NADP-dependent Malate Dehydrogenase Research Articles

Molecular size of enzymes in Trypanosoma cruzi considered in relationship to the genetic interpretation of isozyme patterns

The molecular size of seven Trypanosoma cruzi enzymes, chosen because of their frequent use in studying trypanosome populations, has been found to be similar to that of their mammalian equivalents. Malic enzyme (NADP-dependent malate dehydrogenase, decarboxylating, EC 1.1.1.40) from T. cruzi has an apparent molecular size of only half that of the mammalian enzyme. The probable subunit structure of these T. cruzi enzymes has been deduced from the molecular weights by comparison with mammalian data. The results are compatible with recent interpretations of isozyme data implying the existence of genetic diploidy in trypanosomes.

Intracellular Localization of Enzymes of Carbon Metabolism in Mesembryanthemum crystallinum Exhibiting C3 Photosynthetic Characteristics or Performing Crassulacean Acid Metabolism

Mesembryanthemum crystallinum, a halophilic, inducible Crassulacean acid metabolism (CAM) species, was grown at NaCl concentrations of 20 and 400 millimolar in the rooting medium. Plants from the low salinity treatment showed exclusively C(3)-photosynthetic net CO(2) fixation, whereas plants exposed to the high salinity level exhibited net CO(2) dark fixation involving CAM. Mesophyll protoplasts, isolated from both tissues, were gently ruptured, and the intracellular localization of enzymes was studied following differential centrifugation and Percoll density gradient centrifugation of protoplast extracts. Both centrifugation techniques resulted in the separation of intact chloroplasts, with up to 90% yield, from other organelles and the nonparticulate fraction of cells. Enzymes were identified by determination of activity and by sodium dodecyl sulfate gel electrophoresis of enzyme protein.Experiments established the extraorganellar (cytoplasmic) location of phosphoenolpyruvate carboxylase, enolase, phosphoglyceromutase, and NADP-malic enzyme; the mitochondrial location of NAD-malic enzyme; and the chloroplastic location of pyruvate, Pi dikinase. NAD-glyceraldehyde-3-phosphate dehydrogenase, phosphohexose isomerase, and phosphoglycerate kinase were associated with both cytoplasm and chloroplasts. NADP-dependent malate dehydrogenase activity was found in both the chloroplastic and extrachloroplastic fractions; the activity in the chloroplast showed an optimum at pH 8.0 and was dependent upon preincubation of enzyme with dithiothreitol. The extrachloroplastic activity showed an optimum at pH 6.5 and was independent of pretreatment with dithiothreitol. Protoplast extracts of M. crystallinum performing CAM exhibited higher activities (expressed per mg chlorophyll per min) of phosphoenolpyruvate carboxylase, pyruvate, Pi dikinase, NADP-malic enzyme, NAD-malic enzyme, NADP-malate dehydrogenase, enolase, phosphoglyceromutase, NAD-glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and phosphohexose isomerase than protoplast extracts from M. crystallinum not exhibiting CAM. The increase in total activity of the latter three enzymes following exposure of plants to 400 millimolar NaCl and the development of CAM was due to specific increases in the levels of activity in the cytoplasm.

Dark modulation of NADP-dependent malate dehydrogenase and glucose-6-phosphate dehydrogenase in the chloroplast

The properties of the system which reverses light modulation of NADP-dependent malate dehydrogenase and glucose-6-phosphate dehydrogenase activity in pea chloroplasts were examined. A factor catalyzing dark modulation of these enzymes was found. This factor cochromatographed with thioredoxin in all systems used (Sephacryl S-200, Sephadex G-75, DEAE-cellulose). Inhibition of dithiothreitol-dependent modulation and of dark reversal by antibody against Escherichia coli thioredoxin further suggest that the dark factor is in fact thioredoxin. It appears that the reaction is the reverse of the previously described dithiothreitol-dependent thioredoxin-catalyzed modulation of enzymes. The limiting step in vitro seems to be the oxidation of thioredoxin during the dark period.

