Search

We have previously reported that the catalytic RNA subunit of RNase P of Escherichia coli (M1 RNA) cleaves Drosophila initiator methionine tRNA (tRNA[sup Met][sub i]) within the mature tRNA sequence to produce specific fragments. This cleavage was dependent on the occurrence of an altered conformation of the tRNA substrate. We call this further cleavage hyperprocessing. In the present paper, to search for another tRNA that can be hyperprocessed in vitro, we used total mature tRNAs from Drosophila as substrates for the in vitro M1 RNA reaction. We found that some tRNAs can be hyperprocessed by M1 RNA and that two such tRNAs are an alanine tRNA and a histidine tRNA. Using mutant substrates of these tRNAs, we also show that the hyperprocessing by M1 RNA is dependent on the occurrence of altered conformations of these tRNAs. The altered conformations were very similar to that of tRNA[sup Met][sub i]. We show here that M1 RNA can be used as a powerful tool to detect the alternative conformation of tRNAs. The relationship between these hyperprocessing reactions and stability of the tRNA structure will also be discussed. [ABSTRACT FROM AUTHOR]

Exogenously supplied thymine is only poorly utilized by wild-type cells of Escherichia coli for the synthesis of their DNA. It appears that the lack of incorporation of exogenous thymine is due to a lack of endogenous deoxyribosyl groups, which are required for the synthesis of thymidine. Data to be presented in this paper indicate that the ability of different thymine auxotrophs to grow on progressively lower thymine concentrations is a function of their capacity to increase the internal pool of deoxyribose 1-phosphate and/or the level of thymidine phosphorylase. Thymine incorporation in wild-type cells could be observed if the cells possessed an increased deoxyribose 1-phosphate pool. The kinetics of thymine uptake in wild-type cells, in a mutant lacking thymidine phosphorylase and in thymine auxotrophs show that the initial rates of thymine uptake are almost identical and proportional to the external thymine concentration. The experiments in viva led us to conclude that the incorporation of exogenous thymine occurs via thymidine, which is synthesized from thymine and deoxyribose 1-phosphate, catalyzed by thymidine phosphorylase. In accordance with this studies in vitro with purified thymidine phosphorylase showed that thymidine is synthesized from deoxyribose 1-phosphate and thymine rather than through a deoxyribose transfer from a deoxynucleoside. [ABSTRACT FROM AUTHOR]

In this paper we show, that the in vivo methylation of the elongation factor Tu from Escherichia coli is correlated with the growth phase of the bacterium. Methylation occurs at one position only, i.e. Lys-56, and initially results in monomethylation during logarithmic growth. Upon entering the stationary phase of E. coli, monomethyllysine is gradually converted into dimethyllysine. We have undertaken an extensive comparison between the properties of the highly methylated EF-Tu and unmodified EF-Tu. No gross conformational differences, as measured by the rate of mild tryptic cleavage, were observed. The dissociation rates of the nucleotides GDP and GTP appear likewise to be unaffected by the methylation, just as is the stimulatory effect of the elongation factor Ts upon these rates. Whereas tRNA binding at the classical binding site of EF-Tu (site I) also appears not to be affected by the methylation of the protein, tRNA binding at site II ts. Although the apparent affinity of tRNA for site U remains unaltered upon methylation of EF-Tu, the conformational effects of tRNA binding at this site become different. Both the GTPase activity of the protein and the reactivity of Cys-81 are significantly less stimulated by the tRNA when EF-Tu is methylated. A possible physiological implication of this phenomenon is discussed. [ABSTRACT FROM AUTHOR]

Penicillin-binding protein (pbp) 1b, the main DD-transpeptidase/transglycosylase of Escherichia coli, is normally present in the cell in three molecular forms α, β and γ, differenciated by their mobility in sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The three molecular forms are enzymatically active in vitro and their relative amounts are kept fairly constant in most labelling experiments with radioactive β-lactam antibiotics. In this paper, we have analyzed the expression of ponB (mrcB), the structural gone for pbp 1b, and the relation among the three forms of pbp 1b in ponB strains lysogenyzed by λ540 (ponB+) recombinant bacteriophages. Our data indicate that ponB is transcribed anti-clockwise on the E. coli chromosome and suggest that pbp 1bx is the first membrane-bound form of pbp 1b able to bind labelled β-lactams, and is the precursor of pbp 1bβ which is, in turn, the precursor of pbp 1bγ. [ABSTRACT FROM AUTHOR]

The mannan-synthesizing system of Escherichia coli O9 was studied with membranes of the rfe- mutant 1357, which also lacks phosphomannose isomerase. These membranes did not incorporate mannose from GDP-mannose. Their reconstitution with UDP-glucose/magnesium chloride was studied under different conditions. Simultaneous incubation of rfe- membranes with 10 mM UDP-glucose, 20 mM magnesium chloride and 5 µM GDP-[14C]mannose resulted in the formation of a glycolipid, the carbohydrate moiety of which was characterized as mannosyl-(1→3)-mannosyl-glucose. This glycolipid was not a mannose acceptor when used in membrane reconstitution experiments. Sequential incubation of rfe- membranes first with 10 mM UDP-glucose/20 mM magnesium chloride and then with GDP-[14C]mannose gave rise to the radioactive mannan of E. coli O9. After the incubation of rfe- membranes with 10 mM UDP-glucose/20 mM magnesium chloride in the absence of GDP-mannose, a glucolipid was extracted with butanol and purified by ion-exchange chromatography on DEAE-cellulose. In reconstitution experiments this glucose-containing lipid functions as a mannose acceptor. It is characterized in the accompanying paper [Weisgerber, C. and Jann, K. (1982) Eur. J. Biochem. 127, 165-168] as α-glucosyldiphosphoundecaprenol. The formation of this glucolipid could be reversed with [14C]UMP using rfe- membranes as enzyme source. In the reverse reaction [14C]UDP-glucose was formed in about 20% yield. When rfe- membranes were incubated with 2 µM UDP-[14C]glucose and 20 mM magnesium chloride, a lipid containing [14C]glucose was formed, although no stimulation of mannose incorporation could be observed at this UDP-glucose concentration. The glucose is bound to a lipid moiety (presumably undecaprenol) via a phosphodiester bridge. On the basis of these results the mechanism of the biosynthesis of the E. coli O9 antigen is discussed. We propose that in the initial reversible reaction α-glucosyldiphosphoundecaprenol is formed by the transfer of α-glucosyl 1-phosphate from UDP-glucose to undecaprenol phosphate. The product functions as mannose acceptor in the subsequent sequential transfer of mannose from GDP-mannose directly. The mannan, together with the glucose unit, is finally translocated from undecaprenol diphosphate to core lipid A, with the formation of the complete lipopolysaccharide (O9 antigen). [ABSTRACT FROM AUTHOR]

