Plant Leaf and Stem Proteins. II. Isozymes and Environmental Cabbage

The activity of 10 enzymes separated by acrylamide disc gel electrophoresis of leaf and stem extracts from Dianthus grown under summer and winter conditions was studied. While banding was constant and highly reproducible under each environment, differences between the 3 cultivars and between the tissues were evident. No significant differences in the isozyme patterns of glutamate dehydrogenase, 6-phosphogluconate dehydrogenase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, and catalase were observed between the 2 environments. Loss of activity was observed under winter conditions with amylase and lactate dehydrogenase and loss of certain isozymic components was evident with acid phosphatase and esterase. Prominent changes were observed in peroxidase isozymes, the hardy cultivars developing additional isozymic components under winter conditions. Only minor changes in the total protein banding were seen. The enzymes showed considerable stability in those tissues killed by the freezing conditions.     

Lactate and malate dehydrogenase in the fan-shell associated shrimp, Pontonia pinnophylax (Otto): Effects of temperature and urea

In Pontonia pinnophylax (Otto), a crustacean decapod inhabiting the mantle cavity of Pinna nobilis L. (Bivalvia: Pteriomorpha), the lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) activity, and their electrophoretic patterns, were compared in relation to heat and urea inactivation. Activity was higher in LDH than in MDH, and the electrophoretic patterns showed a predominance of LDH-A4 and the presence of both mitochondrial and cytosolic MDH. Heat incubation reduced both enzymatic activities, but more MDH. Also all isozymes showed different heat sensitivity, with anodic forms more heat-resistant than cathodic ones, either in LDH as in MDH. Urea treatment caused also a higher inactivation of the most cathodic isozymes, but MDH appeared more resistant than LDH at 2 M urea. The high polymorphism of these enzymes suggests an adaptation of Pontonia metabolism to hypoxic conditions; moreover, the different isozyme stability grade should be functional to contrast environmental variability.     

Chorion peroxidase-mediated NADH/O(2) oxidoreduction cooperated by chorion malate dehydrogenase-catalyzed NADH production: a feasible pathway leading to H(2)O(2) formation during chorion hardening in Aedes aegypti mosquitoes

A specific chorion peroxidase is present in Aedes aegypti and this enzyme is responsible for catalyzing chorion protein cross-linking through dityrosine formation during chorion hardening. Peroxidase-mediated dityrosine cross-linking requires H(2)O(2), and this study discusses the possible involvement of the chorion peroxidase in H(2)O(2) formation by mediating NADH/O(2) oxidoreduction during chorion hardening in A. aegypti eggs. Our data show that mosquito chorion peroxidase is able to catalyze pH-dependent NADH oxidation, which is enhanced in the presence of Mn(2+). Molecular oxygen is the electron acceptor during peroxidase-catalyzed NADH oxidation, and reduction of O(2) leads to the production of H(2)O(2), demonstrated by the formation of dityrosine in a NADH/peroxidase reaction mixture following addition of tyrosine. An oxidoreductase capable of catalyzing malate/NAD(+) oxidoreduction is also present in the egg chorion of A. aegypti. The cooperative roles of chorion malate/NAD(+)oxidoreductase and chorion peroxidase on generating H(2)O(2) with NAD(+) and malate as initial substrates were demonstrated by the production of dityrosine after addition of tyrosine to a reaction mixture containing NAD(+) and malate in the presence of both malate dehydrogenase fractions and purified chorion peroxidase. Data suggest that chorion peroxidase-mediated NADH/O(2) oxidoreduction may contribute to the formation of the H(2)O(2) required for chorion protein cross-linking mediated by the same peroxidase, and that the chorion associated malate dehydrogenase may be responsible for the supply of NADH for the H(2)O(2) production.     