Isomers in Thioredoxins of Spinach Chloroplasts

We have developed a method for the concomitant purification of several components of the ferredoxin/thioredoxin system of spinach chloroplasts. By applying this method to spinach-leaf extract or spinach-chloroplast extract we separated and purified three thioredoxins indigenous to chloroplasts. The three thioredoxins, when reduced, will activate certain chloroplast enzymes such as fructose-1,6-bisphosphatase and NADP-dependent malate dehydrogenase. Fructose-1,6-bisphosphatase is activated by thioredoxin f exclusively. Malate dehydrogenase is activated by thioredoxin mb and thioredoxin mc in a similar way, and it is also activated by thioredoxin f but with different kinetics. All three thioredoxins have very similar relative molecular masses of about 12,000 but distinct isoelectric points of 6.1 (thioredoxin f), 5.2 (thioredoxin mb) and 5.0 (thioredoxin mc). The amino acid composition as well as the C-terminal and N-terminal sequences have been determined for each thioredoxin. Thioredoxin f exhibits clear differences in amino acid composition and terminal sequences when compared with the m-type thioredoxins. Thioredoxin mb and thioredoxin mc, however, are very similar, the only difference being an additional lysine residue at the N-terminus of thioredoxin mb. Amino acid analyses, terminal sequences, immunological tests and the activation properties of the thioredoxins support our conclusion that thioredoxins mb and mc are N-terminal redundant isomers coming from one gene whereas thioredoxin f is a different protein coded by a different gene.

Cell organelles from crassulacean acid metabolism (CAM) plants

The intracellular distribution of enzymes involved in the Crassulacean acid metabolism (CAM) has been studied in Bryophyllum calycinum Salisb. and Crassula lycopodioides Lam. After separation of cell organelles by isopycnic centrifugation, enzymes of the Crassulacean acid metabolism were found in the following cell fractions: Phosphoenolpyruvate carboxylase in the chloroplasts; NAD-dependent malate dehydrogenase in the mitochondria and in the supernatant; NADP-dependent malate dehydrogenase and phosphoenolpyruvate carboxykinase in the chloroplasts; NADP-dependent malic enzyme in the supernatant and to a minor extent in the chloroplasts; NAD-dependent malic enzyme in the supernatant and to some degree in the mitochondria; and pyruvate; orthophosphate dikinase in the chloroplasts. The activity of the NAD-dependent malate dehydrogenase was due to three isoenzymes separated by (NH4)2SO4 gradient solubilization. These isoenzymes represented 17, 78, and 5% of the activity recovered, respectively, in the order of elution. The isoenzyme eluting first was associated with the mitochondria and the second isoenzyme was of cytosolic origin, while the intracellular location of the third isoenzyme was probably the peroxisome. Based on these findings, the metabolic path of Crassulacean acid metabolism within cells of CAM plants is discussed.

