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This paper describes the isolation and the properties of DNA-dependent RNA polymerase from Lactobacillus curvatus. The enzyme is highly labile and shows a tendency to dissociate into fragments. Therefore. a special purification procedure also suitable for the isolation of labile RNA polymerases from other pro-karyotes was developed. Three enzyme species, E (core enzyme). Eσ (full enzyme) and Ey were obtained. The subunit composition corresponds to that of other prokaryote RNA polymerases. In contrast to Echerichia coli enzyme. the Β subunit (Mr = 145000) has a lower molecular weight than the Β subunit (Mr = 1.51000). The Β subunit was identified by its capacity to bind [³H]rifamycin. σ is extremely small (Mr = 44000). y is a peptide chain (Mr = 84000) present once in the Ey monomer. By incomplete dissociation, Β α2, Β'σ and Β'y complexes were obtained. y and 5 have never been found together in the same enzyme particle. In contrast to RNA polymerase from E. coli. Eσ from L. curvatus exhibits optimal activity in the presence of Mn2+ as bivalent metal ion. Co2+ and Mg2+ also activate though with considerably lower efficiency. All subunits except y were isolated in pure state. σ was catalytically active. y could only be obtained as a complex with Β'. On single-stranded DNA. Eσ and E are equally active. For the transcription of double-stranded DNA. (σ is absolutely required. Even the transcription of poly[d(A-T)] · [d(A-T)] is strongly stimulated by σ. σ from L. curvatus is able to replace σ from E. coli on E. coli core enzyme even for the formation of the stable preinitiation complex. This effect requires Mg2+, that is conditions optimal for the E. coli system. Thus, the core and not g appears to determine the requirement for the bivalent metal ion. Different double-stranded templates are transcribed with highly different efficiency. The ionic strength optimum is different for different templates. Ey in contrast to E exhibits a low, though significant background activity on double-stranded DNA. It is stimulated by σ to twice the specific activity of Eσ. Thus, σ and y act synergistically though they appear to exclude each other on the core. [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 vivo 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 th3Tnidine is synthesized from deoxyribose 1-phosphate and thymine rather than through a deoxyribose transfer from a deoxynucleoside. [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]

The two types of 30-S · 50-S couples described in the preceding paper, differ markedly in their sensitivity to hydrostatic pressure evoked during centrifugation in sucrose gradients. The sedimentation coefficient of about 60 S recorded with couples formed from native subunits is only apparent, the real a value being 70 S. The latter couples can be stabilized against pressure-induced dissociation by bound tRNA. On the other hand this binding renders couples formed from derived subunits more sensitive to this dissociation. As the factor determining this intriguing difference in pressure sensitivity lies in the 50-S subunit, a comparative analysis of a number of ribosomal constituents has been performed for native and derived 50-S particles. No qualitative differences in the proteins L1–L34 have been detected. Quantitative differences cannot yet be excluded. It is considered unlikely that differences in rRNAs or low-molecular-weight components arc responsible for the distinct association behaviour. Partial reconstitution of ribosomal γ-cores and split proteins detached by CsCl reveals that the determinant factor is located in the core particles. This suggests that native and derived 50-S subunits do not differ in conformation only. The possibility may be envisaged therefore, that the two types of 50-S differ in a ribosomal protein. [ABSTRACT FROM AUTHOR]

In this paper, the successful expression of trypanosomul triosephosphate isomerase (TIM) from Trypanosoma brucei brucei to high yield in Estherichia coli, using a T7-polymerase-based expression system, is described. Overexpressed trypanosomal TIM is fully active. The measured physicochemical properties of this recombinant TIM and TIM purified from trypanosomes are indistinguishable. Crystals of recombinant TIM have been grown in the presence of 2.4 M ammonium sulphate under the same conditions as for trypanosomally expressed TIM. The recombinant TIM crystal structure has been refined at 0.23 nm resolution; no differences were detected between this structure and the original crystal structure. A TIM mutant was made in which a unique dimer-interface histidine residue (His47) was changed into an asparagine. This variant ([H47N]TIM) could he expressed and purified to homogeneity by a procedure which was somewhat different from the purification of recombinant wild-type TIM. It is shown that the [H47N]TIM dimer is considerably less stable than wild-type trypanosomal TIM. The catalytic activity of [H47N]TIM is concentration dependent. The dilution-dependent inactivation is reversible. His47 is involved in a water-mediated hydrogen bond with Asp385 of the other subunit. The lower stability of the [H47N]TIM dimer implies that this water-mediated hydrogen bond is important for the stability of the TIM dimer. [ABSTRACT FROM AUTHOR]

This paper reports the purification and the properties of a thioredoxin from the fungus Aspergillus nidulans. This thioredoxin is an acidic protein which exhibits an unusual fluorescence emission spectrum, characterized by a high contribution of tyrosine residues. Thioredoxin from A. nidulans cannot serve as a substrate for Escherichia coli thioredoxin reductase. Corn NADP-malate dehydrogenase is activated by this thioredoxin in the presence of dithiothreitol, while fructose-1,6-bisphosphatase is not. The amino acid sequence of Aspergillus thioredoxin was determined by automated Edman degradation after cleavage with trypsin, SV8 protease, chymotrypsin and cyanogen bromide. The masses of tryptic peptides were verified by plasma-desorption mass spectrometry. The mass of the protein was determined by electrospray mass spectrometry and shown to be in agreement with the calculated mass derived from the sequence (Mr = 11 564). Compared to thioredoxins from other sources, the protein from A. nidulans displays a maximal sequence similarity with that from yeast (45%). [ABSTRACT FROM AUTHOR]

