alpha-Amylase inhibitor from fungus Cladosporium herbarum F-828.
A strain of fungus Cladosporium herbarum extracellularly produced an inhibitor specific for mammalian alpha-amylase. The inhibitor was purified 81-fold by freeze-thawing, heat treatment, and column chromatography on DEAE-cellulose, Sephadex G-75, DEAE-Sephacel, and Bio-Gel P-100. An apparent molecular weight of approximately 18,000 was estimated for the inhibitor using Bio-Gel P-100 filtration. The purified inhibitor preparation was a glycoprotein containing about 10% carbohydrate. The amino acid analysis of the inhibitor showed abundances of Gly, Asp, Glu, Ser, Ala, and Thr residues. The inhibitor was stable between pH 5 and 12 at 4 degrees C, and below 80 degrees C at pH 7.0. A binary complex formation out of equimolar amounts of the inhibitor and alpha-amylase, was demonstrated by polyacrylamide gel electrophoresis, and Bio-Gel P-100 chromatography. Kinetic studies exhibited that the inhibitor noncompetitively inhibited the enzyme reaction with a Ki value of 2.3 approximately 4.8 x 10(-10) M, by combining with the enzyme molecule at a different site from the substrate binding site.
D000596:Amino Acids / Q000032:analysis; D000681:Amylases / Q000037:antagonists & inhibitors*; D000818:Animals; D001236:Aspergillus oryzae / Q000201:enzymology; D001407:Bacillus / Q000201:enzymology; D002963:Cladosporium / Q000032:analysis*; D005656:Fungal Proteins / Q000302:isolation & purification / Q000494:pharmacology*; D006023:Glycoproteins / Q000302:isolation & purification / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D003904:Mitosporic Fungi / Q000032:analysis*; D010179:Pancreas / Q000201:enzymology; D010944:Plants / Q000201:enzymology; D012463:Saliva / Q000201:enzymology; D013552:Swine; D000516:alpha-Amylases / Q000037:antagonists & inhibitors*
D000596:Amino Acids; D005656:Fungal Proteins; D006023:Glycoproteins; D000681:Amylases; D000516:alpha-Amylases