Disappearance of glyceraldehyde 3-phosphate dehydrogenase from erythrocyte membrane by hemolysis with thermostable direct hemolysin of Vibrio parahaemolyticus or Vibrio cholerae El Tor hemolysin.

It is believed that the thermostable direct hemolysin (TDH) of Vibrio parahaemolyticus and El Tor hemolysin (ETH) of Vibrio cholerae damage erythrocytes and other cells by acting as pore-forming toxins. In this study, we found that a protein band with a molecular weight of 37,000 daltons specifically disappeared from erythrocyte membrane after hemolysis by TDH and ETH, but not after hypotonic hemolysis. The 37 kDa band was identified as glyceraldehyde 3-phosphate dehydrogenase (G3PD), a glycolytic enzyme, based on N-terminal 14 amino acid sequencing. The role of G3PD in hemolysis is discussed.     

Differential expression of the three yeast glyceraldehyde-3-phosphate dehydrogenase genes

Utilizing yeast strains containing insertion mutations in each of the three glyceraldehyde-3-phosphate dehydrogenase structural genes, the level of expression of each gene was determined in logarithmically growing cells. The contribution of the TDH1, TDH2, and TDH3 gene products to the total glyceraldehyde-3-phosphate dehydrogenase activity in wild type cells is 10-15, 25-30, and 50-60%, respectively. The relative proportions of expression of each gene is the same in cells grown in the presence of glucose or ethanol as carbon source although the total glyceraldehyde-3-phosphate dehydrogenase activity in cells grown in the presence of glucose is 2-fold higher than in cells grown on ethanol. The polypeptides encoded by each of the structural genes were identified by two-dimensional polyacrylamide gel electrophoresis. The TDH3 structural gene encodes two resolvable forms of glyceraldehyde-3-phosphate dehydrogenase which differ by their net charge. The apparent specific activity of glyceraldehyde-3-phosphate dehydrogenase encoded by the TDH3 structural gene is severalfold lower than the enzymes encoded by TDH1 or TDH2. The polypeptides encoded by the TDH2 or TDH3 structural genes form catalytically active homotetramers. The apparent Vmax for the homotetramer encoded by TDH3 is 2-3-fold lower than the homotetramer encoded by TDH2. Evidence is presented that isozymes of glyceraldehyde-3-phosphate dehydrogenase exist in yeast cells, however, the number of different isozymes formed was not established. These data confirm that the three yeast glyceraldehyde-3-phosphate dehydrogenase genes encode catalytically active enzyme and that the genes are expressed at different levels during logarithmic cell growth.     

Starvation and temperature upshift cause an increase in the enzymatically active cell wall-associated glyceraldehyde-3-phosphate dehydrogenase protein in yeast

The cell wall-associated glyceraldehyde-3-phosphate dehydrogenase (cwGAPDH) activity in Saccharomyces cerevisiae increases (two- to 10-fold, depending on the strain) in response to starvation and temperature upshift. Assays using transformants carrying pTDH, a yeast centromer derivative plasmid containing the Candida albicans TDH3 gene (encoding GAPDH) fused in frame with the yeast SUC2-coding region for internal invertase, showed that starvation and/or temperature upshift result in a similar increase in both cwGAPDH and cell wall-associated invertase activities. In addition, this incorporation of GAPDH protein into the cell wall in response to stress does not require (i) de novo protein synthesis, indicating that preexisting cytosolic enzyme is incorporated into the cell wall, (ii) nor the participation of the ubiquitin yeast stress response system, as no differences were observed between wild-type and polyubiquitin-depleted (Deltaubi4) strains.     

Proposed Enzymes of Auxin Biosynthesis and Their Regulation II. Tryptophan Dehydrogenase Activity in Plants.

