A serine protease zymogen in insect plasma. Purification and activation by microbial cell wall components.
A protease zymogen present in the plasma fraction of the hemolymph of silkworm, Bombyx mori, was purified to homogeneity as judged by SDS/PAGE and IEF/PAGE. An activating system for the zymogen was also isolated from the plasma fraction and was shown to be triggered by zymosan (yeast cell wall polysaccharide containing beta-1,3-glucan) or peptidoglycan. Using this system, the purified zymogen was activated and the active enzyme was purified to homogeneity. The physiological function of the zymogen or its active form is not yet known, but the active form was shown to have narrower substrate specificity than trypsin. Among 33 peptide derivatives examined, Boc-Gln-Arg-Arg-NH-Mec and Boc-Val-Pro-Arg-NH-Mec (Boc = tert-butoxycarbonyl, NH-Mec = 4-methylcoumaryl-7-amide) were the best and the second best substrates, respectively. The purified zymogen was determined to be a 39-kDa protein consisting of a single polypeptide. The active form of the zymogen was labeled with [3H]diisopropylfluorophosphate and was completely inactivated by (p-amidinophenyl)methanesulfonyl fluoride. The molecular mass of the [3H]-labeled enzyme was determined to be 38 kDa in SDS/PAGE under reducing conditions. These results indicate that the 39-kDa protein purified in the present study is a zymogen of a serine-type protease and that the activation of the zymogen occurs by limited proteolysis.
Katsumi
Y
Y
Biochemical Laboratory, Hokkaido University, Sapporo, Japan.
Kihara
H
H
Ochiai
M
M
Ashida
M
M
D000595:Amino Acid Sequence; D000818:Animals; D012831:Bombyx / Q000201:enzymology*; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism; D004156:Catechol Oxidase / Q000378:metabolism; D002473:Cell Wall / Q000378:metabolism; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000097:blood* / Q000302:isolation & purification / Q000378:metabolism; D006868:Hydrolysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000097:blood* / Q000302:isolation & purification; D013379:Substrate Specificity
D004792:Enzyme Precursors; D011480:Protease Inhibitors; C038451:pro-phenoloxidase; D004156:Catechol Oxidase; D002265:Carboxylic Ester Hydrolases; C019763:benzoylarginine ethyl esterase; D012697:Serine Endopeptidases