We have determined the solution structures and examined thc dynamics of the Escherichia coli trp repressor (a 25-kDa dimer), with and without the co-repressor L-tryptophan, from NMR data. This is the largest protein structure thus far determined by NMR. To obtain a set of data sufficient for a structure determination it was essential to resort to isotopic spectral editing. Line broadening observed in this molecular mass range precludes for the most part the measurement of coupling constants and stereospecific assignments, with the inevitable result that the attainable resolution of the final structure will be somewhat lower than the resolution reported for smaller proteins and peptides. Nevertheless the general topology of the protein can be deduced from the subsets of NOEs defining the secondary and tertiary structure, providing a basis for further refinement using the full set of NOEs and energy minimization. We report here (a) an intermediate resolution structure that can be deduced from NMR data, covalent, angular and van-der-Waals constraints only, without resort to detailed energy calculations, and (b) the limits of uncertainty within which this structure is valid. An examination of trinse structures combined with backbone amide exchange data shows that even at this resolution three important conclusions can be drawn: (a) the protein structure changes upon binding tryptophan; (b) the putative DNA binding region is much more flexible than the core of the molecule, with backbone amide proton exchange rates 1000 times faster than in the core; (c) the binding of tryptophan stabilizes the repressor molecule, which is reflected in both the appearance of additional NOEs, and in the slowing of backbone proton exchange rates by factors of 3-10.Sequence-specific ¹H-NMR assignments and the secondary structure of the holopressor (L-tryptophan-bound form) have been reported previously [C. H. Arrowsmith, R. Pachter, R. B. Altman, S. B. Iyer & O. Jardetzky (1990) Biochemistry 29, 6332-6341]. Those for the trp aporepressor (L-tryptophan-free form), made using the same methods and conditions as described in the cited paper, are reported here. The secondary structure of the aporepressor was calculated from sequential and medium-range NOEs and is the same as reported for the holorepressor except that helix E is shorter. The tertiary solution structures for both forms of the repressor were calculated from long-range NOE data. The two structures are quite similar in overall topology to one another as well as to published crystal structures; however, the DNA binding region appears to be more disordered in solution than in the crystal structures. We propose that the greater flexibility of the DNA binding region observed by us as well as by others [C. L. Lawson, R.-G. Zhang, R. W. Schevitz, Z. Otwinowski, A. Joachimiak & P. B. Sigler (1988) Proteins 3, 18] plays an important role in the mechanism of DNA binding. [ABSTRACT FROM AUTHOR]

Prourokinase is a plasminogen activator of 411 amino acids which displays a clot-lysis activity through a fibrin-dependent mechanism, and which seems to be a promising agent for the treatment of acute myocardial infarction. The preparation of recombinant prourokinase in bacteria has been hampered by its insolubility and by difficulty in refolding the polypeptide chain. In this paper we describe the renaturation process of two recombinant proteins expressed in Escherichia coli as inclusion bodies: prourokinase and a deletion derivative (Δ125-prourokinase) in which 125 amino acids of the N-terminal region have been removed. Deletion of this sequence brings to higher refolding yields and faster kinetics (first-order rate constant of renaturation of 0.57 h-1 for Δ125-prourokinase and 0.25 h-1 for prourokinase). Our process involves sequential steps of denaturation, reduction and controlled refolding of the polypeptide chain. When applied to pure. non-glycosylated and active prourokinase, it gives a refolding yield of about 80%, demonstrating the efficiency of the renaturation procedure. Lower yields (15% and 30%, respectively, for prourokinase and Δ125-prourokinase) were obtained when the same refolding protocol was applied to inclusion bodies from bacteria. After purification to homogeneity (as shown by HPLC and SDS/PAGE) specific activities were 160000 and 250000 IU/mg protein, respectively, for prourokinase and Δ125-prourokinase. As with prourokinase, the deletion mutant 41 25-prourokinase displays a zymogenic nature, being activated by plasmin to the active two-chain form; however, this mutant is approximately fourfold more resistant than prourokinase to plasmin activation, and consequently shows a different fibrinolytic profile. [ABSTRACT FROM AUTHOR]

A second thioredoxin, distinct from the one reported by Meng and Hogenkamp in 1981 (J. Biol. Chem. 256, 9174-9182), has been purified to homogeneity from an Escherichia coli strain containing a plasmid encoding a Corynebacterium nephridii thioredoxin. Thioredoxin genes from C. nephridii were cloned into the plasmid pUC13 and transformants were identified by complementation of a thioredoxin negative (trxA-) E. coli strain. The abilities of the transformants to support the growth of several phages suggested that more than one thioredoxin had been expressed [Lim et al. (1987) J. Biol. Chem. 262, 12114-12119]. In this paper we present the purification and characterization of one of these thioredoxins. The new thioredoxin from C. nephridii, designated thioredoxin C-2, is a heat-stable protein containing three cysteine residues/molecule. It serves as a substrate for C. nephridii thioredoxin reductase and E. coli and Lactobacillus leichmannii ribonucleotide reductases. Thioredoxin C-2 catalyzes the reduction of insulin disulfides by dithiothreitol or by NADPH and thioredoxin reductase and is a hydrogen donor for the methionine sulfoxide reductase of E. coli. Spinach malate dehydrogenase (NADP+) and phosphoribulokinase are activated by this thioredoxin while glyceraldehyde-3-phosphate dehydrogenase (NADP+) is not. Like the thioredoxin first isolated from C. nephridii, this new thioredoxin is not a reducing substrate for the C. nephridii ribonucleotide reductase. The complete primary sequence of this second thioredoxin has been determined. The amino acid sequence shows a high degree of similarity with other thioredoxins. Surprisingly, in contrast to the other sequences, this new thioredoxin contains the tetrapeptide -Cys-Ala-Pro-Cys- at the active site. With the exception of the T4 thioredoxin, this is the first example of a thioredoxin that does not have the sequence -Cys-Gly-Pro-Cys-. Our results suggest that, like plant cells, bacterial cells may utilize more than one thioredoxin. [ABSTRACT FROM AUTHOR]

UTP-modulated attenuation of transcription is involved in regulating the synthesis of pyrimidine nucleotides in Escherichia coli. Thus, expression of two genes, pyrBI and pyrE, was shown to be under this type of control. The genes encode the two subunits of aspartate transcarbamylase and orotate phosphoribosyltransferase respectively. The levels of these enzymes are inversely correlated with the intracellular concentration of UTP. Modulation of attenuation seems to be a consequence of the effect of UTP concentration on the mRNA chain growth rate. Reducing the UTP pool retards RNA polymerase movement. Mechanistically this will couple the ribosomes translating a leader peptide gene more tightly to the elongating RNA polymerase. The ribosomes will then be more prone to prevent the folding of the mRNA chains into terminating hairpin structures when RNA polymerase is at the attenuator and has to decide whether transcription should terminate or continue into the structural genes. This paper described a study of pyrBI and pyrE gene regulation in cells where the ribosomes move slowly as a result of mutation in rpsL. It appears that expression of the two genes is hyper-regulated by the UTP pool in this type of cells. Furthermore, the attenuator model can only account for the results if it is assumed that UTP-concentration dependent pausing of transcription occurs in vivo in the two pry gene leaders such that RNA polymerase waits for the coupled ribosomes before transcribing into the attenuator regions. [ABSTRACT FROM AUTHOR]

