1,3-beta-D-glucanases from Pisum sativum seedlings. I. Isolation and purification

Abstract

Two buffer-soluble endo-1,3-beta-D-glucanases (EC 3.2.1.6) have been purified to within 1% of electrophoretic homogeneity from etiolated Pisum sativum stem tissues. Purified glucanase I and II differ in physical properties, such as electrophoretic mobility in sodium dodecyl sulfate polyacrylamide gels (Mr values were 22 000 and 37 000, respectively) and isoelectric focusing, (pI values were 5.4 and 6.8, respectively). Although the enzymes have similar pH optima (5.5--6.0), Km values for various substrates (0.6--7.4 mg/ml) and thermal inactivation profiles, they are localized in different tissues and they differ markedly in the rates with which they attack the internal linkages of long- vs. short-chain substrates. Glucanase I is concentrated in apical regions of the stem and is most effectively assayed reductometrically (as laminarinase), while glucanase II is localized in mature regions and is relatively more active in viscometric assays (as carboxymethyl-pachymanase).