Malate dehydrogenase of ox kidney. Comparison of the reactivity of sulfhydryl groups and their relationship to activities of the mitochondrial and cytoplasmic enzymes.

Abstract

Out of 10 to 11 cysteine residues per mole of kidney mitochondrial malate dehydrogenase, 3 to 4 exhibit time‐dependent reactivity with 5,5′‐dithio‐bis‐(2‐nitrobenzoic acid) (DTNB) at pH 9.5 to 9.8, when incubated over a period of 3 hours. Under the same conditions, 1.5 to 2.0 out of a total of 6 cysteine residues of the cytoplasmic malate dehydrogenase became DTNB‐reactive. The process of unmasking of unreactive thiols is initiated by a relatively rapid (10 min) reaction on one thiol with DTNB. Beyond this initial reaction, DTNB is not required and unmasking of thiol groups proceeds spontaneously in an alkaline medium. Enzymatic activity is unaffected by the reaction of thiol groups with DTNB. Both the rate and the extent of the thiol‐DTNB reaction is greatly increased in the presence of 2 M urea, which by itself has no effect on the availability of thiol groups. Thiol unmasking in the mitochondrial enzyme is completely inhibited by 1 mM NADH (or to a lesser extent by ADP), whereas the cytoplasmic enzyme does not respond to these agents. Enzymatic activity of the cytoplasmic malate dehydrogenase is more resistant than the mitochondrial enzyme to 4 M urea. ADP or NADH tend to stabilize enzymatic activities of both isoenzymes. There is no simple relationship between enzymatic activity and reactive cysteine thiols. The specific effect of NADH (or ADP) on the mitochondrial malate dehydrogenase by preventing thiol liberation, is interpreted as an expression of a specific nucleotide binding site which controls small scale unfolding of the enzyme protein.