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Z Barak

Publications and source records attributed to Z Barak.

At least 37 records · Page 2Linked to original sources
PubMed1996

On the mechanism of leftward frameshifting at several hungry codons.

We have used lacZ reporter genes to assess leftward ribosome frameshifting on sequences containing the quadruplet U UUC followed by several different triplets coding for lysine, isoleucine, or leucine. Limitation for lysine-tRNA provokes leftward frameshifting when the slippery quadruplet is followed by either lysine codon aag or aaa, but not when followed by an isoleucine or leucine codon. Limitation for isoleucine provokes frameshifting when the quadruplet is followed by either isoleucine codon aua or auc, but not when it is followed by a lysine codon. We conclude that the quadruplet promotes shifting when the ribosome is stalled at any "hungry" codon immediately after it. Changing the quadruplet to U AGC, at which peptidyl-tRNA cognate to the AGC triplet will be mismatched at all three anticodon positions if it slips left, abolishes frameshifting when the ribosome is stalled at the next position. We conclude that the U UUC quadruplet promotes frameshifting by virtue of its ability to pair with a left-slipped peptidyl-tRNA. The frameshift promoted by isoleucine-tRNA limitation of the U UUC aua sequence was analyzed by amino acid sequencing of the protein product. It occurs through reading of the Cau histidine codon overlapping the hungry codon from the left. This result rules out a "simultaneous slippage" type of mechanism. It strongly suggests instead that starvation-promoted frameshifting occurs primarily by slippage of peptidyl-tRNA just upstream of the stall site, followed by decoding of the triplet overlapping the stall site from the left or 5' side. A secondary finding is that the last base of the "hungry" codon has a moderate effect on its shiftiness, aag being shiftier than aaa, and aua being shiftier than auc.

Amino Acid Sequence↗
PubMed1996

Metabolic effects of inhibitors of two enzymes of the branched-chain amino acid pathway in Salmonella typhimurium.

The metabolic effects of inhibitors of two enzymes in the pathway for biosynthesis of branched-chain amino acids were examined in Salmonella typhimurium mutant strain TV105, expressing a single isozyme of acetohydroxy acid synthase (AHAS), AHAS isozyme II. One inhibitor was the sulfonylurea herbicide sulfometuron methyl (SMM), which inhibits this isozyme and AHAS of other organisms, and the other was N-isopropyl oxalylhydroxamate (IpOHA), which inhibits ketol-acid reductoisomerase (KARI). The effects of the inhibitors on growth, levels of several enzymes of the pathway, and levels of intermediates of the pathway were measured. The intracellular concentration of the AHAS substrate 2-ketobutyrate increased on addition of SMM, but a lack of correlation between increased ketobutyrate and growth inhibition suggests that the former is not the immediate cause of the latter. The levels of the keto acid precursor of valine, but not of the precursor of isoleucine, were drastically decreased by SMM, and valine, but not isoleucine, partially overcame SMM inhibition. This apparent stronger effect of SMM on the flux into the valine arm, as opposed to the isoleucine arm, of the branched-chain amino acid pathway is explained by the kinetics of the AHAS reaction, as well as by the different roles of pyruvate, ketobutyrate, and the valine precursor in metabolism. The organization of the pathway thus potentiates the inhibitory effect of SMM. IpOHA has strong initial effects at lower concentrations than does SMM and leads to increases both in the acetohydroxy acid substrates of KARI and, surprisingly, in ketobutyrate. Valine completely protected strain TV105 from IpOHA at the MIC. A number of explanations for this effect can be ruled out, so that some unknown arrangement of the enzymes involved must be suggested. IpOHA led to initial cessation of growth, with partial recovery after a time whose duration increased with the inhibitor concentration. The recovery is apparently due to induction of new KARI synthesis, as well as disappearance of IpOHA from the medium.

Acetolactate Synthase↗
PubMed1996

Raising activity of Bacillus thuringiensis var. israelensis against Anopheles stephensi larvae by encapsulation in Tetrahymena pyriformis (Hymenostomatida:Tetrahymenidae).

