Activation of serine sulphydrase from baker's yeast (Saccharomyces cerevisiae) by vanadate

Vanadate stimulates the liberation of H2S from cysteine in intact cells of baker's yeast (Saccharomyces cerevisiae) with a maximal increase of 60% at 10 microM NH4VO3. Protein separation from crude yeast extract yielded two active protein fractions which were found to catalyze the degradation of cysteine to H2S, pyruvate and ammonia or H2S and serine, respectively, thus characterizing them as cysteine desulphydrase and serine sulphydrase. Only the latter enzyme was found to be activated by vanadate, showing optimal enhancement of about 100% at 10 microM NH4VO3.     

H2O2介导的H2S产生参与干旱诱导的拟南芥气孔关闭

以野生型拟南芥(Arabidopsis thaliana)及其突变体(atrbohD、atrbohF、atrbohD/F、atl-cdes、atd-cdes)和过表达株系(OEL-CDes、OED-CDes)为材料, 利用药理学实验, 结合分光光度法和激光共聚焦显微技术, 探讨硫化氢(hydrogen sulfide, H₂S)在干旱诱导的拟南芥气孔关闭中的作用及其与过氧化氢(hydrogen peroxide, H₂O₂)的关系。结果表明, H₂S清除剂次牛磺酸(hypotaurine, HT)及合成抑制剂氨氧基乙酸(aminooxy acetic acid, AOA)、羟胺(hydroxylamine, NH₂OH)和丙酮酸钾(potasium pyruvate, C₃H₃KO₃)+氨水(ammonia, NH₃)均可不同程度抑制干旱诱导的气孔关闭; 干旱对OEL-CDes和OED-CDes植株气孔关闭的诱导作用明显, 而atl-cdes和atd-cdes叶片气孔对干旱胁迫反应的敏感性下降; 干旱胁迫能明显增加拟南芥保卫细胞中H₂O₂水平及叶片中H₂S含量, 提高D-/L-半胱氨酸脱巯基酶活性及基因表达量, 而对突变体atrbohD、atrbohF和atrbohD/F没有显著影响。清除H₂O₂可减弱干旱胁迫对H₂S含量和D-/L-半胱氨酸脱巯基酶活性的诱导效应。研究 结果表明H₂S位于H₂O₂下游参与干旱诱导拟南芥气孔关闭的信号转导过程。     

Cysteine metabolism in cultured tobacco cells

Transported l-[(35)S]cysteine was rapidly metabolized by cultured tobacco cells when supplied to the cells at 0.02 millimolar or 0.5 millimolar. The internal cysteine pool was expandable to approximately 2400 nmoles per gram fresh weight.The (35)S label derived from cysteine was found in several metabolites. The amount of label in glutathione and sulfate was directly proportional to the internal l-[(35)S]cysteine, while the levels of labeled methionine and protein were apparently independent of internal labeled cysteine. Cysteine was more rapidly metabolized when the external cysteine concentration was low (0.02 millimolar) with up to 90% of the (35)S label present as compounds other than cysteine.The initial step in cysteine degradation yielded pyruvate, sulfide, and presumably NH(4) (+). Stoichiometry studies using extracts prepared from acetone powders of tobacco cells indicated that pyruvate and sulfide were produced in a 1:1 ratio. The catabolic reaction was linear with respect to time and amount of protein and had a pH optimum of 8 in crude extracts. Preliminary kinetic data indicated the K(m) to be approximately 0.2 millimolar. The extractable degradative activity was enhanced 15- to 20-fold by preincubating the cells for 24 hours in 0.5 millimolar cysteine. The extractable specific enzyme activity roughly reflected the growth curve of the cells in culture. Maximal cysteine degradation was observed in extracts prepared from late log phase cultures that were preincubated in cysteine, while little activity was found in similar extracts from stationary phase cultures. These results are consistent with an inducible catabolic enzyme similar to the cysteine desulfhydrase from bacteria.     