In pea, maize and tomato plants a hitherto undescribed L-tryptophan dehydrogenase activity (TDH) has been detected. This enzyme catalyzes the reversible formation of indolepyruvic acid (IPyA) from L-tryptophan (L-trp). TDH and L-glutamate dehydrogenase (GDH), related enzymes in their mode of action, could be separated by gel chromatography. Enzymatic activity of TDH was sustained by both pyridine coenzymes NAD/NADP. With pea TDH the coenzyme NAD displays, at optimum pH 8.5 and at room temperature, only about 40-70 % of the activity of NADP. The amination of IPyA is catalysed more actively than the deamination of L-trp. L-trp/IPyA, L-glu/ketoglutarate, L-ala/pyruvate reacted as dehydrogenase substrates; L-phe/ phenylpyruvate, D-trp and D-phe did not react with pea enzyme extracts. A considerable similarity between the active centres of TDH and GDH has been found using inhibitors: absence of heavy metals, presence of a carbonyl group, indispensibility of bivalent ions for the enzyme activity. Pea TDH and GDH were distinctly inhibited by sodium azide. For the activity of TDH the presence of SH groups is less important than for GDH. The TDH activity in the investigated plants was lower than the GDH activity. The possible role of TDH in the regulation of the IPyA pool is discussed.Doc. RNDr. PhMr. M. Kutáček died on 28 November, 1989. The final form for print was prepared by dr. Ivana Machdckovd of the same Institute, who will also answer the reprint requests. Received June 6, 1990; accepted October 10, 1990     

Woodward's reagent K inactivation of Escherichia coli L-threonine dehydrogenase: increased absorbance at 340-350 nm is due to modification of cysteine and histidine residues, not aspartate or glutamate carboxyl groups.

L-Threonine dehydrogenase (TDH) from Escherichia coli is rapidly inactivated and develops a new absorbance peak at 347 nm when incubated with N-ethyl-5-phenylisoxazolium-3'-sulfonate (Woodward's reagent K, WRK). The cofactors, NAD+ or NADH (1.5 mM), provide complete protection against inactivation; L-threonine (60 mM) is approximately 50% as effective. Tryptic digestion of WRK-modified TDH followed by HPLC fractionation (pH 6.2) yields four 340-nm-absorbing peptides, two of which are absent from enzyme incubated with WRK and NAD+. Peptide I has the sequence TAICGTDVH (TDH residues 35-43), whereas peptide II is TAICGTDVHIY (residues 35-45). Peptides not protected are TMLDTMNHGGR (III, residues 248-258) and NCRGGRTHLCR (IV, residues 98-108). Absorbance spectra of these WRK-peptides were compared with WRK adducts of imidazole, 2-hydroxyethanethiolate, and acetate. Peptides III and IV have pH-dependent lambda max values (340-350 nm), consistent with histidine modification. Peptide I has pH-independent lambda max (350 nm) indicating that a thiol is modified. WRK, therefore, does not react specifically with carboxyl groups in this enzyme, but rather modifies Cys-38 in the active site of TDH; modification of His-105 and His-255 does not affect enzyme activity. These results are the first definitive proof of WRK modifying cysteine and histidine residues of a protein and show that enzyme inactivation by WRK associated with the appearance of new absorptivity at 340-350 nm does not establish modification of aspartate or glutamate residues, as has been assumed in numerous earlier reports.     

Isolation and characterization of yeast strains carrying mutations in the glyceraldehyde-3-phosphate dehydrogenase genes

Mutant yeast strains were constructed which carry insertion mutations in each of the glyceraldehyde-3-phosphate dehydrogenase structural genes which have been designated TDH1, TDH2, and TDH3. Haploid strains carrying mutations in TDH1 and TDH2 as well as TDH1 and TDH3 were isolated from crosses between strains carrying the appropriate single mutations. The three single mutants as well as the two double mutants grow at wild type rates when ethanol is used as carbon source. Mutant strains lacking only a functional TDH2 allele or a TDH3 allele grow at 50 and 75% of the rate observed for wild type cells, respectively, when glucose is used as carbon source. No growth phenotype was observed for strains lacking only a functional TDH1 allele when either fermentable or nonfermentable carbon sources were used. Evidence is presented that strains lacking functional TDH2 and TDH3 alleles are not viable. These data demonstrate that the presence of a functional TDH2 or TDH3 allele is required for cell growth.     

