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H Jakubowski

Publications and source records attributed to H Jakubowski.

At least 55 records · Page 3Linked to original sources
PubMed1989

Effect of variation of charged and uncharged tRNA(Trp) levels on ppGpp synthesis in Escherichia coli.

We introduced into a stringent Escherichia coli tryptophan auxotroph a plasmid bearing the tRNA(Trp) gene under the control of an inducible promoter. This allows us to manipulate the total concentration of tRNA(Trp) in the cell according to whether and when inducer is added to the culture. We also manipulated the concentration of Trp-tRNA(Trp) in vivo since the strain used bears a mutation in the Trp-tRNA synthetase affecting the Km for tryptophan, such that varying the exogenous concentration of tryptophan led to variation in the level of Trp-tRNA(Trp) in the cell. With this system, we found that the signal eliciting ppGpp synthesis during a stringent response triggered by tryptophan limitation did not depend on the absolute concentration of either charged or uncharged tRNA(Trp) but rather depended on a decline in the ratio of charged/uncharged tRNA(Trp). In addition, we found that the amplitude of the response, once triggered by tryptophan limitation, was determined by the total concentration of tRNA(Trp) present in the cell (which is mostly uncharged at that point in time). However, excess uncharged tRNA(Trp) did not amplify ppGpp synthesis triggered by limitation of a different amino acid. These data provide in vivo support for the in vitro-derived model of ppGpp synthesis on ribosomes.

Bacterial Proteins↗
PubMed1988

Negative correlation between the abundance of Escherichia coli aminoacyl-tRNA families and their affinities for elongation factor Tu-GTP.

The number of aminoacyl-tRNA molecules in Escherichia coli cells varies by about one order of magnitude from 730 (glutaminyl-tRNA) to 7900 (valyl-tRNA). Relative affinities of E. coli aminoacyl-tRNA for elongation factor Tu-GTP vary also by about one order of magnitude from 2.08 (glutaminyl-tRNA) to 0.15 (valyl-tRNA). The relationship between the abundance of all 20 aminoacyl-tRNA families in 5 E. coli strains and their affinities for elongation factor Tu-GTP was examined by statistical methods. Negative correlation between the two parameters was found. The correlation coefficient was -0.62 to -0.52 with significance level 0.01. Regression analysis give the following formula for the relation between relative abundance of aminoacyl-tRNA families (x) and their relative affinities for elongation factor Tu-GTP (y): y = 1.25 - 0.25x. The analyses indicate that those aminoacyl-tRNA families that are present in cells in low copy number exhibit higher affinity than the more abundant aminoacyl-tRNA families for elongation factor Tu-GTP. The bacterial protein biosynthetic apparatus evolved in such a way as to compensate for a low copy number of some aminoacyl-tRNAs by tight binding of the aminoacyl-tRNA to elongation factor Tu-GTP. This may assure adequate supply of low copy number aminoacyl-tRNAs under conditions of limitation in elongation factor Tu-GTP, e.g. during stringent response, and is consistent with the idea of elongation factor Tu-GTP modulating translational efficiencies of aminoacyl-tRNAs.

Escherichia coli↗
PubMed1988

Synthesis of diadenosine 5',5'''-P1,P4-tetraphosphate (AppppA) from adenosine 5'-phosphosulfate and adenosine 5'-triphosphate catalyzed by yeast AppppA phosphorylase.

A novel way of enzymatic synthesis of diadenosine 5',5"'-P1,P4-tetraphosphate (AppppA), which does not involve aminoacyl-tRNA synthetases, has been discovered. Yeast AppppA alpha, beta-phosphorylase catalyzes irreversible conversion of adenosine 5'-phosphosulfate (APS) and ATP into AppppA according to the equation APS + ATP----AppppA + sulfate. In this reaction, the enzyme exhibits a broad pH optimum (between 6 and 8) and requires Mn2+, Mg2+, or Ca2+ ions for activity, with Mn2+ being twice as effective as Mg2+ or Ca2+ at optimal concentration (0.5 mM). The Km values computed for APS and ATP are 80 microM and 700 microM, respectively. The rate constant for the AppppA synthesis is 3 s-1 (pH 8.0, 30 degrees C, 0.5 mM MgCl2). Some ATP analogues like ppppA, GTP, adenosine 5'-(alpha, beta-methylenetriphosphate), and adenosine 5'-(beta, gamma-methylenetriphosphate), but not dATP, UTP, or CTP, are also substrates for AppppA phosphorylase and accept adenylate from APS with the formation of AppppA, AppppG, Appp(CH2)pA, and App(CH2)ppA, respectively. Functional versatility of yeast AppppA phosphorylase may provide a link between metabolism of AppppA on one hand and metabolism of APS and phosphate on the other and raises the possibility of participation of AppppA in regulation of metabolism of APS and/or inorganic phosphate in yeast.