This paper reports the purification and characterization of a thioredoxin system (thioredoxin, thioredoxin reductase, NADPH) from the facultative phototroph Rhodobacter sphaeroides Y. Rhodobacter sph. Y thioredoxin was purified to homogeneity with an assay based on the reduction of 5,5′-dithiobis(2-nitrobenzoic acid) by NADPH and Escherichia coli thioredoxin reductase. Rhodobacter sph. Y thioredoxin reductase was purified with the same assay using NADPH and E. coli thioredoxin. Rhodobacter sph. Y thioredoxin contained 102 amino acid residues and had a single disulfide bond. The two half-cystine residues are part of the active site made up of the sequence -Ala-Glu-Trp-Cys-Gly-Pro-Cys-Arg- which is identical to that of E. coli thioredoxin except for the presence of an Arg instead of a Lys. Rhodobacter sph. Y thioredoxin contains two tryptophan residues. The fluorescence intensity of the tryptophan residues is quenched in oxidized thioredoxin; on reduction, a much smaller increase is observed with Rhodobacter sph. Y thioredoxin than with the E. coli protein. However, the presence of 5 M guanidine · HCl results in the complete exposure of the two tryptophan residues. Rhodobacter sph. Y thioredoxin reductase has structural and functional similarities to E. coli thioredoxin reductase; it has a molecular mass of 68 kDa, and consists of two, probably identical, subunits. Each subunit has one bound FAD molecule. The enzyme is highly specific for NADPH; it is also highly specific for Rhodobacter sph. Y thioredoxin with a Km value of 3.3 ± 0.6 µM. A kinetic study of the two thioredoxin systems shows that they have a high degree of cross-reactivity. [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]

In the present paper laser desorption mass spectrometry (LDMS) was applied to dephosphorylated free lipid A preparations obtained from lipopolysaccharides of Re mutants of Salmonella minnesota, Escherichia coli and Proteus mirabilis. The purpose of this study was to elucidate the location of (R)-3-hydroxytetradecanoic acid and 3-O-acylated (R)-3-hydroxytetradecanoic acid residues which are bound to amino and hydroxyl groups of the glucosamine disaccharide backbone of lipid A. Based on the previous finding from biochemical analyses that the amino group of the nonreducing glucosamine residue (GlcN II) of the backbone carries, in S. minnesota and E. coli, 3-dodecanoyloxytetradecanoic acid and, in P. mirabilis, 3-tetradecanoyloxytetradecanoic acid, a self-consistent interpretation of the LDMS was possible. It was found that: (a) in all three lipids A GlcN II is, besides the amide-linked 3-acyloxyacyl residue, substituted by ester-linked 3-tetradecanoyloxytetradecanoic acid; (b) the reducing glucosamine (GlcN I) is substituted by ester-linked 3-hydroxytetradecanoic acid; (c) the amino group of GlcN I carries a 3-hydroxytetradecanoic acid which is non-acylated in E. coli and which is partially acylated by hexadecanoic acid in S. minnesota and P. mirabilis. In lipids A which were obtained from the P. mirabilis Re mutant grown at low temperature (12°C) LDMS analysis revealed that specifically the one fatty acid bound to the 3-hydroxyl group of amide-linked 3-hydroxytetradecanoic acid at GlcN II is positionally replaced by Δ9-hexadecenoic acid (palmitoleic acid). It appears, therefore, that enterobacterial lipids A resemble each other in that the 3-hydroxyl groups of the two 3-hydroxytetradecanoic acid residues linked to Glecn II are fully acylated, while those of the two 3-hydroxytetradecanoic acid groups attached to Glen I are free or only partially substituted. [ABSTRACT FROM AUTHOR]

An eighteen-amino-acid peptide having the linear amino acid sequence of human heat-stable enterotoxin (ST) has been synthesized by solid peptide synthesis. The purified peptide could be obtained in yields approaching 25% after purification by size, charge, and high-performance ligand chromatography, amino acid analysis, paper electrophoresis and thin-layer chromatography. The formation of the disulfide bonds was critical for biological and immunological activity and were tentatively determined to be between cysteines 5 and 14, 6 and 10, and 9 and 17. This synthetic peptide had full immunological and biological activity when compared to native ST by enzyme-linked immunosorbent assay and the sucking mouse assay respectively. [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]

Enzyme-IIIgle is part of the glucose phosphotransferase system of Escherichia coli and Salmonella typhimturium and is phosphorylated by phosphoenolpyruvate in a reduction requiring enzyme 1 (phosphoeno/pyruvate-protein phospho- transferase), and the histidine-containing phospho-carrier protein HPr. In this paper we report the isolation of IIIgle from E. coil and the characterization of the activc center. Alkaline hydrolysis of [32P]P-IIIgle and chromatography of the hydrolysate suggested that the phosphoryl group is bound to a histidyl residue in P-IIIgle of S. typhimurium. Here we present 1H-NMR measurements of IIIgle and P-IIIgle from E. coli which further substantiate that the phosphoryl group in P-IIIgle is linked to the N-3 position of a histidyl residue. After phosporylation of IIIgle with [32P]Phosphoenolpyruvate, enzyme I and HPr, the phosphorylated protein was cleaved with either alkaline protease from Streptomyces griseus or subtilisin from Bacillus subtilis. According to amino acid analysis both proteases produced the same peptide carrying the phosphoryl group. The amino acid sequence of this peptide was found to be Val-His-Phe-Gly-Ile-Asp. The lower electrophoretic mobility of P-IIIgle on dodecylsulfute/polyacrylamide gels and its stronger binding to the hydrophobic matrix of a reversed-phase column compared to uuphosphorylated protein may indicate a structural change following phosphoenol/pyruvate-dependent phosphorylation. [ABSTRACT FROM AUTHOR]

