Membrane-bound and soluble extracellular alpha-amylase from Bacillus subtilis.
Extracellular alpha-amylase was purified to homogeneity from a Marburg strain of Bacillus subtilis. The enzyme is a single polypeptide chain of molecular weight approximately 67,000. Its NH2-terminal amino acid sequence is Leu-Thr-Ala-Pro-Ser-Ile-Lys. A membrane-derived alpha-amylase was solubilizing from membrane vesicles by treatment with Triton X-100 and was highly purified by chromatography on an anti-alpha-amylase-protein A-Sepharose column. Membrane-derived alpha-amylase was indistinguishable from the soluble extracellular enzyme by sodium dodecyl sulfate-gel electrophoresis and radioimmunoassay. The membrane-derived enzyme contains phospholipid. Approximately 30 to 80% of the phospholipid was extracted from the purified enzyme by chloroform:methanol. The extracted phospholipid was predominately phosphatidylethanolamine. Treatment with phospholipase D released phosphatidic acid. Membrane-bound alpha-amylase was latent in membrane vesicles. Release of membrane-bound alpha-amylase from vesicles by an endogenous enzyme was maximal at pH 8.5, was inhibited by metal chelators and diisopropyl fluorophosphate and was stimulated by Ca2+ and Mg2+. The amount of membrane-bound alpha-amylase was related to the level of secretion.
D000595:Amino Acid Sequence; D000681:Amylases / Q000302:isolation & purification*; D001412:Bacillus subtilis / Q000201:enzymology*; D002462:Cell Membrane / Q000201:enzymology; D003429:Cross Reactions; D007700:Kinetics; D008970:Molecular Weight; D011863:Radioimmunoassay; D013045:Species Specificity; D000516:alpha-Amylases / Q000302:isolation & purification* / Q000378:metabolism
D000681:Amylases; D000516:alpha-Amylases