Toxicity of Bacillus thuringiensis var israelensis (B.t.i.) against surface-feeding mosquito larvae of Anopheles stephensi was enhanced by encapsulation in the protozoan Tetrahymena pyriformis. In the laboratory, larvae died about 8 times faster when exposed to protozoan cells filled with B.t.i. than when exposed to the same concentrations of B.t.i. alone. Best larvicidal activities were achieved with ratios of 1:200-1:500 T. pyriformis cells to B.t.i. spores. The concentration of B.t.i. needed to kill 50% of exposed populations was 4-fold lower with T. pyriformis than with B.t.i. alone in 100 ml-test cups. Toxicity enhancement is very likely a consequence of concentrating B.t.i. insecticidal crystal proteins in T. pyriformis cells and floating them to the water surface in the larval feeding zone. Reduction in the exposure time of B.t.i. to unfavorable field conditions, as a result of the decrease in larval mortality time, might improve the persistence of this biological control agent in nature.

Animals↗
PubMed1994

Protozoan-enhanced toxicity of Bacillus thuringiensis var. israelensis delta-endotoxin against Aedes aegypti larvae.

The toxicity of Bacillus thuringiensis var. israelensis (Bti) in mosquito larvae was enhanced by encapsulation in the protozoan Tetrahymena pyriformis. Aedes aegypti larvae which fed on T. pyriformis loaded with Bti died about three times faster than when fed on the same concentrations of Bti alone due to ingestion of higher toxin concentrations, reflected by shorter death times of exposed populations. The best larvicidal activities were achieved at ratios of cell/spore numbers in the range of 1:200 to 1:500. This enhancement of mortality by preincubation with T. pyriformis was higher at low Bti concentrations or in late third-instar larvae. Ninety minutes of preincubation yielded the best enhancement effect. Toxicity enhancement is very likely a consequence of concentrating large quantities of Bti spores and crystals (containing delta-endotoxin) by T. pyriformis cells and delivering them to the larvae. Shortening larval mortality time by encapsulation in T. pyriformis should reduce the exposure time of Bti to unfavorable field conditions that inactivate its larvicidal activity. Whether this method will indeed improve Bti efficacy is still to be determined.

Aedes↗
PubMed1993

Subunit association in acetohydroxy acid synthase isozyme III.

Acetohydroxy acid synthase isozyme III (AHAS III) from Escherichia coli is composed of large and small subunits (encoded by the genes ilvI and ilvH) in an alpha 2 beta 2 structure. The large (61-kDa) subunit apparently contains the catalytic machinery of the enzyme, while the small (17-kDa) subunit is required for specific stabilization of the active conformation of the large subunit as well as for valine sensitivity. The interaction between subunits has been studied by using purified enzyme and extracts containing subcloned subunits. The association between large and small subunits is reversible, with a dissociation constant sufficiently high to have important experimental consequences: the activity of the enzyme shows a concentration dependence curve which is concave upward, and this dependence becomes linear upon the addition of excess large or small subunits. We estimate that at a concentration of 10(-7) M for each subunit (7 micrograms of enzyme ml-1), the large subunits are only half associated as the I2H2 active holoenzyme. This dissociation constant is high enough to cause underestimation of the activity of AHAS III in bacterial extracts. The true activity of this isozyme in extracts is observed in the presence of excess small subunits, which maintain the enzyme in its associated form. Reexamination of an E. coli K-12 ilvBN+ ilvIH+ strain grown in glucose indicates that AHAS III is the major isozyme expressed. As an excess of small subunits does not influence the apparent Ki for valine inhibition of the purified enzyme, it is likely that valine binds to and inhibits I2H2 rather than inducing dissociation. AHAS I and II seem to show a much lower tendency to dissociate than does AHAS III.

Acetolactate Synthase↗
PubMed1992

Properties of subcloned subunits of bacterial acetohydroxy acid synthases.

