The regulation of subtilisin-cleaved actin by tropomyosin/troponin.
Vertebrate striated muscle contraction is regulated in a Ca(2+)-dependent fashion by tropomyosin (Tm) and troponin (Tn). This regulation involves shifts in the position of Tm and Tn on actin filaments and may include conformational changes in actin that are then communicated to myosin subfragment 1 (S1). To determine whether subdomain 2 of actin plays a role in this regulation, the DNase-I loop 38-52 of this subdomain was cleaved by subtilisin between residues Met(47) and Gly(48). Despite impaired unregulated function, the potentiation and regulation of cleaved actin movement in the in vitro motility assay was not significantly different from that of uncleaved actin. Stopped-flow measurements of ADP release from regulated and unregulated cleaved acto-S1 showed a marked increase in ADP release from acto-S1 in the presence of the regulatory complex. The enhancement of the actin affinity for S1 in the presence of regulatory proteins was greater for uncleaved than for cleaved F-actin. Finally, both cleaved and uncleaved actins protect myosin loop 1 from papain cleavage equally well. Our results suggest that the potentiation of actin function in the in vitro motility assay by regulatory proteins stems from changes in cross-bridge cycle kinetics. In addition, the unimpaired calcium-sensitive regulation of cleaved actin indicates that subdomain 2 conformation does not play an essential role in the regulation process.
Pavlov
Dmitry
D
Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095, USA.
Gerson
Jack H
JH
Yu
Tianwei
T
Tobacman
Larry S
LS
Homsher
Earl
E
Reisler
Emil
E
D000199:Actins / Q000737:chemistry* / Q000378:metabolism*; D000205:Actomyosin / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D003850:Deoxyribonuclease I; D007700:Kinetics; D009119:Muscle Contraction / Q000502:physiology; D018482:Muscle, Skeletal / Q000502:physiology; D015879:Myosin Subfragments / Q000737:chemistry / Q000378:metabolism*; D010206:Papain / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D010907:Pituitary Hormones / Q000378:metabolism; D011817:Rabbits; D020860:Subtilisin / Q000378:metabolism*; D014335:Tropomyosin / Q000378:metabolism*; D014336:Troponin / Q000378:metabolism*
D000199:Actins; D015879:Myosin Subfragments; D010446:Peptide Fragments; D010907:Pituitary Hormones; D014335:Tropomyosin; D014336:Troponin; C010912:diabetogenic factor, human; D000205:Actomyosin; D003850:Deoxyribonuclease I; D020860:Subtilisin; D010206:Papain