Biochemical characteristics and function of a threonine dehydrogenase encoded by ste11 in Ebosin biosynthesis of Streptomyces sp. 139

Aims:  Ebosin, a novel exopolysaccharide (EPS) produced by Streptomyces sp. 139 has antagonistic activity for interleukin-1 receptor (IL-1R) in vitro and remarkable anti-rheumatic arthritis activity in vivo. Ebosin biosynthesis gene ( ste ) cluster has been identified in our laboratory. This paper reports our effort to characterize the function of ste11 gene.
Methods and Results:  After the ste11 gene was cloned and expressed in Escherichia coli BL21, the recombinant Ste11 was purified and found capable of catalyzing NAD⁺ and l -threonine to NADH and 2-amino-3-ketobutyrate, hence identified as a threonine dehydrogenase (TDH). To investigate its function in the biosynthesis of Ebosin, the ste11 gene was knocked out with a double crossover via homologous recombination. The monosaccharide composition of EPS produced by the mutant strain (EPS-m) was altered from that of Ebosin. The analysis of IL-1R antagonist activity for EPSs showed that the bioactivity of EPS-m was lower than Ebosin.
Conclusions:  ste11 gene encoding a TDH may function as a modifier gene of Ebosin during its biosynthesis.
Significance and Impact of the Study:  TDH encoded by ste11 is functional in Ebosin biosynthesis. It is the first characterized TDH in Streptomyces .     

Interaction of the GTS1 gene product with glyceraldehyde- 3-phosphate dehydrogenase 1 required for the maintenance of the metabolic oscillations of the yeast Saccharomyces cerevisiae.

We previously reported that GTS1 is involved in regulating ultradian oscillations of the glycolytic pathway induced by cyanide in cell suspensions as well as oscillations of energy metabolism in aerobic continuous cultures. Here, we screened a yeast cDNA library for proteins that bind to Gts1p using the yeast two-hybrid system and cloned multiple TDH cDNAs encoding the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We found that the zinc-finger and dimerization sites of Gts1p were required for full ability to bind GAPDH, and Gts1ps mutated at these sites lost the ability to regulate both aerobic and unaerobic ultradian oscillations of energy metabolism. Of the three TDH genes, only TDH1 fluctuated at the mRNA level in continuous culture and its deletion resulted in the disappearance of the oscillation without any affect on growth rate. This loss of biological rhythms in the TDH1-deleted mutant was rescued by the expression of TDH1 but not of TDH2 or TDH3 under the control of the TDH1 promoter. Thus, we hypothesized that Gts1p plays a role in the regulation of metabolic oscillation by interacting with the TDH1 product, GAPDH1, in yeast.     

Changes in enzyme activity during differentiation in Chlamydomonas reinhardtii

The patterns of alanine dehydrogenase, glutamate dehydrogenase and malate dehydrogenase activity were studied during the normal vegetative cell cycle and during the process of gametic differentiation and dedifferentiation in synchronized cultures of Chlamydomonas reinhardtii. During all three phases of growth and differentiation the synthesis of DNA was also measured. During gametic differentiation all three enzyme levels were suppressed compared to vegetative cells although DNA and cell number were comparable. During gametic dedifferentiation no DNA synthesis occurred during the first 24 h cycle and only a doubling during the second. It was not until the third cycle that a normal 4-fold increase in DNA was observed. Cell number followed a similar pattern. Athough the levels of alanine dehydrogenase and malate dehydrogenase were uniformly low during the first cycle when glutamate dehydrogenase increased 4-fold, during the second cycle the patterns of these enzymes changed markedly. The enzymes did not attain levels characteristic of vegetative cells until the third cycle.     

