Mitochondrial malate dehydrogenase from corn : purification of multiple forms

A method to fractionate corn (Zea mays L. B73) mitochondria into soluble proteins, high molecular weight soluble proteins, and membrane proteins was developed. These fractions were analyzed by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and assays of mitochondrial enzyme activities. The Krebs cycle enzymes were enriched in the soluble fraction. Malate dehydrogenase has been purified from the soluble fraction by a two-step fast protein liquid chromatography method. Six different malate dehydrogenase peaks were obtained from the Mono Q column. These peaks were individually purified using a Phenyl Superose column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified peaks showed that three of the isoenzymes consisted of different homodimers (I, III, VI) and three were different heterodimers (II, IV, V). Apparent molecular masses of the three different monomer subunits were 37, 38, and 39 kilodaltons. Nondenaturing gel analysis of the malate dehydrogenase peaks showed that each Mono Q peak contained a band of malate dehydrogenase activity with different mobility. These observations are consistent with three nuclear genes encoding corn mitochondrial malate dehydrogenase. Polyclonal antibodies raised against purified malate dehydrogenase were used to identify the gene products using Western blots of two-dimensional gels.     

Affinity chromatography of malic enzyme from grape berries

NADP-dependent malic enzyme from grape berries is associated with NAD-dependent malate dehydrogenase. A two step procedure, involving affinity chromatography on 2′,5′-ADP-Sepharose 4B, followed by gel- permeation on Bio-Gel A- 1.5 m, was used to separate malic enzyme from malate dehydrogenase and other proteins. The yield was ca 60% Malic enzyme and malate dehydrogenase migrated respectively as three bands and one band during disc electrophoresis in polyacrylamide gel. The MW resulting from gel-permeation was 220 000 for malic enzyme and 53 000 for malate dehydrogenase.     

The Effect of Ethylene and Temperature on ATP Accumulation from Various Substrates in Peanut (Arachis hypogaea L. ) Seeds

Peanut (Arachis hypogaea L. ) seed powder accumulated ATP fromAMP and phosphoenolpyruvate (PEP) at a rate of approx. 100 pmolmin^–1mg^– powder at 35° C. When peanut seed powderwas incubated with various substrates, which may result in PEPor AMP (ADP) synthesis, then ATP accumulated. The best substratecombinations examined so far were AMP + succinate, NADH₂, andAMP + malate + NAD, with activities of 33, 12 and 12 pmol min^–mg^–1powder,respectively; AMP + malate showed very low activity. Some combinationsexhibited linear activities with time, while others had an exponential-typeprofile. The temperature dependence of the ATP accumulationdemonstrated by the Ahrrenius plot had a double phase with atransition point at 25° C. The Ea values between 15°C and 25° C were 25 000–50 000 cal/mol, while above25° C the Ea values fluctuated between 6000 and 8000 cal/mol(depending on the substrate). The AMP + PEP combination exhibiteda single-phase profile between 15° C and 40° C, withan Ea value of 22 000 cal/mol. In the presence of some substrates,ethephon (ethylene) had a stimulatory effect and caused an increasein the Ea values at the high temperature phase. A comparisonof seed powder from dormant seeds with that from non-dormantseeds revealed that some substrate combinations accumulate ATPfaster in non-dormant seeds and others do so in dormant seeds. Key words: Arachis hypogaea, ATP, Ethylene, Dormancy, Peanut, Seed     

Δ5-3β-Hydroxysteroid dehydrogenase from Digitalis lanata Ehrh. – a multifunctional enzyme in steroid metabolism?

Δ⁵-3β-Ηydroxysteroid dehydrogenase (Δ⁵-3β-HSD; EC 1.1.1.145), an enzyme converting pregn-5-ene-3β-ol-20-one (pregnenolone) to pregn-5-ene-3,20-dione (isoprogesterone), was isolated from the soluble fraction of suspension-cultured cells of Digitalis lanata L. strain VIII. Starting with acetone dry powder the enzyme was purified in three steps using column chromatography on Fractogel-TSK DEAE, hydroxyapatite and Sephacryl G-200. Fractions with highest Δ⁵-3β-HSD activity were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After in-situ digestion the resulting bands were sequenced N-terminally. The 29-kDa band yielded three fragments with high sequence homology to members of the superfamily of short-chain dehydrogenases/reductases. High similarity was found to microbial hydroxysteroid dehydrogenases. The band may therefore represent the Δ⁵-3β-HSD. The purified enzyme was characterized with respect to kinetic parameters, substrate specificity and localization. The function of the enzyme in steroid metabolism is discussed. Received: 20 January 1999 / Accepted: 5 May 1999     

