Limited proteolysis of complement protein C3b by regulatory enzyme C3b inactivator: isolation and characterization of a biologically active fragment, C3d,g.
Limited proteolysis of C3b by C3b inactivator (factor I) consists of a two-step reaction; rapid cleavage of C3b to yield a nicked C3b derivative, iC3b, and followed by slow cleavage of iC3b to yield two antigenically distinct fragments, C3c and C3d,g. Using a fluorescence-labeled C3b as a substrate for I, we have investigated in detail the optimal conditions for the sequential cleavages of C3b by I. The pH optimum for the first cleavage was markedly affected by the ionic strength of buffers. The cleavage was maximum at pH 6.0 under physiological ionic strength but at pH 8.5 under low ionic strength (such as 1.7 mS). The second cleavage was a slow reaction and occurred only under low ionic strength and within a narrow pH range around pH 6.0. One of the products of the second cleavage, C3d,g, was isolated and shown to be a single polypeptide chain of 41,000 daltons with pI 5.0. C3d,g had leucocytosis-inducing activity, like C3d-k, which is a C3d fragment released by the action of plasma kallikrein. Trypsin digestion of C3d,g produced two fragments of 30,000 and 10,000 daltons and the 10,000-dalton fragment retained the leucocytosis inducing activity.
Seya
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Nagasawa
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D000818:Animals; D003179:Complement C3b / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology*; D003180:Complement C3b Inactivator Proteins / Q000378:metabolism*; D005341:Fibrinolysin / Q000378:metabolism; D005456:Fluorescent Dyes; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007610:Kallikreins / Q000378:metabolism; D007964:Leukocytosis / Q000139:chemically induced; D008301:Maleimides; D008970:Molecular Weight; D009994:Osmolar Concentration; D010196:Pancreatic Elastase / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification / Q000502:physiology*; D051381:Rats; D014357:Trypsin / Q000378:metabolism
D003180:Complement C3b Inactivator Proteins; D005456:Fluorescent Dyes; D008301:Maleimides; D010446:Peptide Fragments; C045477:complement C3d,g; C019347:N-(7-dimethylamino-4-methylcoumarinyl)maleimide; D003179:Complement C3b; D007610:Kallikreins; D010196:Pancreatic Elastase; D014357:Trypsin; D005341:Fibrinolysin