Purification and characterization of a novel serine proteinase from the microsomal fraction of bovine pancreas.
A novel trypsin-like serine proteinase was purified to homogeneity from the bovine pancreas microsome fraction. The enzyme was solubilized with 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS), and purified by a series of column chromatographic steps on Ultrogel AcA-34, trypsin inhibitor-Sepharose 4B, and arginine-Sepharose 4B. The molecular mass of this pancreas trypsin-like proteinase (bPTLP) was estimated to be 29.5 kDa by SDS-PAGE under reducing conditions. The NH2-terminal sequence of bPTLP is very homologous, but not identical to those of other serine proteinases, especially such as elastases IV, II, and III. Substrate specificity studies involving a synthetic substrate and glucagon indicated that the enzyme hydrolyzes Arg-X, Lys-X, and Leu-X bonds. The best synthetic substrate for bPTLP was t-butyloxycarbonyl Gln-Arg-Arg-4-methylcoumaryl 7-amide. The enzyme failed to hydrolyze the substrate for chymotrypsin and elastase. The enzyme activity was inhibited by diisopropyl fluorophosphate, p-amidinophenylmethane sulfonylfluoride, and leupeptin, indicating that it is a serine-proteinase. These findings show that bPTLP is a novel serine-proteinase which differs from all known proteinases. The physiological function of the enzyme has yet to be determined.
Department of Biological Science and Technology, The University of Tokushima.
A
A
Tsuji
K
K
Edazawa
K
K
Sakiyama
K
K
Nagata
Y
Y
Sasaki
H
H
Nagamune
Y
Y
Matsuda
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D005591:Chemical Fractionation; D004795:Enzyme Stability; D005934:Glucagon / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008861:Microsomes / Q000201:enzymology*; D008969:Molecular Sequence Data; D010179:Pancreas / Q000737:chemistry / Q000201:enzymology* / Q000648:ultrastructure; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity
D000596:Amino Acids; D015842:Serine Proteinase Inhibitors; D005934:Glucagon; D012697:Serine Endopeptidases