H2O2介导的H2S产生参与干旱诱导的拟南芥气孔关闭

以野生型拟南芥(Arabidopsis thaliana)及其突变体(atrbohD、atrbohF、atrbohD/F、atl-cdes、atd-cdes)和过表达株系(OEL-CDes、OED-CDes)为材料,利用药理学实验,结合分光光度法和激光共聚焦显微技术,探讨硫化氢(hydrogen sulfide,H2S)在干旱诱导的拟南芥气孔关闭中的作用及其与过氧化氢(hydrogen peroxide,H2O2)的关系.结果表明,H2S清除剂次牛磺酸(hypotaurine,HT)及合成抑制剂氨氧基乙酸(aminooxy acetic acid,AOA)、羟胺(hydroxylamine,NH2OH)和丙酮酸钾(potasium pyruvate,C3H3KO3)+氨水(ammonia,NH3)均可不同程度抑制干旱诱导的气孔关闭;干旱对OEL-CDes和OED-CDes植株气孔关闭的诱导作用明显,而atl-cdes和atd-cdes叶片气孔对干旱胁迫反应的敏感性下降;干旱胁迫能明显增加拟南芥保卫细胞中H2O2水平及叶片中H2S含量,提高D-/L-半胱氨酸脱巯基酶活性及基因表达量,而对突变体atrbohD、atrbohF和atrbohD/F没有显著影响.清除H2O2可减弱干旱胁迫对H2S含量和D-/L-半胱氨酸脱巯基酶活性的诱导效应.研究结果表明H2S位于H2O2下游参与干旱诱导拟南芥气孔关闭的信号转导过程.     

Cysteine desulfhydrase activity in cucurbit plants: Stimulation by preincubation with l- or d-cysteine

Cysteine desulfhydrase activity in leaf discs of cucurbit plants is enhanced 2–4-fold by preincubation with l or d-cysteine. Preincubation with structural analogs of cysteine also stimulated the activity of the enzyme, but to a smaller extent. Maximal increase in cysteine desulfhydrase activity was observed by preincubation with 5 mM or higher concentrations of cysteine. Although not caused by activation, stimulation of the enzyme activity was half-maximal within less than 15 min. Whereas the increase in cysteine desulfhydrase activity by preincubation of leaf discs with cysteine was light independent, pretreatment of the entire plant with light or dark determined the leaf discs' potential for stimulation of the enzyme. Exposure to darkness for 4 hr reduced this potential by 60%. It is concluded that the potential for stimulation of cysteine desulfhydrase activity by preincubation with cysteine is regulated by a compound not synthesized, but metabolized, in the leaf tissue. This regulatory compound may be supplied to the leaves by long-distance transport.     

Role of D-cysteine desulfhydrase in the adaptation of Escherichia coli to D-cysteine

D-cysteine, a powerful inhibitor of Escherichia coli growth, is decomposed in vitro into pyruvate, H2S, and NH3 by D-cysteine desulfhydrase. To assess the role of this reaction in the adaptation of the bacterium to growth on D-cysteine, the gene of the desulfhydrase was cloned. It corresponds to the open reading frame yedO at 43.03 min on the genetic map of E. coli. The amino acid sequence deduced from this gene is homologous to those of several 1-aminocyclopropane-carboxylate deaminases. However, the E. coli desulfhydrase does not use 1-aminocyclopropane-1-carboxylate as substrate. Various mutants in which the yedO gene was inactivated or overexpressed were constructed. They exhibited hypersensitivity or resistance, respectively, to the presence of d-cysteine in the culture medium. Growth protection against D-cysteine in minimal medium was conferred by the simultaneous addition of isoleucine, leucine, and valine. In agreement with this behavior, D-cysteine inhibited the activity of threonine deaminase, a key enzyme of the isoleucine, leucine, and valine pathway. Finally, in the presence of the intact yedO gene, E. coli growth was improved by addition of D-cysteine as the sole sulfur source. In agreement with a role of the desulfhydrase in sulfur metabolism, yedO expression was induced under conditions of sulfate limitation.     

Impact of elevated H(2)S on metabolite levels, activity of enzymes and expression of genes involved in cysteine metabolism.

