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Articles

Peptidohydrolases of Soybean Root Nodules ^1,2

IDENTIFICATION, SEPARATION, AND PARTIAL CHARACTERIZATION OF ENZYMES FROM BACTEROID-FREE EXTRACTS

Department of Agricultural Biochemistry, University of Nebraska-Lincoln, Lincoln, Nebraska 68583

Nodule extracts prepared from Glycine max var Woodworth possessed endopeptidase, aminopeptidase, and carboxypeptidase activities. Three distinct endopeptidase activities could be resolved by disc-gel electrophoresis at pH 8.8. According to their order of increasing electrophoretic mobility, the first of these enzymes hydrolyzed azocasein and n-benzoyl-L-Leu--naphthylamide, while the second hydrolyzed n-benzoyl-L-Arg--naphthylamine (Bz-L-Arg-NA), n-benzoyl-L-Arg-p-nitroanilide (Bz-L-Arg-pNA), and azocasein. The third endopeptidase hydrolyzed Bz-L-Arg-NA, Bz-L-Arg-pNA, and hemoglobin. Fractions of these enzymes extracted from electrophoresis gels were shown to have pH optima from 7.5 to 9.8. All of the endopeptidases were completely inhibited by diisopropylphosphorofluoridate, demonstrating that they were serine proteases.

Aminopeptidase activity was measured using amino acyl--naphthylamides. Electrophoresis of nodule extracts at pH 6.8 resolved the aminopeptidase activity of nodule extracts into at least four fractions based on mobility and on activities toward amino acyl--naphthylamides. The major activity of two of the aminopeptidases was directed toward L-Leu- and L-Met--naphthylamide, while the other two aminopeptidases exhibited broader specificity and were capable of hydrolyzing a large number of amino acyl--naphthylamides. Two of the aminopeptidases extracted from electrophoresis gels were classified as thiol type enzymes, and all four aminopeptidases had neutral to basic pH optima.

¹ This work was supported by United States Department of Agriculture, Science and Education Administration-CRGO grant 509-0410-9-0233 and by a grant from the Nebraska Soybean Development, Utilization and Marketing Board.

² Published as Paper No. 6089, Journal Series, Nebraska Agricultural Experiment Station.