Detection and Evaluation of Serine Enzymes by [^3H]-DFP Affinity Labeling in Spinach Plants

概要

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Serine enzymes were detected in spinach plants by affinity labeling with [^3H]-di-isopropyl phosphorofluoridate (DFP) and SDS-polyacrylamide gel electrophoresis. Two serine enzymes were detected in the dry seeds, and another 4 major and 3 minor serine enzymes were detected in 48-hr soaked seeds, especially in the cotyledons. Fourteen serine enzymes, including 9 enzymes in the cotyledons, were detected in mature leaves. [^3H]-DFP binding with some serine enzymes in mature leaves was inhibited by a prior treatment with phenylmethylsulfonyl fluoride, a more specific probe of serine proteases. Other affinity labeling reagents for serine proteases, L-1-tosylamide-2-phenylethyl chloromethyl ketone and N-p-tosyl-L-lysine chlorbmethyl ketone also decreased DFP-binding to some serine enzymes. These results are evidence that the enzymes found are serine proteases. Natural inhibitors for serine proteases, leupeptin, aprotinin and soybean trypsin inhibitor had no effect on [^3H]-DFP binding. DFP-binding with all the serine enzymes detected in the mature leaves was decreased by p-chloromercuric benzoate but not by EDTA.
日本植物生理学会の論文