Nathan O. Kaplan

Starch-gel electrophoresis of malate dehydrogenase

Abstract Malate dehydrogenase preparations were subjected to electrophoresis on starch gel at pH 7.0. Purified preparations of mitochondrial malate dehydrogenase were shown to exist in up to six separable forms on the gel. The distribution pattern of these forms was not influenced by the age of the tissue of origin, the purification procedures used or by a number of degradative treatments, although methyl iodide and urea treatment had some effect on the preparations. After elution from the gel, the components of the mitochondrial enzymes were shown to be relatively similar by some catalytic criteria, but to differ collectively from the “supernatant” (cytoplasmic) enzyme from pig heart.

SIGNIFICANCE OF SUBSTRATE INHIBITION OF DEHYDROGENASES

The recent expansive interest in multiple molecular forms of enzymes has brought about an increase in interest in the function of multiple forms catalyzing the same function. Several years ago, we postulated, on the basis of the distribution of the Hand M-lactate dehydrogenases (LDH’s)t as well as their difference in catalytic properties, that the two types of enzyme have different functional roles.’-3 We suggested that the role of the H-type LDH was to function in cells with a high degree of aerobic metabolism, whereas the properties of the M type were such as to indicate that it could operate in cells with low oxygen uptake or high rate of glycolysis. Hence, the H-type enzyme would be found in muscles geared to perform sustained activity, whereas the M form would be a constituent of voluntary muscles geared for sudden activity. In general, there has been an excellent correlation between the composition of LDH and the relative degree of oxygen uptake in a given muscle or cell type. These results have been summarized e l s e ~ h e r e . ~ ~ In several instances, exceptions have been pointed out with respect to the composition of LDH; for example, certain higher-vertebrate erythrocytes have a relatively high percentage of H-type LDH, even though the red cells show a low oxygen uptake. I t should be pointed out that the amount of glycolysis or total metabolism in the erythrocyte is relatively small, as is the total level of LDH, and hence the type of LDH may not be significant because of the quantitative aspects. This may also be true of the chicken lens, which has the H-type of LDH and relies, for most of its energy, on glycolysi~.~ The erythrocytes and lens can in no way be compared quantitatively to voluntary, striated muscles in their rates of glycolysis and in amounts of pyruvate formed. Liver, an aerobic tissue, contains mainly M4 enzyme in most species; the LDH composition of liver does vary in certain vertebrates as discussed by us elsewhere (Cahn et 0 1 . ~ ) . The importance of rates of glycolysis as factors in regulation will be discussed below. We believed that the difference in inhibition with excess pyruvate was an indication of physiologically distinct roles for the two types of LDH. We thought that this inhibition might be important since, in voluntary, striated muscles, pyruvate accumulates rapidly. If the H-type enzyme existed in such

Immobilization of hydrogenase on glass beads.

Abstract Hydrogenase from Clostridium pasteurianum was immobilized on glass beads by four different methods. The sensitivity of the native and bound enzyme to oxygen was examined. Hydrogenase bound to succinyl glass proved to be the most stable to oxygen. All bound enzymes were active with ferredoxin as a substrate and evolved hydrogen in a chloroplast-ferredoxin-hydrogenase system driven by light.

8-(6-Aminohexyl)-amino-adenine nucleotide derivatives for affinity chromatography

Abstract 8-(6-Aminohexyl)-amino-AMP and 8-(6-aminohexyl)-amino-NAD(H) are synthesized from AMP and NAD + , respectively, in a two-step procedure via the 8-Br-adenine nucleotides. The AMP derivatives and the NAD + derivatives possess some biological activity as substrates in the adenylate kinase and as coenzymes in the alcohol and lactate dehydrogenase systems, respectively. The synthesized derivatives may be coupled to Sepharose by cyanogen bromide activation. The covalently attached ligands show strong affinities for various dehydrogenases. Examples of the selective purification of several dehydrogenases using the new affinity columns are presented.

D-Lactate Specific Pyridine Nucleotide Lactate Dehydrogenase in Animals

A survey of representative invertebrates has revealed the presence of pyridine nucleotide-linked D-lactate dehydrogenase in a number of groups. All species studied contained either DD-or LL-lactate dehydrogenase, but no species contained both enzymes. The D-lactate dehydrogenase from Limulus polyphemus has been purified and has a molecular weight of 65,000.

Control of synthesis of lactic acid dehydrogenases

The rate of synthesis of lactic dehydrogenase, particularly of the “muscle-type” of subunits of this enzyme, was regulated by the concentration of oxygen in the environment of some cells in tissue culture and some tissues in chicken embryos. This regulation was most evident when synthesis of “muscle-type” subunits was compared to synthesis of “heart-type” subunits. The rate of synthesis of M subunits was increased when the oxygen tension fell below 0·1 atmosphere (76 mm Hg). This increase was blocked by the presence of actinomycin D, puromycin, actidione or cold during exposure to reduced oxygen tensions. The stimulating effect of low oxygen concentrations on enzyme synthesis persisted after a brief exposure of cultured cells to anoxia. This persistent increase was abbreviated but not abolished by actinomycin. The effect of oxygen tension on enzyme production appeared to be distinct from its immediate effect on carbohydrate metabolism, since lactate production was not correlated with the enzyme change, and chemical agents which caused a shift to anaerobic metabolism did not cause an increase in synthesis of M subunits of lactic dehydrogenase. Some chelating agents increased the rate of enzyme synthesis despite exposure to high oxygen concentrations. This is taken as evidence that oxygen exerts a repressive effect on lactic dehydrogenase synthesis, mediated by heavy metals. Based on results with actinomycin, and the prolonged effect of a brief exposure to anoxia, it is postulated that low oxygen tensions may result in increased synthesis of messenger RNA for M subunits, and that this messenger RNA survives for at least nine hours. The results also indicate that oxygen tension is not the sole regulator of lactic dehydrogenase synthesis. Other regulation is suggested by the rigid limits for the response to altered oxygen tension, by cells which failed to respond at all to changes in oxygen tension and by changes in rate of enzyme synthesis with age of the culture.