Chlorophyll a forms in mesophyll and bundle sheath chloroplasts of Zea mays leaves grown at low light intensity

Mesophyll and bundle sheath chloroplasts were prepared fromleaves of Zea mays grown at light intensities of 1.1 and 240µW/cm², respectively. The mesophyll chloroplasts thatdeveloped at the low intensity and bundle sheath chloroplatsthat developed at both low and high intensities showed higherratios of chlorophyll a/b and P700/chlorophylls compared withthe normal ratios found for the mesophyll chloroplasts thathad developed at the high intensity. Derivative absorption spectrophotometryat 77?K revealed that the low intensity mesophyll chloroplastscontained more of chlorophyll a forms with longer wavelengthred bands than high intensity mesophyll chloroplasts. More ofthe longer wavelength forms of chlorophyll a were also presentin the bundle sheath chloroplasts that had developed at lowand high intensities. All these four types of chloroplasts showedtwo peaks of fluorescence, one at 687 hra and the other at 733or 738 nm. In addition to these peaks, the high intensity mesophyllchloroplasts showed a shoulder at 697 nm, and the two typesof bundle sheath chloroplasts showed a shoulder at 680 nm. (Received June 17, 1974; )     

Mitochondrial malate dehydrogenase of watermelon cotyledons: Time course and mode of enzyme activity changes during germination

Summary Specific antibodies were prepared against the purified mitochondrial malate dehydrogenase (EC 1.1.1.37) from cotyledons of watermelon seedlings (Citrullus vulgaris Schrad.). The isoenzyme was assayed by means of quantitative radial immunodiffusion. Cotyledons of ungerminated seeds were found to contain mitochondrial MDH. During the first 4 days of germination the enzyme activity increased threefold finally contributing 16% to the total MDH activity extracted from cotyledon tissue. Isopycnic CsCl density centrifugation was used to investigate the mode of activity increase. After a four-day period of labelling with deuterium oxide and purification of the mitochondrial isoenzyme, a density shift of 0.021kgx1^-1, accompanied by considerable band broadening of the enzyme profile was observed. These findings are evidence for the de novo synthesis of mitochondrial MDH and its relatively slow turnover in germinating seeds.Abbreviations mMDH mitochondrial malate dehydrogenase - D₂O deuterium oxide     

Electrophoretic study of heating in situ on proteins and enzymes in germinated Arachis hypogaea cotyledons: Comparison with dormant seed

-Cotyledons from 5-day germinated seed of Arachis hypogaea were heated in a moisturized chamber at temperatures from 25 to 121°. Proteins were extracted in phosphate buffer and analyzed with horizontal starch gel electrophoresis to determine the effect of heat on migration patterns of soluble proteins, malate dehydrogenase, glutamate dehydrogenase, leucine aminopeptidase, peroxidases and nonspecific esterases. The intensity of staining of soluble proteins from 5-day cotyledons began decreasing at 80–90°; very little staining occurred at 100° with the exception of a distinct band at R_f 1·0. Glutamate dehydrogenase and benzidine peroxidase retained some activity at 80° but other enzymes were inactivated at temperatures near 65°. Differential heat sensitivities of isoenzymes were obvious. Heat did not alter the R_f values of the bands of soluble proteins or enzymes but influenced the intensity of staining. Two-year storage at 4° of viable seed and 33-month storage at -10° of frozen extracts from dormant seed had no influence upon migration patterns of soluble proteins and enzymes assayed.     

Purification and properties of a new cathepsin from rat liver.