Considering enzymatic activities found in bacteria and in animal cells, there are two possible mechanisms for repair of N-methylated purines produced by methylating agents such as the mutagen and carcinogen N-methyl-N′-nitro-N-nitrosoguanidine. Both mechanisms involve first an enzymatic removal of the methylated bases by glycosylases. The resulting apurinic sites could then be repaired by (a) direct insertion of the correct bases purine insertases or (b) opening of the polynucleotide chain by apurinic endonuclease followed by repair synthesis. As the methods commonly used to detect lesions induced by methylating agents involve alkali, it was thus far not possible to decide between the above possibilities because apurinic sites are by themselves alkali labile. In this paper I describe two methods which avoid alkali and therefore allow the clarification of some aspects of the repair reaction. One of these methods makes use of 95% formamide at 40 °C in place of alkali to denature DNA with pre-existing single-strand breaks, the other measures the capacity of DNA scissions with free 3′-OH groups to act as primer for Escherichia coli DNA polymerase I. Results obtained with both methods make it unlikely that purine insertases play a major role in the repair of apurinic sites. Kinetics of production and repair of single-strand breaks, produced in 3T6 mouse fibroblasts by incubation with N-methyl-N′-nitro-N-nitrosoguanidine, were also examined using the methods of alkaline elution and alkaline sucrose gradient centrifugation. [ABSTRACT FROM AUTHOR]

A total rate equation was used to calculate the discrimination of valine by the isoleucyl-tRNA synthetase from Escherichia coli. The PPi present in the cell makes the backward reaction or the pyrophosphorolysis of the E · aa-AMP possible. If the E · Ile-AMP has been corrected for wrong aminoacyl adenylation by the pretransfer proofreading, the pyrophosphorolysis rapidly equilibrates the corrected E · Ile-AMP with E · Ile and thus spoils the effect of the proofreading. The loss of the corrected species is avoided if there is a barrier (perhaps conformational) formed by a slow reaction step between the noncorrected E · Ile-AMP and the corrected *EtRNAIle-AMP. If such a slow conformational change exists, the increase in accuracy from the pretransfer proofreading would be beneficial, and, in addition, the PPi increases the accuracy by optimizing the initial discrimination of the wrong amino acid. [ABSTRACT FROM AUTHOR]

The activity of the Escherichia coli outer-membrane phospholipase (OM PLA) is strictly regulated in its natural habitat, the E. coli outer membrane. OM PLA can be reconstituted in phospholipid bilayers, resulting in low specific activity of the enzyme compared to its activity on mixed lipid/detergent micelles. The enzyme can be activated by the addition to these vesicles of the membrane-perturbing peptides polymyxin B, melittin or cardiotoxin resulting in hydrolysis of mainly the sn-1 ester bond of the phospholipids as is also observed in vivo. We used the affinity label hexadecanesulfonyl fluoride to probe the influence of lipid environment on the activity of OM PLA. In detergent and substrate micelles, the rate constant for the sulfonylation of the active-center serine of the purified OM PLA by the affinity label hexadecanesulfonyl fluoride depends on amphiphile concentration. We have reported a similar influence of amphiphile concentration on the activity of the enzyme [Horrevoets, A. J. G. et al. (1989) Biochemistry 28, 1139-1147]. Analysis of the rates of inactivation of OM PLA by hexadecanesulfonyl fluoride in vesicles composed of various phospholipids indicated that activation of the enzyme by membrane-perturbing peptides can be accurately quantified with this affinity label. Our results show that the affinity label hexadecanesulfonyl fluoride can be used to monitor the state of activation of OM PLA in different lipid environments, including non-hydrolyzable substrate analogues. Implications for the in vivo situation are discussed. [ABSTRACT FROM AUTHOR]

The citrate-transport determinant of plasmid pES1 from Klebsiella pneumoniae [Schwarz, E. & Oesterhelt, D. (1985) EMBO J. 4, 1599 – 1603] has been subcloned in Escherichia coli DH1. Uptake of citrate in E. coli membrane vesicles via this uptake system is an electrogenic process, although the pH gradient is the main driving force for citrate uptake. The rate of citrate uptake, driven by artificially imposed ion-gradients, is high in the presence of an artificial ΔpH and low in the presence of an artificial ΔΨ. Citrate transport does not depend on the presence of Na+ or Mg2+ as has been observed for other citrate-transport systems. Citrate has three pK values: 3.14, 4.77 and 5.40. Citrate forms a stable complex with Mg2+ with a stability constant of 3.2. Kinetic parameters and calculations of the different citrate (Cit) species at a given pH, indicate that the HCit2- is the species transported and that transport occurs in symport with three protons. This citrate-transport system is thus a unique example of a 3H+/solute symport system. [ABSTRACT FROM AUTHOR]

The reaction of isoleucyl-tRNA synthetase from Escherichia coli B was analysed by deriving total steady-state rate equations for the ATP/PP1 exchange reaction and for the aminoacylation of tRNA, and by fitting these rate equations to series of experimental results. The analysis suggests that (a) a Mg2+ inhibits the aminoacylation of tRNA but not the activation of the amino acid. In the chosen mechanism, this enzyme-bound Mg2+ is required at the activation step. (b) Another Mg2+ is required at ATP, but the MgATP apparently can be replaced by the spermidine · ATP complex. Spermidine · ATP is a weaker substrate. The role of spermidine · ATP is especially suggested by the relative rates of the aminoacylation of tRNA when the spermidine and magnesium concentrations are varied. The aminoacylation measurements still suggest that (c) two (or more) Mg2+ are bound to the tRNA molecule and are required for enzyme activity at the transfer step, and that these Mg2+ can be replaced by spermidines. [ABSTRACT FROM AUTHOR]

A resistance gene, carB, originally isolated from the carbomycin-producing organism. Streptomyces thermotolerans, confers on Streptomyces lividans high-level resistance to the drug. However, ribosomes from S. lividans expressing carB show only moderate resistance to this macrolide in vitro, although they are highly resistant to the action of lincosamide antibiotics. The carB product monomethylates the amino group of the adenosine residue located at position 2058 in 23S ribosomal RNA. In contrast, ribosomes from S. lividans expressing ermE, in which 23S RNA is dimethylated at this same position, are much more highly resistant to macrolides and insensitive to lincosamides. [ABSTRACT FROM AUTHOR]

