Rat Liver Lactate Dehydrogenase

AMINO-TERMINAL AND ACETYLATION STATUS

From the ¹ From the Department of Biochemistry, University of Iowa, Iowa City, Iowa 52241

The amino-terminal sequence of rat liver M₄ lactate dehydrogenase has been investigated in an attempt to determine the number of polypeptide chains in this protein. Quantitative dinitrophenylation and phenylthiocarbamylation experiments carried out under a wide variety of conditions yielded only submolar quantities of amino-terminal residues. The conditions used included those which lead to the dissociation of lactate dehydrogenases and those which cause extensive unfolding of the polypeptide chains.

The acetylation status of rat liver M₄ lactate dehydrogenase was investigated by means of a microenzymatic method developed during this study. The presence of 6 to 8 moles of acetate per mole of rat liver lactate dehydrogenase was shown. Bovine heart H₄ and bovine heart MH₃ lactate dehydrogenases also contained 7 to 8 moles of acetate per mole. Chicken heart H₄ lactate dehydrogenase contained 3 to 4 moles of acetate per mole. All the acetyl groups in rat liver M₄ lactate dehydrogenase are N-acetyl residues. Rat liver M₄ lactate dehydrogenase also contains a quantity of a carbohydrate-like material which is not easily removed by dialysis, precipitation with ammonium sulfate, or repeated Bio-Gel chromatography.

These data, combined with the lack of reactivity of rat liver M₄ lactate dehydrogenase toward leucine aminopeptidase (62), lead to the conclusion that the amino-terminal residues of rat liver M₄ lactate dehydrogenase are not readily available for reaction with amino-terminal labeling reagents and are probably acetylated. The large number of acetyl residues suggests the presence of more than one polypeptide chain per subunit.

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