Fv) antibodies (scAb) have been constructed based on the phagemid pSEX, which expresses DNA encoding a scAb fused to the gene III product of filamentous phage {Breitling et al., Gene 104 (1991) 147-153} . A smaller version of this phagemid, pSEX20, was made by removing an unnecessary cat . To produce a vector for the surface display of other proteins and peptides, the scAb of pSEX20 was substituted by a polycloning site (MCS) to give pSEX40 . For the presentation of Ab on the surface of Escherichia coli, phagemid pAP10 was derived from pSEX20 by substituting gene III with a gene encoding the peptidoglycan-associated lipoprotein (PAL) . Vectors for producing scAb that can be purified by antibody and metal affinity chromatography were constructed by substituting gene III in the vector pSEX20 with DNA encoding a peptide with a C-terminal epitope recognised by a monoclonal antibody (phagemid pOPE40) or with five C-terminal histidines (pOPE 90). Gene, 1993 Jun 15, 128(1), 43 - 9 Identification of a peptide which binds to the carbohydrate-specific monoclonal antibody B3; Hoess R et al.; The monoclonal antibody (mAb) B3 recognizes an antigen found on the surface of many adenocarcinoma cells . While the structure of the cellular antigen is unknown, epitope mapping using neoglycoproteins with known carbohydrate moieties indicates that the mAb B3 reacts with the LewisY (LeY) antigen {Pastan et al., Cancer Res . 51 (1991) 3781-3787} . We have used mAb B3 to select for peptides that mimic the carbohydrate structure using libraries of filamentous phage displaying random peptides on their surface . Phage that were selected coded for the sequence APWLYGPA . The corresponding peptide was synthesized and tested for its ability to bind to mAb B3 . The peptide was found to inhibit specifically the binding of 111In-labeled mAb B3 to A431 adenocarcinoma cells, as well as to inhibit killing of these cells by a B3 immunotoxin . In addition, the LeY carbohydrate, lactodifucotetraose, was able to compete with the phage displaying this peptide for binding to mAb B3 . Alanine-scanning mutagenesis of the sequence coding for this peptide indicates that four residues, PWLY, were critical for binding to the mAb . The sequence is similar to other sequences known to mimic carbohydrate structures. Blood, 1993 Jun 15, 81(12), 3313 - 7 Activation of the kallikrein-kinin system after endotoxin administration to normal human volunteers; DeLa Cadena RA et al.; The objective of this study was to determine the role of the kallikrein-kinin system in healthy humans after intravenous administration of either Escherichia coli endotoxin or saline . We studied a total of 18 healthy nonsmoking volunteers, 23 to 38 years old, in an open-label study at the Critical Care Medicine Department, Clinical Center, National Institutes of Health (Bethesda, MD) in which some of the patients served as their own controls . After baseline data collection, the subjects received intravenously either E coli endotoxin (n = 15, 4 ng/kg of body weight) or saline (n = 8, controls) . Signs, symptoms, systemic blood pressure, factor XII, plasma prekallikrein (PK), factor XI (FXI), antithrombin III (AT-III), high molecular weight kininogen (HK), and alpha 2-macroglobulin-kallikrein complexes were measured at baseline and 1, 2, 3, 5, and 24 hours after injection of either saline or endotoxin . After infusion of endotoxin, we found the functional plasma levels of FXI decreased at 2 hours (P < .05) and at 5 hours (P < .05) . Functional PK was significantly depressed by 2 hours (P < .05), at 5 hours (P < .05), and at 24 hours (P < .01), whereas the PK antigen was only low at 5 hours (P < .05) . These changes were accompanied by a significant increase in circulating alpha 2-macroglobulin-kallikrein complexes at 3 hours (P < .05) and 5 hours (P < .01) . No significant changes occurred in the plasma levels of factor XII or HK . We concluded that clinical response to intravenous endotoxin in healthy human volunteers is associated with activation of the kallikrein-kinin systems . Further investigation is needed with specific inhibitors of the kallikrein-kinin system to define its primary or secondary role in the endotoxin-mediated reactions. Blood, 1993 Jun 15, 81(12), 3226 - 33 Recombinant human leukemia inhibitory factor induces acute phase proteins and raises the blood platelet counts in nonhuman primates; Mayer P et al.; Recombinant