The chemical structure of the O-antigen of a proposed new provisional serotype of Shigella flexneri has been determined. Methylation analysis, GLC-MS, ¹H-NMR and 13C-NMR showed that the linear O-antigenic polysaccharide is the same as for all S. flexneri [Kenae, L., Lindberg, B., Petersson, K. & Romanowska, E. (1977) Carbohydr. Res. 56, 363-370]. A novel structural feature is that the disaccharide α-D-Glcp-(1→2)-α-D-Glcp is linked to O4 of the N-acetyl-glucosamine residue. →2)-α-L-Rhap-(1 → 2)-α-L-Rhap-(1 → 3)-α-1-Rhap-(1 → 3)--β-D-GlcpNAc--(1 → ¦(4 ↑ 1) ¦ α-D-Glcp ¦ (2 ↑ 1) α-D-Glcp. Western blotting of the lipopolysaccharide with an E. coli R3 core-specific monoclonal antibody, suggested the presence of an E. coli R3 core. [ABSTRACT FROM AUTHOR]

The inhibitory effects of hexasodium sym-bis(m-aminobenzoyl-m-amino-p-methylbenzoyl-1-naphthylamino-4,6,8-trisulfonate)carbamide (trivial name: suramin) on the activities of various deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) polymerases from mammalian cells, bacteria and retrovirus were examined and compared with each other. Among the various DNA and RNA polymerases tested, the activities of DNA primase, DNA polymerase α, reverse transcriptase and Escherichia coli RNA polymerase were strongly inhibited by suramin, while the activities of other enzymes including DNA polymerases β and γ, terminal deoxynucleotidyl-transferase and DNA polymerase I were relatively resistant to inhibition by this drug. The inhibition by suramin of DNA polymerase α from KB cells and Rauscher murine leukemia virus (RLV) reverse transcriptase was due to competition with the respective template, primer (activated DNA for α polymerase and (rA)n · (dT)12-18 for reverse transcriptase) for the template · primer-binding site of the enzyme, while the inhibition of DNA primase and E. coli RNA polymerase was due to competition with the ribonucleoside triphosphate substrate. The inhibition constants (Ki) of suramin were determined to be 2.6 µM, 0.35 µM, 0.54 µM and 0.70 µM for DNA primase, DNA polymerase α, RLV reverse transcriptase and E. coli RNA polymerase respectively. The observed inhibitions of these polynucleotide-synthesizing enzymes by suramin seem to explain, at least in part, an as yet unknown mechanism of trypanocidal action of this drug. [ABSTRACT FROM AUTHOR]

Amidation of the 5′-phosphate group of the heptanucleotide pdApdApdApdTpdCpdGprC and of its derivatives of the general formula (pdN)npdGprC (n = 0-5) with imidazole, N-methylimidazole, and 4-dimethylaminopyridine afforded a series of phosphorylating affinity reagents. The parent oligonucleotides of this series complementary to promoter A2 of T7 phage over the region (-5 to + 2) are known to be efficient primers of the synthesis of RNA by Escherichia coli RNA polymerase with promoter A2 as template. Treatment of the complex RNA-polymerase - promoter-A2 with affinity reagents followed by addition of [α- 32P]UTP resulted in labelling of RNA polymerase by the residues -(pdN)npdGprCU (p = radioactive phosphate). This affinity labelling was highly selective because elongation of the covalently bound residues (pdN)npdGprC by prU residues was catalyzed by the active center of RNA polymerase. The most efficient reagents were N-methylimidazolides. A dramatic change of the pattern of labelling of the subunits β, β', and σ took place with changing n. Maximum labelling of the β subunit occurred at n = 1 and of the σ subunit at n = 5. The targets in both the subunits were His residues. The α subunit was not specifically labelled. [ABSTRACT FROM AUTHOR]

The expression of the enzymatically active 6-hydroxy-D-nicotine oxidase (6-HDNO) from Arthrobacter oxidans requires the covalent attachment of FAD to the polypeptide chain. How this modification takes place and at what time during the synthesis of the polypeptide is not known. We investigated the possibility of cotranslational flavinylation by generating various deletions of the 6-HDNO gene carried on appropriate plasmid vectors. The polypeptides expressed from these plasmids were analyzed for their ability to incorporate [14C]FAD covalently in an Escherichia coli-derived coupled transcription/translation system. The data show that removal of approximately 40% from the carboxy-terminal part of the 6-HDNO polypeptide did not inhibit the covalent flavinylation of the truncated protein. A fusion protein, consisting of the truncated 6-HDNO polypeptide and the β-lactamase of pBR322, was also covalently flavinylated. The amino acid sequence surrounding the histidine residue, assumed to bind FAD, was shown to be situated approximately 70 amino acid residues from the amino-terminal end of the 6-HDNO polypeptide. Removal of the first 30 amino acids did not abolish covalent flavinylation. Flavinylation could no longer be detected, however, if a short amino acid sequence, consisting of seven residues, replaced the amino acid sequence upstream of the histidine. These findings prove, in our opinion, that cotranslational flavinylation takes place in the synthesis of 6-HDNO. [ABSTRACT FROM AUTHOR]

The isolation of bacterially synthesized, recombinant-DNA-derived, bovine growth hormone (r-bGH) with native structure is described. The r-bGH is found in insoluble form, in a pellet fraction, after cell breakage and centrifugation. Cell envelope components (protein, lipid, endotoxin) and nucleic acids are selectively removed from the pellet fraction by an EDTA/lysozyme/deoxycholate extraction. We demonstrate that the r-bGH is largely reduced until solubilized using 6 M guanidine/HCl. Air oxidation is then carried out, in the presence of the guanidine/HCl. The oxidation results in a mixture of about one-third disulfide-linked oligomers and two-thirds oxidized monomer. The latter may include some incorrectly oxidized material, but appears to be mostly correctly oxidized. The oxidized monomer is isolated by gel filtration in the presence of guanidine/HCl. Subsequent guanidine/HCl removal leads to refolded, oxidized r-bGH. All steps in the procedure, in particular the oxidation and refolding steps, can be carried out at relatively high protein concentrations. [ABSTRACT FROM AUTHOR]

The amino acid sequences of mutationally altered ribosomal protein L7/L12 from four different rplL mutants of Escherichia coli were determined and correlated with some features of the mutant ribosomes. Two of the rplL mutations are deletions around position 40, which give rise to a shortened hinge region between the two domains of L7/L12. The other two mutants harbor point mutations at position 74 (Gly→Asp) or at position 82 (Glu→Lys), which are in or close to an evolutionarily conserved sequence in the C-terminal domain. The two latter mutations are associated with decreased rates of growth and translational elongation. All four mutants show increased nonsense codon read-through in vivo. Ribosomes from one of the deletion mutants show clearly increased missense error rates in vitro. [ABSTRACT FROM AUTHOR]

High-resolution proton nuclear magnetic resonance spectra of the trp repressor of Escherichia coli under various conditions are reported and analysed .The spectrum of the denatured state agrees with that predicted from the amino acid composition, with the exception of the two histidine residues, which have different chemical shifts although they titrate normally. The spectrum of the native protein shows the presence of extensive secondary and tertiary structure. Using information from chemical shifts, numbers of protons, titration behaviour, homonuclear chemical-shift-correlated spectroscopy and nuclear Overhauser enhancement correlated spectroscopy, most of the aromatic protons have been assigned to residue type. Further, about 30% of the aliphatic protons have been assigned to residue type by two-dimensional spectroscopy. Nuclear Overhauser enhancements establish that high field methyl groups belonging to a valine residue lie directly over an aromatic ring. [ABSTRACT FROM AUTHOR]

