Myosin subfragment 1 and structural elements of G-actin: effects of S-1(A2) on sequences 39-52 and 61-69 in subdomain 2 of G-actin.
The effect of myosin on the structure of two sequences on G-actin, a loop between residues 39 and 52 and a segment between residues 61 and 69 from the NH2-terminus, was probed by limited proteolytic digestions of G-actin in the presence of the myosin subfragment 1 isozyme S-1(A2). Under the experimental conditions of this work, no polymerization of actin was induced by S-1(A2) [Chen & Reisler (1991) Biochemistry 30, 4546-4552]. S-1(A2) did not change the rates of subtilisin and chymotryptic digestion of G-actin at loop 39-52. In contrast to this, the second protease-sensitive region on G-actin, segment 61-69, was protected strongly by S-1(A2) from tryptic cleavage. The minor if any involvement of loop 39-52 in S-1 binding was confirmed by determining the binding constants of S-1(A2) for pyrene-labeled G-actin (1.2 x 10(6) M-1), subtilisin-cleaved pyrenyl G-actin (0.3 x 10(6) M-1), and DNase I-pyrenyl G-actin complexes (0.3 x 10(6) M-1). Consistent with this, the activity of DNase I, which binds to actin loop 39-52 [Kabsch et al. (1990) Nature 347, 37-44], was inhibited almost equally well by actin in the presence and absence of S-1(A2). These results confirm the observation that DNase I and S-1(A2) bind to distinct sites on actin [Bettache et al. (1990) Biochemistry 29, 9085-9091] and demonstrate myosin-induced changes in segment 61-69 of G-actin.
Chen
T
T
Department of Chemistry and Biochemistry, University of California, Los Angeles 90024.
Haigentz
M
M
Reisler
E
E
D000199:Actins / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002918:Chymotrypsin / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism / Q000494:pharmacology*; D008969:Molecular Sequence Data; D015879:Myosin Subfragments / Q000378:metabolism / Q000494:pharmacology*; D010446:Peptide Fragments / Q000737:chemistry*; D011817:Rabbits; D013381:Subtilisins / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D000199:Actins; D007527:Isoenzymes; D015879:Myosin Subfragments; D010446:Peptide Fragments; D003850:Deoxyribonuclease I; D013381:Subtilisins; D002918:Chymotrypsin; D014357:Trypsin