human leukemia inhibitory factor (rhLIF) produced by Escherichia coli was administered subcutaneously (sc) to rhesus monkeys at doses of 2, 10, and 50 micrograms/kg body weight/d for 14 days to assess its biologic activities in vivo . Serum levels of positively regulated acute phase proteins (APP) (C-reactive protein, alpha 1-antitrypsin, haptoglobin, and ceruloplasmin) were increased, whereas the negatively regulated APP prealbumin decreased in response to rhLIF treatment . During the second week of treatment, blood platelet counts began to increase, resulting in a maximum of a twofold increase above normal levels a week after termination of the rhLIF treatment . No changes were seen in total and differential white blood cell counts in blood progenitor levels and in red blood cell numbers . The low- and medium-dose rhLIF treatments were tolerated without significant side effects . The animals treated with the high dose showed a reduction in body weight of approximately 10% . In conclusion, rhLIF was shown to stimulate APP and to increase the number of platelets in circulation in nonhuman primates. FEBS Lett, 1993 Jun 14, 324(2), 167 - 70 Crystallization of the seryl-tRNA synthetase:tRNAS(ser) complex of Escherichia coli; Price S et al.; Crystals of the complex between seryl-tRNA synthetase and tRNA(2ser) from Escherichia coli have been obtained from ammonium sulphate solutions . The crystals are of the 1:2 enzyme:tRNA complex, belong to the space group C222(1), have cell dimensions of a = 128.9 A, b = 164.9 A, c = 127.3 A and diffract anisotropically from 3.5 to 4.5 A . An X-ray diffraction data set to 4 A has been collected . The combination of molecular replacement using the refined structure of the catalytic domain of the native enzyme, data from a heavy atom derivative and solvent flattening was used to produce a map at 4 A resolution . This shows that a tRNA molecule binds across the dimer, the anticodon stem and loop do not contact the protein and the helical arm of the enzyme contacts the T psi C loop and the long extra arm of the tRNA. FEBS Lett, 1993 Jun 14, 324(2), 131 - 5 The in vivo assembly and function of the N- and C-terminal halves of the Tn10-encoded TetA protein in Escherichia coli; Yamaguchi A et al.; The tetA gene was cut into its N- and C-terminal halves at the central EcoRI site and the two halves were subcloned individually or together under a separate lac promoter/operator . The expression of the C-terminal half was detected with a C-terminal-specific antibody . The amount of the N-terminal half in the cytoplasmic membrane was not affected by the presence of the C-terminal half . In contrast, the amount of the C-terminal half in the membrane was increased in the presence of the N-terminal half, indicating that the N-terminal half helps the stable folding of the C-terminal half in the membrane . Each half individually showed no tetracycline transport activity, however, when both halves were expressed together, the resultant complex showed about 40% of the tetracycline transport activity of the wild-type per number of the C-terminals of TetA protein in the membrane. FEBS Lett, 1993 Jun 14, 324(2), 162 - 6 In vivo overexpression and purification of Escherichia coli tRNA(ser); Borel F et al.; DNA fragments corresponding to the sequences of Escherichia coli tRNA(2ser) and amber suppressor tRNA(ser), were synthesized from overlapping oligonucleotides . These were interposed between a strong promotor and a synthetic transcriptional terminator to ensure the production of a transcript of the correct size . The genes of promotor, fragment and terminator were cloned into a conditional runaway replication plasmid . At temperatures below 37 degrees C this vector has a low copy number but, following a temperature shift to 42 degrees C, the copy number is no longer regulated . Using these constructs an overexpression of tRNA(ser) of about 20 times the level of the wild-type pool could be obtained (corresponding e.g . to 200 times the expression tRNA(2ser)) . From these systems 10 mg quantities of tRNA(ser)s could be isolated with a serine acceptance of 1,100 pmol/A280 unit. Science, 1993 Jun 11, 260(5114), 1646 - 9 Evidence of DNA bending in transcription complexes imaged by scanning force microscopy; Rees WA et al.; Complexes of Escherichia coli RNA polymerase with DNA containing the lambda PL promoter have been deposited on mica and imaged in air with