Active uptake of sucrose by plant plasma membrane vesicles: determination of some important physical and energetical parameters

Changes in general protease activity and the levels of the zein storage proteins were monitored during germination and early seedling growth of maize ( Zea mays L . inbred A636). General endosperm endoprotease activity, measured in vitro using azocasein as a substrate, increased continuously to day 5 and remained high thereafter. The increase was in parallel with the loss of zein protein as determined by immunoblot analysis, with a total loss of detectable zein by 10 days after inhibition of the seeds. A method was developed for the specific in vitro assay of zein degrading activity by monitoring the release of soluble radioactivity from the immobilized storage protein. The in vitro pH optimum for both general protease and zein degrading activities in homogenates prepared from isolated endosperm was 4.0. However, the curve for general protease activity was asymmetrical suggesting the presence of more than 1 protease. This was verified by activity staining of gelatin-containing polyacrylamide gels, which suggested the presence of 3 major and 3 minor protease activities. Both general protease and zein-specific activities were enhanced more than 2-fold when 2-mercaptoethanol (ME) was included in the assays, and both were inhibited by fhiol-protease directed compounds. Most of the general protease activity but none of the zein-specific protease activity bound to con A-Sepharose. Both the con A-binding and nonbinding fractions were analyzed using gelatin activity gels. The 3 minor activity bands were present in the fraction which was specifically eluted from the con A column by a-methyl-mannoside, while the 3 major activity bands, corresponding to the zein-specific protease activity, did not bind to the immobilized lectin.