The malate dehydrogenase isoforms from Trypanosoma brucei: subcellular localization and differential expression in bloodstream and procyclic forms

Trypanosoma brucei procyclic forms possess three different malate dehydrogenase isozymes that could be separated by hydrophobic interaction chromatography and were recognized as the mitochondrial, glycosomal and cytosolic malate dehydrogenase isozymes. The latter is the only malate dehydrogenase expressed in the bloodstream forms, thus confirming that the expression of malate dehydrogenase isozymes is regulated during the T. brucei life cycle. To achieve further biochemical characterization, the genes encoding mitochondrial and glycosomal malate dehydrogenase were cloned on the basis of previously reported nucleotide sequences and the recombinant enzymes were functionally expressed in Escherichia coli cultures. Mitochondrial malate dehydrogenase showed to be more active than glycosomal malate dehydrogenase in the reduction of oxaloacetate; nearly 80% of the total activity in procyclic crude extracts corresponds to the former isozyme which also catalyzes, although less efficiently, the reduction of p-hydroxyphenyl-pyruvate. The rabbit antisera raised against each of the recombinant isozymes showed that the three malate dehydrogenases do not cross-react immunologically. Immunofluorescence experiments using these antisera confirmed the glycosomal and mitochondrial localization of glycosomal and mitochondrial malate dehydrogenase, as well as a cytosolic localization for the third malate dehydrogenase isozyme. These results clearly distinguish Trypanosoma brucei from Trypanosoma cruzi, since in the latter parasite a cytosolic malate dehydrogenase is not present and mitochondrial malate dehydrogenase specifically reduces oxaloacetate.     

Isozymal differention between two species of Prosopis

The patterns of five multilocus isozyme systems were investigated in seed, shoot and cotyledon tissue of two species of mesquite, Prosopis glandulosa var. glandulosa and P. pallida. The isozymes of malate dehydrogenase, peroxidase, esterase, alcohol dehydrogenase and acid phosphatase from each of these tissues were analysed by starch gel electrophoresis and specific histochemical stains. In the case of each enzyme system examined, there were distinctly different isozymes which could be utilized to differentiate between these two species.     

Biochemical Properties of Mitochondrial Membrane from Dry Pea Seeds and Changes in the Properties during Imbibition

An attempt to isolate intact mitochondria from dry pea seeds (Pisum sativum var. Alaska) ended in failure. Cytochrome oxidase in crude mitochondrial fraction from dry seeds was separated into three fractions by sucrose density gradient centrifugation. Two of the fractions contained malate dehydrogenase, whereas the other did not. Equilibrium centrifugation of mitochondrial membrane on sucrose gradients revealed that the membrane from the fraction without malate dehydrogenase was lighter than that from the others. Differences were observed in relative content of phospholipid to protein and in polypeptide composition analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis among the membranes from three fractions and imbibed cotyledons. Membrane from the fraction without malate dehydrogenase was rich in phospholipid and lacking in polypeptides with relatively high molecular weights as compared with that from others. During imbibition, the fraction without malate dehydrogenase and one of the other two disappeared rapidly after a lag phase lasting for at least 1 hour. Concomitantly, active and stable mitochondria increased in the cotyledons. The results were interpreted to indicate that there were at least three types of mitochondria in dry seeds, the membranes of which differed in their biochemical properties, and that the mitochondria became active and stable through assembly of protein into the membranes during imbibition.     

Rapid Development of Mitochondria in Pea Cotyledons during the Early Stage of Germination

Rapid increases in activities and components of mitochondrial particles isolated from cotyledons of Pisum sativum var. Alaska during the early stage of germination are described. Respiratory rate of the cotyledons increased rapidly as hydration proceeded. A similar but slightly delayed increase in respiratory activity of the isolated mitochondrial fraction was observed. The respiratory control ratio and adenosine 5′-pyrophosphate/oxygen ratio rose during imbibition. Cytochrome oxidase and malate dehydrogenase activities in the mitochondrial fraction increased during the initial phase of imbibition. The increase seemed to precede that in respiratory activity. A significant activity of cytochrome oxidase and most of the malate dehydrogenase activity in the cotyledons were present in the postmitochondrial fraction in the case of the dry seeds. Mitochondrial protein and phospholipid also increased during imbibition, and the rise in the components seemed to concur with that in respiratory activity. The mechanism of mitochondrial development during imbibition is discussed.     

