Solubility of β-Glucosidase in Homogenates of Sweetclover Leaves and Bean Hypocotyls

Abstract

In a recent paper Eberhart and Beck (4) suggested that the aryl-,B-glucosidase of Neurospora conidia is a mural enzyme. Similarly, Nevins (11) reported that ,-glucosidase is strongly associated with cell walls during extraction of bean hypocotyls at pH 4.6 and suggested that this enzyme is wall-bound in situ, and Keegstra and Albersheim (9) ,B-Glucosidase was extracted from the hypocotyls of beans (Phaseolus vulgaris L. var. Red Kidney) which had been allowed to germinate for 5 days at room temperature between moist paper towels. The bean seeds were obtained from a local market. Extraction and centrifugation procedures were similar to those used for sweetclover preparations.The cellular debris used in some preincubation experiments was prepared from sweetclover leaves which had been frozen in liquid nitrogen, pulverized in a mortar, lyophilized, and stored in a freezer. A 1-g portion of this material was suspended in 20 ml of 50 mm borate buffer, pH 8.5, and the suspension was centrifuged at 17,000g for 10 min. The pellet was subjected to three additional washings with buffer, one with water, and a final washing with acetone. The washed pellet was air-dried.Preincubation mixtures consisted of 1.0 ml of deionized enzyme preparation; 0.5 ml of water, 81 mm acetate buffer, pH 5.0, or 81 mm borate buffer, pH 8.5; and, in some instances, 5 mg of cellular debris. Mixtures were allowed to stand for 5 min at room temperature and were then centrifuged. Supernatant fractions from this centrifugation were assayed for ,Bglucosidase activity. Pellets were resuspended in 1.5 ml of 50 mM borate buffer, pH 8.5, and recentrifuged. Assay of BO-glucosidase activity in the supernatant fractions from this centrifugation provided a measure of the activity which was associated with the pellet at the end of the preincubation treatment.

Contamination of oat mesophyll protoplasts by apoplastic polyamine oxidase

“…Although the binding of cytoplasmic enzymes to ceil walls has been reported (Jaynes et al 1972, Nagahashi 1985, to our knowledge the contamination of isolated protoplasts by apoplastic enzymes has not been previously described. One reason for the lack of previous reports of this phenomenon is that until recently no definitive technique has been available to extract soluble and weakly ionicaily bound apoplastic enzymes without contamination by cytoplasuiic components.…”