1. In Escherichia coli ML308 isocitrate dehydrogenase is partially inactivated during growth on acetate [Bennett, P. M. and Holms, W. H. (1975) J. Gen. Micribiol. 87, 37-51]. 2. The active form of isocitrate dehydrogenase was purified to homogeneity from cells grown on glycerol. The key step in the procedure was chromatography on procion-red-Sepharose, from which the enzyme was specifically eluted with NADP+. 3. Two forms of isocitrate dehydrogenase were purified to homogeneity from cells grown on acetate. One form did not bind to procion-red-Sepharose and was essentially inactive; this form could be resolved from the active form by non-denaturing gel electrophoresis. The other form was specifically eluted from procion-red-Sepharose and was partially active; analysis of this form by non-denaturing gel electrophoresis suggested that it was a mixture of the active and inactive forms. 4. The three forms comigrated on denaturing gel electrophoresis and were identical by the criterion of onedimensional peptide mapping. 5. Analysis of the active and inactive forms by sedimentation equilibrium centrifugation and non-denaturing gel electrophoresis showed that they differed in charge but not in size. Amino acid analysis and two-dimensional peptide mapping showed that both forms were directs of identical subunits. 6. The active form of the enzyme contained no detectable alkali-labile phosphate, the inactive form contained 0.8 molecule subunit and the partially active form contained an intermediate amount. 7. The data suggest that the active and inactive forms of isocitrate dehydrogenase differ only in the presence of one phosphate group per subunit in the latter form; this is consistent with our results from phosphorylation of isocitrate dehydrogenase in vitro (Following paper in this journal). 8. The nature of the partially active form of isocitrate dehydrogenase and the significance of the results are discussed. [ABSTRACT FROM AUTHOR]

Escherichia coli ribosomal proteins S4 and S17 were specifically labelled at their thiol groups with the acetylaminoethyl-dansyland/or bimane fluorophores. Each formed a complex with 16-S RNA and, when the other 30-S ribosomal proteins were added, a complete 30-S subunit with at least partial activity. If the 3′ end of the RNA was also labelled (with fluorescein) then the distance between the two fluorophores could be measured by Förster-type energy transfer. The result for S4 was 6.0 nm (60 Å) in the ribonucleoprotein complex and 5.6 nm (56 Å) in the 30-S subunit, and for S17 6.3 nm (63 Å) in the complex and 6.2 nm (62 Å) in the subunit. There is no evidence for a major change in the relative disposition of the 3′ and 5′ ends of the 16-S RNA during formation of the 30-S subunit. Sources of error are discussed, including the question of multiple labelling. In order to measure more accurately the extent of energy transfer a procedure based upon enzymic digestion was developed and is detailed in this paper. [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]

We have studied the ribonucleosidediphosphate reductase of a deoxyuridine-requiring mutant LD195 of Escherichia coli K. This enzyme was purified 50 fold from the mutant and a control strain. In all steps of the purification the mutant strain yielded normal activity of the Bi subunit of the enzyme but approximately 10 % of the wild-type activity of the B2 subunit The activity is similarly reduced for all three substrates tested CDP, UDP, and GDP. The mutation appears to affect the physical structure of the B2 subunit, since addition of high concentrations of sodium acetate can restore enzymatic activity to the partially purified B2 subunit from the mutant strain. In contrast, the same concentrations strongly inhibit the wild- type B2 subunit. Although the mutant has a requirement of deoxyuridine at 42 °C but not at 30 °C (as shown in an accompanying paper), mutant protein B2 is not more thermo-labile than wild-type protein B2 This requirement therefore probably reflects an increased demand of deoxyribonucleotides at higher temperatures. Hydroxyurea inhibits mutant and wild-type ribonucleosidediphosphate reductase to the same extent in vitro. In spite of this, hydroxyurea inhibits growth of the mutant strain at a 20 fold lower concentration than that required for comparable inhibition of growth of the parent. This is explained by the greatly decreased protein B2 activity of the mutant. [ABSTRACT FROM AUTHOR]

A procedure to isolate Escherichia coli mutants defective in ribonucleotide reduction is described. Using a parental strain defective in dCTP deaminase (paxA) and thus unable to synthesize dUMP from deoxycytidine nucleotides, we selected for cells unable to reduce UDP at a rate sufficient to satisfy the cell's need for thymidine nucleotides. This was accomplished by selecting for cells resistant to 2,4-diamino-5-(3,4,5-trimethoxybenzyl)pyrimidine (trimethoprim) in the presence of thymidine and screening these cells for a thymidine requirement that could be satisfied by deoxyuridine. Genetic analysis as well as enzyme assays of four such mutants chosen for study indicated that three had a defective thymidylate synthetase. One, LD195, which had normal thymidylate synthetase activity, was found to have a conditional deoxyuridine requirement that is only expressed under favourable growth conditions. Since restoration of a functional dCTP deaminase to derivatives of LD195 eliminated the deoxyuridine requirement, it was concluded that a combination of two mutations is responsible for the expression of this phenotype. Analysis of nucleotide pools of LD195 grown exponentially under permissive conditions revealed that both the dTMP and dTTP pools were 6-fold lower than in the parent indicating a deficiency in the production of a thymidine nucleotide precursor. Enzyme assays of crude extracts of LD195 indicated that this deficiency was due to a defective ribonucleosidediphosphate reductase. This conclusion is proven by an extensive enzymatic analysis presented in an accompanying paper. [ABSTRACT FROM AUTHOR]

Initiation of protein synthesis in Escherichia coli is a catalytic process, provided that conditions for hydrolysis of GTP are present. This means that the initiation factors IF-1, IF-2 and IF-3 are released at a certain stage of initiation complex formation, whereafter they recycle and are reutilized in another round of initiation. Experiments presented in this paper provide evidence for the mechanism of recycling, in which the hydrolysis of GTP and IF-1 both play a predominant role. We demonstrate that binding of fMet-tRNA, which is dependent on IF-2, is a stoichiometric process in a system devoid of IF-1. Addition of IF-1 converts it into a catalytic process, however, only if GTP can be hydrolysed. No such conversion takes place with 5′-guanylylmethylenediphosphonate. The same observation is valid for the uncoupled IF-2 dependent GTPase activity. In the process of initiation IF-1 acts as a recycling promoting factor, which displays analogous properties to elongation factor EF-Ts. We postulate that a ribosomal-bound IF-2 · GDP complex is converted into an IF-2 · GTP complex, which is released and is capable of starting a new cycle of initiation. Furthermore, kinetic evidence is presented that IF-3 recycles even at the level of 30-S initiation complex formation, irrespective of whether conditions for hydrolysis of GTP are provided. [ABSTRACT FROM AUTHOR]