Adenine Nucleotides↗
PubMed1988

Evidence for cooperation between cells during sporulation of the yeast Saccharomyces cerevisiae.

Diploid Saccharomyces cerevisiae cells heterozygous for the mating type locus (MATa/MAT alpha) undergo meiosis and sporulation when starved for nitrogen in the presence of a poor carbon source such as potassium acetate. Diploid yeast adenine auxotrophs sporulated well at high cell density (10(7) cells per ml) under these conditions but failed to differentiate at low cell density (10(5) cells per ml). The conditional sporulation-deficient phenotype of adenine auxotrophs could be complemented by wild-type yeast cells, by medium from cultures that sporulate at high cell density, or by exogenously added adenine (or hypoxanthine with some mutants). Adenine and hypoxanthine in addition to guanine, adenosine, and numerous nucleotides were secreted into the medium, each in its unique temporal pattern, by sporulating auxotrophic and prototrophic yeast strains. The major source of these compounds was degradation of RNA. The data indicated that differentiating yeast cells cooperate during sporulation in maintaining sufficiently high concentrations of extracellular purines which are absolutely required for sporulation of adenine auxotrophs. Yeast prototrophs, which also sporulated less efficiently at low cell density (10(3) cells per ml), reutilized secreted purines in preference to de novo-made purine nucleotides whose synthesis was in fact inhibited during sporulation at high cell density. Adenine enhanced sporulation of yeast prototrophs at low cell density. The behavior of adenine auxotrophs bearing additional mutations in purine salvage pathway genes (ade apt1, ade aah1 apt1, ade hpt1) supports a model in which secretion of degradation products, uptake, and reutilization of these products is a signal between cells synchronizing the sporulation process.

Adenine↗
PubMed1987

Phosphonate analogues of diadenosine 5',5'''-P1,P4-tetraphosphate as substrates or inhibitors of procaryotic and eucaryotic enzymes degrading dinucleoside tetraphosphates.

The substrate specificity of procaryotic and eucaryotic AppppA-degrading enzymes was investigated with phosphonate analogues of diadenosine 5',5'''-P1,P4-tetraphosphate (AppppA). App(CH2)ppA (I), App(CHBr)ppA (II), and Appp(CH2)pA (III), but not Ap(CH2)pp(CH2)pA (IV), are substrates for lupin AppppA hydrolase (EC 3.6.1.17) and phosphodiesterase I (EC 3.1.4.1). None of the four analogues is hydrolyzed by bacterial AppppA hydrolase (EC 3.6.1.41), and only analogue III is degraded by yeast AppppA phosphorylase (EC 2.7.7.53). The analogues are competitive inhibitors of all four enzymes. The affinity of analogue IV is 3-40-fold lower than that of analogues I-III for all four enzymes. Introduction of one methylene (as in I and III) [or bromomethylene (as in II)] group into AppppA results in a 3-15-fold increase of its affinity for lupin and Escherichia coli AppppA hydrolases. The same modifications only negligibly (10-30%) affect its affinity for yeast AppppA phosphorylase and decrease its affinity for lupin phosphodiesterase I about 2.5-fold. The data provide further evidence for the heterogeneity among catalytic sites of all four AppppA-degrading enzymes.

Acid Anhydride Hydrolases↗
PubMed1986

Sporulation of the yeast Saccharomyces cerevisiae is accompanied by synthesis of adenosine 5'-tetraphosphate and adenosine 5'-pentaphosphate.

