Isolation, characterization, and mechanism of action of rat beta 1H.
beta 1Hrat was purified to homogeneity from fresh rat plasma by precipitation with 28.6% ammonium sulfate followed by sequential chromatography on DEAE-Sephacel, Biorex-70, and gel filtration on Sephacryl-S300. The final material was homogeneous on SDS-PAGE analysis and had an apparent m.w. of 150,000. Reduction with dithiothreitol did not affect its m.w., suggesting that the molecule is composed of one polypeptide chain. The recovery of beta 1H was approximately 10%. A monospecific antibody against beta 1Hrat was obtained from immunized rabbits, which recognized beta 1Hrat as a protein with beta-electrophoretic mobility upon immunoelectrophoresis of fresh rat plasma. The concentration of beta 1H in plasma of normal 4-mo-old Wistar rats was 243.5 +/- 36.3 micrograms/ml (mean +/- S.D.). beta 1Hrat in this study was detected by its capacity to inhibit formation of the P-stabilized cell-bound amplification C3/C5 convertase composed of cell-bound C3bhu and Bbhu. Purified beta 1Hrat produced a dose-related, first-order loss of convertase function and release of 126I-Bbhu from the P-stabilized C3bhuBbhu convertase, indicating a mechanism of action by decay-dissociation of Bbhu from the complex C3bhuBbhuP. beta 1Hrat was at least four times less effective than beta 1Hhu in release of 125I-Bbhu from the homologous convertase composed of C3bhu and Bbhu. On the other hand beta 1H was twice as effective in releasing 125I-Bbrat from the convertase composed of C3bratBbratP when compared to beta 1Hhu. These differences are presumably dependent upon the species-specific affinity of beta 1H from humans or rats for C3bhu or C3brat, respectively.
D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D050577:Complement C3-C5 Convertases / Q000378:metabolism; D003180:Complement C3b Inactivator Proteins / Q000302:isolation & purification*; D011415:Complement Factor B / Q000378:metabolism; D017242:Complement Factor H; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D051381:Rats; D011919:Rats, Inbred Strains
C493495:CFH protein, human; D003180:Complement C3b Inactivator Proteins; D017242:Complement Factor H; D050577:Complement C3-C5 Convertases; D011415:Complement Factor B