Purification and biochemical properties of genetically defined malate dehydrogenase in maize

Malate dehydrogenase of maize exists in multiple molecular forms (isozymes). In strain W64A, two soluble forms (s-MDH), five mitochondrial forms (m-MDH), and two glyoxysomal forms (g-MDH) were found in etiolated seedlings. The s-MDHs and m-MDHs were prepared in highly purified form. Using these purified isozymes, experiments with reducing agents (100 mm mercaptoethanol), low pH (2.0), and high salt cocn (7.5 m guanidine-HCl), along with genetic data, have eliminated the possibility of conformational alterations as an explanation for MDH multiplicity in maize; the MDH isozymes are genetically determined. Biochemical properties for each of the seven MDH isozymes were examined. Molecular weight, pI, pH optimum, thermolability, and K_m for oxaloacetate, malate, NAD, and NADH at different pH values were determined for each isozyme. Different kinetics of substrate inhibition (oxaloacetate) and coenzyme inhibition (NAD) were observed for the different isozymes. Effects of NAD analogs, chelating agents, reducing agents, metal ions, and TCA cycle acids on the enzymatic activity of these isozymes were tested. Based on the physical and kinetic properties observed, the maize malate dehydrogenase isozymes can be classified into four groups: s-MDH¹; s-MDH²; the two most anodal m-MDHs; and the three most cathodal m-MDHs. Since strain W64A is highly inbred, our data along with our previous and simultaneous genetic analysis suggest that multiple genes are involved in the expression of maize malate dehydrogenase isozymes.     

Characterization of malate dehydrogenase isozymes in wheat germ.

Malate dehydrogenase of wheat germ exists in multiple molecular forms (isozymes). Comparisons of some physical properties such as Stoke's radii, sedimentation constants, electrophoretic mobilities on polyacrylamide gel, chromatographic behaviors on DEAE-cellulose, stabilities to heat and iodacetamide inactivation, as well as kinetic parameters were described. When all these properties are considered together, at least five isozymes were found to associate with cytoplasm, mitochondria, glyoxysomes and proplastids of wheat germ. Wheat germ malate dehydrogenases are specific for the reduction of oxaloacetate and its monoesters. At least one carboxylic group of oxaloacetate must be free, in order to exhibit substrate activity, and maximum binding of oxaloacetate is achieved when both carboxylic groups are free. Soluble malate dehydrogenase and organelle-associated malate dehydrogenase can be differentiated readily in that the former can not utilize 4-ethyl oxaloaceode of ATP inhibition.     

Cyclic-amp control of some oxido-reductases during pine pollen germination and tube growth

Cyclic-AMP markedly increased the activities of peroxidase, malate dehydrogenase and succinate dehydrogenase but not glucose-6-phosphate dehydrogenase. Using inhibitors of protein and RNA synthesis, it was found that a part of enzyme activity increase caused by cyclic-AMP required fresh protein synthesis. The question of specificity of enzyme induction by cyclic-AMP has been examined.     

Electrophoretic variation in glutamate dehydrogenase and other isozymes in wild carrot cells cultured in the presence and absence of 2,4-dichlorophenoxyacetic acid

Summary Electrophoretic analysis of isozymal differences was performed with extracts of wild carrot (Daucus carota L.) cells, grown in the presence and absence of 2,4-dichlorophenoxyacetic acid (2,4-D). There were no differences in the patterns of malate dehydrogenase, acid phosphatase, aspartate aminotransferase, and γ-glutamyl transferase. Quantitative differences in peroxidase isozymes were detected, the plus 2,4-D cultures having lower activities. Esterase patterns were similar, but there were differences in individual isozyme activities and an additional form present in the minus 2,4-D cells. the greatest differences were in patterns of glutamate dehydrogenase with the minus 2,4-D cultures containing only the slowly migrating isozymes. The changes in glutamate dehydrogenase, as revealed by isozyme changes, together with the requirement for ammonia in embryogenesis, suggests that this enzyme may be associated with differentiation in wild carrot cells.     

