Preparation of monoclonal antibodies against human ventricular myosin light chain 1 (HVMLC1) for functional studies.
Using purified recombinant human ventricular myosin light chain 1 (HVMLC1) as the antigen, three monoclonal antibodies, designated C8, C9 and B12, were prepared. Immunoblot experiments demonstrated that all monoclonal antibodies could react with the ventricular myosin light chain 1 isolated from different sources, such as human, rat or pig. It was also demonstrated that C8 was directed against the NN part of the N-fragment (amino acid 1-40) of HVMLC1, and both C9 and B12 against the C-fragment (amino acid 99-195). The affinity constants of C8, C9 and B12 were 3.20 x 10(8), 8.600 x 10(7) and 1.770 x 10(8) M(-1), respectively, determined by non-competitive ELISA. The isotype of B12 was determined as IgG2a, whereas the isotype of both C8 and C9 were IgG1. In the presence of C9 or B12, the actin-activated Mg(2+)ATPase activity of myosin was greatly inhibited, but there was almost no effect on the Mg(2+) ATPase activity for C8. B12 and C9 also inhibited the superprecipitation of porcine cardiac native actomyosin (myosin B) and reconstituted actomyosin, but C8 did not. The results indicate that all three monoclonal antibodies could bind the intact myosin molecule, but B12 and C9 might more easily react with epitopes located in the C-fragment of HVMLC1. The inhibitory effects of B12 and C9 on ATPase activity and superprecipitation assays show that light chain 1, particularly the C-fragment domain, is involved in the modulation of the actin-activated Mg(2+) ATPase activity of myosin and, as a consequence, plays an essential role in the interaction of actin and myosin.
Fu
Zhen-Yan
ZY
Cardiovascular Research Center, First Clinical College of Xinjiang Medical University, Urumqi 830054, China.
Xie
Bao-Tong
BT
Ma
Yi-Tong
YT
Gong
Zu-Xun
ZX
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology*; D017301:Ca(2+) Mg(2+)-ATPase / Q000276:immunology*; D002478:Cells, Cultured; D006352:Heart Ventricles / Q000276:immunology*; D006801:Humans; D018994:Myosin Light Chains / Q000276:immunology*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology; D013552:Swine
D000911:Antibodies, Monoclonal; D018994:Myosin Light Chains; D011994:Recombinant Proteins; C076659:myosin light chain I; D017301:Ca(2+) Mg(2+)-ATPase