Two-dimensional TLC analysis of 32P-labeled nucleotides extracted from the yeast Saccharomyces cerevisiae reveals that two highly phosphorylated nucleotides are synthesized during sporulation. These nucleotides have been identified as adenosine 5'-tetraphosphate (ppppA) and adenosine 5'-pentaphosphate (pppppA). The synthesis of ppppA and pppppA commences late in sporulation and follows formation of ascospores. The maximum concentration of ppppA and pppppA in sporulating yeast cultures was 2% and 1.5%, respectively, that of ATP. Adenosine 5'-tetraphosphate and 5'-pentaphosphate are unique to this stage of yeast development and are absent in vegetative yeast cells. Since these nucleotides are also absent in asporogenous a/a and alpha/alpha cells, it is reasonable to propose that they are signal nucleotides marking one of the stages of yeast development--i.e., ascospore formation.

Adenine Nucleotides↗
PubMed1984

Quantities of individual aminoacyl-tRNA families and their turnover in Escherichia coli.

The cellular content of all 20 aminoacyl-tRNA species was determined in small cultures of Escherichia coli by labeling cells with 3H-amino acids and extraction of 3H-amino acid-labeled nucleic acid by standard procedures. Of 3H-amino acid-labeled material, 25 to 90% was identified as 3H-aminoacyl-tRNA by the following criteria: sensitivity to base hydrolysis with expected kinetics; association of 3H counts released by base treatment of the 3H-amino acid-labeled nucleic acid with amino acid standards upon paper chromatography of the hydrolysate; and changes in the amount of 3H-amino acid-labeled nucleic acid recovered from cells as a function of time. Individual aminoacyl-tRNA content was determined with as few as 8 X 10(7) to 4 X 10(8) E. coli cells. Although the total number of aminoacyl-tRNA molecules per cell varied only by 10 to 20% among various strains of E. coli, some individual aminoacyl-tRNA families varied two- to threefold among strains. For a given amino acid, the number of aminoacyl-tRNA molecules per cell in E. coli strain K38 growing with a doubling time of 60 min varied from 730 (glutamyl-tRNA) to 7,910 (valyl-tRNA) with a mean value of 3,200. The total number of aminoacyl-tRNA molecules per cell (6.4 X 10(4)) in E. coli K38 was 5.5-fold higher than the number of ribosomes and was equal to 84% of the amount of elongation factor Tu molecules per cell. The ratio of aminoacyl-tRNA to synthetase for 10 amino acids varied from about 1 to 15 with a mean value of 4.7. The turnover of individual aminoacyl-tRNA families in E. coli cells was estimated to be in the range of 1.7 to 8.1 s-1 with a mean value of 3.7 s-1. An estimate of minimum in vivo molecular activity of aminoacyl-tRNA synthetases gives values of 2 to 48 s-1 for individual enzymes.

Amino Acids↗
PubMed1983

Catabolism of diadenosine 5',5"'-P1,P4-tetraphosphate in procaryotes. Purification and properties of diadenosine 5',5"'-P1,P4-tetraphosphate (symmetrical) pyrophosphohydrolase from Escherichia coli K12.

Enzymatic activity which hydrolyzes diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) yielding ADP has been identified in extracts of eubacteria, Escherichia coli and Acidaminococcus fermentans, and of a highly thermophilic archaebacterium, Pyrodictum occultum. Specific Ap4A (symmetric) pyrophosphohydrolase from Escherichia coli K12 has been purified almost 400-fold. The preparation was free of phosphatase, ATPase, phosphodiesterase, AMP-nucleosidase, and adenylate kinase. The Ap4A pyrophosphohydrolase molecular weight estimated by gel filtration is 27,000 +/- 1,000. Activity maximum is at pH 8.3. The Km value computed for Ap4A is 25 +/- 3 microM. The sulfhydryl group(s) is essential for enzyme activity. Metal chelators, EDTA, and o-phenanthroline, inhibit Ap4A hydrolysis; I0.5 values are 3 and 50 microM, respectively. Co2+ is a strong stimulator with an almost 100-fold increase in rate of Ap4A hydrolysis and a plateau in the range of 100-500 microM Co2+, when compared with the nonstimulated hydrolysis. Other transition metal ions, Mn2+, Cd2+, and Ni2+, stimulate by factors of 8, 3.5, and 3.5, respectively, with optimal concentrations in the range 200-500, 2-5, and 4-8 microM, respectively. Zn2+, Cu2+, and Fe2+, up to 30 microM, are without effect and they inhibit at higher concentrations. Mg2+ or Ca2+, in the absence of other divalent metal ions, are weak stimulators (1.5-fold stimulation occurs at 1-2 mM concentration), but act synergistically with Co2+ at its suboptimal concentrations. Stimulation in the presence of 10 microM Co2+ and either 1 mM MgCl2 or CaCl2 increases up to 75-fold. The same degree of synergy is found at 10 microM Co2+ and either 2-5 mM spermidine or 0.5-1.5 mM spermine. Besides Ap4A, bacterial Ap4A pyrophosphohydrolase hydrolyzes effectively Ap5A and Gp4G, and, to some extent, p4A, Ap6A, and Ap3A yielding in each case corresponding nucleoside diphosphate as one of the products.

