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Plant and Cell Physiology, 1982, Vol. 23, No. 8 1383-1389
© 1982

Article

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

Shinobu Satoh and Tadashi Fujii

Institute of Biological Sciences, The University of Tsukuba Sakura-mura, Ibaraki 305, Japan

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 chloromethyl 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.

(Received June 12, 1982; Accepted September 28, 1982)
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