Tryptophan aminotransferase and tryptophan dehydrogenase activities in some cell compartments of spinach leaves: The effect of calcium ions on tryptophan dehydrogenase

The content of spinach-leaf cells was compartmented by differential centrifugation. Three fractions were obtained,i.e. chloroplasts, pellet of remaining organelles sedimenting at 97 000g and cytosol. Enzyme activities of L-tryptophan aminotransferase (TAT) as well as L-tryptophan dehydrogenase (TDH) were demonstrated in all cell fractions. The highest activities of both enzymes were found in the pellet of organelles followed by the enzyme activities in the chloroplasts. The cytosol had the lowest enzyme activities. Chloroplasts are characterized by a relatively higher TDH activity, organelles sedimenting at 97 000g were marked by a relatively higher TAT activity. In all fractions both pyridine nucleotide coenzymes catalyzed the TDH activity. Ca²⁺ in a concentration of 0.8 minol l⁻¹ increased markedly the TDH activity in both directions of its activity. An erratum to this article is available at .     

Riboflavin deficiency in cultured rat hepatoma cells: A model for studying the hepatic effects of riboflavin deficiency

Summary The acyl-CoA dehydrogenases are a family of mitochondrial flavoenzymes required for fatty acid beta-oxidation and branched-chain amino acid degradation. The hepatic activity of these enzymes, particularly the short-chain acyl-coenzyme A (CoA) dehydrogenase, is markedly decreased in riboflavin deficient rats. We now report that the in vivo effects of riboflavin deficiency on the beta-oxidation enzymes of this group are reproduced in FAO rat hepatoma cells cultured in riboflavin-deficient medium. Although it has been long known that hepatic short-chain acyl-CoA dehydrogenase activity is the most severely affected of the straight-chain specific enzymes in riboflavin deficiency, the mechanism by which its activity is decreased has not been reported. We have used this new cell culture system to characterize further this mechanism. Whole cell extracts from riboflavin-deficient and control cells were subjected to analysis by denaturing polyacrylamide gel electrophoresis. The contents of the gels were then electroblotted onto nitrocellulose filters and probed with short-chain acyl-CoA dehydrogenase-specific antiserum. The relative abundance of enzyme antigen was estimated autoradiographically. Our findings indicate that short-chain acyl-CoA dehydrogenase activity changes in parallel with its antigen, suggesting that riboflavin deprivation does not affect the activity of individual enzyme molecules. Further, no evidence of extramitochondrial enzyme precursor was found on the blots, making unlikely a significant block in the mitochondrial uptake process. These findings suggest that changes in short-chain acyl-CoA dehydrogenase activity in riboflavin deficiency result from either increased synthesis or decreased degradation of the enzyme. This work was supported by grants from the VA Medical Research Service, the Diabetes Association of Greater Cleveland, and the National Institutes of Health (HD25299), Bethesda, MD. Portions of the work presented here were presented at the 71st meeting of the Endocrine Society, Seattle, WA.     

The role of glutamate in modulating the synthesis and degradation of NADP-glutamate dehydrogenase from Saccharomyces cerevisiae

Abstract NADP-glutamate dehydrogenase (NADP-GDH) from Saccharomyces cerevisiae has a lower activity in yeast grown on glutamate as nitrogen source than when grown on ammonium. With the use of the immunotitration method, it was found that the difference in activity was parallel to the difference in immunoprecipitable material. By isotope incorporation studies, it was established that the decrease in NADP-glutamate dehydrogenase levels in glutamate-grown cells was brought about by an increase in the degradation rate and a decrease in the synthesis constant of the enzyme. The degradation rate of NADP-glutamate dehydrogenase is further increased in carbon-starved cells. The possible role of internal metabolites in modulating NADP-glutamate dehydrogenase degradation is discussed.     