a scanning force microscope . The topographic images reveal the gross spatial relations of the polymerase relative to the DNA template . The DNA appears bent in open promoter complexes containing RNA polymerase bound to the promoter and appears more severely bent in elongation complexes in which RNA polymerase has synthesized a 15-nucleotide transcript . This difference could be related to the conformational changes that accompany the maturation of open promoter complexes into elongation complexes and suggests that formation of the elongation complex involves a considerable modification of the spatial relations between the polymerase and the DNA template. Nucleic Acids Res, 1993 Jun 11, 21(11), 2709 - 14 Elimination of band compression in sequencing gels by the use of N4-methyl-2'-deoxycytidine 5'-triphosphate; Li S et al.; Taq DNA polymerase, Sequenase, and the large fragment of E.coli polymerase I effectively utilize N4-methyl-2'-deoxycytidine 5'-triphosphate (N4-methyl-dCTP) in the place of dCTP in dideoxynucleotide terminator sequencing reactions on single-stranded templates . When the resulting fragment mixtures are resolved on sequencing gels, they are found to be free of band compressions even in cases where such compressions remain unresolved by the substitution of 7-deaza-dGTP for dGTP . Sequencing reactions using N4-methyl-dCTP instead of dCTP are somewhat more prone to false stops than are sequencing reactions using 7-deaza-dGTP instead of dGTP; this difference is more pronounced when sequencing with Sequenase at 37 degrees C than when sequencing with Taq DNA polymerase at 72 degrees C . For the three polymerases investigated, replacement of dCTP by N4-methyl-dCTP does not fundamentally change the characteristic variations in band intensities seen in the C-lane . N4-methyl-dCTP can also be used for sequencing double-stranded DNA and for DNA amplification by the polymerase chain reaction. Nucleic Acids Res, 1993 Jun 11, 21(11), 2627 - 31 A PCR-based method for high stringency screening of DNA libraries; Israel DI; A rapid method for cloning genomic DNA utilizing a PCR-based screening protocol is described . A murine genomic library in lambda phage was subdivided into 64 wells, each containing 1000 clones, and propagated in bacteria . Amplified phage from each of 8 wells across columns, and each of 8 wells down rows, were pooled . The pooled phage were screened for the presence of murine M-CSF DNA by PCR using specific oligonucleotide primers . A single well that contained an M-CSF genomic clone was identified by the synthesis of a PCR product of the correct size that hybridized to an internal M-CSF oligonucleotide probe . This well was subdivided into 64 wells, each containing approximately 30 individual phage, reamplified, and rescreened utilizing the same protocol . A positive well was then subdivided and amplified a third time starting with an average of 2 phage per well, and rescreened for M-CSF DNA by PCR . Phage from a PCR-positive well, now highly enriched for M-CSF DNA, were grown as individual plaques . PCR-screening of randomly picked plaques demonstrated that the majority contained an M-CSF genomic insert . This method obviates the more labor and time intensive method of plaque hybridization screening of DNA libraries, and is more stringent since three oligonucleotides (the two PCR primers, and the hybridization probe) are required to give a true positive signal . Similar methodology has also been used to clone a cDNA gene contained within a plasmid library. Nucleic Acids Res, 1993 Jun 11, 21(11), 2579 - 84 Nuclear and mitochondrial forms of human uracil-DNA glycosylase are encoded by the same gene; Slupphaug G et al.; Recent cloning of a cDNA (UNG15) encoding human uracil-DNA glycosylase (UDG), indicated that the gene product of M(r) = 33,800 contains an N-terminal sequence of 77 amino acids not present in the presumed mature form of M(r) = 25,800 . This led to the hypothesis that the N-terminal sequence might be involved in intracellular targeting . To examine this hypothesis, we analysed UDG from nuclei, mitochondria and cytosol by western blotting and high resolution gel filtration . An antibody that recognises a sequence in the mature form of the UNG protein detected all three forms, indicating that they are products of the same gene . The nuclear and mitochondrial form had an apparent M(r) = 27,500 and the cytosolic form an apparent