Acid Anhydride Hydrolases↗
PubMed1983

Control of RNA and protein synthesis by the concentration of Trp-tRNATrp in Escherichia coli infected with bacteriophage MS2.

The effect of varying the concentration of Trp-tRNATrp in Escherichia coli infected with bacteriophage MS2 has been studied by varying the amount of exogenously added tryptophan (Trp) to cells bearing a mutation which results in a tryptophanyl-tRNA synthetase with a higher Km value for Trp. The phenotype of the mutant has been confirmed by measuring the level of tRNATrp which can be aminoacylated in vivo, and the mutant has also been shown to have elevated tRNATrp levels compared to wild-type. The growth of MS2 decreases continuously with decreasing Trp concentration (and hence, decreasing Trp-tRNATrp concentration). This appears to be due to reduced gene expression, since at later times in infection the amount of MS2 coat protein synthesized likewise decreases continuously with decreasing Trp concentration. However, there is little decrease in the amount of coat protein or replicase synthesized during the first few minutes after the Trp concentration shift. A continuous increase in the average polysome size distribution is seen as the Trp concentration is decreased. MS2 RNA synthesis also decreases continuously with decreasing Trp concentration, and is shut off in the absence of Trp. This does not seem to be due to ppGpp as these cells are functionally relaxed under these conditions, nor does it seem to be due to degradation of pre-existing template. Addition of chloramphenicol abolishes the effect of Trp concentration on RNA synthesis. The data are consistent with a model in which ribosomes pause at Trp codons in the absence of Trp-tRNATrp, while other ribosomes queue behind and continue to load onto the message. The reduction of RNA synthesis would then be a consequence of coupling to translation.

Coliphages↗
PubMed1983

Enzymes hydrolyzing ApppA and/or AppppA in higher plants. Purification and some properties of diadenosine triphosphatase, diadenosine tetraphosphatase, and phosphodiesterase from yellow lupin (Lupinus luteus) seeds.