1) A lysosomal protease, a new cathepsin that inactivates glucose-6-phosphate dehydrogenase [EC 1.1.1.49] and some other enzymes and differs from cathepsin B [EC 3.4.22.1] was purified about 2,200-fold from crude extracts of rat liver by cell-fractionation, freezing and thawing, acetone treatment, gel filtration, and DEAE Sephadex and CM-Sephadex column chromatographies. 2) The new cathepsin was markedly activated by the thiol-reagent, 2-mercaptoethanol and inhibited by monoiodoacetate. 3) The molecular weight of the new cathepsin was found by Sephadex G-75 column chromatography to be 22,000, which is smaller than that of cathepsin B. 4) The optimum pH of the enzyme for inactivation of glucose-6-phosphate dehydrogenase was pH 5.0--5.5. The enzyme was unstable in alkali and on heat treatment. 5) The rates of inactivation of glucose-6-phosphate dehydrogenase, apo-ornithine aminotransferase [EC 2.6.1.13], apo-tyrosine aminotransferase [EC 2.6.1.5], apo-cystathionase [EC 4.4.1.1], glucokinase [EC 2.7.1.2], glyceraldehyde-3-phosphate dehydrogenase [EC 1.2.1.12], and malate dehydrogenase [EC 1.1.1.37] by the new cathepsin were higher than those by cathepsin B. However aldolase [EC 4.1.2.13] was inactivated more rapidly by cathepsin B than by the new cathepsin. Lactate dehydrogenase [EC 1.1.1.27], glutamate dehydrogenase [EC 1.4.1.2] and alcohol dehydrogenase [EC 1.1.1.1] were not inactivated by either cathepsin. Unlike cathepsin B, the new cathepsin scarcely hydrolyzes N-substituted derivatives of arginine.     

Localization of Glucose-6-phosphate Dehydrogenase Activity in Seeds and its Possible Involvement in Dormancy Breakage

A quantitative cytochemical analysis of glucose-6-phosphatedehydrogenase activity of deeply dormant seeds of Avena fatuashowed that although the enzyme activity is present in mostcell types of the embryo and seed, it is only in the embryothat activity is increased on treatment with GA₃ to break dormancy.This would appear to happen prior to any measurable embryonicaxis growth, and supports the idea that activation of the pentosephosphate pathway is an early event in dormancy break. A similar,though less marked, change occurred in less dormant seeds ofA. fatua, but could not be detected in dormant seeds of Lactucasaliva. Dry seeds of L. sativa and weakly dormant A. fatua containedtwice the activity seen in seeds imbibed with either water orGA₃, indicating that this might be a marker of low levels ofdormancy. Avena fatua, Lactuca sativa, seeds, dormancy, pentose phosphate pathway, cytochemistry, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase     

The Effect of Hypoxia and Sulphide on Culture-Grown Wetland and Non-Wetland Plants: II. METABOLIC AND PHYSIOLOGICAL CHANGES

Pearson, J. and Havill, D. C. 1988. The effect of hypoxia andsulphide on culture-grown wetland and non-wetland plants. II.Metabolic and physiological changes.—J. exp. Bot. 39:431–439. Two non-wetland (Agropyron pungens, Hordeum vulgare) and fivewetland species (Oryza sativa, Aster tripolium, three Salicorniaspp.) were grown in aerated, unaerated and sulphide-treatedculture solution. Changes in the activity of alcohol dehydrogenase(ADH) and cytochrome oxidase (COase) in the roots were measured.In the non-wetland species, treatment with hypoxia or sulphideincreased ADH activity by 900–1 800%, whereas COase activitydecreased by 80–92% of the aerated control. In the wetlandspecies, except S. europaea which was not affected, hypoxiaincreased ADH activity by 350–550%, while COase activitywas little affected. Generally, when treated with sulphide theactivity of ADH increased to about 750% in most of the wetlandspecies, but increases as low as 175% (S. europaea) and as highas 1400% (S.fragilis) were recorded. The effect of sulphideon the COase activity in the wetland plants was not as markedas in the non-wetland plants. The Salicornia spp. were the leastaffected by the sulphide treatment and they also had intrinsicallyhigher levels of COase activity than the other species sampled.Of the wetland plants the Salicornia species had the lowestvalue for root aerenchyma, 3–6%. Therefore, there wasno correlation between the possession of aerenchyma and thephysiological changes measured. Measurement of malate, lactateand ethanol in roots of the first four species listed abovegave no evidence for alternative anaerobic fermentation pathways.While in the flood-intolerant species, high ADH activities werenot able to maintain the energy charge. It is suggested thatmaintenance of relatively high COase activity in wetland plantsmay help to ‘scavenge’ any available oxygen withinroots and thus help reduce energy loss. Key words: Cytochrome oxidase, alcohol dehydrogenase, metabolic adaptation     

