Enzymatic characterization and functional domain mapping of brain myosin-V.
The actin binding and ATPase properties, as well as the functional domain structure of chick brain myosin-V, a two-headed, unconventional myosin, is reported here. Compared to conventional myosin from skeletal muscle, brain myosin-V exhibits low K-EDTA- and Ca-ATPase activities (1.8 and 0.8 ATP/s per head). The physiologically relevant Mg-ATPase is also low (approximately 0.3 ATP/s), unless activated by the presence of both F-actin and Ca2+ (Vmax of 27 ATP/s). Ca2+ stimulates the actin-activated Mg-ATPase over a narrow concentration range between 1 and 3 microM. In the presence of saturating Ca2+ and 75 mM KCl, surprisingly low concentrations of F-actin activate the Mg-ATPase in a hyperbolic manner (KATPase of 1.3 microM). Brain myosin-V also binds with relatively high affinity (compared to other known myosins) to F-actin in the presence of ATP, as assayed by cosedimentation. Digestion of brain myosin-V with calpain yielded a 65-kDa head domain fragment that cosediments with actin in an ATP-sensitive manner and a 80-kDa tail fragment that does not interact with F-actin. The 80-kDa fragment results from cleavage one residue beyond the proline-, glutamate-, serine-, threonine-rich region. Our data indicate that the Mg-ATPase cycle of brain myosin-V is tightly regulated by Ca2+, probably via direct binding to the calmodulin light chains in the neck domain, which like brush border myosin-I, results in partial (approximately 30%) dissociation of the calmodulin associated with brain myosin-V. The effect of Ca2+ binding, which appears to relieve suppression by the neck domain, can be mimicked by calpain cleavage near the head/neck junction.
Nascimento
A A
AA
Department of Biology, Yale University, New Haven, Connecticut 06511, USA.
Cheney
R E
RE
Tauhata
S B
SB
Larson
R E
RE
Mooseker
M S
MS
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002154:Calpain; D002645:Chickens; D007700:Kinetics; D008969:Molecular Sequence Data; D018994:Myosin Light Chains / Q000378:metabolism; D024701:Myosin Type V; D009218:Myosins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification
D000199:Actins; D000596:Amino Acids; D002148:Calmodulin-Binding Proteins; D018994:Myosin Light Chains; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; C078829:chicken brain myosin-V p190; D002154:Calpain; D017301:Ca(2+) Mg(2+)-ATPase; D024701:Myosin Type V; D009218:Myosins