The chemical structure of the K52 antigenic capsular polysaccharide (K 52 antigen) of Escherichia coli O4:K 52: H- was elucidated by composition, nuclear magnetic resonance spectroscopy, methylation, periodate oxidation before and after graded acid hydrolysis and by oligosaccharide analysis. The polysaccharide consists of a backbone of α-galactose units interlinked between C1 and C3 by phosphodiester bridges. This poly(α-galactosyl-phosphate) is substituted at C2 of each galactose unit by β-fructofuranose residues. About 80% of the galactose units are O-acetylated at C4 and about 10% of the fructose units are both O-acetylated and O-propionylated at C1. The K 52 polysaccharide has an average molecular mass of 34 kDa, thus consisting of approximately 65 fructosyl-galactosyl-phosphate repeating units. [ABSTRACT FROM AUTHOR]

The arginine-ornithine periplasmic binding protein, an essential component of the arginine-ornithine transport system of Escherichia coli was isolated in a phosphorylated form and in a non-phosphorylated form from the periplasmic fluid, after incubation of intact cells with (32P)orthophosphate under conditions similar to those used for arginine transport studies. The binding protein could also be labeled with 32Pi by incubation in vitro of the periplasmic fluid with [γ-32P]ATP or by incubation in vitro of the purified binding protein, with radioactive ATP, Mg2+ and a phosphokinase enzyme released by osmotic-shock treatment. The two forms of the protein were separated by DEAE-Sephacel chromatography. By several different criteria, which included binding studies, analyses of the amino acid composition of the forms of the protein, analysis by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and testing for other components of the periplasmic space with affinities for inorganic phosphate, it was concluded that the 32P. labeled protein corresponds to a phosphorylated form of the arginine-ornithine-binding protein. The phosphorylation reaction required Mg2+ and a phosphokinase from the periplasmic fluid. The dissociation constant of the phosphorylated protein for arginine was 5.0 μM (dissociation constant of the unmodified protein equals 0.1 μM), suggesting that the chemically modified protein is the active form of the molecule which releases the ligand for its translocation through the cytoplasmic membrane. The pH-stability profile of the phosphoprotein has a ‘U’-shape characteristic Of acyl phosphates. Reaction of the phosphorylated binding protein with hydroxylamine at pH 5.4, also released Pi from the phosphoprotein. These properties suggest that the phosphoryl group of the phosphoprotein is linked covalently to a carboxyl transaction of the protein. This information indicates that ATP is a direct energy donor for the active transport of arginine and ornithine in E. coli, and a step of phosphorylation of the arginine-ornithine-binding protein appears to be involved in the utilization of the phosphate bond energy by the arginine-ornithine transport system. [ABSTRACT FROM AUTHOR]

The accuracy of Qβ translation by Escherichia coli extracts in polymix and a conventional Tris/Mg2+ system has been followed. Misinsertions of histidine and of tryptophan into the phage coat protein were less frequent in polymix than in Tris/Mg2+, as were errors leading to u change in the coat protein pI. Even the lowest Qβ error rates, however, were still an order of magnitude greater than those for poly(U) or poly(U-G) translation. Comparing Qβ translational errors made in vitro to those found in whole cells, histidine misinsertions were almost twice as frequent, errors leading to a coat protein charge change six times more frequent and tryptophan misinsertions at least 15 times more frequent in vitro. The relation of these findings to measurements of translational accuracy and to factors affecting fidelity is discussed. [ABSTRACT FROM AUTHOR]

1. Isocitrate dehydrogenase kinase and isocitrate dehydrogenase phosphatase were purified over 1000-fold from Escherichia coli ML308 by a procedure involving fractionation with (NH4)2SO4 and chromatography on DEAEcellulose, blue-dextran-Sepharose and Sephadex G150. 2. The kinase and phosphatase activities copurified, in agreement with the observation [Laporte, D. C. and Koshland, D. E. (1982) Nature (Lond.) 300, 458-460] that a single protein bears both activities. 3. Isocitrate dehydrogenase kinase catalysed the phosphorylation of homogeneous active isocitrate dehydrogenase with a stoichiometry of just under one phosphate group incorporated per subunit. This almost completely inactivated the dehydrogenase. There was a good correlation between phosphorylation and inactivation. 4. Analysis of a partial acid hydrolysate of phosphorylated isocitrate dehydrogenase showed that the only phosphoamino acid present was phosphoserine. 5. Isocitrate dehydrogenase phosphatase catalysed the release of 32P from 32P-phosphorylated isocitrate dehydrogenase; it required either ADP or ATP for activity. In the presence of ADP, or ATP plus an inhibitor of the kinase, the phosphatase catalysed full reactivation of isocitrate dehydrogenase and there was n good correlation between reactivation and the release of phosphate. In the presence of ATP alone the phosphatase catalysed the release of 32P from phosphorylated isocitrate dehydrogenase but the activity of the dehydrogenase remained low, indicating that the kinase and phosphatase were active simultaneously in these conditions. 6. The active and inactive forms of isocitrate dehydrogenase can be resolved by non-denaturing gel electrophoresis; the two forms of the enzyme were interconverted by phosphorylation and dephosphorylation in vitro. The extent of the interconversion correlated well with the changes in isocitrate dehydrogenase activity. [ABSTRACT FROM AUTHOR]

The nucleotide sequence of a 3180-base-pair segment of DNA, containing the sucA gene encoding the 2-oxoglutarate dehydrogenase component (Elo) of the 2-oxoglutarate dehydrogenase complex of Escherichia coli, has been determined by the dideoxy chain-termination method. The sucA structural gene contains 2796 base pairs 1932 codons, excluding the initiation codon AUG) and encodes a polypeptide having a glutamine residue at the amino terminus, a glutamate residue at the carboxy-terminus and a calculated Mr = 104905. The predicted amino acid composition is in good agreement with published information obtained by hydrolysis of the purified enzyme. There is a striking lack of sequence homology between the 2-oxoglutarate dehydrogenase (E1o) and the corresponding pyruvate dehydrogenase (E1p) which suggests that the two components are not closely related in evolutionary terms. The location and polarity of the sucA gene, relative to the restriction map of the corresponding segment of DNA, are consistent with it being the proximal gene of the suc operon, as defined in previous genetic and post-infection labelling studies, but it could also form part of a more complex regulatory unit. The sucA gene is preceded by a segment of DNA thai contains many substantial regions of hyphenated dyad symmetry including an IS-like sequence of the type that is thought to function as an intercistronic regulatory element. This segment also contains three putative RNA polymerase binding sites and a good ribosome binding site. [ABSTRACT FROM AUTHOR]