After the recent investigation on the biochemical properties of the 2-keto-3-deoxy-gluconokinase and 2-keto-3-deoxy-6-phospho-gluconate aldolase in Escherichia coli K12, we report in this paper the induction mechanism of these two enzymes and the genetic control of a new enzymatic activity: the 2-keto-3-deoxy-gluconate transport system. The D-glucuronate and D-galacturonate raise the high basal levels of the kinase and aldolase on glycerol by a factor of 5 and 3-4, respectively, the kinase and aldolase being enzymes of the common degradative pathway of D-glucuronate and D-galacturonate. The differential rates of synthesis of these two enzymes are constant from the addition of the galacturonate. Under a variety of conditions (different inducers and substrates of growth) kinase and aldolase have been found to be synthesized non coordinately. The strongest decoordination is carried out by gluconate: this compound which is a good inducer of the aldolase, not only is unable to induce the kinase but also represses it. Moreover, the aldolase seems to be less sensitive to the catabolic repression than the kinase. By means of appropriate negative mutants we have established that the two hexuronates and two keto-uronates do not induce directly kinase and aldolase, but by sequential conversion into 2-keto-3-deoxy-gluconate. Furthermore, the fact that the hexuronates induce the kinase in aldolase-negative mutants (kdg A) and besides, "over" induce the aldolase in kinase negative mutants (kdg K) strongly suggests that, 2-keto-3-deoxy-gluconate is a true inducer for both enzymes. In addition, the gluconate which still induces the aldolase in an edd strain suggests that, this compound and/or the 6-phospho-gluconate are/is also inducer(s) of this enzyme. Moreover, we have isolated spontaneous mutants able to utilize the 2-keto-3-deoxy-gluconate as a unique carbon source. Two classes have been identified. The mutants of these classes which arise at high frequency (10-5 to 10-6) are either simultaneously constitutive for a specific 2-keto3-deoxy-gluconate transport system, kinase and aldolase (class kdg Rc) or only constitutive for the transport system (class kdg Pc). The 2-leto-3-deoxy-gluconate as exogenous substrate, which cannot induce its transport system in the wild type, behaves like a non-inducer substrate. So the strains kdg Rc and kdg Pc are respectively i- and oc lac-like mutants. These findings and the fact that we have found thermosensitive mutants mapping in the kdg R locus (34.5 min) and which are simultaneously derepressed for the permease, kinase and aldolase at 42 °C but not at 28 °C, strongly suggest that the synthesis of the three sequential enzymes degrading the 2-keto-3-deoxy-gluconate is negatively controlled by a common regulator gene product. The decoordinated syntheses of these three enzymes are in agreement with the scattering of the corresponding operons on the chromosome: transport system operon (76.5 min), kinase operon (69 min), aldolase operon (34 min). [ABSTRACT FROM AUTHOR]

D-Mannonate : NAD oxidoreductase of Escherichia coli K12 has been purified. The purification consists of four steps: precipitation of nucleic acids with protamine sulfate; ammonium sulfate precipitation; ion-exchange chromatography on DEAE-cellulose and Sephadex G-200 gel filtration. The overall procedure results in a 53-fold purification with a yield of 70%. Mannonate oxidoreductase is an inducible enzyme: glucuronate and fructuronate are good inducers, galacturonate is not. Optimal activity is obtained at pH 6.3 fro fructuronate reduction, and at pH 8.4 for mannonate oxidation. The apparent Michaelis constants for the purified enzyme have been determined: for fructuronate, the Km is 0.5 mM; for NADH 0.03 mM; for mannonate, 1.8 mM and for NAD+, 0.2 mM. Mannonate oxidoreductase and altronate oxidoreductase have been separated on DEAE-Sephadex in order to study mannonate-oxidoreductase specificity. After separation, mannonate oxidoreductase is found to be active with both fructuronate and tagaturonate as substrates. Kinetic studies, differential inhibition of activities by p-chloromercuribenzoate and thermal inactivation studies confirm this double specificity. Other carbohydrates tested are unsuitable as substrates. Mannonate oxidoreductase is active with NADPH as coenzyme and the apparent Km value for NADPH is 0.7 mM. Kinetic inhibition studies of the enzyme by direct and reverse reactional products suggest a random mechanism for mannonate oxidoreductase. Enzyme inhibition specificity appears to be restricted. The value of the energy of activation is 34 kJ/mol (8 kcal/mol); the Q10 is 1.5. The results of thermal inactivation of mannonate oxidoreductase (53 °C) indicate homogeneous-enzyme activity fructuronate in the crude extract. In the absence of inducer, basal activity is dependent on mannonate oxidoreductase only. Thermal inactivations also indicate that the same enzyme, mannonate oxidoreductase, catalyzes the direct reactions with NADH or NADPH, and the reverse reaction. Enzyme substrates, such as NADH, NADPH, fructuronate, tagaturonate, NAD+, mannonate and altronate protect enzymatic activity from degradation by heat. NADH in particular, is effective as a stabilizer; this ability is proportional to its concentration. p-Chloromercuribenzoate is an active inhibitor: at 5 μM, activity is 100% inhibited. Inhibition is non-competitive, with an apparent Ki of 0.1 μM. The end product of the reaction of mannonate oxidoreductase with fructuronate has been identified by means of paper chromatography as D-mannonate. [ABSTRACT FROM AUTHOR]