Genetic variation in supernatant malate dehydrogenase of birds and reptiles

Electrophoretic variants of supernatant malate dehydrogenase were found within populations of a brid (Pitohui kirhocephalus) and of a reptile (Chrysemys picta). Upon starch gel electrophoresis, tissue extracts from variant individuals exhibit three equally spaced bands of supernatant malate dehydrogenase activity, whose staining intensities are approximately in a ratio of 1:2:1. Normal individuals, on the other hand, exhibit a single major band which is identical in mobility with the fastest of the variant bands. Immunological tests show that the Pitohui and Chrysemys enzymes are homologous with the well-characterized supernatant malate dehydrogenase of the domestic chicken. These results along with those obtained on other species imply that birds, reptiles, mammals, and certain invertebrates may possess a single locus coding for supernatant malate dehydrogenase. By contrast, some species of fishes and invertebrates possess multiple loci for this enzyme. A notable feature of the present investigation was the use of phenoxyethanol for extracting malate dehydrogenases from bird tissues. Extracts were prepared simply by soaking tissue in an aqueous solution of this reagent, no homogenization or centrifugation being required.This investigation was supported by funds from a research grant awarded by the National Science Foundation (GB-13119) and a U.S. Public Health Service Training Grant (GM 31-12).     

Mechanism of the Increase in Ribonuclease Activity in Potato Tuber Slices

Upon wounding of potato tubers (Solanum tuberosum L. cv. Spunta)RNase activity increases, peaks in about 16 hours, then declines.To see if the increase of the activity is due to de novo synthesisof the enzyme protein, the extracts were compared for theirability to react with a rabbit antibody prepared against thewound activated RNase. The enzyme was purified by polyacrylamidegel electrophoresis of a RNase preparation, which had been partiallypurified from aged potato slices by ammonium sulfate precipitation,carboxymethyl-Sephadex column chromatography and gel filtrationthrough Sephadex G-100. Using rocket immunoelectrophoresis RNase-proteinimmunoprecipitated by the antibody increased in wounded tissue.This observation implies that the activity increase involvesenzyme synthesis. The increase was inhibited by actinomycinD and cordycepin, but not by 5-fluorouracil, suggesting a requirementfor mRNA synthesis. (Received April 9, 1985; Accepted December 16, 1985)     

Diurnal regulation of phosphoenolpyruvate carboxylase from crassula

Phosphoenolpyruvate carboxylase appears to be located in or associated with the chloroplasts of Crassula. As has been found with this enzyme in other CAM plants, a crude extract of leaves gathered during darkness and rapidly assayed for phosphoenolpyruvate carboxylase (PEPc) activity is relatively insensitive to inhibition by malate. After illumination begins, the PEPc activity becomes progressively more sensitive to malate. This enzyme also shows a diurnal change in activation by glucose-6-phosphate, with the enzyme from dark leaves more strongly activated than that from leaves in the light.

When the enzyme is partially purified in the presence of malate, the characteristic sensitivity of the day leaf enzyme is largely retained. Partial purification of the enzyme from dark leaves results in a small increase in sensitivity to malate inhibition.

Partially purified enzyme is found by polyacrylamide gel electrophoresis analysis to have two bands of PEPc activity. In enzymes from dark leaves, the slower moving band predominates, but in the light, the faster moving band is preponderant. Both of these bands are shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be composed of the same subunit of 103,000 daltons.

The enzyme partially purified from night leaves has a pH optimum of 5.6, and is relatively insensitive to malate inhibition over the range from pH 4.5 to 8. The enzyme from day leaves has a pH optimum of 6.6 and is strongly inhibited by malate at pH values below 7, but becomes insensitive at higher pH values.

Gel filtration of partially purified PEPc showed two activity peaks, one corresponding approximately to a dimer of the single subunit, and the other twice as large. The larger protein was relatively insensitive to malate inhibition, the smaller was strongly inhibited by malate.

Kinetic studies showed that malate is a mixed type inhibitor of the sensitive, day, enzyme, increasing K_m for phosphoenolpyruvate and reducing V_max. With the insensitive, night, enzyme, malate is a K type inhibitor, reducing the K_m for phosphoenolpyruvate, but having little effect on V_max. The inhibition of the insensitive enzyme by malate appears to be hysteretic, taking several minutes to be expressed during assay, probably indicating a change in the conformation or aggregation state of the enzyme.