On the biochemical systematics of three apodemus species

-1. Electrophoretic patterns of various proteins were studied in samples of Apodemus silvaticus, Apodemus flavicollis and Apodemus microps populations. 2. According to electrophoretic results Apodemus microps is closer related to Apodemusflavicollis than to Apodemus sylvaticus. 3. From the data of 26 loci, Nei's coefficient of the overall genetic distance between Apodemus flavicollis and Apodemus sylvaticus was calculated and resulted in a divergence time of some 423,000 yr between the two species. I N T R O D U C T I O N Protein electrophoresis has proved to be a powerful tool for resolving systematic problems at the species and sub-species level. The advantages and disadvantages of this method have been discussed (Avise, 1974, 1976; Mayr 1975; Selander 1976). It is the aim of this study, using biochemical markers, to elucidate the systematic relationship between three species of the sub-genus Sylvaemus, a group of mice that are very difficult to classify morphologically, especially at younger age. Zimmermann (1962) has grouped into this sub-genus the three species Apodemus sylvaticus (L.), Apodemus flavicollis (Melchior), and Apodemus microps (Kratochvil et Rosicky). Karyotype analyses (Matthey, 1936, 1962; Kral 1970; Engel et al., 1973) show the close relationship among these species. As yet there is no single morphological feature known to the authors by which these species could be separated. The members of this species triad often can be distinguished only by correlation of some morphological features (Steiner 1968; Csaiki, manuscript in preparation). Together with the systematic results a new catching locality for Apodemus microps is described which seems to represent one of the most westerly points of the distribution of this species. MATERIALS AND METHODS Collection of material All the animals examined were caught live by means of wooden traps in the Steinfeld, a part of the Wiener Becken in Lower Austria. Apodemus sylvaticus (n = 23) and Apodemus microps (n = 2) were caught in the southwest of the village Tattendoff along hedges and windbreaks in the autumn of 1977. Apodemusflavicollis (n = 21) was caught at the same time in the woodlands of the river Leitha near the town Ebenfurt. Ecological studies on the fauna of these biotopes carried out by one of the authors (F.C.) revealed that Apo~ demus microps occurs in low densities and has a very large migration rate. The earlier work of Steiner (1968) also showed for other biotopes the low densities and the unsteady character of this species. 411 This may be the reason that among the normally very successful capture rates of Apodemus sylvaticus only two Apodemus microps individuals were caught. Electrophoretic methods Blood was taken by puncture of the sinus retro-orbitalis following anaesthesia of the animals. An equal volume of a 1.47~o glucose-0.44~ o citric acid-l.32~o sodium citrate solution was added, and after centrifugation at 2000 g for 2 min at 4°C, the supernatant serum was removed with a pipette. and frozen at -20°C. This provided the material for the investigation of serum proteins. To the erythrocytes was added an equal volume of 0.01 M phosphate buffer (pH = 7.4) containing 0.1 mM MgCI2. After freezing and thawing the hemolysates were centrifuged at 20,000 g for 30 min at 4°C. The clear supernatant was absorbed to filter pads and inserted into gel slots. Organs were homogenized in a glass/glass PotterElvenhjem homogenizer using the same phosphate buffer as described above. The samples were treated further as described for the lysate. For electrophoresis four different buffer systems were used: (1) gel: 13Yo starch in 0.006M potassium phosphate, pH = 7,4; bridge: 0.1 M potassium phosphate, pH = 7.4; for lactate dehydrogenase (LDH), 6-phosphogluconate dehydrogenase (6-PGD), glucose-6-phosphate dehydrogenase (G-6-PD), adenosine deaminase (ADA), superoxide dismutase (SOD), aspartate aminotransferase (AAT), glyoxalase I (GLO I), NAD-dependent malate dehydrogenase (NAD-MDH), NADP-dependent malate dehydrogenase (NADP-MDH), esterase D (Est D), phosphoglucose isomerase (PGI), haemoglobin (Hb) (2) gel: 12Yo starch in 0.008M Tris-0.003 Citrate. pH = 8.0; bridge: 0.223 M Tris-0.068 M Citrate, pH = 8.0, for NADPH-dependent isocitrate dehydrogenase (NADPIDH). (3) gel: 12~o starch in 0.01 M Tris-0.01 M Malate0.001 M MgCI2, pH = 7.7; bridge: 0.1 M Tris-0.1 Malate0.01 M MgCI2, pH = 7.7, for creatine phosphokinase (CPK), adenylate kinase (AK), aldolase (Ald). (4) gel: 13~o starch in 0.043 M Tris-0.012 Citrate-0.05 M LiOH-0.0015 M Borate, pH = 7.4; bridge: 0.2 M Borate, pH = 8.0, for transferrine (TO and postalbumine (PA). After electrophoresis the gels were sliced and stained for Est D according to Hopkinson et al. (1973), for ADA according to Spencer et al. (1968), for GLO I according to KiSmpf et al. (1975) and according to Shaw & Prasad (1970) for all other enzymes.