4-Dihydromethyltrisporate dehydrogenase, an enzyme of the sex hormone pathway in Mucor mucedo, is constitutively transcribed but its activity is differently regulated in (+) and (-) mating types

4-Dihydromethyltrisporate dehydrogenase (TDH) converts the (+) mating type sex pheromone 4-dihydromethyltrisporate into methyltrisporate. In Mucor mucedo, this conversion is required only in the (-) mating type. Expression of the TDH encoding TSP1 gene was analyzed qualitatively using reverse-transcribed PCR. TSP1 is constitutively transcribed in the (+) and in the (-) mating type, irrespective of the mating situation. By immunodetection, the translation product is also formed constitutively. In contrast to gene expression, TDH enzyme activity depends on the sexual status of the mycelium. Activity is restricted to the sexually stimulated (-) mating type. Non-stimulated (-), as well as stimulated and non-stimulated (+) mycelia exhibit no activity and do not influence activity in stimulated (-) mycelia. Time course analysis shows strongly increased enzyme activity at 80 min after stimulation. Low activity exists from the onset of stimulation, indicating that additional regulation mechanisms are involved in TDH function.     

The effects of recessive lethal Notch mutations of Drosophila melanogaster on flavoprotein enzyme activities whose inhibitions cause Notch-like phenocopies

The biochemical action of the Notch locus whose mutants cause morphological aberrations in flies, viz., notches of wings and bristle multiplication, has been analyzed (1) by the addition to the food medium of enzyme inhibitors causing phenocopies of Notch and (2) by comparison of enzyme activity patterns of Notch mutants with different degrees of phenotypic expression. Notch phenocopies were induced by inhibitors of enzyme activities in two biochemical pathways: (1) the de novo pyrimidine synthesis by 5-methylorotate (inhibitor of dihydroorotate dehydrogenase) and (2) the choline shunt by amobarbital (inhibits choline dehydrogenase) and methoxyacetate (inhibits sarcosine dehydrogenase). The inhibition of de novo pyrimidine synthesis prevents the production of deoxyuridine-5-phosphate, the substrate for the synthesis of thymidine-5-phosphate via thymidylate synthase, whereas the inhibition of the choline shunt prevents the production of HCHO groups and glycine, both of which are involved in the synthesis of 5,10-methylenetetrahydrofolate, which is a cofactor of thymidylate synthase. It was already known that the inhibition of the latter enzyme in vivo induces Notch phenocopies. Notch mutants with a strong morphological expression show low enzyme activities for dihydroorotate dehydrogenase and choline dehydrogenase. Both are flavoprotein enzymes linked to the respiratory chain. The correspondence between the low enzyme activities in Notch mutants with a strong morphological expression and the phenocopying effect of antimetabolites on these enzymes in the two biochemical pathways involved strongly suggests that the morphological effects of Notch on flies are a consequence of lowered activities of choline dehydrogenase and dihydroorotate dehydrogenase.     

Ca(2+) modulates respiratory and steroidogenic activities of human term placental mitochondria

We investigated the effects of calcium on the oxidative metabolism and steroidogenic activity of human term placental mitochondria. Submicromolar Ca(2+) concentrations stimulated state 3 oxygen consumption with 2-oxoglutarate and isocitrate and activated the 2-oxoglutarate and the NAD-isocitrate dehydrogenases by diminishing their Michaelis-Menten constants. Ca(2+) inhibited NADP-isocitrate dehydrogenase (NADP-ICDH) and the synthesis of progesterone. The NADP-ICDH maximal velocity was threefold higher than that of NAD-ICDH and had a threefold lower K(m) for isocitrate than NAD-ICDH. Isocitrate but not malate or 2-oxoglutarate supported progesterone synthesis. Calcium inhibition of progesterone synthesis was observed with isocitrate but not with malate or 2-oxoglutarate. Tight regulation of NADP-isocitrate dehydrogenase by calcium ions suggests that this enzyme plays an important role in placental mitochondrial metabolism.     