M(r) = 38,000 by western blotting . Gel filtration gave essentially similar estimates . An antibody with specificity towards the presequence recognised the cytosolic form of M(r) = 38,000 only, indicating that the difference in size is due to the presequence . Immunofluorescence studies of HeLa cells clearly demonstrated that the major part of the UDG activity was localised in the nuclei . Transfection experiments with plasmids carrying full-length UNG15 cDNA or a truncated form of UNG15 encoding the presumed mature UNG protein demonstrated that the UNG presequence mediated sorting to the mitochondria, whereas UNG lacking the presequence was translocated to the nuclei . We conclude that the same gene encodes nuclear and mitochondrial uracil-DNA glycosylase and that the signals for mitochondrial translocation resides in the presequence, whereas signals for nuclear import are within the mature protein. Nucleic Acids Res, 1993 Jun 11, 21(11), 2747 - 54 Synthetic gene for the hepatitis C virus nucleocapsid protein; Khudyakov YE et al.; A synthetic gene encoding the hepatitis C virus (HCV) nucleocapsid protein was constructed and expressed in E . coli . To synthesize this gene, we developed a new method that results in the enzymatic synthesis of long polydeoxyribonucleotides from oligodeoxyribonucleotides . The method, designated as the 'Exchangeable Template Reaction' (ETR), uses oligonucleotides as templates for DNA polymerase . A special mechanism was designed to exchange the templates during the polymerase reaction . The mechanism relies on the formation of a single-stranded 3'-protrusion at the 'growing point' of the elongating DNA such that it can be subsequently annealed, in a sequence-specific manner, with the next synthetic oligonucleotide . When annealed to the 3'-protrusion, the added oligonucleotide becomes a template for DNA polymerase, and the protruding 3'-end of the double-stranded DNA is used as the primer . The HCV nucleocapsid gene was assembled with DNA ligase from three fragments synthesized by ETR . The data verify that this method is efficient . The main advantage of ETR is the ability to combine more than two oligonucleotides in one tube together with polymerase and an enzymatic activity that produces a 3'-protrusion (e.g., BstXI) rather than the sequential addition of each component . The data demonstrate that as many as five oligonucleotides can be used simultaneously, resulting in a synthesized DNA fragment of designed sequence . The synthetic gene expressed in E . coli produced a 27 kDa protein that specifically interacted with antibodies from sera obtained from HCV-infected individuals. Biochim Biophys Acta, 1993 Jun 10, 1143(1), 62 - 6 ATP-driven potassium transport in right-side-out membrane vesicles via the Kdp system of Escherichia coli; Kollmann R et al.; The ATP-generating system described by Hugenholtz, J., Hong, J.-S . and Kaback, H.R . ((1981) Proc . Natl . Acad . Sci . USA 78, 3446-3449) has been used to synthesize ATP up to 1.8 mM in right-side-out membrane vesicles from Escherichia coli . This ATP level was sufficient to drive uptake of potassium ions via the Kdp-ATPase . In the kdp wild type strain about 110 nmoles K+/mg membrane protein were accumulated . This process was still partially sensitive to the well-known inhibitors of P-type ATPases, orthovanadate and bafilomycin B1. Biochim Biophys Acta, 1993 Jun 10, 1143(1), 109 - 11 PCR cloning, sequence analysis and expression of the cybC genes encoding soluble cytochrome b-562 from Escherichia coli B strain OP7 and K strain NM522; Trower MK; The cybC genes encoding cytochrome b-562 from B and K strains of Escherichia coli were shown to differ in size following their isolation by in vitro amplification using the polymerase chain reaction . Nucleotide sequencing of the genes and flanking regions revealed the discrepancy was due to a 26 bp deletion at the 5' end of the K strain sequence that included the initiation codon . Expression studies confirmed that the K strain gene was untranslated . These data indicate that the cybC gene product is non-essential in E . coli. Mol Cell Biochem, 1993 Jun 9-23, 123(1-2), 39 - 44 Identification of high affinity membrane-bound fatty acid-binding proteins using a photoreactive fatty acid; Gerber GE et al.; A photoaffinity labeling method was developed to identify and characterize high affinity fatty acid-binding proteins in membranes . The