On the Mechanism of Activation by Light of the NADP-dependent Malate Dehydrogenase in Spinach Chloroplasts

With intact spinach (Spinacia oleracea L. cv. Vital R) chloroplasts, the activity of the NADP-dependent malate dehydrogenase after activation by light was 30 micromoles of malate formed per milligram of chlorophyll per hour; an identical rate of O(2) evolution was obtained upon oxaloacetate reduction by the intact plastids. However, when the activity of NADP-dependent malate dehydrogenase was measured subsequently to maximal activation of the enzyme by dithiothreitol (DTT) an average rate of 113 micromoles per milligram of chlorophyll per hour was obtained. When membranes and stroma were separated after osmotic disruption of the chloroplasts, 28% of NADP-dependent malate dehydrogenase activity inducible by DTT was found with the membranes and 72% was found in the stromal fraction. The membrane-associated portion of the enzyme corresponds well with the activity achieved after activation by light. About 64% of an activator system was found to be associated also with the membrane fraction. Washing the membranes with buffer removed more activator than enzyme. However, both were removed almost completely by ethylenediaminetetraacetate. It was concluded that both a portion of the enzyme and the total activator system are associated with the chloroplast membranes in vivo and that the activator is more loosely bound than the enzyme. A model describing the partial activation of chloroplastic NADP-dependent malate dehydrogenase by light and the total activation by DTT is presented.

Improved in vitro light activation and assay systems for two spinach chloroplast enzymes

An improved system for the in vitro light activation of the chloroplast enzymes fructose-1,6-bisphosphatase and NADP-dependent malate dehydrogenase is described. Through the presence of the monothiol β-mercapto-ethanol in the reaction mixtures the activated forms of the enzymes can be stabilized and their activity determined spectrophotometrically.

Intracellular distribution of enzymes associated with lipogenesis and gluconeogenesis in fat body of the adult cockroach, Periplaneta

The intracellular distribution and maximal activities of some enzymes associated with lipogenesis, gluconeogenesis and fatty acid oxidation have been determined in the fat body of the adult male cockroach Periplaneta americana. Of the enzymes of lipogenesis, acetyl-CoA synthase, acetyl-CoA carboxylase, glucose 6-phosphate dehydrogenase, and ATP citrate lyase are located entirely in the cytosol. Of the other enzymes of citrate metabolism studied, citrate synthase and NAD-dependent isocitrate dehydrogenase are almost exclusively mitochondrial, whereas NADP-dependent isocitrate dehydrogenase and aconitase are predominantly cytosolic although significant mitochondrial activity is also present. The latter subcellular distribution is also observed for ‘malic enzyme’ and NAD and NADP dependent malate dehydrogenase. The enzyme of fatty acid oxidation studied, 3-hydroxyacyl-CoA dehydrogenase is entirely mitochondrial. Of the enzymes possibly involved in gluconeogenesis, glucose 6-phosphate is microsomal, fructose 1,6-diphosphatase cytosolic, pyruvate carboxylase mitochondrial and phosphoenolpyruvate carboxykinase predominantly cytosolic. Of the enzymes of amino acid metabolism studied, glutamate dehydrogenase is NAD-dependent and located in the mitochondria whereas glutamate/oxalacetate and glutamate/pyruvate transaminases are predominantly cytosolic but with significant activity in the mitochondria. Glycerol kinase and sorbitol dehydrogenase are cytosolic and the glyoxylate cycle enzymes malate synthetase and isocitrate lyase are not detected in the fat body. The distribution and relative activities of the enzymes are discussed in relation to fat body biosynthesis and in comparison to mammalian liver and adipose tissue.

