Malate dehydrogenase preparation

Compartmentation of these reactions to prevent photorespiration involves the interaction of two cell types, mescrphyll cells and bundle sheath cells. The meso-phyll cells take up COg at the leaf surface, where Og is abundant, and use it to carboxylate phosphoenolpyruvate to yield OAA in a reaction catalyzed by PEP carboxylase (Figure 22.30). This four-carbon dicarboxylic acid is then either reduced to malate by an NADPH-specific malate dehydrogenase or transaminated to give aspartate in the mesophyll cells. The 4-C COg carrier (malate or aspartate) then is transported to the bundle sheath cells, where it is decarboxylated to yield COg and a 3-C product. The COg is then fixed into organic carbon by the Calvin cycle localized within the bundle sheath cells, and the 3-C product is returned to the mesophyll cells, where it is reconverted to PEP in preparation to accept another COg (Figure 22.30). Plants that use the C-4 pathway are termed C4 plants, in contrast to those plants with the conventional pathway of COg uptake (C3 plants). 

Students will isolate intact mitochondria from beef heart and fractionate them to prepare submitochondrial particles. Each fraction will be characterized by protein estimation by the biuret method and measurement of malate dehydrogenase and monoamine oxidase activity. 

Lindbladh, C., Rault, M., Hagglund, C., Small, W. C., Mosbach, K., Bulow, L., Evans, C., and Srere, P. A. (1994). Preparation and kinetic characterization of a fusion protein of yeast mitochondrial citrate synthase and malate dehydrogenase. Biochemistry, 33, 11692-11698. 

A second type of EMIT has been developed using the enzyme malate dehydrogenase as the enzymatic label. Research has shown that thyroxine competitively inhibits malate dehydrogenase. A conjugate prepared with thyroxine covalently bound close to the enzyme s active site shows very low specific activity that can be restored by binding of the thyroxine to arcP -thyroxine antibody. In this very specific assay for thyroxine, enzyme activity increases upon antibody binding, so that in a competitive assay for free thyroxine, activity decreases with increasing free thyroxine concentration. 

A single polypeptide chain can in theory exist in an infinite number of different conformations. However, one specific conformation generally appears to be the most stable for any given sequence of amino acids, and this conformation is assumed by the chain as it is synthesized within the cell. Thus, the primary structure of the polypeptide chain also determines its three-dimensional secondary and tertiary structures. It is conceivable that in some cases there may be several alternative conformations ("conforraers ) of a single chain that are of nearly equal stabilities and therefore these alternative forms may coexist. This possibility was first suggested to account for the heterogeneity noted in preparations of the cytoplasmic and mitochondrial isoenzymes of malate dehydrogenase and has also been proposed as an explanation of the multiple electrophoretic zones of erythrocyte acid phosphatase. However, no multiple enzyme forms have been shown unequivocally to be due to conformational isomerism. 

Fig. 6.3. Carbon dioxide-fixing pathway in the C4-plants (prepared mainly on the basis of Hatch et al., 1967). Circled numbers 1, phosphoenolpyruvate carboxylase 2, malate dehydrogenase (NADP ) 3, malate dehydrogenase (NADP ) (OAA decarboxylating) (= malic enzyme) 4, Rubisco 5, pyruvate orthophosphate dikinase. Pi, phosphate PPi, diphosphate...

Malate dehydrogenase has been identified in a wide variety of sources and purified to homogeneity from a number of them. The majority of studies have been carried out with enzyme isolated from either pig or beef heart. The first apparently pure preparation was obtained by Wolfe and Neilands (7). It differs from previously reported procedures (S,9)... 

Homogeneous preparations of malate dehydrogenase have also been isolated from chicken heart (27), horse heart (28), Drosophila virilis... 

The transamination of the a-amino group to a keto acid acceptor (reaction 2) has been demonstrated in a number of higher plant studies (Nahler and Ruis, 1973 Streeter, 1977 Lloyd and Joy, 1978). The product of the transamination is 2-oxosuccinamate. This can be deamidated to oxaloacetate by lettuce and spinach leaf preparations (Meister, 1953). A similar reaction was reported by Streeter (1977) in soybean and pea leaf extracts. On the other hand, Joy (1978) reported that the 2-oxosuccinamate is reduced to 2-hydroxysuccinamate in these leaves in vivo. The apparent discrepancy between the results of Streeter (1977) and those of Joy (1978) may be due to the enzyme assay used by the former. It consisted of the oxidation of NADH in the presence of the enzyme extract and 2-oxosuccinamate. The assumption was that deamidation occurred leading to oxaloacetate which then acted as the substrate of endogenous malate dehydrogenase. The work of Davies (1961) showed that plant malate dehydrogenase is not specific for oxaloacetate, and it is possible that the 2-oxosuccinamate may act as a substrate. Meister (1953). actually measured the production of ammonia from 2-oxosuccinamate by his leaf preparations. 