Three distinct enzymes hydrolyzing either ApppA or AppppA, or both, were separated and purified from yellow lupin seed extracts. Two of the enzymes were purified to homogeneity. These enzymes differ greatly in their catalytic and physical properties. One hydrolase, with a native molecular weight of 41,000, exhibits broad pH (from 5-8) optimum for activity, requires Mg2+ for activity, is inhibited by zinc ions (I0.5 = 25 microM) and hydrolyses ApppA (V = 1), ApppC (V = 0.38), ApppG (V = 0.2), and ribose(5')pppA (V = 0.2). The enzyme exhibits much lower activity with AppppA (V = 0.1), and ApppppA, AppppppA, ppppA, and ATP are hydrolyzed 25- to 100-fold slower then ApppA. ADP was always one of the products of the reactions catalyzed by the enzyme. AppA, NAD, NADP, FAD, cAMP, and p-nitrophenyl-thymidine 5'-phosphate were not hydrolyzed by the enzyme. The enzyme is diadenosine 5',5"'-P1, P3-triphosphatase. The second hydrolase, composed of one polypeptide chain of a molecular weight 18,000-18,500, exhibits optimal activity in the pH range from 7.5-9, requires Mg2+ for activity, is inhibited by calcium ions (I0.5 for calcium depends on the concentration of Mg2+ and is 35-180 microM in the presence of 0.5-10 mM Mg2+, respectively), and hydrolyzes AppppA (V = 1, Km = 1 microM), ApppppA (V = 0.42, Km = 1.8 microM), AppppppA (V = 0.34), AppppU (V = 0.73), AppppC (V = 0.67), AppppG (V = 0.27), and ppppA. ATP was always one of the products of the reactions catalyzed by the enzyme. Dinucleoside di- and triphosphates, ATP, cAMP, and p-nitrophenylthymidine 5'-phosphate were not hydrolyzed by the enzyme. This enzyme is diadenosine 5',5"'-P1,P4-tetraphosphatase (EC 3.6.1.17). The third hydrolase, composed of one polypeptide chain of a molecular weight of 56,000, exhibits maximal activity at pH 9-10.5, does not require Mg2+ ions for activity, is inhibited neither by divalent cations (Mg2+, Ca2+, Zn2+, Co2+, Mn2+, or Ni2+) nor by EDTA, and uses as substrates all compounds which are substrates for the diadenosine 5',5"'-P1,P3-triphosphatase and diadenosine 5',5"'-P1,P4-tetraphosphatase. In addition, the enzyme hydrolyzes p-nitrophenyl-thymidine 5'-phosphate, p-nitrophenylthymidine 3'-phosphate, bis-p-nitrophenylphosphate, ADP, AppA, NAD, NADP, and FAD, but not cAMP. With the exception of p-nitrophenylphosphate derivatives all other substrates of the enzyme yield AMP as one of the products of hydrolysis. This enzyme has a specificity similar to that of phosphodiesterases (EC 3.1.4.1) from other sources. With the lupin phosphodiesterase, ApppA (V = 1, Km = 2.2 microM) and AppppA (V = 1, Km = 2.0 microM) are better substrates than NAD (V = 0.8, Km = 9.6 microM), AppA (V = 0.4), ApppppA (V = 0.6), and AppppppA (V = 0.34).

Acid Anhydride Hydrolases↗
PubMed1983

Substrate specificity of S-adenosylhomocysteinase. Cysteine is a substrate of the plant and mammalian enzymes.

Substrate specificity of S-adenosylhomocysteinases (S-adenosyl-L-homocysteine hydrolase, EC 3.3.1.1) with respect to amino acid has been studied using homogeneous preparations of the enzymes from yellow lupin (Lupinus luteus) seeds and bovine liver. Both enzymes use cysteine, in addition to homocysteine, as a substrate. Homoserine, serine, pinicillamine, reduced glutathione and 2-mercaptoethanol are not substrates. In the presence of cysteine, the reaction of S-adenosylthio-amino acid synthesis is characterized by 20-40-fold lower kcat values (kcat = 0.23 s-1 or 0.11 s-1 in the presence of cysteine and either bovine or lupin enzyme) and 270-250-fold higher Km values (Km for cysteine is 15 mM and 35 mM with bovine and lupin enzyme, respectively) than the reaction in the presence of the normal substrate, homocysteine. In the reverse reaction, S-adenosylcysteine is hydrolyzed by the mammalian enzyme much faster than by the plant one. Specificity (kcat/Km) towards S-adenosylcysteine and S-adenosylhomocysteine is 0.9 M-1 . s-1 and 60 000 M-1 . s-1, respectively, with the plant enzyme and 15.3 M-1 . s-1 and 70 000 M-1 . s-1, respectively, with the mammalian enzyme. With plant enzyme, the reactions with cysteine and homocysteine are not competitive, i.e., cysteine does not inhibit the synthesis of S-adenosylhomocysteine, and homocysteine does not inhibit the synthesis of S-adenosylcysteine. This is consistent with independent binding of cysteine and homocysteine to both enzyme subunits. Using adenosine analogs and the mammalian S-adenosylhomocysteinase we were able to synthesize a number of novel S-adenosylcysteine analogs. These included: S-N6-hydroxyadenosyl-L-cysteine, S-2-aminoadenosyl-L-cysteine, S-nebularyl-L-cysteine, S-3-deazaadenosyl-L-cysteine, S-formycyl-L-cysteine, S-N6-methyladenosyl-L-cysteine and S-N1-oxideadenosyl-L-cysteine.