The problem of the coordination between cyclic events in the DNA assembly line and the cell envelope assembly tine was approached with the technique of synchronized cultures. Escherichia co/i strains ML 30, K12 3300, K12 PC2, K12 BB2014 and B/rF were synchronized by repeated cycles of mass doubling followed by short phosphate starvation periods. Steady-state balanced growth was obtained by subsequent incubation in non-limiting growth conditions for one or more generation times. Several successive cell cycles were monitored for mass increase and cell number, while the rate of DNA synthesis and the rate of phospholipid synthesis were usually measured with more than one method. In all strains, and in strain ML 30 in five different growth media giving doubling times from 20-110 mm, a discontinuity in the rate of synthesis of phosphatidylethanolamine and of phosphatidylglycerol was observed. These two major phospholipid components of inner and outer membranes were synthesized at a constant rate per cell for a large portion of the cell cycle and the rate of synthesis of both increased twofold at the same time. This cyclic program was reproducible not only in successive cell cycles, but also in separate experiments with the same strain, in the same medium. In contrast, differences in timing were observed with different strains, and in the same strain with different carbon sources. In particular, the simultaneity of the increase in phospholipid synthesis either with DNA initiation or with cell division could not be observed as a rule. [ABSTRACT FROM AUTHOR]

Three dimeric glutamyl-tRNA synthetases (GluRS) were isolated from extracts of quiescent wheat germ and wheat chloroplasts. 1. The chloroplast enzyme (Mr = 110000), called GluRS C, exhibits a prokaryotic (Escherichia coli) tRNA specificity. 2. Two enzymes were found in the quiescent germ and were separated on phosphocellulose P11: one called GluRS P, probably the mitochondrial enzyme, has the same tRNA specificity as GluRS C; the other, called GIuRS E, has eukaryotic (wheat germ) tRHA specificity. Both enzymes exhibit a molecular weight close to 160000. 3. Each of these enzymes co-eluate on hydroxyapatite and phosphocellulose chromatographies with an unstab|e active monomer whose molecular weight is approximately half that of the corresponding dimer. Two assumptions are discussed about these monomers. [ABSTRACT FROM AUTHOR]

The O-specific polysaccharide of the 0114 antigen (lipopolysaccharide) of Escherichia coli 0114 and oligosaccharides obtained from it by Smith degradation and hydrogen fluoride solvolysis were analyzed, using proton and 13C nuclear magnetic resonance spectroscopy and methylation. The results indicated that the 0114 polysaccharide has the tetrasaccharide repeating unit α-N-acetylglucosamine(1→4)β-3, 6-dideoxy-3-(N-acetylL-seryl)aminoglucose(1→3)β-ribofuranose(1→4)galactose. In the polysaccharide the repeating units are joined through β1→3-galactosyl linkages. This structure is compared with that of the serologically cross-reacting Shigella boydii 08 antigen and the serological similarity is discussed. [ABSTRACT FROM AUTHOR]

The primary structure of the K12 antigenic capsular polysaccharide (K12 antigen) of Escherichia coli O4:K12:H- was elucidated by composition, nuclear magnetic resonance spectroscopy, methylation, periodate oxidation and oligosaccharide analysis. The polysaccharide consists of repeating trisaccharide α-rhamnosyl-1.2-α-rhamnosyl-1.5-dOclA units (dOclA=2-keto-3-deoxy-D-manno-octonic acid) which are joined through β-2,3-linkages. About 50% of the dOclA units are O-acetylated at C7 or C8. The sequence of acetylated and non-acetylated dOclA resides is not known. As had been reported before, the polysaccharide is linked to a phosphatidic acid at the reducing end (dOclA) via a phosphodiester bridge. The serologically specific part of the K12 antigen is its polysaccharide moiety. [ABSTRACT FROM AUTHOR]

Upon incubation of the envelope fraction of Escherichia coli a precursor of the major outer membrane lipoprotein that accumulates in the cytoplasmic membrane of the globomycin-treated cell is processed to the mature form [Hussain. M., Ichihara, S., and Mizushima, S. (1980) J. Biol. Chem. 255, 3707–3712; (1982) J. Biol. Chem. 257, 5177–5182], When this precursor-containing envelope fraction was incubated in the presence of protease inhibitors such as antipain, leupeptin, chymostatin and elastatinal, a new peptide appeared on a polyacrylamide gel at the position where the signal peptide was expected to appear. This was proved to be the signal peptide of the lipoprotein from the following facts: (a) its appearance is in proportion to the appearance of the lipoprotein and disappearance of the precursor; (b) when the cleavage of the signal peptide from the precursor was inhibited by globomycin, the peptide did not appear on the gel: and (c) the results of labeling of the peptide with [3H]leucine, [35S]methionine and [3H]arginine were consistent with the amino acid composition of the signal peptide. The signal peptide thus accumulated in the envelope fraction was hydrolyzed by an enzyme named ‘signal peptide peptidase’ when the envelope fraction was washed to remove the inhibitors. The hydrolysis was inhibited by re-addition of these inhibitors. The signal peptide peptidase hydrolyzed the signal peptide only after its cleavage from the lipoprotein precursor. [ABSTRACT FROM AUTHOR]

A setisitive fluorimetric assay was developed for bacterial aminopeptidase P, based on intramolecularly quenched fluorogenic substrates. Two substrates were synthesized, Phe(NO2)-Pro-HN-CH2-CH2-NH-ABz (substrate I) and Phe(NO2)-Pro-Pro-HN-CH2-CH2-NH-ABz (substrate II), in which the Phe(NO2) group (p-nitro- L-phenylalanyl) quenches the fluorescence of the ABz group (o-aminobenzoyl). Both substrates were readily cleaved by aminopeptidase P from Escherichia coli, releasing p-nitro-L-phenylalanine and causing a proportional increase in fluorescence. Complete hydrolysis of the two substrates resulted in a 7.5-fold and 3.4-fold fluorescence increase, respectively. Applying this fluorogenic assay, we were able to detect and measure quantitatively aminopeptidase P-like activity in the human serum and calf-lung extracts. Substrate II was shown to be specifically cleaved by aminopeptidase P in these preparations, while substrate I was apparently cleaved by other enzymes as well. In both preparations, the enzyme activity was independent of Co2+ ions, and Pro-HN-CH2-CH2-NH-ABz( Cbz) was inhibitory. The kinetic constant Km was determined as 0.35 mM and 0.28 mM for the human serum and the calf-lung enzymes respectively. The enzyme activity was only slightly dependent on pH in the range 7.0-8.4. [ABSTRACT FROM AUTHOR]