Corn leaf NADP- malate dehydrogenase (NADP- MDH), spinach ferredoxin, ferredoxin thioredoxin reductase and thioredoxin m were purified to homogeneity as described earlier (1). Pig ad enal ferredoxin was prepared as in (1). Human lymphocyte thioredoxin was cloned, overexpressed and purified from E coli as described in (2). 

E. coli thioredoxin reductase was purified on 2, 5 -ADP Sepharose (4). E. coli ribonucleotide reductase was a generous gift of B-M. Sjoberg, Stockholm. Ribonucleotide reductase from Anabaena 7119 was purified as previously described (6). NADP-malate dehydrogenase and fructose-1,6-bisphosphatse were partially purified from spinach chloroplasts (7). Thioredoxins m and f were homogeneous preparations from spinach chloroplasts (8). 

Leaf extracts were prepared by boiling leaf discs (8.5 mm in diameter) in 20% (v/v) ethanol. Titratable acidity was determined by addition of 5 mM KOH to pH 7.0 (photoperiod study) or pH 8.0 (hormone study). Malate in the supernatant was determined spectrophotometrically with NAD-malate dehydrogenase. 

Mitochondrial malate dehydrogenase (MDH) from several species has been shown to exist in several enzymically active forms which also appear to be conformational isoenzymes (Kitto et al, 1966, 1970). Kitto et al (1970) showed, in contrast to Epstein and Schechter (1968), that these MDH s were interconvertible in vitro, had the same amino acid compositions and molecular weights, but differed, once reversibly denatured, considerably in their heat stability. Similar interconversion of isoenzymes has also been observed with purified preparations of horse liver alcohol dehydrogenase (Lutstorf and von Wartburg, 1969). The question arises if such conformers are of any physiological or functional significance. It is possible that beeause of their differences in surface charge, the various conformative isoenzymes are differently bound within a cell. 

From the fact that the glyoxylate and TCA cycles have several enzymes (citrate synthetase, aconitase, malate dehydrogenase) in common it appeared axiomatic that they must operate together in the same intracellular compartment, the mitochondrion. Early experiments with crude particulate pellets containing mitochondria gave only partial support to this view since, although malate synthetase was present in such preparations, most of the isocitrate lyase was present in the supernatant fraction (Yamamoto and Beevers, 1961 Marcus and Velasco, 1960). 

The NADP-specific malate dehydrogenase is known to be light-activated (Hatch and Slack, 1969 Johnson and Hatch, 1970), and indeed must be prepared and assayed in the presence of dithiothreitol or other powerful sulfhydryl reagents. In spinach tissue, we observed that the naturally occurring lipoate would also activate and could be an in vivo regulator of this enzyme. Since the protein is apparently light-activated, its role in dark CO2 fixation is nil. 

Poly(ethylene glycol)-NAD+ Fig. 22 Preparation of a malate dehydrogenase-polyethylene glycol-NAD complex. 

NADH as an end product. This implicates oxidized malic acid, either pyruvic or oxaloacetic acid, as another end product. By adding commercial preparations of L-lactic dehydrogenase or malic dehydrogenase to the reaction mixture, Morenzoni (90) concluded that the end product was pyruvic acid. Attempts were then made to show whether two enzymes—malate carboxy lyase and the classic malic enzyme, malate oxidoreductase (decarboxylating), were involved or if the two activities were on the same enzyme. The preponderance of evidence indicated that only one enzyme is involved. This evidence came from temperature inactivation studies, heavy-metal inhibition studies, and ratio measurements of the two activities of partially purified preparations of Schiitz and Radlers malo-lactic enzyme (76, 90). This is not the first case of a single enzyme having two different activities (91). 

Matsumoto et al (41) prepared a multi-enzyme electrode using glucose oxidase, invertase, mutarotase, fructose-5-dehydrogenase, and catalase to simultaneously detect glucose, fructose, and sucrose in fruit juices and soft drinks. Detection of multi-components by enzyme sensors was also reported in analysis of sucrose and glucose in honey (42) and drinks (43), and L-malate and L-lactate in wines (44). 

According to Veeger et al. 149), succinate dehydrogenase can also oxidize L-chlorosuccinate, L-methyl succinate, D-malate, and L-malate. Brodie and Nicholls 174) have reported that monofluorosuccinate and 2,2-difluorosuccinate are also oxidized by Keilin-Hartree preparations yielding oxaloacetate as a final product. D-Chlorosuccinate, D-methyl suc-... 

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