D-Altronate: NAD-oxidoreductase of Escherichia coli K12 has been purified. The purification consists of four steps: nucleic acids precipitation with protamine sulfate; ammonium sulfate precipitation; ion exchange chromatography on DEAE-cellulose and Sephadex G-150 gel filtration. The overall procedure results in a 53-fold purification with a yield of 60%. Altronate oxidoreductase is an inducible enzyme: galacturonate, tagaturonate, glucuronate and fructuronate are good inducers. The activity remains stable at 4 °C or at — 20 °C for several months. Optimal activity is obtained at pH 6.3 for tagaturonate reduction, and at pH 8.9 for altronate oxidation. Altronate oxidoreductase does not require any cofactor, except NADH. The apparent Michaelis constants have been determined: for tagaturonate, the Km is 350 μM; for NADH, 60 μM; for altronate, 90 μM, and for NAD+, 110 μM. Moreover, altronate oxidoreductase is active with NADPH as coenzyme and the apparent Km value for NADPH is 400 μM. Other carbohydrates tested are unsuitable as substrates. Many other carbohydrates tested do not prove to be inhibitors. The value for the energy of activation is 38 kJ/mol (9 kcal/mol); the Q10 is 1.5. The results of heat inactivation of altronate oxidoreductase (58.5 °C) indicate homogeneous enzyme activity in crude extract. Thermal inactivations also indicate that the same enzyme, altronate oxidoreductase, is induced by galacturonate and glucuronate, and catalyzes the direct reactions with NADH or NADPH. Finally, it has been confirmed by heat inactivation that there is no interference between specific assays of altronate and mannonate oxidoreduetases. Compounds such as NADH, NADPH, altronate and mannonate protect enzymatic activity from degradation by heat, whereas NAD+, tagaturonate and fructuronate do not. NADH, in particular, is effective as a stabilizer; this ability is proportional to its concentration. Enzyme inhibition with para-chloromercuribenzoate is non-competitive and the apparent Ki is 1 mM. para-Chloromercuribenzoate also inactivates altronate oxidoreductase indicating homogeneous activity and specificity of the assay method in crude extracts. Chromatographic properties of the enzyme are identical whether the inducer is galacturonate or glucuronate. Using paper chromatography, the reaction product of enzyme action on D-tagaturonate is identified as D-altronate. [ABSTRACT FROM AUTHOR]

In this paper we report on the properties of thc purified isoleucyl-tRNA synthetase from Bacillus stearothermophilus. The enzyme which was purified approximately 500-fold, has a molecular weight of about 110 000. The effect of temperature on the activation and transfer activities has been studied. The thermostable enzyme shows an optimal activity at 65 °C for the isoleucine-dependent ATP-PPi exchange reaction and at 45 °C for the formation of isoleucyl-tRNA. Attempts to heat inactivate the amino-acid-transfer activity selectively, i.e. without loss of the activation reaction, were unsuccessful. The protection of the enzyme against heat inactivation in presence of tRNA makes it possible to study the binding of this molecule to the protein. An association constant of 0.11 nM-1 was determined at 70.5 °C. Formation of this tRNA · enzyme complex was also demonstrated by binding onto nitrocellulose filters. By this technique one tRNA site per enzyme molecule was detected and an affinity constant of 0.4 nM-1 measured. The same results were obtained either with tRNA from Escherichia coli or B. stearothermophilus, showing close resemblance of the two nucleic acids. Periodate-oxidation of the 3'-terminal adenosine of the tRNA does not perceptibly affect the binding or the degree of protection of the enzyme against heat inactivation. These effects are still strictly dependent on magnesium ions and a clearcut requirement of these ions for the formation and/or stabilization of the tRNA · enzyme complex has been demonstrated. The intermediate isoleucine · AMP · enzyme complex with a molar ratio of 1:1:1 was isolated by molecular sieving and by chromatography on DEAE-cellulose. Mg2+ ions are required for the formation of the complex but are not necessary to stabilize the isoleucyl-adenylate once this is bound to the enzyme. The transfer of activated isoleucine to tRNA again requires magnesium ions and must probably be related to the formation of a tRNA enzyme complex. Part only of the activated isoleucine is transferred to tRNA; this results essentially from the breakdown of a fraction of the isoleucine · AMP · enzyme complex, induced by tRNA and Mg2+ ions, acting together. Periodate-oxidized tRNA of B. stearothermophilus or of E. coli in presence of Mg2+ ions completely releases isoleucine from the complex. [ABSTRACT FROM AUTHOR]

The 09 antigen from Escherichia coli 09:K30 (A):H12 contains mannose (90%) and glucose (10%). Incorporation of [14C]mannose from guanosine diphosphate [14C]mannose (GDP-[14C]Man) into acetic-acid precipitates has been studied with a preparation from cells ground with alumina. The time-course of mannose incorporation was biphasic (rapid reaction for 2-5m in followed by a slower, linear reaction for 5-20 rain) and the incorporation was stimulated by uridine diphosphate glucose (UDP-Glc) during the early part of the reaction. The reactions (mannose incorporation after 2 rain and 15 min, ± UDP-Glc) were nevertheless proportional to protein concentration. [14C]Glucose was incorporated into acetic-acid precipitates from UDP[14C]Glc and this reaction was slightly stimulated by GDP-Man. Radioactivity from GDP[14C]Man was incorporated into butanol extracta and the early part of the reaction (2-5 min) was markedly stimulated by UDP-Glc. Bacitracin did not inhibit the incorporation of mannose into acetic-acid precipitates in the early part of the reaction, but did inhibit incorporation about 20% between 5 and 30 min. Larger-scale incubation mixtures were extracted with 45% aqueous phenol. The product was of high molecular weight as indicated by Sephadex G-100 chromatography and yielded a single radioactive peak corresponding to mannose after total hydrolysis and paper chromatography. This high-molecular-weight product precipitated with anti-09 antiserum in an antigen-binding assay, indicating that it was serologically active. Thus, it is likely that the 09 antigen of E. coli 09:K30(A):H12 is synthesized from GDP-Man and UDP-Glc through a butanol-extractable (lipid-linked) intermediate. Reactions appear to be analogous to those described for lipopolysaccharide biosynthesis in Salmonella. [ABSTRACT FROM AUTHOR]