The effects of elevated atmospheric hydrogen sulfide (H(2)S) levels (0.25, 0.5, and 0.75 microl l(-1)) have been investigated in a short-term exposure experiment (3-48 h) on the model plant Arabidopsis thaliana (L.) Heynh. in comparison to untreated control plants. The most pronounced effects of H(2)S fumigation could be observed on the metabolite level: the contents of the thiols cysteine and glutathione were increased up to 20- and fourfold, respectively. A direct positive correlation of the thiol contents with the H(2)S concentrations applied was observed. To elucidate the molecular basis for the increased thiol levels, enzyme activities, messenger RNA and protein steady-state levels of cysteine-synthesizing and degrading pathways have been determined. The enzyme activities of O-acetyl-l-serine(thiol)lyase (OAS-TL) (EC 4.2.99.8) and l-cysteine desulfhydrase (EC 4.4.1.-) proteins were not significantly higher at elevated H(2)S levels in comparison to untreated control plants. 3-Mercaptopyruvate sulfurtransferase (EC 2.8.1.2) activity was slightly higher after the longest H(2)S exposure times. Elevated H(2)S levels of 0.25 and 0.5 microl l(-1) had promoting effects on both mRNA and protein levels of cysteine-synthesizing and degrading enzymes whereas the highest H(2)S concentrations caused lower levels of expression combined with mild symptoms of oxidative stress, as the consequence of its phytotoxicity. The differences in the expression of the three different OAS-TL isoforms (cytoplasmic, plastidic and mitochondrial) by H(2)S were very small. Increasing concentrations of H(2)S and longer exposure times to H(2)S let to a reduction in the pool of O-acetyl-l-serine, the second precursor of cysteine, and N-acetyl-l-serine in the leaves and shoots, indicating a substrate depletion in agreement with the increased thiol levels.     

Distribution of Cysteine Desulfhydrase in Microorganisms

The distribution of cysteine desulfhydrase activity in microorganisms was studied with intact cells. The enzyme activity was found mainly in strains belonging to Enterobacteriaceae, especially to genus Aerobacter (Enterobacter). Aerobacter cloacae IFO 12009 showed markedly high activity.

l-Cysteine was essential as an inducer of the enzyme formation, of which 0.2% in the medium is appropriate.

Intact cells of bacteria containing high cysteine desulfhydrase activity, prepared from broth cultured for 19hr, catalyzed the synthesis of l-cysteine from pyruvate, ammonia and hydrogen sulfide.     

Metabolic engineering of an aerobic sulfate reduction pathway and its application to precipitation of cadmium on the cell surface

The conversion of sulfate to an excess of free sulfide requires stringent reductive conditions. Dissimilatory sulfate reduction is used in nature by sulfate-reducing bacteria for respiration and results in the conversion of sulfate to sulfide. However, this dissimilatory sulfate reduction pathway is inhibited by oxygen and is thus limited to anaerobic environments. As an alternative, we have metabolically engineered a novel aerobic sulfate reduction pathway for the secretion of sulfides. The assimilatory sulfate reduction pathway was redirected to overproduce cysteine, and excess cysteine was converted to sulfide by cysteine desulfhydrase. As a potential application for this pathway, a bacterium was engineered with this pathway and was used to aerobically precipitate cadmium as cadmium sulfide, which was deposited on the cell surface. To maximize sulfide production and cadmium precipitation, the production of cysteine desulfhydrase was modulated to achieve an optimal balance between the production and degradation of cysteine.     

Metabolic Engineering of an Aerobic Sulfate Reduction Pathway and Its Application to Precipitation of Cadmium on the Cell Surface

The conversion of sulfate to an excess of free sulfide requires stringent reductive conditions. Dissimilatory sulfate reduction is used in nature by sulfate-reducing bacteria for respiration and results in the conversion of sulfate to sulfide. However, this dissimilatory sulfate reduction pathway is inhibited by oxygen and is thus limited to anaerobic environments. As an alternative, we have metabolically engineered a novel aerobic sulfate reduction pathway for the secretion of sulfides. The assimilatory sulfate reduction pathway was redirected to overproduce cysteine, and excess cysteine was converted to sulfide by cysteine desulfhydrase. As a potential application for this pathway, a bacterium was engineered with this pathway and was used to aerobically precipitate cadmium as cadmium sulfide, which was deposited on the cell surface. To maximize sulfide production and cadmium precipitation, the production of cysteine desulfhydrase was modulated to achieve an optimal balance between the production and degradation of cysteine.     

