PURIFICATION OF THE MAJOR SOYBEAN LEAF ACID-PHOSPHATASE THAT IS INCREASED BY SEED-POD REMOVAL

Fruit removal for 5 weeks after flowering increased acid phosphatase a ctivity 10-fold in soybean (Glycine max L. Merr. var Hobbit) leaves co mpared with normal seed-pod-bearing plants. The major acid phosphatase activity in leaves was purified over 2700-fold, yielding a single pol ypeptide of 51 kD with a specific activity of 1353 units/mg protein us ing p-nitrophenylphosphate as the substrate. Isoelectric focusing demo nstrated that the purified protein co-migrated with a majority of the activity that increased in leaves following seed-pod removal. Immunobl ot analysis demonstrated that at least part of the increased activity was due to an increased abundance of the phosphatase protein. In situ enzyme activity staining localized most of the total phosphatase activ ity to vascular tissues, the leaf paraveinal mesophyll cell layer, and the lower epidermis. This distribution and the response to seed-pod r emoval paralleled previous results for soybean vegetative storage prot ein (VSP) alpha and beta. However, in a native polyacrylamide gel the VSP detected by immunological staining of electrophoretically transfer red protein did not migrate with the majority of the phosphatase activ ity. Fractionation of crude leaf protein on concanavalin A-Sepharose y ielded a fraction containing 97% of the total VSP but only 0.1% of the total acid phosphatase activity.