Adenosylhomocysteinase↗
PubMed1982

The mechanism of inhibition of valyl-tRNA synthetase by S-adenosylhomocysteine.

S-Adenosylhomocysteine inhibits ATP-PPi exchange and tRNA aminoacylation reactions catalysed by pure lupin valyl-tRNA synthetase. The reactions catalysed by phenylalanyl-tRNA, tyrosyl-tRNA, seryl-tRNA, arginyl-tRNA and tryptophanyl-tRNA synthetases are not inhibited by S-adenosylhomocysteine. The inhibition is proportional to first power of S-adenosylhomocysteine concentration. S-Adenosylhomocysteine is a competitive inhibitor of the lupin valyl-tRNA synthetase with respect to ATP and valine in the ATP-PPi exchange reaction, noncompetitive with respect to ATP and valine and uncompetitive with respect to tRNA in the tRNA aminoacylation reaction. Inhibition constant for S-adenosylhomocysteine determined from the reciprocal of Km for tRNA vs. the inhibitor concentration (at saturating ATP and not saturating valine concentration) is 0.35 mM. Inhibition constants for S-adenosylhomocysteine determined from the ATP-PPi exchange are 0.21 mM at saturating valine concentration with variable ATP and 0.33 mM at saturating ATP concentration with variable valine. The data are consistent with the binding of S-adenosylhomocysteine to the valyl adenylate site on the enzyme.

Amino Acyl-tRNA Synthetases↗
PubMed1981

S-Adenosylhomocysteinase from yellow lupin seeds: stoichiometry and reactions of the enzyme-adenosine complex.

Plant (Lupinus luteus) S-adenosylhomocysteinase, an alpha 2 dimer, forms a 1:2 enzyme-adenosine complex. The binding sites for adenosine are not equivalent. Binding of the first molecule of adenosine is fast (k greater than 7 x 10(5) M-1 s-1), whereas the second molecule of adenosine binds in a slow process with a half-life of 5 min. Adenosine in the 1:1 and 1:2 enzyme--substrate complexes reacts slowly (k = 0.05 min-1) to give finally free enzyme, adenine, and ribose. The enzyme does not lose its ability to catalyze the synthesis of S-adenosylhomocysteine during the reactions. The relevance of the data to the catalytic functioning of the plant S-adenosylhomocysteinase is discussed.

Adenosine↗
PubMed1981

Alternative pathways for editing non-cognate amino acids by aminoacyl-tRNA synthetases.

Evidence is presented that the editing mechanisms of aminoacyl-tRNA synthetase operate by two alternative pathways: pre-transfer, by hydrolysis of the non-cognate aminoacyl adenylate; post-transfer, by hydrolysis of the mischarged tRNA. The methionyl-tRNA synthetases from Escherichia coli and Bacillus stearothermophilus and isoleucyl-tRNA synthetase from E. coli, for example, are shown to reject misactivated homocysteine rapidly by the pre-transfer route. A novel feature of this reaction is that homocysteine thiolactone is formed by the facile cyclisation of the homocysteinyl adenylate. Valyl-tRNA synthetases, on the other hand, reject the more readily activated non-cognate amino acids by primarily the post-transfer route. The features governing the choice of pathway are discussed.

Amino Acids↗
PubMed1981

Gel mapping of wheat and lupin tRNAs.

Transfer RNAs were isolated from plants representing mono- and dicotyledons: wheat embryos and lupin seeds. The two tRNA preparations were compared by polyacrylamide-gel-electrophoresis mapping. Transfer RNAs extracted from the separated spots were tested for acceptance of 11 amino acids. Comparison of electrophoretic mobility of respective tRNA species indicates the overall similarity of tRNA populations in the two plants studied. Especially, isoaccepting tRNAs for glycine, tyrosine and valine and some of isoacceptors of tRNAArg, tRNAAsp, tRNALeu, tRNALys and tRNAPhe occupy identical or closely similar positions on both polyacrylamide-gel maps. However, some tRNA isoacceptors from one population have no counterparts in the second one, which may indicate differences in their primary structures.

Electrophoresis, Polyacrylamide Gel↗