The capsular polysaccharide was isolated from Escherichia coli 010:K5:H4: it could not be obtained from a uncapsulated (K5-) mutant. It contains N-acetylglucosamine and glucuronic acid in a molar ratio of 1:1. Acid hydrolysis of the acidic polysaccharide as well as Smith degradation and degradation by deamination of the carboxyl-reduced polysaccharide suggested that the polysaccharide is composed of a disaccharide repeating unit. The data obtained by methylation analysis and nuclear magnetic resonance spectroscopy indicated that the repeating sequence of the capsular polysaccharide is the 4-β-glucuronyl-l,4-α-N-acetylglucosaminyl unit. This structure is similar to that of desulfo-heparin. [ABSTRACT FROM AUTHOR]

Partially purified enzymatic fractions from extracts of Escherichia coli B/r catalyse transfer of the isotope label from [adenine-2,8-3H]NAD+ to some bacterial proteins, as well as to hen egg-white lysozyme. The radio-active group in the modified lysozyme was identified as mono(ADP-ribose). Several bacterial proteins were labelled in vivo with 32P; the presence of the label in the form of an ADP-ribosyl group was shown in one of them. [ABSTRACT FROM AUTHOR]

Reports on the development of a procedure for the simultaneous purification of bacteriophage-T4 induced enzymes. Polynucleotide kinase; DNA ligase; RNA ligase; DNA polymerase; Bacterial cell disruption; Growth of Escherichia coli B-23 cells infected with T4amN82 phage.

The complete primary structure of elongation factor Tu from Escherichia coli has been elucidated. The protein, which is a mixture of two gene products, consists of a single polypeptide chain of 393 residues. After tryptic digestion of S-carboxymethylated protein, 50 tryptic peptides were isolated covering the complete protein chain. Their alignment was established with overlapping peptides obtained by chemical cleavage with cyanogen bromide and subsequent enzymic subdigestion with Staphylococcus aureus protease, chymotrypsin, elastase and thermolysin. Peptides were sequenced by manual dansyl-Edman and direct Edman degradation procedures. The N-terminal amino acid of EF-Tu is serine and is N-acetylated. The lysine residue at position 56, in the polypeptide chain is partly methylated. The C-terminal residue is a mixture of serine and glycine, and this was the only heterogeneity found in the EF-Tu preparation used in this study. [ABSTRACT FROM AUTHOR]

A ribonucleoprotein prepared by strong ribonuclease digestion of a complex of 16-S ribosomal RNA and proteins S4 and S20 from Escherichia coli has been characterized; its nucleotide sequence, the positions of enzyme cuts and the sequence excisions have been placed in the completed sequence of 16-S RNA. The positions and yields of enzyme cuts, and excisions of sequence, are compared with those of various ribonucleoproteins prepared with S4 or S20 alone, and with the ribonuclease-resistant S4 RNA prepared from renatured 16-S RNA in the absence of ribosomal protein. These data yield important information on the topography and organization of the 5′ third of the 16-S RNA which is selectively maintained in its native conformation by the bound proteins; they also provide criteria for testing secondary structural models of this region of 16-S RNA. [ABSTRACT FROM AUTHOR]

Methionyl-tRNA synthetase from Escherichia coli can react with periodate-treated tRNA to form a Schiff's base through the ε-amino group of a lysine within the enzymic active center and the 2′,3′-aldehyde groups created at the 3′-terminal ribose of tRNA. At alkaline pH, the Schiff's base equilibrium can be continuously and specifically displaced by reduction in situ with sodium cyanohydridoborate, which on the other hand leaves intact the reacting aldehyde groups of oxidized tRNA. The effects of temperature, pH and of reducing agent concentration on the rate and extent of reduction of the Schiff's base are analysed. Conditions are described (37°C, pH 8.0, in the presence of 1 mM cyanohydridoborate) which allowed rapid and complete conversion of the monomeric trypsin-modified methionyl-tRNA synthetase into its 1 : 1 covalent complex with tRNAfMet. [ABSTRACT FROM AUTHOR]

[32P]tRNALys, from Escherichia coli, was modified with kethoxal, in the presence and absence of the oligonucleotide codon (A)4. The presence of the codon resulted in a faster modification rate of the tRNA at three guanine sites which were identified by a diagonal fingerprint method. A large increase in the modification rate occurred at the 7-methylguanosine residue 46 (m7G-46) in the presence of the codon; weakly enhanced modification was observed at G-15 and G-57. It is concluded that the formation of a codon-anticodon complex induces, primarily, a conformationai change involving disruption of the m7G-46 from the m7G46 · G-22 · C-13 base triple. Subsequently, the guanines of G-15 and G-57, in the D and T loops, respectively, become slightly more reactive, suggesting a weak tendency for these two interacting arms to unfold. The results are interpreted in terms of an equilibrium between two main conformers and a third minor one; the possible significance of these conformers, in protein biosynthesis, is considered. [ABSTRACT FROM AUTHOR]

The effect of bacteriocin (cloacin DF13) treatment of Escherichia coli ribosomes on initiation of protein synthesis has been studied in detail. In agreement with out previous findings [Baan et al. (1976) Proc. Natl Acad. Sci. U.S.A, 73, 702–706] it is shown that 70-S initiation complexes can be formed with cloacin-treated ribosomes, but that the initiation factor IF-1 does not function properly. The following pleiotropic effects of this factor have been studied: (a) the acceleration of ribosomal subunit exchange with 70-S couples; (b) the stimulation of the IF-3-mediated dissociation of 70-S ribosomes; (c) the stimulation of 30-S initiation complex formation; (d) the enhancement of the rate of release of IF-2 from 70-S initiation complexes. The effects (a) and (b) are virtually abolished after cleavage of 16-S rRNA. The effect (d) is only partially reduced whereas effect (c) seems to be unimpaired. It is concluded that 70-S initiation complex formation with cloacin-treated ribosomes suffers from improper functioning of IF-1 in the generation of active subunits from 70-S tight couples. This is the only effect on initiation. It can he compensated for by adding more IF-3. The data provide functional evidence that 16-S rRNA is involved in ribosomal subnnit interaction. [ABSTRACT FROM AUTHOR]

Envelopes from regions of the cell which in vivo show very little, if any, murein synthesis were isolated using the minicell-producing strain P678-54. Envelopes from minicells, representing in fact cell ends, were able to synthesize murein and to carry out transpeptidation in vitro; also all four murein hydrolase activities tested, carboxypeptidase, endopeptidase, amidase and transglycosylase, were found to be present. The specific activities of the murein synthesizing and degrading enzymes in envelopes derived from cell poles and from actively growing cells were similar. The topological distribution of murein-synthesizing enzymes and of murein hydrolases over the cell envelope is discussed. [ABSTRACT FROM AUTHOR]

