BIOSYNTHESIS AND SECRETION OF ALPHA-AMYLASE BY RICE SUSPENSION-CULTURED CELLS - PURIFICATION AND CHARACTERIZATION OF ALPHA-AMYLASE ISOZYME-H

A major alpha-amylase isozyme H synthesized in suspension-cultured cel ls of rice (Oryza sativa L. cv. Nipponkai) was purified and characteri zed. The molecular mass of isozyme H was estimated by sodium dodecyl s ulfate-polyacrylamide gel electrophoresis to be 44,000 Da. Isozyme H h ad an isoelectric point of pH 5.7. Isozyme H was stained with periodic acid-Schiff reagents and concanavalin A-peroxidase, indicating that i sozyme H was a glycoprotein. The carbohydrate content of isozyme H was determined by phenol-sulfuric acid reagents to be 2% (w/w). Specific polyclonal antibodies against isozyme H did not bind to the isozyme A + B, while anti-isozyme A + B antibodies did not recognise isozyme H. Isozyme H required greater than 80 nM Ca2+ for activity and was maxima lly activated by 500 nM Ca2+. The activity of isozyme H exhibited a st ringent divalent cation dependency on Ca2+, Sr2+ and Ba2+. These resul ts support the conclusion that isozyme H is a novel rice alpha-amylase isozyme.