Two methods of measuring protein breakdown resultiag from self-digeston during incubation in extracts of soybean leaves were examined. The release of free a-amino-nitrogen was measured with ninhydrin, and the disappearance of the large subunit of ribulose bisphosphate carboxylase (RuBPcase) was foDlowed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Rates of protein breakdown were measured as a function of temperature, pH, and leaf developmental stage and in the presence of various proteinase inhibitors. These treatments had differential effects on apparent proteolysis, depending on the method used. Determination of the ratio of a-amino-nitrogen plus peptide bond-nitrogen to a-amino-nitrogen indicated that the ninhydrin method detected the activity of exopeptidases preferentiaDy. The disappearance of the large subunit of RuBPCase as shown on gels was due primarily to the activity of endopeptidases. The sensitivity of the two types of proteolytic degradation to proteinase inhibitors differed.Determination of temporal changes in proteolytic activity during leaf development showed that total proteolytic activity, measured by either method, increased during leaf expansion and maturation and decreased during senescence. Incubation of intact isolated chloroplasts at 37 C resulted in the breakdown of the large subunit of RuBPcase, although the chloroplasts contained no measurable proteinase activity as determined by the release of a-amino-nitrogen during the incubation. No acid proteinase (pH 4.5) activity was detected in the chloroplasts when hemoglobin was used as a substrate. These results indicate that the proteinases which break down RuBPCase in isolated chloroplasts may not be detectable by conventional assay procedures.Leaf senescence in many plants, and especially in monocarpic crop plants, is accompanied by breakdown of leaf protein and reutilization of the hydrolysis products by the developing seeds (9-11, 21, 26). It has been suggested that the amount of protein which accumulates in the seeds is related to the extent of leaf protein breakdown (1 1, 19). Attempts have been made to correlate leaf protein breakdown with the levels of various proteinases in the leaves and, in some cases, a correlation was found (9,11,26,37), whereas, in others, there appeared to be no relation between proteinase levels and protein breakdown (1,28, 32 (10)(11)(12)23), by the loss of immunochemical determinants (35-37), or by the breakdown of polypeptides as ascertained by SDS-acrylamide gel electrophoresis (33).As part of our study on the role of proteinases in protein breakdown during leaf senescence, we have examined the hydrolysis of the LS of RuBPCase during the autodigestion of crude leaf extracts. Proteolysis was measured by the release of ninhydrinreactive material and the disappearance of the LS of RuBPCase on SDS-polyacrylamide gels. Our results show that the two methods probably measure different complements of proteinases. The discrepancy between the two methods was most obvious in isolated ch...
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