The acetohydroxy acid synthase (AHAS) isozymes from enterobacteria are each composed of a large and small subunit in an alpha 2 beta 2 structure. It has been generally accepted that the large (ca. 60-kDa) subunits are catalytic, while the small ones are regulatory. In order to further characterize the roles of the subunits as well as the nature and the specificities of their interactions, we have constructed plasmids encoding the large or small subunits of isozymes AHAS I and AHAS III, each with limited remnants of the other peptide. The catalytic properties of the large subunits have been characterized and compared with those of extracts containing the intact enzyme or of purified enzymes. Antisera to the isolated subunits have been used in Western blot (immunoblot) analyses for qualitative and semiquantitative determinations of the presence of the polypeptides in extracts. The large subunits of AHAS isozymes I and III have lower activities than the intact enzymes: Vmax/Km is 20 to 50 times lower in both cases. However, for AHAS I, most of this difference is due to the raised Km of the large subunit alone, while for AHAS III, it is due to a lowered Vmax. The substrate specificities, R, of large subunits are close to those of the intact enzymes. The catalytic activity of the large subunits of AHAS I is dependent on flavin adenine dinucleotide (FAD), as is that of the intact enzyme, although the apparent affinities of the large subunits alone for FAD are 10-fold lower. Isolated subunits are insensitive to valine inhibition. Nearly all of the properties of the intact AHAS isozyme I or III can be reconstituted by mixing extracts containing the respective large and small subunits. The mixing of subunits from different enzymes does not lead to activation of the large subunits. It is concluded that the catalytic machinery of these AHAS isozymes is entirely contained within the large subunits. The small subunits are required, however, for specific stabilization of an active conformation of the large subunits as well as for value sensitivity.

Acetolactate Synthase↗
PubMed1990

Determination of products of acetohydroxy acid synthase by the colorimetric method, revisited.

The enzyme acetohydroxy acid synthase (AHAS, EC 4.1.3.18) catalyzes two competing reactions of physiological importance: condensation of two molecules of pyruvate to form acetolactate (AL) or condensation of pyruvate and 2-ketobutyrate to form acetohydroxybutyrate (AHB). The activity of AHAS is most frequently analyzed using the Westerfeld method, in which the acetoin formed upon decarboxylation of AL is determined by colorimetric reaction with creatine and alpha-naphthol. However, there has been confusion as to the interpretation of the results of this assay in the presence of both substrates, conditions which lead to formation of both AL and AHB. By applying this assay to enzymatically prepared samples of AL and AHB which have also been analyzed by two other independent methods, we show here that the color yield for AHB in the commonly used assay is 35-40% that for equivalent amounts of acetoin or AL. The relative color yield is not significantly affected by varying the time or temperature of various steps in the color-forming reaction. This information could in principle be used, together with an independent specific assay for AHB, to determine the composition of an AHAS product mixture; it would, however, be less accurate than a simultaneous chromatographic method.

Acetoin↗
PubMed1990

Induction of an ATP-polymerizing enzyme in TMV-infected tobacco and its homology to the human 2'-5' A synthetase.

Several reports have indicated that tobacco carries an enzyme (APE) that, in the presence of poly (rI):(rC), polymerizes ATP to oligoadenylates. This paper demonstrates that the tobacco APE system comprises several proteins (estimated sizes: 32, 42, 67, and 84 +/- 10% kD). Only one of these proteins (the "67-kD" form) binds to poly (rI):(rC). This APE form has been purified by affinity chromatography on a synthetic ds-RNA column. Four tobacco proteins, including the purified one, crossreact with antibodies against the human enzyme, 2'-5' A synthetase. The ATP-binding capacity of some of these proteins has also been demonstrated. The amount of plant oligoadenylates obtained by polymerizing ATP with the purified APE form allows, for the first time, their direct analysis by TLC. The TLC analysis indicated that the oligomer produced by APE is not identical to the 2'-5' oligoadenylate. The appearance of the 2'-5' A-related proteins correlates with the build up of TMV infection, and the pattern of their stimulation and turnover was established. Nucleic acid hybridization indicates homology of tobacco DNA and RNA sequences with cloned cDNA of the human 2'-5' A synthetase gene. The stimulation in tobacco, upon TMV infection, of mRNA species homologous to the above human cDNA has been demonstrated. The analogy between the plant and the human system is discussed.

