Rapid preparation of mitochondrial malate dehydrogenase from rat liver and heart

Abstract

Mitochondrial malate dehydrogenase (L-ma-1ate:NAD' oxidoreductase, EC 1.1.1.37) has been purified from both rat liver and rat heart by using Sepharose-Blue Dextran pseudoaffinity chromatography. Under the conditions employed most of the mitochondrial malate dehydrogenase and some cytosolic malate dehydrogenase are adsorbed, whereas many proteins simply pass through. The blue column is then subjected to several treatments which elute a variety of other proteins (notably residual cytosolic malate dehydrogenase and lactate dehydrogenase) which also have an affinity for the bound dye Cibacron Blue F3GA. Mitochondrial malate dehydrogenase is then eluted by forming an abortive ternary complex with reduced nicotinamide adenine dinucleotide-sodium D( +)-malate. Pooled active fractions are passed through a preequilibrated diethylaminoethyl-Sephadex column which retains contaminating proteins. The active fractions are concentrated, and residual contaminants are removed by very small stepwise pH changes on a CM-52 cellulose column. The procedure is mild and rapid and yields enzyme which is homogeneous by the applied criteria. In M i t o c h o n d r i a l malate dehydrogenase (m-MDH) has been isolated and partially characterized from various sources (Ochoa

Comparison of the precursor and mature forms of rat heart mitochondrial malate dehydrogenase

“…Mitochondrial malate dehydrogenase was purified from rat hearts obtained from 150-g male Sprague-Dawley rats. Pure enzyme was obtained after ammonium sulfate precipitation from a crude homogenate and consecutive chromatography on (carboxymethyl)-Sepharose, blue Sepharose, and 5'-AMP-Sepharose as previously described (Glatthaar et al, 1974;Kuan et al, 1979). Sixty-six milligrams of mMDH with a specific activity of 183 units/mg was obtained from 400 g of rat hearts.…”

“…To this solution, a 5-fold molar excess of dithiothreitol 1 Abbreviations: NAD+, oxidized nicotinamide adenine dinucleotide; mMDH, mitochondrial malate dehydrogenase; sMDH, cytoplasmic malate dehydrogenase; MDH, malate dehydrogenase; LDH, lactate dehydrogenase; HPLC, high-performance liquid chromatography; TPCK, TV-tosyl-L-phenylalanine chloromethyl ketone; SDS, sodium dodecyl sulfate; TFA, trifluoroacetic acid; PTH, phenylthiohydantoin; Tris, tris(hydroxymethyl)aminomethane. was added relative to the estimated cysteine content (Kuan et al, 1979). The reduction reaction was incubated for 3 h at 25 °C.…”

A Reinvestigation of the Purity, Isoelectric Points and Some Kinetic Properties of the Aldehyde Dehydrogenases from Sheep Liver

“…Marangos and Constantinides [22] have shown multiple forms of glyceraldehyde-3-phosphate dehydrogenase on electrofocusing and suggested that these could arise from distinct conformational forms of a multimeric enzyme composed of a single subunit type. Alternatively, Kuan et al [23] have proposed that multiple forms of mitochondrial malate dehydrogenase seen in isoelectric focusing may arise through masking of ionic side-chain groups by tightly bound phospholipid. Further work is necessary to decide which, if any, of the above explanations applies in the case of the multiple forms of aldchyde dehydrogenase.…”

Kinetic Characterization of Mitochondrial Malate Dehydrogenase from Dictyostelium discoideum

“…The mitochondrial MDH activity was found to have a pH optimum of 9.3-9-5 using Tris/HCI and bicarbonate buffers in assays over a pH range of 7-10. This was much higher than the pH optimum (6.5-7.5) reported from other sources (Flury et al, 1974;Kuan et al, 1979). Both thyroxine and hydroxymalonate inhibited the M D H activity.…”