Isolation and characterization of different forms of C1 [formula omitted], a subcomponent of the first component of human complement

Abstract

C1 r , a subunit of the first component of complement, was purified from the euglobulin fraction of human serum by successive chromatographies on DEAE-cellulose, QAE-Sephadex, hydroxylapatite, Bio-Gel A-5m and Sephadex G-200 column. The final preparation of C1 r gave a single protein band on sodium dodecyl sulfate gel electrophoresis, and its molecular weight was calculated to be about 110 000. On treatment with 2-mercaptoethanol, C1 r split into two subunits with molecular weights of 68 000 and 41 000. Degradation of C1 r occurred during incubation of C1 r alone, resulting in formation of smaller C1 r , having a molecular weight of 64 000. This smaller C1 r , designated as C1 r (II), had N α -acetylarginine methylester hydrolyzing and C1s activating activities. Further, C1 r (II) split into two subunits, with molecular weights of 41 000 and 21 000, on treatment with 2-mercaptoethanol. The isoelectric point of C1 r (II) was 6.3, whereas that of native C1 r , designated as C1 r (I), was 4.9 C1 r (I) and C1 r (II) had similar affinities for N α -acetylarginine methylester, with K m values of 2.0 · 10 −2 M and 2.8 · 10 −2 M, respectively. Both components were strongly inhibited by l-leupeptin and N, N-dimethylamino p-( p′-guanidino benzoyloxy)-benzyloxy glucolate ( p′-GB-DBiG), which are strong inhibitors of tryptic enzymes, and the K i values of these inhibitors were similar: those with l-leupeptin were 4.3 · 10 −4 M for C1 r (I) and 7.9 · 10 −4 M for C1 r (II), and those with p′-GB-DBiG were 5.0 · 10 −6 M for C1 r (I) and 4.3 · 10 −6 M for C1 r (II). Therefore, C1 r (II) was presumably produced by cleavage of the heavy chain of C1 r (I).