Characterization of human DHRS4: an inducible short-chain dehydrogenase/reductase enzyme with 3beta-hydroxysteroid dehydrogenase activity

Human DHRS4 is a peroxisomal member of the short-chain dehydrogenase/reductase superfamily, but its enzymatic properties, except for displaying NADP(H)-dependent retinol dehydrogenase/reductase activity, are unknown. We show that the human enzyme, a tetramer composed of 27 kDa subunits, is inactivated at low temperature without dissociation into subunits. The cold inactivation was prevented by a mutation of Thr177 with the corresponding residue, Asn, in cold-stable pig DHRS4, where this residue is hydrogen-bonded to Asn165 in a substrate-binding loop of other subunit. Human DHRS4 reduced various aromatic ketones and α-dicarbonyl compounds including cytotoxic 9,10-phenanthrenequinone. The overexpression of the peroxisomal enzyme in cultured cells did not increase the cytotoxicity of 9,10-phenanthrenequinone. While its activity towards all-trans-retinal was low, human DHRS4 efficiently reduced 3-keto-C₁₉/C₂₁-steroids into 3β-hydroxysteroids. The stereospecific conversion to 3β-hydroxysteroids was observed in endothelial cells transfected with vectors expressing the enzyme. The mRNA for the enzyme was ubiquitously expressed in human tissues and several cancer cells, and the enzyme in HepG2 cells was induced by peroxisome-proliferator-activated receptor α ligands. The results suggest a novel mechanism of cold inactivation and role of the inducible human DHRS4 in 3β-hydroxysteroid synthesis and xenobiotic carbonyl metabolism.     

The human L-threonine 3-dehydrogenase gene is an expressed pseudogene

Background  

L-threonine is an indispensable amino acid. One of the major L-threonine degradation pathways is the conversion of L-threonine via 2-amino-3-ketobutyrate to glycine. L-threonine dehydrogenase (EC 1.1.1.103) is the first enzyme in the pathway and catalyses the reaction: L-threonine + NAD⁺ = 2-amino-3-ketobutyrate + NADH. The murine and porcine L-threonine dehydrogenase genes (TDH) have been identified previously, but the human gene has not been identified.     

7-脱氢胆甾醇合成功能模块与底盘细胞的适配性

利用合成生物技术生产7-脱氢胆甾醇的挑战性在于获得合成功能模块与底盘细胞的适配关系。从更换不同调控强度的启动子和不同改造的酵母底盘两方面,对二者的适配性进行研究,以增加7-脱氢胆甾醇产量:过表达酵母固醇合成途径中的内源基因tHMGR和ERG1,敲除非必需基因ERG6和ERG5以抑制酵母固醇向麦角固醇的转化,得到改造后的酵母底盘SyBE_000956;利用由强到弱依次为TDH3p、PGK1p和TDH1p的启动子,引入人源C-24还原酶基因DHCR24,构建3种强度的外源功能模块,并分别导入3种底盘中,得到9种人工合成细胞。结果表明,TDH3p调控的功能模块与底盘细胞SyBE_000956具备较好的适配性,实现7-脱氢胆甾醇产量的提高。为后续的适配性研究提供了理性设计的依据。     

Comparison of the developmental patterns of mitochondrial malate dehydrogenase between mung bean and cucumber cotyledons during and following germination

The development of mitochondrial NAD⁺-malate dehydrogenase (EC 1.1.1.37) in mung bean and cucumber cotyledons was followed. using the antibody raised against it, during and following germination. The developmental patterns were quite different between the two. In cucumber, the content of mitochondrial malate dehydrogenase continued to increase through 3–4 days after the beginning of imbibition. This was, at least in part, due to active synthesis of the enzyme protein, and the synthesis seemed to be regulated by the availability of the translatable mRNA for the enzyme. In mung bean, on the other hand, the enzyme was present in dry cotyledons at a rather high concentration, and remained at a constant level between day 1 and day 3 after the reduction of the content to one-half its initial level during the first day. De novo synthesis of the enzyme could not be detected in mung bean cotyledons by pulse-labeling experiment.