Thiol Accumulation and Cysteine Desulfhydrase Activity in H2S-Fumigated Leaves and Leaf Homogenates of Cucurbit Plants

Fumigation of both, cucurbit plants and cucurbit leaf homogenateswith hydrogen sulfide (H₂S) resulted in an increase in solublethiol, mainly glutathione and cysteine. In leaf homogenatesthis increase was counteracted or prevented by the additionat 1 mM of inhibitors of pyridoxalphosphate dependent enzymesor of products of the cysteine desulfhydrase reaction. Thesecompounds inhibited cysteine desulfhydrase activity, but didnot severely affect O-acetylserine sulfhydrylase activity atthis concentration. These results provide circumstantial evidencethat cysteine desulfhydrase in its reverse reaction, but notO-acetylserine sulfhydrylase participates in the assimilationof atmospheric H₂S. (Received February 4, 1991; Accepted April 30, 1991)     

Characterization of cysteine-degrading and H2S-releasing enzymes of higher plants - from the field to the test tube and back

Due to the clean air acts and subsequent reduction of emission of gaseous sulfur compounds sulfur deficiency became one of the major nutrient disorders in Northern Europe. Typical sulfur deficiency symptoms can be diagnosed. Especially plants of the Cruciferae family are more susceptible against pathogen attack. Sulfur fertilization can in part recover or even increase resistance against pathogens in comparison to sulfur-deficient plants. The term sulfur-induced resistance (SIR) was introduced, however, the molecular basis for SIR is largely unknown. There are several sulfur-containing compounds in plants which might be involved in SIR, such as high levels of thiols, glucosinolates, cysteine-rich proteins, phytoalexins, elemental sulfur, or H2S. Probably more than one strategy is used by plants. Species- or even variety-dependent differences in the development of SIR are probably used. Our research focussed mainly on the release of H2S as defence strategy. In field experiments using different BRASSICA NAPUS genotypes it was shown that the genetic differences among BRASSICA genotypes lead to differences in sulfur content and L-cysteine desulfhydrase activity. Another field experiment demonstrated that sulfur supply and infection with PYRENOPEZIZA BRASSICA influenced L-cysteine desulfhydrase activity in BRASSICA NAPUS. Cysteine-degrading enzymes such as cysteine desulfhydrases are hypothesized to be involved in H2S release. Several L- and D-cysteine-specific desulfhydrase candidates have been isolated and partially analyzed from the model plant ARABIDOPSIS THALIANA. However, it cannot be excluded that H2S is also released in a partial back reaction of O-acetyl-L-serine(thiol)lyase or enzymes not yet characterized. For the exact determination of the H2S concentration in the cell a H2S-specific microsensor was used the first time for plant cells. The transfer of the results obtained for application back on BRASSICA was initiated.     

Precipitation of cadmium by Clostridium thermoaceticum.

Cadmium at an initial concentration of 1 mM was completely precipitated by cultures of Clostridium thermoaceticum in complex medium. The precipitation was energy dependent and required cysteine, although cysteine alone did not act as a growth substrate. Electron microscopic analysis revealed localized areas of precipitation at the surfaces of nonstarved cells as well as precipitate in the surrounding medium. The addition of cadmium had no apparent effect on growth or acetogenesis. However, nickel and cadmium were synergistically toxic at a concentration (1 mM) at which neither alone was toxic. The amount of protein extracted from cadmium-treated cultures was twofold higher than that in control extracts, and the amount of total sulfide was fourfold higher in cultures containing cadmium than in control cultures. Comparable levels of cysteine desulfhydrase activity were observed in extracts of both cadmium-treated and control cultures, but the enzyme activity was expressed maximally about 24 h earlier in the cadmium-treated cultures than in the untreated controls.     

Production of the neuromodulator H2S by cystathionine beta-synthase via the condensation of cysteine and homocysteine

Hydrogen sulfide (H2S) has been observed in relatively high concentrations in the mammalian brain and has been shown to act as a neuromodulator. However, there is confusion in the literature regarding the actual source of H2S production. Reactions catalyzed by the cystathionine beta-synthase enzyme (CBS) are one possible source for the production of H2S. Here we show that the CBS enzyme can efficiently produce H2S via a beta-replacement reaction in which cysteine is condensed with homocysteine to form cystathionine and H2S. The production of H2S by this reaction is at least 50 times more efficient than that produced by hydrolysis of cysteine alone via beta-elimination. Kinetic studies demonstrate that the Km and Kcat for cysteine is 3-fold higher and 2-fold lower, respectively, than that for serine. Consistent with these data, in vitro reconstitution studies show that at physiologically relevant concentrations of serine, homocysteine, and cysteine, about 5% of the cystathionine formed is from cysteine. We also show that AdoMet stimulates this H2S producing reaction but that there is no evidence for stimulation by calcium and calmodulin as reported previously. In summary, these results confirm the ability of CBS to produce H2S, but show in contrast to prior reports that the major mechanism is via beta-replacement and not cysteine hydrolysis. In addition, these studies provide a biochemical explanation for the previously inexplicable homocysteine-lowering effects of N-acetylcysteine treatments in humans.     