Enzyme stereospecificities for nicotinamide nucleotides

This is a summary of 25 years of research on the stereospecificity of pyridine nucleotide-linked dehydrogenases. It records the stereoselectivity of 127 dehydrogenases for pyridine nucleotides.

Addition products of diphosphopyridine nucleotides with substrates of pyridine nucleotide-linked dehydrogenases☆

Abstract Preparation of the reduced and oxidized forms of addition compounds from diphosphopyridine nucleotide and a number of carbonyl compounds are described; these include pyruvate, pyruvic ethylester, oxaloacetate, α-ketoglutarate, acetaldehyde, butyraldehyde, and α-ketobutyrate. The adducts are closely related to the DPN-acetone and the DPN-dihydroxyacetone adducts, both in their chemical and spectral properties. The chemical structures of the reduced and oxidized adducts have been determined and information concerning the mechanism of formation of the adducts is presented. The lactate, malate, glutamate, and alcohol dehydrogenases show a unique specificity for the reduced adducts; the specificity was found to be related to the normal oxidized substrate utilized by the various enzymes. The inhibition is competitive with DPNH and noncompetitive with the substrate. Oxidized adducts produce considerably less inhibitory effects than the reduced adducts. Evidence is presented showing that the reduced adducts bind to the enzymes in a manner similar to that of the reduced coenzyme. On the basis of the high selectivity of the dehydrogenases for the adducts, a model is proposed which shows the spatial relationship between the reduced coenzyme and the oxidized substrate on the enzyme in the transition state. From this model, a general reaction mechanism is postulated for the pyridine nucleotide-dependent dehydrogenases, which may explain the stereo-specificity of the hydrogen transfer with respect to the coenzyme as well as the substrate.

Characteristics of 8-substituted adenine nucleotide derivatives utilized in affinity chromatography.

Abstract Improved methods for the preparation of several 8-substituted adenine nucleotide derivatives are described. Enzymatic properties of these 8-substituted derivatives were investigated by steady state kinetic and inhibition studies. It was found that 8-(6-aminohexyl)-amino DPN+ and TPN+ exhibit relatively high affinity for most DPN+ and TPN+ dependent dehydrogenases. Preliminary nmr studies indicate that the 8-substituted adenine nucleotide derivatives may exist in slightly different ribosyl as well as glycosyl conformations from those of the natural adenine nucleotides. The chemical shift difference between geminal C4 protons of dihydropyridine moiety of DPN+ and TPN+ changes from 0.1 to 0.2 ppm upon the 8-hexyl substitution of the natural coenzymes, indicating a strong interaction between 8-hexyl side chain of adenine moiety and the dihydropyridine moiety of these coenzyme derivatives. However, the folding and fluorescence properties of 8-(6-aminohexyl)-amino DPN+ and TPN+ as well as their reduced analogs in aqueous solutions are not significantly altered as compared to those of natural DPN+ and TPN+. Purification of glucose-6-phosphate dehydrogenase from yeast extracts and human erythrocytes using 8-(6-aminohexyl)-amino-TPN+ -Sepharose column is reported. Preliminary studies on the purification of various kinases using 8-substituted ADP and ATP Sepharose columns are also presented.

The formation of ternary complexes by diphosphopyridine nucleotide-dependent dehydrogenases

Abstract Formation of abortive ternary complexes between enzyme, coenzyme, and substrate has been demonstrated for several pyridine nucleotide-dependent dehydrogenases by measuring the quenching of protein fluorescence. Evidence is presented suggesting the participation of a ternary complex consisting of lactate dehydrogenase, DPN + , and pyruvate in the phenomenon of substrate inhibition observed with lactate dehydrogenases. This complex is more readily formed with the H-type LDH than with the M-type enzyme under identical conditions. The ternary complex of LDH-DPN + -pyruvate can be formed at physiological levels of pyruvate. Information concerning the structure of the ternary complex has been derived from its absorption and fluorescence spectra, as well as from its kinetic and chemical properties. The pyridine ring in the complex appears to be in the reduced form, similar to that found in DPNH. The ternary complex can be precipitated with ammonium sulfate. Denaturation of the enzyme results in a product which is easily oxidized. Evidence is presented indicating that this product consists of DPN which is covalently bonded to pyruvate. Similar products can be obtained with analogs of DPN, but the reaction for lactate dehydrogenase is specific for pyruvate. Related ternary complexes have been observed from malate dehydrogenase, glutamic dehydrogenase, and alcohol dehydrogenase. The significance of ternary complex formation is discussed in relation to the reaction mechanism of pyridine nucleotide-linked dehydrogenases.