A host contribution to the regeneration of muscle grafts

Isoenzymic forms of NADP-dependent malate dehydrogenase (MDH) have been used as markers in experiments to discover whether the source(s) of the new muscle fibres which form in grafts are of host, donor or mixed origin in regenerating muscle. Grafts of minced tibialis anterior muscle were made from CBA donor mice into C3H hosts. These 2 strains produce different isoenzymic forms of NADP-MDH. C3H hosts were made tolerant to the donor strain by neonatal injection of CBA × C3H lymphoid cells. Some 73% of grafts of CBA muscle made into tolerant C3H hosts contained newly formed muscle fibres. Pools of such grafts yielded on extraction MDH isoenzymes which were shown by electrophoresis to be largely of host type: traces were found of donor-type but not of hybrid isoenzymes. Histochemical and other evidence suggested that MDH activity was derived mainly from the muscle component of grafts (but in part from fibrous connective tissue). The absence from 150–220 day-old grafts of hybrid isoenzymes suggests that few, if any, muscle fibres had formed by fusion of host with donor cells. The predominance of host isoenzymes suggests that little or none of the muscle formed was of donor origin.

Etude de l'evolution de la malate deshydrogenase a NADP durant le verdissement des feuilles de Phaseolus vulgaris L

The evolution of NADP-dependent malate dehydrogenase (MDH) activity was examined during the greening of primary leaves of Phaseolus vulgaris. The enzyme was present in material grown in darkness; upon illumination, the specific activity reached a level two to six times higher than of etiolated material. During the same time, NAD dependent MDH activity remained unchanged whereas the level of NADP dependent G-3P-DH increased tenfold. After the dark-light transition, D-threo-chloramphenicol had no effect on the evolution of enzyme actvities. This suggests that the enhancement of the catalytic power of these chloroplastic enzymes does not result from de novo protein synthesis but more probably from the activation, under light control, of pre-existing molecules.

Changes in enzyme pattern of ehrlich ascites tumor cells following serial cultivation in media with increased (hypertonic) NaCl content

Adaptation of Ehrlich ascites tumor cells to serial cultivation in media with progressively elevated (hypertonic) NaCl content (“high NaCl”-tolerant cells) has resulted in progressive increases of the cellular activities of NAD-dependent glycerol-3-phosohate dehydrogenase (EC 1.1.1.8), NAD-dependent malate dehydrogenase (EC 1.1.1.37), glutamate—oxalacetate transaminase (EC 2.6.1.1.), NAD(P)-dependent glutamate dehydrogenase (EC 1.4.1.3), NADP-dependent malate dehydrogenase (EC 1.1.1.40, “malic enzyme”) and NADP-dependent isocitrate dehydrogenase (EC 1.1.1.42). The activities of glutamate—pyruvate transaminase (EC 2.6.1.2.) and of glycolytic enzymes as phosphofructokinase (EC 2.7.1.11), glyceradehydephosphate dehydrogenase (EC 1.2.1.12) and lactate dehydrogenase (EC 1.1.1.27) were only slightly and not in progressive manner (in response to the progressive increase of the environmental NaCl concentration) affected. These changes are discussed with respect to a metabolic pattern of these “high NaCl”-tolerant cells which is compatible with increased energy requirements, especially for active cation transport. It is suggested that these increased cellular enzyme activitees reflect an increased transfer of reducing equivalents across mitochondrial membranes (via the “glycerophosphate cycle and the malate—aspartate shuttle”) and possibly a stimulated lipid metabolism. These alterations in the level of enzyme activities must be regarded as an adaptive cellular response to the “high NaCl” enviromment, since readaptation to growth in regular isotonic media resulted in a reversion to the enzyme pattern characteristic of the parent cells.