Ketomethylthiobutyric acid formation from methylthioadenosine: A diffusion assay

Typical enzyme kinetics were observed when 5′-methylthioadenosine was used as substrate with extracts of malignant murine cells in a diffusion assay. The volatile product was measured after diffusion into a solution of the sulfhydryl reagent, 5,5′-dithiobis(2-nitrobenzoic acid), which it reduced to a yellow chromophore. Cysteine was required in the system. The volatile product was identified as H₂S derived from the cysteine. The yield of H₂S was similar to the amount of 2-keto-4-methylthiobutyric acid (KMTB) formed from methylthioadenosine when the KMTB was measured simultaneously in an ether extraction assay. KMTB could replace methylthioadenosine as a substrate capable of causing the formation of the diffusible product from cysteine. It is concluded that the following sequence of reactions takes place in the diffusion assay system: (1) 5′-methylthioadenosine + P_i → adenine + 5-methylthioribose-1-P, (2) 5-methylthioribose-1-P → KMTB, (3) KMTB + cysteine → methionine + 3-mercaptopyruvate, (4) 3-mercaptopyruvate + excess R-SH → pyruvate + H₂S, (5) H₂S + 5,5′-dithiobis(2-nitrobenzoic acid) → 5-mercapto-2-nitrobenzoic acid. Thus, the diffusion assay measures the amount of KMTB formed. The key enzyme, cysteine aminotransferase, EC 2.6.1.3, was partially purified from malignant cells and from liver and several of its characteristics are described. The diffusion assay using this enzyme is useful in measuring de novo synthesis of α-keto acids and it is applicable to crude enzyme preparations. The sensitivity is about 5 nmol of keto acid and the accurate range is 5 to 100 nmol.     

Isolation and characterization of a D-cysteine desulfhydrase protein from Arabidopsis thaliana

In several organisms D-cysteine desulfhydrase (D-CDes) activity (EC 4.1.99.4) was measured; this enzyme decomposes D-cysteine into pyruvate, H2S, and NH3. A gene encoding a putative D-CDes protein was identified in Arabidopsis thaliana (L) Heynh. based on high homology to an Escherichia coli protein called YedO that has D-CDes activity. The deduced Arabidopsis protein consists of 401 amino acids and has a molecular mass of 43.9 kDa. It contains a pyridoxal-5'-phosphate binding site. The purified recombinant mature protein had a Km for D-cysteine of 0.25 mm. Only D-cysteine but not L-cysteine was converted by D-CDes to pyruvate, H2S, and NH3. The activity was inhibited by aminooxy acetic acid and hydroxylamine, inhibitors specific for pyridoxal-5'-phosphate dependent proteins, at low micromolar concentrations. The protein did not exhibit 1-aminocyclopropane-1-carboxylate deaminase activity (EC 3.5.99.7) as homologous bacterial proteins. Western blot analysis of isolated organelles and localization studies using fusion constructs with the green fluorescent protein indicated an intracellular localization of the nuclear encoded D-CDes protein in the mitochondria. D-CDes RNA levels increased with proceeding development of Arabidopsis but decreased in senescent plants; D-CDes protein levels remained almost unchanged in the same plants whereas specific D-CDes activity was highest in senescent plants. In plants grown in a 12-h light/12-h dark rhythm D-CDes RNA levels were highest in the dark, whereas protein levels and enzyme activity were lower in the dark period than in the light indicating post-translational regulation. Plants grown under low sulfate concentration showed an accumulation of D-CDes RNA and increased protein levels, the D-CDes activity was almost unchanged. Putative in vivo functions of the Arabidopsisd-CDes protein are discussed.     

Partial purification and characterization of 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase from rat spleen.