Adenine Nucleotides↗
PubMed1990

The fate of Bacillus thuringiensis var. israelensis in B. thuringiensis var. israelensis-killed pupae of Aedes aegypti.

Carcasses of mosquito larvae killed by Bacillus thuringiensis var. israelensis allow its complete growth cycle (germination, vegetative growth, and sporulation), thus becoming toxic themselves to scavenging larvae. In this study, we demonstrate that the bacterium is capable of inducing death of Aedes aegypti pupae and of recycling in the resulting carcasses. B. thuringiensis var. israelensis-killed pupae were obtained by treating 40-hr-old synchronized fourth instar larvae with a low dose of spores (8000/ml). The fraction of dead pupae was reduced by higher or lower spore concentrations as well as by treating younger or older larval populations (both fourth instar): Increased proportions of dead larvae were obtained at higher concentration or by earlier treatment, whereas lower concentrations or later treatment resulted in more living pupae. Multiplication of B. thuringiensis var. israelensis is shown to occur in the carcasses of dead pupae. The number of spores in each pupal carcass followed a similar kinetic as in larval carcasses, but the final yield was about 10-fold higher, apparently reflecting the difference in dry weight between the two mosquito developmental stages (426 micrograms vs 83 micrograms, respectively). The specific larvicidal activity in a homogenized dead pupa was similar to that of B. thuringiensis var. israelensis powder, LC50 of about 600 spores/ml.

Aedes↗
PubMed1990

Acetohydroxy Acid Synthase Activity in Chlorella emersonii under Auto- and Heterotrophic Growth Conditions.

Acetohydroxyacid synthase (AHAS) activity was studied in the green unicellular alga Chlorella emersonii. This activity and its regulation was compared in the algae grown autotrophically and heterotrophically on glucose in the dark. No evidence for the existence of more than one enzyme was found. The activity in crude extracts from either heterotrophically or autotrophically grown cells showed a K(m) for pyruvate of 9 millimolar, a 22-fold preference for 2-ketobutyrate over pyruvate as the second substrate, 50% inhibition by 0.5 millimolar valine, and 50% inhibition by 0.3 micromolar sulfometuron methyl (SMM). Spontaneous mutants of the alga resistant to SMM were isolated, which appeared to be single gene mutants containing SMM-resistant AHAS activity. Hence, AHAS appears to be the sole direct target site of SMM in C. emersonii. The fact that the mutants had equivalent SMM resistance under auto- and heterotrophic conditions further supports the conclusion that the same enzyme functions under both physiological regimes. The addition of valine and isoleucine leads to partial relief of SMM inhibition of biomass increase, but not of SMM inhibition of cell division.

Journal Article↗
PubMed1990

Physiological implications of the substrate specificities of acetohydroxy acid synthases from varied organisms.

Acetohydroxy acid synthase (AHAS; EC 4.1.3.18) catalyzes the following two parallel, physiologically important reactions: condensation of two molecules of pyruvate to form acetolactate (AL), in the pathway to valine and leucine, and condensation of pyruvate plus 2-ketobutyrate to form acetohydroxybutyrate (AHB), in the pathway to isoleucine. We have determined the specificity ratio R with regard to these two reactions (where VAHB and VAL are rates of formation of the respective products) as follows: VAHB/VAL = R [2-ketobutyrate]/[pyruvate] for 14 enzymes from 10 procaryotic and eucaryotic organisms. Each organism considered has at least one AHAS of R greater than 20, and some appear to contain but a single biosynthetic AHAS. The implications of this for the design of the pathway are discussed. The selective pressure for high specificity for 2-ketobutyrate versus pyruvate implies that the 2-ketobutyrate concentration is much lower than the pyruvate concentration in all these organisms. It seems important for 2-ketobutyrate levels to be relatively low to avoid a variety of metabolic interferences. These results also reinforce the conclusion that biosynthetic AHAS isozymes of low R (1 to 2) are a special adaptation for heterotrophic growth on certain poor carbon sources. Two catabolic "pH 6 AL-synthesizing enzymes" are shown to be highly specific for AL formation only (R less than 0.1).

Acetolactate Synthase↗
PubMed1989

Kinetics and mechanism of acetohydroxy acid synthase isozyme III from Escherichia coli.