Activation by glucose-6-phosphate is of the mixed type for the day form of the enzyme, causing both a decreased K_m for phosphoenolpyruvate and an increased V_max, but the night, or insensitive, form shows only an increase in V_max in response to glucose-6-phosphate.

     

Immunochemical Investigations with Highly Purified Glutamate Dehydrogenase from Pea Seeds by Means of the Ouchterlony Test

Monospecific antibodies have been prepared with a homogeneousprotein fraction of the main activity band of pea seed glutamatedehydrogenase. This protein precipitates with its antibodiesin a single band with complete fusion as seen by the Ouchterlonydouble-diffusion test. Identical behaviour is observed withthe protein of the adjacent activity bands of the multiple molecularforms of this enzyme and the antibodies to the former fraction.Organ-specific ‘isoenzymes’ of glutamate dehydrogenasewith preparations of pea roots and epicotyls are not detectedby this procedure. Partially purified glutamate dehydrogenasepreparations from Lemna perpusilla, Zea mays, and Oryza sativaalso precipitate with the antibodies to the pea protein. TheLemna protein is shown to be different from the pea enzyme asjudged from immunological behaviour. The pea antibodies¹ donot cross-react with glutamate dehydrogenases from Candida orbeef liver, nor do the beef liver antibodies react with thepea and Candida enzymes.     

Some Biochemical Characteristics of Guard Cell and Mesophyll Cell Protoplasts from Commelina communis L.

Guard cell and mesophyll cell protoplasts of Commelina communisL., were isolated and used to investigate their various biochemicalcharacteristics. Contamination of the samples by other celltypes was very low and viability of the protoplasts, assessedby the use of neutral red, Evans blue and fluorescein diacetate,was high (89–98%). Mesophyll cell protoplasts containedmore chlorophyll (x 47), more soluble protein (x 10), more totalN (x 36) and more DNA (x 9) than guard cell protoplasts. Theabsorption spectra of protoplast extracts were similar for bothcell types except that below 400 nm there was a large increasein absorption by the guard cell protoplast extract. In guardcell protoplast extracts, high levels of activity of phosphoenolpyruvatecarboxylase (E.C. 4.1.1.31 [EC] ), NAD malate dehydrogenase (E.C.1.1,1.37), NADP malic enzyme (E.C. 1.1.1.40 [EC] ) and carbonic anhydrase(E.C. 4.2.1.1 [EC] ) were detected while only low levels of pyruvate-orthophosphatedikinase (E.C. 2.7.9.1 [EC] ) activity were detected. Glycollate oxidase(E.C. 1.1.3.1 [EC] ), ribulose-l,5-bisphosphate carboxylase (E.C 4.1.1.39 [EC] ),NADP malate dehydrogenase (E.C. 1.1.1.82 [EC] ) and NAD malic enzyme(E.C. 1.1.1.39 [EC] ) were not detected in guard cell protoplast extracts.High levels of ribulose-1, 5-bisphosphate carboxylase, glycollateoxidase, NAD malate dehydrogenase and carbonic anhydrase weredetected in mesophyll cell protoplast extracts which is typicalof C₃ plants. A pathway of carbon flow during stomatal openingand closing is proposed. Key words: Carbon metabolism, Commelina communis, guard cell protoplasts, mesophyll cell protoplasts, stomata     

Effect of growth temperature on reserve mobilization and hydrolytic enzyme activities in Vigna mungo cotyledons

Seeds of Vigna mungo were allowed to germinate at 27, 18 and15°C, and time-course changes of hydrolytic enzyme activitiesand the mobilization rate of reserve components in cotyledonswere studied. The seeds germinated at 27 and 18°C grew normally,whereas the growth at 15°C was markedly retarded. In cotyledonsof seedlings grown at 27 and 18°C, amylolytic and proteolyticenzyme activities increased at early stages of growth and therates of starch and protein mobilization changed correspondingto the hydrolytic enzyme activities. At 15°C the enzymeactivities increased gradually during the experimental periodof 16 days, but the reserves in cotyledons remained almost unchangeduntil the end of the experimental period. Changes of zymogram patterns of amylolytic and proteolytic activitiesin cotyledons of seedlings grownat 27, 18 and 15°C wereexamined using polyacrylamide gel electrophoresis. The intensitiesof a main band of a-amylase and at least two bands of protease(gelatin-hydrolyzing activity) increased concurrently with invitro activities of amylolytic and proteolytic enzymes. At leastthree bands of starch phosphorylase were present in cotyledonsat early stages of germination and their intensities decreasedduring the growth of seedlings at 27, 18 and 15°C. (Received June 4, 1980; )     