The binding of purified Gly3-Phe-tRNA to Escherichia coli ribosomes was compared to that of crude Gly3-Phe-tRNA (containing bulk tRNA). Gly3-phe-tRNA from the purified preparation binds preferentially to the P site (peptidyl site) at both a high (30 mM) and a low (10 mM) magnesium acetate concentration, while peptidyl-tRNA from the crude preparation bound more to A site (aminoacyl site) than to the P site at 30 mM magnesium acetate under the same experimental conditions. The addition of tRNAPhe to the purified Gly3-Phe-tRNA preparation strongly inhibited the binding at 10 mM. At 30 mM Mg2+ the addition of the same amount of tRNAPhe caused the Gly3-Phe-tRNA to bind partially to the A site, but did not inhibit the total amount of Gly3-Phe-tRNA which binds to the ribosomes. The binding of purified Phe-tRNA in the presence of T factor and GTP is not inhibited by tetracycline. However, tetracycline strongly inhibited the enzymatic binding of Phe-tRNA when tRNAPhe was added. From the results presented in this paper it is proposed that in the presence of poly(U), Phe-tRNA or peptidyl-tRNAPhe can bind to A sites only of those ribosomes which have their P site occupied by tRNA or one of its derivatives (aminoacyl-tRNA or peptidyl-tRNA). [ABSTRACT FROM AUTHOR]

1. 1. Acapsular (K-) forms were isolated from Escherichia colt strain E56b (serotype O8 :K27(A): H-). A number of different R strains were obtained from the K- forms by selection of spontaneous mutants and by phage selection. 2. The lipopolysaccharides of these R strains (consisting of core and lipid A) were subjected to mild acid degradation and, after removal of insoluble lipid A, the mixtures were fractionated by gel chromatography. Results of chemical analyses showed that the core oligosaccharides of different R mutants differed with respect to their sugar constituents as well as to the molar ratios of these constituents. A group of R mutants did not contain phosphate in their core oligosaccharides (P- mutants). Enzyme determinations indicated that one R mutant had a block involving UDP glucose pyrophosphorylase, whereas the enzymes of activation and interconversion of component sugars were intact in the other R mutants. 4. By allelic exchange (mating with S-form Hfr strains)it could be demonstrated that with one exception the chromosomal site of the S→ R mutation of all mutants maps close to mtl (mannitol utilization). In Salmonella the rfa locus maps fun the same region. All R mutants had an unimpaired (his-linked) rfb locus. 5. The groups of mutants which differed in the sugar composition of their core oligosaccharides could also be differentiated serologically and by their sensitivity to phages. There was serological cross reactivity between the lipopolysaccharides of some R mutants with R mutants of Salmonella minnesota which also have a very incomplete core. It is concluded that the lipopolysaccharides of the R mutants described in this paper have incomplete core structures which result from blocks at successive stages in the synthesis of the coli R1 core. in analogy to Salmonella these E. coli R mutants shoed, with one exception, be termed rfa type mutants. [ABSTRACT FROM AUTHOR]

The experiments reported in this paper lead to the following conclusions: 1. An endonucleolytic activity is associated with the "replication site" in Escherichia coli cells; this endonucleolytic activity attacks the complementary strand of the ψx replicative form DNA only. 2. the viral strand of the ψx-replicative form DNA remains circularly closed during the replication cycle; 3. In the immediate replication product both composing strands are linear; in circularization no strand is preferred; 4. A replication product with both strands closed is transiently found in a structural state which can be, under our experimental conditions, only partially denatured by alkali. Subsequently it is transformed to a typical supercoiled RFI structure. [ABSTRACT FROM AUTHOR]

It is well know that protein synthesis stopped either by chloromycetin or by starvation of an essential amino acid for a RC relstrain, ribosomal RNA synthesis continues, but products do not appear in 50 S and 30 ribosomes. It has also been shown that 5 S RNA synthesis is altered by the addition of chloromycetin but in an unknown manner. In this paper we report that: (a) during amino-acid starvation of a starvation of RCrel strain of Escherichia coli, 5 S RNA is transcribed as 5 S relax RNA located outside the ribosomes, in the supernatant fraction; (b) this 5 S RNA which is slightly longer than the normal product is convertible into a true 5 S RNA when protein synthesis is restored after blockage of RNA synthesis by actinomycin ;(c) part of the 23 S RNA from a 25 S particle appears after resumption of protein synthesis in a 40 S parcel which contains normal 5S RNA; (d) a pool of 5 S precursor RNA exists outside the 50 S ribosome. This 5 S precursor RNA is isolated; (e) this precursor which is isolated from the 105000xg supernatant fraction after pulse labeling, migrates on gel electrophoresis between 5 S relax RNA and true 5 S RNA. [ABSTRACT FROM AUTHOR]

Multiple species of Escherichia coli transfer RNA for valine and leucine were separated from each other by countercurrent distribution. Each transfer RNA species was charged with the corresponding radioactive amino acid and then allowed to transfer its label into hemoglobin peptides in the ribosomal system from rabbit reticulocytes. Earlier experiments had shown that the minor tRNAIIbLeu would only label one position, no. 48 of the α-hemoglobin chain under these conditions. In this paper we report the coding properties of the other separated leucine tRNAs and of multiple valine tRNAs with the hemoglobin messenger. The leucine codons in the α-chain messenger can be divided into three classes with respect to their tRNA response : (a) position 48 is recognized only by tRNAIIbLeu. (b) The majority of the leucine codons can be recognized by both the two major leucine tRNAs. (c) Some leucine codons can only be recognized by one of the major leucine tRNAs. Similarly, two classes of valine codons can be distinguished in the α-chain messenger. [ABSTRACT FROM AUTHOR]