The ionization constants of each individual free amino group of Escherichia coli ribosomal protein L12 as it exists on the native intact 50-S subunit has been determined. All these amino groups are exposed and have pK values varying from 9,7 to 11,0, The amino terminus, on the other hand, is unreactive and therefore appears to be buried. In addition, the reactivities indicate that the aminoterminal region of residues 1-59 undergoes a pH-dependent conformational change on the 50-S subunit. [ABSTRACT FROM AUTHOR]

The NAD+:protein ADP-ribosyltransferase responsible for the bacteriophage-T4-induced modification of RNA polymerase has been purified 100-200-fold from Escherichia coli B/r infected with the phage T4. The enzyme has a molecular weight around 26 000. It does not require magnesium ions and is protected by sulfhydryl reagents, The temperature optimum is around 20 °C. The reaction is inhibited at increased ionic strength and by the reaction products nicotinamide and T4-modified RNA polymerase. The reaction is irreversible. The only reaction products formed to a significant extent in a crude extract are T4-modified α subunit and one smaller ADP-ribosylated peptide. As in vivo, the reaction is complete. Both α subunits in the RNA polymerase monomer are ADP-ribosylated in one and the same position. The product of modification in vitro appears to be identical with that of modification in vivo. ADP-ribose has been obtained by hydrolysis both of T4-modified α subunit and of the reaction product of egg-white lysozyme, NAD+ and the transferase responsible for bacteriophage-T4-induced alteration of host proteins [Rohrer, H., Zillig, W., and Mailhammer, R. (1975) Eur. J. Biochem. 60, 227-238]. The amino acid carrying the ADP-ribosyl residue appears to be arginine. T4-modified α subunit and the major species of T4-altered α subunit are identical. An NAD+:protein ADP-ribosyltransferase using E. coli proteins and egg-white lysozyme but no RNA polymerase subunit as acceptor(s) has been purified from uninfected E. coli cells. [ABSTRACT FROM AUTHOR]

A DNA-stimulated ATP-γ-phosphohydrolase of molecular weight 75 000 was purified from Escherichia coli cells. The ATPase, a globular molecule (identical probably with an ATPase described previously by Richet and Kohiyama in 1976) shows specificity for adenine nucleotides, it prefers single-stranded DNA as the cofactor, it exhibits a complicated mode of response to variations of the cofactor concentration and it is devoid of nuclease activity. Preparations derived from rep3 mutant cells yield widely varying amounts of an apparently normal ATPase. [ABSTRACT FROM AUTHOR]

1. The reaction of native chorismate mutase/prephenate dehydratase with 5,5′-dithio-bis(2nitrobenzoic acid) (Nbs2) caused a differential inactivation of the two enzymic activities. The dehydratase activity was completely inactivated by the reaction of 1.2 mol of sulphydryl per mol of enzyme subunit, while the mutase activity was inhibited only 5%. The Michaelis constant for chorismate increased about 2.5-fold while the maximum rate for the mutase activity was unaffected. Reaction of an average of two more moles of sulphydryl per mole of enzyme subunit caused a further 25% inhibition of the mutase activity. 2. Circular dichroic measurements indicated that native chorismate mutase/prephenate dehydratase has 25% α-helix and that modification of one sulphydryl group per subunit with Nbs2 caused significant changes in the secondary structure of the enzyme and in the environment of one or more tyrosine residues. 3. Prephenate is a competitive inhibitor of the mutase activity, with an inhibition constant of 0.047 ± 0.008 mM. Modification of one sulphydryl group per subunit with Nbs2 increased the value of the inhibition constant and the Michaelis constant for chorismate by the same amount. 4. At concentrations from 5-1000 µM the allosteric inhibitor phenylalanine did not markedly affect the reactivity of the most reactive sulphydryl group with Nbs2, but decreased the reactivity of the other sulphydryl groups. 5. Prephenate protected the most reactive sulphydryl group against reaction with Nbs2; phenylpyruvate had no effect. 6. It was concluded that one sulphydryl group is involved directly in the dehydratase active site. 7. The results are interpreted as favouring the theory that chorismate mutase/prephenate dehydratase has separate active sites for the two activities, although the possibility of partially overlapping sites has not been eliminated. [ABSTRACT FROM AUTHOR]

The kinetics of thymidine uptake by Escherichia coli and Bacillus subtilis cells in the presence of adenine and guanine nucleosides was investigated. The initial concentration of thymidine in the growth medium was 0.35 µg/ml while the initial concentration of purine nucleosides ranged from 25 to 250 µg/ml. Adenine nucleosides when present at a concentration more than 50 µg/ml strongly inhibit thymidine uptake by the bacteria. The duration of the inhibition depends on the initial concentration of adenine nucleoside in the growth medium. At an initial concentration of deoxyadenosine (or adenosine) of 250 µg/ml the time of inhibition of thymidine uptake was about 60 min. During this period thymidine is almost completely preserved from the action of bacterial thymidine phosphorylase. Guanine nucleosides (guanosine or deoxyguanosine) do not markedly inhibit thymidine uptake by bacteria even at a concentration of 250 µg/ml. It is shown that they do protect thymidine from the phosphorolytic action of the thymidine phosphorylase although much less effectively than adenine nucleosides. It is suggested that some areas in the bacterial membrane where thymidine phosphorylase is located are not available to guanine nucleosides. [ABSTRACT FROM AUTHOR]

The binding of tRNAIle to isoleucyl-tRNA synthetase in the presence of isoleucine or ATP was investigated using the equilibrium partition method. Isoleucine decreased the affinity of tRNAIle for the enzyme by a factor of about 5. For the free standard energy of interaction a value of about 1 kcal/mol (4.2 kJ/mol) was calculated. ATP exhibits qualitatively the same effect as isoleucine. A binding of two molecules isoleucine per molecule of enzyme could not be demonstrated even in the presence of ATP and pyrophosphatase. [ABSTRACT FROM AUTHOR]

The almost complete sequence of a region of the 23-S ribosomal RNA from Escherichia coli, 175 nucleotides in length, located between the 550th and the 1000th nucleotide at the 3′ end of 23-S RNA and containing the binding site of protein L1, is given. Possible models of secondary structure are proposed. [ABSTRACT FROM AUTHOR]

Ribonucleoproteins were obtained by T1 ribonuclease digestion of reconstituted complexes of ribosomal protein L1 and 23-S RNA from Escherichia coli. The RNA region of the main ribonucleoprotein 2 was totally digested with Tl ribonuclease. The oligonucleotide products were characterised and they showed that this region comprises 148 nucleotides located between the 550th and 1000th nucleotides from the 3′ end of the 23-S RNA. Of the other two ribonucleoproteins, the largest ribonucleoprotein 1 contained an extra RNA sequence, of at least 15 nucleotides, that was located at the 5′ end of the RNA region. The smallest ribonucleoprotein 3 lacked an RNA section towards the 3′ end of the region. The order of the RNA subfragments and the enzymic cutting positions in the whole RNA region are given for the ribonucleoproteins. It is shown that protein L1 most strongly protects a continuous section of 115 nucleotides at the 5′ end of the main RNA region. Finally, evidence is presented for a methylated base, and for two sequence heterogeneities, in this region of the 23-S RNA. [ABSTRACT FROM AUTHOR]