Enzymes of malate metabolism in Mesorhizobium ciceri CC 1192

Electrophoretic studies were performed on enzymes concerned with the oxidation of malate in free-living and bacteroid cells of Mesorhizobium ciceri CC 1192, which forms nitrogen-fixing symbioses with chickpea (Cicer arietinum L.) plants. Two malate dehydrogenases were detected in extracts from both types of cells in native polyacrylamide electrophoresis gels that were stained for enzyme activity. One band of malate dehydrogenase activity was stained only in the presence of NADP+, whereas the other band was revealed with NAD+ but not NADP+. Further evidence for the occurrence of separate NAD- and NADP-dependent malate dehydrogenases was obtained from preliminary enzyme kinetic studies with crude extracts from free-living M. ciceri CC 1192 cells. Activity staining of electrophoretic gels also indicated the presence of two malic enzymes in free-living and bacteroid cells of M. ciceri CC 1192. One malic enzyme was active with both NAD+ and NADP+, whereas the other was specific for NADP+. Possible roles of the multiple forms of malate dehydrogenase and malic enzyme in nitrogen-fixing symbioses are discussed.     

Nardilysin facilitates complex formation between mitochondrial malate dehydrogenase and citrate synthase

Gel filtration chromatography showed that nardilysin activity in a rat testis or rat brain extract exhibited an apparent molecular weight of approximately 300 kDa compared to approximately 187 kDa for the purified enzyme. The addition of purified nardilysin to a rat brain extract, but not to an E. coli extract, produced the higher molecular species. The addition of a GST fusion protein containing the acidic domain of nardilysin eliminated the higher molecular weight nardilysin forms, suggesting that oligomerization involves the acidic domain of nardilysin. Using an immobilized nardilysin column, mitochondrial malate dehydrogenase (mMDH) and citrate synthase (CS) were isolated from a fractionated rat brain extract. Porcine mMDH, but not porcine cytosolic MDH, was shown to form a heterodimer with nardilysin. Mitochondrial MDH increased nardilysin activity about 50%, while nardilysin stabilized mMDH towards heat inactivation. CS was co-immunoprecipitated with mMDH only in the presence of nardilysin showing that nardilysin facilitates complex formation.     

Purification of Chlorella Malate Dehydrogenase

Our prior investigation of Chlorella malate dehydrogenase (MDH) revealed supernatant and particulate isoenzymes which were immunologically, chromatographically, and electro-phoretically distinct. By means of ammonium sulfate fractionation and column chromatography a crystalline preparation of the particulate isoenzyme, specific activity of approximately 2000 has been obtained. It appears homogeneous in the analytical ultracentrifuge, by column chromatography and by electrophoresis. This preparation migrates as a single band of activity when subjected to starch gel electrophoresis and its mobility and activity (on the gel) are unaffected by prior incubation with citric acid, pH 2.0, EDTA, β-mercapto-ethanol, substrates or products; however, new bands appear when preincubated with exogenous proteins (RNase, Ovalbumin, Aldolase).     

STUDIES ON HOMOSERINE DEHYDROGENASE IN PEA SEEDLINGS

Partially purified homoserine dehydrogenase was prepared frompea seedlings. The optimum pH for this enzyme is approximately 5.4. The K_mvaluesfor ASA and TPNH are 4.6xl0^–4Af and 7.7xl0^–5M, respectively.This enzyme can also utilize DPNH but less effectively thanTPNH. In contrast with yeast homoserine dehydrogenase whichis insensitive to — SH reagents, the pea enzyme is inhibitedalmost completely by 10^–4MPCMB and 10^–5MHgCl₂, theinhibition being removed by 10^–2M thioglycolate. Homoserinedehydrogenase was found not only in decotylized seedlings, butalso in cotyledons. The significance of this enzyme in homoserine biosynthesis ingerminating pea seeds has been discussed. (Received February 20, 1961; )