Action of sulphite on plant malate dehydrogenase

SO 3 2− acts on NAD- and NADP-dependent malate dehydrogenase in several ways. Firstly, SO 3 2− favours the appearance of low MW species (65 000 and 39 000 daltons) in Sephadex gel chromatography. Secondly, the enzyme form which is obtained by gel chromatography with dithioerythritol plus nucleotide cofactor is changed in the presence of SO 3 2−. This is indicated by the appearance of a linear reaction (instead of curvilinear), and by the abolition of the biphasic sigmoidal kinetics on varying substrate and cofactor concentrations. SO 3 2− causes the loss of negative cooperativity at low substrate or cofactor concentrations. Thus the inhibition of initial velocity at high substrate or cofactor concentrations is even more marked than at lower ones. Thirdly, SO 3 2− strongly reduces the activity in substrate saturating conditions.

Histochemical Observations of Steroid Dehydrogenases and Related Enzymes in the Livers of Laying and Moulting Hens

The livers of laying White Leghorn hens showed moderate 17β-hydroxysteroid dehydrogenase activity, whereas those of moulting hens had none or were weak. This activity had an intimate relationship with ovarian hormone synthesis. No Δ5-3β-hydroxysteroid dehydrogenase activity was found in the livers of laying or moulting hens which indicated an absence of steroid hormone synthesis in chicken livers. Moderate NADP-dependent malate dehydrogenase activity appeared in the livers of laying hens but there was little in those of moulting ones. No glucose-6-phosphate dehydrogenase activity was found. From these results, it was assumed that the NADPH2 utilized in steroid metabolism must be supplied from NADP-dependent malate dehydrogenase. Although NADP-dependent isocitrate dehydrogenase was strong in chicken livers, it is doubtful that it participates in steroid synthesis because this enzyme is located only in mitochondria. The lactate and succinate dehydrogenase activities were strong in the livers of laying hens but weak in those that were moulting. NAD-dependent malate dehydrogenase activity was strong, and that of NAD-dependent isocitrate dehydrogenase weak, with no differences between laying and moulting hens.

Separation of Mesophyll Protoplasts and Bundle Sheath Cells from Maize Leaves for Photosynthetic Studies

Mesophyll protoplasts and bundle sheath strands of maize (Zea mays L.) leaves have been isolated by enzymatic digestion with cellulase. Mesophyll protoplasts, enzymatically released from maize leaf segments, were further purified by use of a polyethylene glycol-dextran liquid-liquid two phase system. Bundle sheath strands released from the leaf segments were isolated using filtration techniques. Light and electron microscopy show separation of the mesophyll cell protoplasts from bundle sheath strands. Two varieties of maize isolated mesophyll protoplasts had chlorophyll a/b ratios of 3.1 and 3.3, whereas isolated bundle sheath strands had chlorophyll a/b ratios of 6.2 and 6.6. Based on the chlorophyll a/b ratios in mesophyll protoplasts, bundle sheath cells, and whole leaf extracts, approximately 60% of the chlorophyll in the maize leaves would be in mesophyll cells and 40% in bundle sheath cells. The purity of the preparations was also evident from the exclusive localization of phosphopyruvate carboxylase (EC 4.1.1.31) and NADP-dependent malate dehydrogenase (EC 1.1.1) in mesophyll cells and ribulose 1,5-diphosphate carboxylase (EC 4.1.1.39), phosphoribulokinase (EC 2.7.1.19), and "malic enzyme" (EC 1.1.1.40) in bundle sheath cells. NADP-glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.13) was found in both mesophyll and bundle sheath cells, while ribose 5-phosphate isomerase (EC 5.3.1.6) was primarily found in bundle sheath cells. In comparison to the enzyme activities in the whole leaf extract, there was about 90% recovery of the mesophyll enzymes and 65% recovery of the bundle sheath enzymes in the cellular preparations.

Synchronous activation of the alleles coding for the S-form of the NADP-dependent malate dehydrogenase during mouse embryogenesis.