The NADPH-dependent enzymic reduction of disulfide bonds in human choriogonadotropin and its two subunits, α and β, was examined with thioredoxin and thioredoxin reductase from Escherichia coli. With 12 μM thioredoxin and 0.1 μM thioredoxin reductase at pH 7 all disulfide bonds in the α subunit could be reduced in 15 min. The reduction of disulfide bonds was recorded by a simple spectrophotometric assay at 340 nm, which allowed quantitation of the reduction rate and the number of disulfide bonds reduced. Partial reduction of the α subunit with thioredoxin followed by S-carboxymethylation with iodo[2-3H]acetic acid and analysis of tryptic peptides indicated that all S-S bonds in the α subunit were surface oriented and equally reactive. The usefulness of thioredoxin reduction of disulfide bonds as a chemical probe of protein structure was shown by the much slower reaction of disulfide bonds in the intact hormone as compared to its two biologically inactive subunits. [ABSTRACT FROM AUTHOR]

Amylomaltase, the inducible 4-α-glucanotransferase of Escherichia coli strain ML, has been purified to homogeneity. Its specific activity with a commercial maltose substrate was 500 mkat/kg protein (30 μmol glucose formed rain-1 mg protein-1). The purified enzyme, dependent on buffer concentration, exists in interconvertible low-molecular-weight (apparent molecular weight 71000) and high-molecular-weight (apparent molecular weight 370000) forms. The specificity of amylomaltase has been redefined. Hitherto, the enzyme was thought to be a glucosyltransferase, catalysing the transfer of single glucosyl units, and maltose has been regarded as its most important substrate. Amylomaltase is now shown to exhibit both glucosyl-transfer and 4-α-glucanosyl-transfer specificity. 4-α-Glucanosyl chains containing up to at least nine glucosyl units can be transferred. However, it is concluded that the transfer reaction by which amylomaltase action was originally expressed, does not take place, i.e., Maltose + maltose [This symbol cannot be presented in ASCII format] Maltotriose + glucose and that maltose has a restricted role as a substrate. This may be due to the inability of maltose to function as a donor substrate, serving only as an acceptor substrate. It is confirmed that when a maltodextrin serves as a donor, that portion of the molecule transfered by the enzyme is that containing the nonreducing-end-group. Enzyme action on chromatographically pure maltose is characterized by a lag phase in the time course of glucose release. The lag phase is overcome by addition of ‘priming’ (catalytic) concentrations of maltotriose or higher maltodextrins. An autocatalytic reaction mechanism involving the generation of primer molecules is proposed to explain the action of the enzyme on maltose. The redefined action pattern of amylomaltase is consistent with the redefined role of the enzyme in the utilization of exogenous and endogenous 1.4-α-glucans by E. coli. [ABSTRACT FROM AUTHOR]

Lipopolysaccharide was isolated from Chromatium vinosum by phenol/water extraction. The lipopolysaccharide is found exclusively in the phenol phase and can be cleaved into a sugar moiety and a lipid A fraction by hydrolysis in 10% acetic acid at 100 °C for 3–4 h. The sugar moiety contains the neutral sugars 3-O-methyl-D-ribose, D-ribose, L-arabinose, mannosamine and glucose, and smaller quantities of D-rhamnose, D-glycero-D-manno-heptose (tentatively identified), quinovosamine and 2-keto-3-deoxyoctonate. L-glycero-D-manno-heptose was not detected. The 2-keto-3-deoxyoctonate linkage in C. vinosum lipopolysaccharide is more resistant to acid hydrolysis than that of Escherichia coli. The lipid A fraction contains glucosamine, mannose and the fatty acids of the lipopolysaccharide. The major fatty acid is β-hydroxymyristic acid, with smaller amounts of lauric and palmitic acids as well as a 14-carbon mono-unsaturated fatty acid, also being present. The phosphorous content of the C. vinosum lipopolysaccharide was found to be approximately 0.1 %. Erythrocytes sensitized with alkali-treated C. vinosum lipopolysaccharide were agglutinated by antisera prepared against heat-killed cells. Untreated or heat-treated lipopolysaccharide did not sensitize erythrocytes. The lethal toxicity to mice of the C. vinosum lipopolysaccharide is about one-tenth as that from Salmonella abortus equi. [ABSTRACT FROM AUTHOR]

The interaction between phenylalanyl-tRNA synthetase from yeast and Escherichia coli and tRNAPhe (yeast), tRNASer (yeast), tRNAVal1 (E. coli) and tRNATyr (E. coli) has been investigated by ultracentrifugation analysis, fluorescence titrations and fast kinetic techniques. The fluorescence of the Y-base of tRNAPhe and the intrinsic fluorescence of the synthetases have been used as optical indicators. I. Specific complexes between phenylalanyl-tRNA synthetase and tRNAPhe from yeast are formed in a two-step mechanism: a nearly diffusion-controlled recombination is followed by a fast con- formational transition. Binding constants, rate constants and changes in the quantum yield of the Y-base fluorescence upon binding are given under a variety of conditions with respect to pH, added salt. concentration of Mg2+ ions and temperature. 2. Heterologous complexes between phenylalanyl-tRNA synthetase (E. coli) and tRNAPhe (yeast) are formed in a similar two-step mechanism as the specific complexes: the conformational transition, however, is slower by a factor 4–5. 3. Formation of non-specific complexes between phenylalanyl-tRNA synthetase (yeast) and tRNATyr (E. coli) proceeds in a one-step mechanism. Phenylalanyl-tRNA synthetase (yeast) binds either two molecules of tRNAPhe (yeast) or only one molecule of tRNATyr (E. coli); tRNAVal1 (E. coli) or tRNASer (yeast) are also bound in a 1:1 stoichiometry. Binding constants for complexes of phenyl- alanyl-tRNA synthetase (yeast) and tRNATyr (E. coli) are determined under a variety of conditions. In contrast to specific complex formation, non-specific binding is disfavoured by the presence of Mg2+ ions, and is not affected by pH and the presence of pyrophosphate. The difference in the stabilities of specific and non-specific complexes can be varied by a factor of 2–100 depending on the ionic conditions. Discrimination of cognate and non-cognate tRNA by phenylalanyl-tRNA synthetase (yeast) is discussed in terms of the binding mechanism, the topology of the binding sites, the nature of interacting forces and the relation between specificity and ionic conditions. [ABSTRACT FROM AUTHOR]