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Abstract
Soybean root nodule peptidohydrolases of both host cell cytosol and bacteroids were identified and biochemically characterized. Three endopeptidases active toward n-benzoyl-L-Arg-(beta)-naphthylamide (Bz-L-Arg-(beta)NA) in nodule cytosol were resolved by polyacrylamide gel electrophoresis. All had pH optima between pH 7.5 to 9.8, and were classified as serine proteases. The major endopeptidase identified in bacteroid extracts exhibited the same electrophoretic mobility, isoelectric pH, and sensitivity to DFP as one of the host cytosolic endopeptidases. Azocasein-digesting activity was identified in both cytosol and bacteroid extracts. Hemoglobin-digesting activity and autolytic activity were characterized in the bacteroids.
At least four distinct aminopeptidases were identified in nodule cytosol on the basis of electrophoretic mobility and substrate specificity. Two aminopeptidases were most active twoard L-Leu- and L-Met-(beta)-naphthylamide, while the other two exhibited broader specificity. Two of the aminopeptidases from nodule cytosol were classified as thiol-type enzymes, and all four aminopeptidases had neutral to basic pH optima. A single aminopeptidase having broad substrate specificity against various amino acyl-(beta)-naphthylamides was found in bacteroid extracts. This aminopeptidase had an alkaline pH optimum and was classified as a metalloenzyme.
The activities of the peptidohydrolases identified above were monitored throughout the developmental cycle of soybean nodules and during reversible dark-induced senescence of soybean nodules. Aminopeptidase activity and endopeptidase activity toward Bz-L-Arg-(beta)NA in the cytosol were maximal during early pod development and decreased during the period of natural nodule senescence and during dark-induced senescence. In contrast, an increase in proteinase activity toward azocasein in the cytosol was closely correlated with cytosolic protein and leghemoglobin loss during both natural and dark-induced senescence. Bacteroid protein content, on the other hand, did not decrease during the period of nodule deterioration, and proteolytic activity in bacteroid extracts did not increase. Thus it appeared that nodule deterioration was confined to cells of the legume host, since no degradative changes were detected in the bacteroids.
