Functional effects of LC1-reassociation with cardiac papain Mg.S1.
The effect of LC1 on cardiac myosin structure and activity was investigated using as a model S1 prepared by papain digestion in the presence of Mg2+. The resulting S1 contained LC2 but a part of the N-terminal region of LC1 was cleaved. Sequencing the N-terminal part of the band migrating below LC1 on SDS gels revealed it to consist of alternating alanyl and prolyl residues thus establishing LC1 as the origin of this band. However, Western blots did not reveal any LC1 while radioimmunoassays indicated it to be present at the 5% level suggesting the anti-LC1 antibody used in these experiments did not recognize the C-terminal portion of LC1 still attached to Mg.S1. Mixing a 10-15 M excess of isolated light chains with Mg.S1 in the presence of 10 mM ATP, 12 mM MgCl2, 4.7 M NH4Cl allowed LC1 to recombine with LC1-deficient Mg.S1. Equilibrium ultracentrifugation analysis revealed a highly heterogeneous LC1-deficient S1 which upon recombination with intact LC1 became monodisperse as indicated by the superimposition of molecular weight averages all across the centrifuge cell. LC1-deficient Mg.S1 had a Vm of 0.4 s-1, Ka of 30 microM and a Kbind of 28 microM. In the presence of intact LC1, Vm rose to 0.8 s-1 while Ka and Kbind were reduced to 7.5 and 12 microM, respectively. The fourfold decrease in Ka strongly indicated an increased affinity for actin by Mg.S1 in the presence of uncleaved LC1. Also, Ca(2+)-regulation of dog heart myofibrils was suppressed when Ca(2+)-activated MgATPase assays, as a function of Ca2+, were performed in the presence of anti-LC1 antibodies. These observations suggest the presence of intact, uncleaved LC1 in S1 is required for the stability of S1 heavy chains and proper Ca(2+)-regulation.
Margossian
S S
SS
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10467.
White
H D
HD
Lefford
J
J
Holt
J C
JC
Malhotra
A
A
Stafford
W F
WF
Slayter
H S
HS
D000199:Actins / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism*; D002918:Chymotrypsin; D004285:Dogs; D006321:Heart; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D009206:Myocardium / Q000737:chemistry; D018994:Myosin Light Chains; D015879:Myosin Subfragments / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism; D010206:Papain
D000199:Actins; D018994:Myosin Light Chains; D015879:Myosin Subfragments; C076659:myosin light chain I; D002918:Chymotrypsin; D010206:Papain; D000251:Adenosine Triphosphatases; D009218:Myosins; D008274:Magnesium; D002118:Calcium