The interaction of yeast citrate synthase with yeast mitochondrial inner membranes

The specific interaction of yeast citrate synthase with yeast mitochondrial inner membranes was characterized with respect to saturability of binding, pH optimum, effect of ionic strength, temperature response, and inhibition by oxalacetate. The binding ability of the inner membranes is inhibited by proteolysis and heat treatment, which implies that the membrane component(s) responsible for binding is a protein. A protein fraction from inner membranes when added to liposomes will bind citrate synthase. In addition, the binding of yeast fumarase, mitochondrial malate dehydrogenase, and cytosolic malate dehydrogenase to yeast inner membranes was examined. For these studies the yeast mitochondrial matrix enzymes, citrate synthase (from two types of yeast), malate dehydrogenase, and fumarase, as well as cytosolic malate dehydrogenase, were purified using rapid new techniques.     

黑鲷不同组织的9种同工酶谱

对黑鲷（Sparus macrocephalus）9种组织的9种同工酶采用垂直聚丙烯酰胺平板电泳技术进行研究。结果表明,乳酸脱氢酶（LDH）、醇脱氢酶（ADH）、苹果酸脱氢酶（MDH）、苹果酸酶（ME）、超氧化物歧化酶（SOD）、过氧化物酶（POD）、酯酶（EST）、半乳糖脱氢酶（GAD）、甲酸脱氢酶（FDH）等酶在表型、分布和活性上组织特异性明显。还对眼和肠组织同工酶表达特性的生理意义进行了讨论。     

Isoenzyme makeup of the malate dehydrogenase in 2 species of Acetabularia]

The isozymes of malate dehydrogenase (MDH) were studied by means of electrophoresis in polyacrilamide gel in Acetabularia crenulata and A. mediterranea. The isozyme profile of MDH was shown to be variable in different parts of the plant. Distinct differences in isozyme profiles of MDH between A. crenulata and A. mediteranea were found when studying the cell fractions which consisted mainly of chloroplasts. The chloroplast fraction of A. mediterranea contained 8 isozymes which form 2 groups with different electrophoretic mobility. The chloroplast fraction of A. crenulata contained 9 isozymes. All the isozymes of the first group were common for both the species under study.     

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.     

Glycerol phosphate dehydrogenase in mammalian peroxisomes

Peroxisomes isolated on sucrose density gradients from homogenates of rat, chicken, or dog livers and rat kidney contained NAD⁺:α-glycerol phosphate dehydrogenase. Since the amount of sucrose in the peroxisomal fraction inhibited the enzyme activity about 70%, it was necessary to remove the sucrose by dialysis. About 8.4% of the total dehydrogenase of rat livers was in the surviving intact peroxisomes after homogenation. If corrected for particle breakage, this represented approximately 21% of the total activity. About 9.5% of the total enzyme was isolated in rat kidney peroxisomes, and because of severe particle rupture may represent over half of the total activity. No glycerol phosphate dehydrogenase was found in spinach leaf peroxisomes. A specific activity of 326 nmoles min^?1 mg^?1 protein in the rat liver peroxisomal fraction was at least twice that in the cytoplasm. NAD⁺:α-glycerol phosphate dehydrogenase was also present in a membrane fraction which was not identified, but none was in the mitochondria. The liver peroxisomal and cytoplasmic NAD⁺:α-glycerol phosphate dehydrogenase moved similarly on polyacrylamide gels and each resolved into two adjacent bands.Malate dehydrogenase was not found in peroxisomes from liver and kidney of rats and pigs, but 1–2% of the total particulate malate dehydrogenase was present in the peroxisomal area of the gradient from dog livers. However, this malate dehydrogenase in dog peroxisomal fractions did not exactly coincide with the peroxisomal marker, catalase. Malate dehydrogenase in dog liver mitochondria and in the peroxisomal fraction had similar pH optima and K_m values and migrated similarly to the anode at pH 6.5 on starch gels as a major and a minor band. The cytoplasmic malate dehydrogenase had a different pH optimum and K_m value and resolved into five different isoenzymes by electrophoresis. It is concluded that NAD⁺:α-glycerol phosphate dehydrogenase is in peroxisomes of liver and kidney, whereas malate dehydrogenase, present in peroxisomes of plants, is apparently absent in animal peroxisomes.     