The behavior of N-acetylphenylalanyl-tRNA as an initiator of phenylalanine polymerisation in an Escherichia coli cell-free system shows some analogy with the behavior of formylmethionyl- tRNA during the synthesis of natural proteins. At low Mg++ concentrations, polyphenylalanine chain initiation depends on N-acetylphenylalanyl-tRNA and the same initiation factors which are required for protein chain initiation with formylmethionyl-tRNA. However in a rabbit reticulocyte cell-free system, N-acetylphenylalanine is not incorporated into polyphénylalanine at any Mg++ concentration. In this paper, we showed that : a) N-acetylphenylalanyl-tRNA did bind to reticulocyte ribosomes in the presence of poly U. This binding was GTP dependent and needed a factor which was extracted by washing ribosomes with salt (NH4CI 1 M); b) the bound N-acetylphenylalanyl-tRNA was partially released by puromycin or by addition of phenylalanyl-tRNA. In this last case, incorporation of N-acetylphenylalanine into polyphenyl- alanine was noticed (ratio N-acetylphenylalanine/phenylalanine = 1/20); c) the ribosomal wash fluid also greatly enhanced phenylalanyl-tRNA binding to salt- treated ribosomes, in the presence of poly U and GTP. The maximum binding was obtained with the same concentrations of Mg++ and K+ as for N-acetylphenylalanyl-tRNA binding. It was not possible to separate on a DEAE-cellulose column the factors that were required for phenyl- alanyl-tRNA and acetylphenylalanyl-tRNA binding. On the other hand, a preparation of the binding enzyme from reticulocyte supernatant enhanced both phenylalanyl-tRNA and acetyl- phenylalanyl-tRNA binding to ribosomes; d) the optimum Mg+÷ concentration for phenylalanine polymerisation was higher with salt- washed ribosomes than with sucrose-washed ribosomes. Adding the wash fluid shifted the Mg++ optimum back to the lower value. N-acetylphenylalanyl-tRNA was not required for this shift as noticed in Escherichia coli. One likely explanation of our results with reticulocytes is the possibility that the ‘binding enzyme’ of Schweet et al., which was present in our ribosome wash fluid, could work with both N-acetyl as well as with phenylalanyl-tRNA. Thus, the inability to incorporate N-acetylphenyl- alanine might be due to a competition between these two compounds for the same enzyme and the same ribosomal site. If this is indeed the case, it is in marked contrast to results obtained with Escherichia coti and could reflect a basic difference in the mechanism of protein chain initiation. [ABSTRACT FROM AUTHOR]

Alkaline phosphatase (Escherichia coli) was found to catalyze the synthesis of monophosphate esters of serine, ethanolamine, propanolamine, butanol, glycerol, L-glucose, and tris(hydroxymethyl)aminomethane by phosphoryl transfer from cysteamine S-phosphate (donor) to the respective alcohols (acceptors). Maximum enzymic synthesis of the above esters occurred at pH 7.8. The newly-formed phosphate esters were measured quantitatively after separation of the products by the aid of paper high voltage electrophoresis techniques or by an amino acid analyzer. The percentage of the enzyme-catalyzed synthesis of the new phosphate esters varied from 15-39 0/0 using different acceptors under otherwise identical experimental conditions. It is pertinent to note that the same compounds were phosphorylated to essentially the same extent when the donor (substrate) compound was serine O-phosphate, aminoethanol O-phosphate or p-nitrophenyl O-phosphate. The rates of enzymic consumption of cysteamine S-phosphate (vh + va) were measured at different pH values and at different concentrations of acceptor (Tris or aminoethanol) in the presence of 1.5 M NaCl using 3 mM substrate (cysteamine S-phosphate). The presence of either Tris or aminoethanol gave the same pH profile for the rate of consumption (vh + va) of cysteamine S-phosphate with a maximum value around pH 7.8. This value differs from that found for the same reaction in barbital buffer (pH 9.0). The rate of enzymic consumption of cysteamine S-phosphate was also measured at various substrate and Tris concentrations at pH 7.8 under constant ionic strength (Γ/2 = 1.0). The same Km value (0.24 mM) was obtained at Tris concentrations varying from 0.02 M to 0.5 M. The Vmax values derived from these experiments were linearly related to the Tris concentrations up to 0.5 M Tris. The ph profile of the rate of consumption of p-nitrophenyl phosphate as well as the Km value (0.26 mM) were similar to the corresponding values obtained for cysteamine S-phosphate. The rate of hydrolysis (vh) could not be measured when cysteamine S-phosphate served as the donor compound due to the instability of cysteamine S-phosphate under the conditions required for inorganic phosphate assay. Therefore, the rate of transfer (va = vRSH - vP1) could not be estimated. The pH dependence of the rate of transfer, va, was calculated from experimental data obtained when p-nitrophenyl phosphate served as the donor compound and Tris served as the acceptor. The pH profile for the rate of transfer, va, obtained from these measurements was very similar to that obtained by direct measurement of Tris O-phosphate formation using cysteamine as the donor. It is suggested that phosphoryl transfer occurs through a phosphorylated enzyme intermediate, and therefore, the types of compounds that could serve as acceptors are independent of the donor compounds and depend only on the bond energy of the particular phosphorylated enzyme intermediate. [ABSTRACT FROM AUTHOR]

It is known from previous work that the regulation of Glutaminesynthetase in coli is effected by two mechanisms. First, the synthesis of GS is repressed by NH4+; and secondly, the activity of GS is regulated by inactivation and reactivation of the enzyme molecule. In the present paper we demonstrate and characterize the reactivation of inactivated GS in intact cells of E. coli. GS is inactivated after addition of NH4+ to the culture medium. Experiments with a cell flee extract from E. coli have shown, that the inactivation is mediated by a specific "inactivating enzyme", ATP, Mg2+, and Glutamine which is probably formed from the added NH4+, The reactivation is observed, when the inactivated ceils are centrifuged from the NH4+ containing medium and then resuspended in an NH4+ free medium. In about 5–7 minutes the activity increases 5–30-fold. Inhibitors of protein synthesis such as chloramphenicol and p-fluoro-phenylalanine do not affect the reactivation process. Only a slight retardation of the reactivation is observed. A carbon source is required for reactivation. When inactivated cells are incubated in a medium containing only salts and buffer no reactivation is observed. Probably the carbon sources is metabolized to form intermediates and energy, which are needed for glutamine catabolizing reactions or for prevention of glutamine synthesis. Reactivation does not take place m the presence of NH4+ i.e. under inactivating conditions. However if inactivation is inhabited by fluoride, reactivation can be observed in the presence of NH4+. When inactivated cells are incubated with fluoride only, without a carbon source in the reactivation medium, a reactivation is found. Inactivation as well as reactivation arc inhibited by thiol-group reagents. From these results we conclude that: (a) Reactivation is independent of protein synthesis; (b) Reactivation does not require energy; (c) Probably thiol-groups are involuted in the reactivation process. [ABSTRACT FROM AUTHOR]

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]