The first appearance during mouse development of different allelic products was studied using the supernatant (S-)form of NADP-dependent MDH. Heterozygote offspring were produced by cross-breeding the strains C3H and CBA, and the embryos were taken at various developmental stages. The electrophoretic examination revealed no enzyme activity in unfertilized ova and preimplantation stages. After implantation, an isoenzyme pattern was seen for the first time at day 10 (228 hrs after fertilization), representing the 3 different heteromers of this tetrameric enzyme in binomial distribution. 240 hrs after fertilization, the 5-banded pattern to be expected in the heterozygotes was complete. Measurements of enzyme activity revealed a strong activity increase at about 240 hrs. The results indicate that the S-form of NADP-MDH appears rather late in development and that the two alleles tested at this gene locus commence functioning synchronously.

Interspecific hybridization between human and mouse somatic cells: enzyme and linkage studies.

Human-mouse somatic cell hybrids have been isolated and examined for enzyme and chromosome constitution. The enzymes assayed were lactate dehydrogenase (LDH), isocitrate dehydrogenase (IDH), NADP-dependent malate dehydrogenase (MDH), glucose 6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), phosphoglucomutase (PGM), and several esterases. Coexpression of mouse and human genomes and formation of heteropolymeric enzymes were observed in seven different hybrid populations for the enzymes LDH, IDH, MDH, and G6PD. Evidence predicated on the absence of chromosomal rearrangements is provided for the lack of genetic linkage in the human genome for these four enzymes, as well as for thymidine kinase.

Subunit structure and gene control of mouse NADP-malate dehydrogenase.

The NADP-dependent malate dehydrogenase (MDH) in the supernatant fraction of mouse tissues is known to occur in two allelic forms which are electrophoretically distinguishable; each produces a single band in starch gel. We have investigated the subunit structure and synthesis of NADP—MDH through electrophoretic patterns obtained from several experimental sources. (1) Heterozygotes containing both alleles yield a five-banded pattern. The bands are in an approximate frequency of 1:4:6:4:1; the two extremes correspond to the pure types and the three intermediates are presumably hybrid enzymes. The NADP—MDH molecule therefore appears to be a tetramer. (2) In muscle heterokaryons of allophenic mice (with homozygous nuclei of each genotype within a common cytoplasm), hybrid enzymes are formed; they are not formed in other allophenic tissues. Therefore the gene at this locus codes only for monomeric subunits and the tetramer is assembled in a second step in the cytoplasm. Also, both genes must function in a nucleus when the locus is active (e.g., in F1 uninucleated cells). (3) Dissociation in vitro of mixtures of both pure types of enzymes, followed by reassociation among fragments, leads to a three-banded pattern, even after repeated cycles. Thus the tetramer must cleave in a fixed plane, to form dimers, which reassociate, rather than in a random fashion to form monomers. The most likely interpretation is that mouse NADP—MDH is an example of the type of tetramer postulated by Monod et al. (1965) and termed “isologous.” The dimers are held symmetrically in the tetrameric conformation by relatively weak forces; the monomeric subunits comprising the dimer are held together by stronger forces.

NAD- and NADP-dependent malate dehydrogenase in wheat.

NAD- and NADP-dependent malate dehydrogenase in wheat.

Isozymes and genetic control of NADP-malate dehydrogenase in mice

NADP-dependent malate dehydrogenase (MDH) has been found to exist in two isozymic forms in mouse tissues. The two isozymes differ in electrophoretic mobility and tissue and subcellular location. The isozyme predominating in heart tissue is located in the mitochondrial fraction and the isozyme predominating in liver tissue is located in the supernatant fraction of tissue homogenates. In an examination of inbred strains of mice, a genetic variant of the supernatant isozyme was found. The mitochondrial isozyme was not affected by this mutation. Preliminary genetic data indicate that the gene controlling supernatant NADP-MDH is in mouse linkage group II. Animals heterozygous for the alleles controlling the synthesis of NADP-MDH contain at least one hybrid enzyme, intermediate between the two parental forms in electrophoretic mobility. This indicates that the enzyme is composed of subunits.