Presence of Serine Enzymes in Endoplasmic Reticulum of Spinach Callus

Two serine enzymes were detected in microsomes of spinach callusby labeling with [³H]-di-isopropyl phosphorofluoridate (DFP)and examination by SDS-polyacrylamide gel electrophoresis. Thecontents of the larger (mol wt 44,000) and the smaller (molwt 39,000) DFP-binding protein (proteins which have a DFP-reactivesite) were maximum at the third and the second week after cellinoculation, respectively. The positions of both proteins onthe continuous sucrose gradient coincided with that of NADH-cytochromec reductase, a marker enzyme of the endoplasmic reticulum. Thesmaller protein was released from microsomes treated with 0.05%deoxycholate. The larger protein was solubilized with 0.5% cholate,but not with 0.05% deoxycholate, and the apparent molecularweight of the solubilized protein was about 90,000 in 0.5% cholateon Sephacryl S-200 column. [³H]-DFP-binding with the largerprotein was strongly inhibited by DFP, phenylmethylsulfonylfluoride, N-p-tosyl-L-lysine chloromethyl ketone and L-1-tosylamide-2-phenylethylchloromethyl ketone, and slightly by p-chloromercuric benzoateand o-phenanthroline. DFP binding with the smaller one was stronglyinhibited by DFP and PMSF, but not by other reagents. (Received February 12, 1985; Accepted May 27, 1985)