specific labeling of these sites requires the use of low concentrations (nanomolar) of the photoreactive fatty acid 11-m-diazirinophenoxy-{11-3H}undecanoate . It was delivered as a bovine serum albumin (BSA) complex which serves as a reservoir for fatty acid and thus allows precise control of unbound fatty acid concentrations . The fadL protein of E . coli, which is required for fatty acid permeation of its outer membrane, was labeled by the photoreactive fatty acid neither specifically nor saturably when the probe was added in the absence of BSA; however when a nanomolar concentration of the uncomplexed probe was maintained in the presence of BSA, the labeling of the fadL protein was highly specific and saturable . This photoaffinity labeling method was also used to characterize a 22 kDa, high affinity fatty acid-binding protein which we have recently identified in the plasma membrane of 3T3-L1 adipocytes . This protein bound the probe with a Kd of 216 nM . The approach described is easily capable of identifying membrane-bound fatty acid-binding proteins and can distinguish between those of high and low affinities for fatty acids . It represents a general method for the identification and characterization of fatty acid-binding proteins. Mol Cell Biochem, 1993 Jun 9-23, 123(1-2), 3 - 13 High resolution X-ray studies of mammalian intestinal and muscle fatty acid-binding proteins provide an opportunity for defining the chemical nature of fatty acid: protein interactions; Scapin G et al.; The structure of E . coli-derived rat intestinal fatty acid-binding protein has recently been refined to 1.2 A without bound fatty acid and to 2.0 A and 1.75 A with bound hexadecanoate (palmitate) and 9Z-octadecenoate (oleate), respectively . The structure of E . coli-derived human muscle fatty acid-binding protein has also been solved to 2.1 A with a C16 bacterial fatty acid . Both proteins contain 10 anti-parallel beta-strands in a +1, +1, +1.. . motif . The strands are arranged in two beta-pleated sheets that are orthogonally oriented . In each case, the fatty acid is enclosed by the beta-sheets and is bound to the proteins by feeble forces . These feeble forces consist of (i) a hydrogen bonding network between the fatty acid's carboxylate group, ordered solvent, and side chains of polar/ionizable amino acid residues; (ii) van der Waals contacts between the methylene chain of the fatty acid and the side chain atoms of hydrophobic and aromatic residues; (iii) van der Waals interactions between the omega-terminal methyl and the component methenyls of the phenyl side chain of a Phe which serves as an adjustable terminal sensor situated over a surface opening or portal connecting interior and exterior solvent; and (iv) van der Waals contacts between methylenes of the alkyl chain and oxygens of ordered waters that have been located inside the binding cavity . These waters are positioned over one face of the ligand and are held in place by hydrogen bonding with one another and with the side chains of protein's polar and ionizable residues . Binding of the fatty acid ligand is associated with minimal adjustments of the positions of main chain or side chain atoms . However, acquisition of ligand is associated with removal of ordered interior solvent suggesting that the free energy of dehydration of the binding site may be as important for the energy of the binding reaction as the free energy of stabilization of the fatty acid: protein complex. Biochemistry, 1993 Jun 8, 32(22), 5913 - 6 Phosphoryl transfer between phosphorylated histidine-containing protein and histidine-containing protein is not autocatalytic; Anderson JW et al.; Histidine-containing protein, HPr, is a phosphocarrier protein that is part of the bacterial phosphoenolpyruvate:sugar phosphotransferase system . HPr is phosphorylated by enzyme I, and P-HPr transfers the phosphoryl group to the IIA domain of a number of sugar-specific enzyme II complexes . Autocatalytic phosphoryl transfer between P-HPr and HPr has recently been reported {van Dijk, A . A., Eisermann, R., Hengstenberg, W., & Robillard, G . T . (1991) Biochemistry 30, 2876-2882} . Our results show that this phosphoryl transfer is due to an unidentified contaminant of HPr preparations . The phosphoryl transfer activity is not present in all HPr preparations . When present, the phosphoryl transfer activity can be removed by further purification or destroyed