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Using purified recombinant human ventricular myosin light chain 1 (HVMLC 1) as the antigen,three monoclonal antibodies,designated C8,C9 and B 12,were prepared.Immunoblot experiments demonstratedthat all monoclonal antibodies could react with the ventricular myosin light chain 1 isolated from differentsources,such as human,rat or pig.It was also demonstrated that C8 was directed against the NN part of theN-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 3.20×10~8,8.60×10~7 and 1.77×10~8 M~(-1),respectively,determined by non-competitive ELISA.The isotype of B12 was determined as lgG2a,whereas the isotype ofboth C8 and C9 were IgG1.In the presence of C9 or B12,the actin-activated Mg~(2+)ATPase activity of myosinwas greatly inhibited,but there was almost no effect on the Mg~(2+)ATPase activity for C8.B12 and C9 alsoinhibited 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 inhibitoryeffects of B 12 and C9 on ATPase activity and superprecipitation assays show that light chain 1,particularlythe C-fragment domain,is involved in the modulation of the actin-activated Mg~(2+)ATPase activity of myosinand,as a consequence,plays an essential role in the interaction of actin and myosin.
Using purified recombinant human ventricular myosin light chain 1 (HVMLC 1) as the antigen, three monoclonal antibodies, designated C8, C9 and B 12, were prepared. Immunoblot tests demonstrated that all monoclonal antibodies could react with the ventricular myosin light chain 1 isolated from differents , such as human, rat or pig. It was also displayed 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 195). The affinity constants of C8, C9 and B12 were 3.20 × 10 -8, 8.6 × 10 -7 and 1.77 × 10 -8 M -1, respectively, determined by non-competitive ELISA. The isotype of B12 was determined as lgG2a, while the isotype ofboth C8 and C9 were IgG1.In the presence of C9 or B12, the actin-activated Mg ~ (2+) ATPase activity of myosinwas greatly inhibited, but there was almost no effect on the Mg ~ (2+) ATPase activity for C8.B12 and C9 also inhibits the superprecipitation of porcine cardiac native actomyosin (myosin B) and reconstituted actomyosin, but C8 did not. The results that all three monoclonal antibodies could bind the intact myosin molecule, but B12 and C9 might more readily react with epitopes located in the C-fragment of HVMLC1. Inhibitory effects of B 12 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 myosinand, as a consequence, plays an essential role in the interaction of actin and myosin.