Substrate channeling of NADH and binding of dehydrogenases to complex I

The binding of porcine heart mitochondrial malate dehydrogenase and beta-hydroxyacyl-CoA dehydrogenase to bovine heart NADH:ubiquinone oxidoreductase (complex I), but not that of bovine heart alpha-ketoglutarate dehydrogenase complex, is virtually abolished by 0.1 mM NADH. The malate dehydrogenase and beta-hydroxyacyl-CoA enzymes compete in part for the same binding site(s) on complex I as do the malate dehydrogenase and alpha-ketoglutarate dehydrogenase complex enzymes. Associations between mitochondrial malate dehydrogenase and bovine serum albumin were observed. Subtle convection artifacts in short-time centrifugation tests of enzyme association with the Beckman Airfuge are described. Substrate channeling of NADH from both the mitochondrial and cytoplasmic malate dehydrogenase isozymes to complex I and reduction of ubiquinone-1 were shown to occur in vitro by transient enzyme-enzyme complex formation. Excess apoenzyme causes little inhibition of the substrate channeling reaction with both malate dehydrogenase isozymes in spite of tighter equilibrium binding than the holoenzyme to complex I. This substrate channeling could, in principle, provide a dynamic microcompartmentation of mitochondrial NADH.     

Isolation and properties of malate dehydrogenase from Meso-and thermophilic bacteria

A scheme of purification of malate dehydrogenase from Macromonas bipunctata strain D-405 and Vulcanithermus medioatlanticus DSM 14978^T was developed. This scheme was used to obtain electrophoretically homogeneous enzyme preparations of the mesophilic bacterium M. bipunctata (specific activity, 26.9 ± 0.8 U/mg protein; yield, 10.9%) and the thermophilic bacterium V. medioatlanticus (specific activity, 5.0 ± 0.2 U/mg protein; yield, 19.2%). Using these high-purity enzymatic preparations, the physicochemical and regulatory properties of malate dehydrogenase were studied and the differences in kinetic characteristics and thermal stability of the preparations were determined.     

Cytoplasmic synthesis of soluble and mitochondrial malate dehydrogenase isozymes in maize.

The effects of cycloheximide and chloramphenicol on the incorporation of radioactive leucine into trichloroacetic acid-insoluble material and on malate dehydrogenase (MDH) activity in maize scutella were studied. In 40 h of treatment, chloramphenicol (0.5 – 2.0 mg/ml) does not inhibit the increase of either soluble (s) or mitochondrial (m) malate dehydrogenase isozymes. However, 8 h following the addition of cycloheximide (2–10 μg/ml), the usual increase of total malate dehydrogenase activity is reduced by more than 70%. The reduction in the activity of the soluble and the mitochondrial malate dehydrogenase isozymes is similar. From these observations, and from our former studies on this system, we conclude that both the soluble and the mitochondrial malate dehydrogenases are synthesized on cytoplasmic ribosomes.     

Origin of cytokinin-and auxin-autonomy and changes in specific proteins in tobacco callus tissue

On the basis of earlier data it was suggested that the induction of cytokinin autonomy might be accompanied by disorders in plastid function and a decrease in cytokinin utilization. In the work presented below the formation of chlorophyll and the isozyme patterns of nine enzymes, some of which are known to be localized in plastids, were compared in tobacco callus tissues differing in their hormonal requirements. Tissues either not requiring cytokinin or both auxin and cytokinin for their growth, contained a lower amount of chlorophyll than the cytokinin-and auxin-dependent strain. The number of isozymes of glucose-6-phosphate and NADP-malate dehydrogenase (i.e. enzymes which are known to be located in plastids) was reduced from four in the cytokinin-and auxin-dependent strain to two and one in the two cytokinin-autonomous strains, respectively. The fully habituated tissue contained an additional isozyme of NADP-malate dehydrogenase. The total number of isozymes of the remaining enzymes (NAD-malate dehydrogenase, peroxidase, esterase and a-and β-galactosidase) either was decreased or not changed in the cytokinin autonomous strains. The exception was an additional anodic peroxidase in one strain. The number of these isozymes in tissue habituated with respect to both auxin and cytokinin either remained the same or increased. Tobacco callus strains with altered requirements for growth regulators contained some new isozymes which were not present in any other strain and some isozymes present in other strains were absent. These differences are discussed in relation to the possible role of plastid function disorder associated with habituation.