Acetohydroxy acid synthase (AHAS, EC 4.1.3.18) isozyme III from Escherichia coli has been studied in steady-state kinetic experiments in which the rates of formation of acetolactate (AL) and acetohydroxybutyrate (AHB) have been determined simultaneously. The ratio between the rates of production of the two alternative products and the concentrations of the substrates pyruvate and 2-ketobutyrate (2KB) leading to them, R, VAHB/VAL = R[( 2KB]/[pyruvate]), was found to be 40 +/- 3 under a wide variety of conditions. Because pyruvate is a common substrate in the reactions leading to both products and competes with 2-ketobutyrate to determine whether AL or AHB is formed, steady-state kinetic studies are unusually informative for this enzyme. At a given pyruvate concentration, the sum of the rates of formation of AL and AHB was nearly independent of the 2-ketobutyrate concentration. On the basis of these results, a mechanism is proposed for the enzyme that involves irreversible and rate-determining reaction of pyruvate, at a site which accepts 2-ketobutyrate poorly, if at all, to form an intermediate common to all the reactions. In the second phase of the reaction, various 2-keto acids can compete for this intermediate to form the respective acetohydroxy acids. 2-Keto acids other than the natural substrates pyruvate and 2-ketobutyrate may also compete, to a greater or lesser extent, in the second phase of the reaction to yield alternative products, e.g., 2-ketovalerate is preferred by about 2.5-fold over pyruvate. However, the presence of an additional keto acid does not affect the relative specificity of the enzyme for pyruvate and 2-ketobutyrate; this further supports the proposed mechanism. The substrate specificity in the second phase is an intrinsic property of the enzyme, unaffected by pH or feedback inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetolactate Synthase↗
PubMed1989

Structural correlates of a regulatory idiotope.

The A48RI expressed on the ABPC48 and UPC10 beta 2----6 fructosan-binding myeloma proteins is a conformational antigenic determinant encoded by V genes deriving from the VHX24 and VK10 families. In the preimmune repertoire the clones using VHX24 genes rarely express A48 idiotopes, clearly demonstrating that this regulatory idiotope is a minor or silent idiotope. Furthermore, these same VHX24-utilizing preimmune clones are frequently associated with the VK1 gene family which is highly represented in the neonatal and adult repertoires. The clonal expansion occurring subsequent to neonatal injection of minute amounts of anti-Id antibodies leads to selective expansion of A48Id+ clones associated with class switching. Few somatic mutations are observed in preimmune clones, or in those expanded by anti-Id antibodies. The fact that few mutations were observed in the IgG1 clones obtained from animals injected with anti-A48Id antibodies after birth indicates that, in contrast to antigen-induced class-switching, the anti-Id-induced switching is not associated with a highly active mutational process. In contrast to the preimmune clones, or those expanded by anti-Id (in the absence of antigenic stimulation) in which VHX24 is associated with VK regions deriving from various gene families, the clones expanded by anti-Id and fructan resemble A48 by using VHX24 and VK10 genes. Few apparent mutations were also observed in these IgM or IgG3 clones expressing A48 idiotopes. The A48 RI can be expressed on clones producing antibodies specific for various self and foreign antigens, and encoded by V genes deriving from various VH and VK families. These results indicate that key contacting residues bearing A48 conformational idiotypic determinants can be made up by various VH-VK combinations. A comparison of the VH and VL sequences of A48 RI+ mAbs showed that many of the observed somatic mutations could be correlated to decreased IDA10 binding. This comparison allowed identification of specific idiotope-determining regions of VH and VK which could represent contacting residues with anti-idiotypic antibodies. The contributions of these regions to the expression of the A48Id was tested by generating a transfectoma antibody expressing the rearranged VHJ558 gene of the ricin 45 hybridoma and the VK10-Ars-a gene of the 36-65 hybridoma. This transfectoma antibody expresses the idiotope recognized by IDA10 and confirms the conformational nature of this idiotope. There are three amino acid residues shared by VHX24 and VHJ558 antibodies expressing the A48 RI which are important for its expression.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