The enzyme 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine: acetyl-CoA acetyltransferase (EC 2.3.1.67) was purified from rat spleen approx. 1500-fold in 1.6% yield. The specific activity of the purified enzyme was 0.317 +/- 0.089 mumol/min per mg of protein (mean +/- S.D., n = 6). The Km for the substrate acetyl-CoA was 137 +/- 13 microM and the pH optimum was about 8. Incubation of the purified enzyme was 1-O-[3H]octadecyl-2-lyso-sn-glycero-3-phosphocholine followed by electrophoresis resulted in the incorporation of radioactivity into a protein of Mr 29,000. The enzyme was most active towards 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine as substrate, 1-palmitoyl-2-lyso-glycero-3-phosphocholine being a poor substrate. In addition, the enzyme preferred acetyl-CoA to palmitoyl-CoA or oleoyl-CoA as substrate.     

Purification and characterization of (2S)-flavanone 3-hydroxylase from Petunia hybrida

(2S)-Flavanone 3-hydroxylase from flowers of Petunia hybrida catalyses the conversion of (2S)-naringenin to (2R,3R)-dihydrokaempferol. The enzyme could be partially stabilized under anaerobic conditions in the presence of ascorbate. For purification, 2-oxoglutarate and Fe2+ had to be added to the buffers. The hydroxylase was purified about 200-fold by a six-step procedure with low recovery. The Mr of the enzyme was estimated by gel filtration to be about 74,000. The hydroxylase reaction has a pH optimum at pH 8.5 and requires as cofactors oxygen, 2-oxoglutarate, Fe2+ and ascorbate. With 2-oxo[1-14C]glutarate in the enzyme assay dihydrokaempferol and 14CO2 are formed in a molar ratio of 1:1. Catalase stimulates the reaction. The product was unequivocally identified as (+)-(2R,3R)-dihydrokaempferol. (2S)-Naringenin, but not the (2R)-enantiomer is a substrate of the hydroxylase. (2S)-Eriodictyol is converted to (2R,3R)-dihydroquercetin. In contrast, 5,7,3',4',5'-pentahydroxy-flavanone is not a substrate. Apparent Michaelis constants for (2S)-naringenin and 2-oxoglutarate were determined to be respectively 5.6 mumol X l-1 and 20 mumol X l-1 at pH 8.5. The Km for (2S)-eriodictyol is 12 mumol X l-1 at pH 8.0. Pyridine 2,4-dicarboxylate and 2,5-dicarboxylate are strong competitive inhibitors with respect to 2-oxoglutarate with Ki values of 1.2 mumol X l-1 and 40 mumol X l-1, respectively.     

Cytophotometric analysis of reaction rates of succinate and lactate dehydrogenase activity in rat liver, heart muscle and tracheal epithelium

Reaction rates of succinate and lactate dehydrogenase activity in cryostat sections of rat liver, tracheal epithelium and heart muscle were monitored by continuous measurement of formazan formation by cytophotometry at room temperature. Incubation media contained polyvinyl alcohol as tissue protectant and Tetranitro BT as final electron acceptor. Control media lacked either substrate or substrate and coenzyme. Controls were also performed by adding malonate (a competitive inhibitor of succinate dehydrogenase), pyruvate (a non-competitive inhibitor of lactate dehydrogenase), oxalate (a competitive inhibitor of lactate dehydrogenase) or N-ethylmaleimide (a blocker of SH groups). A specific malonate-sensitive linear test minus control response for succinate dehydrogenase activity was obtained in liver (1.6 mumol H2cm-3 min-1) and tracheal epithelium (0.8 mumol H2cm-3 min-1) but not in heart muscle. All variations in the incubation conditions tested did not result in a linear test minus control response in the latter tissue. Because the reaction was sensitive to malonate, it was concluded that the initial reaction rate was the specific rate of succinate dehydrogenase activity in heart muscle (9.1 mumol H2 cm-3 min-1). Test minus control reactions for lactate dehydrogenase activity were distinctly non-linear for all tissues tested. This appeared to be due to product inhibition by pyruvate generated during the reaction and therefore it was concluded that the appropriate control reaction was the test reaction in the presence of 20 mM pyruvate. The initial rate of the test minus this control was the true rate of lactate dehydrogenase activity. The lactate dehydrogenase activity thus found in liver parenchyma was 5.0 mumol of H2 generated per cm3 liver tissue per min.