Preparation and characterization of heavy meromyosin and subfragment 1 from vertebrate cytoplasmic myosins.
The soluble fragments of myosin, heavy meromyosin (HMM), and subfragment 1 (S-1) have been instrumental in elucidating the kinetic mechanisms of the actin-activated MgATPase activity of both skeletal and smooth muscle myosin. To date, relatively little has been published on these fragments from vertebrate cytoplasmic myosins. We now describe the preparation and steady-state kinetic characterization of S-1 and HMM from human platelet and avian intestinal epithelial brush border myosin. The HMM prepared from each of these tissues was similar both in their SDS-polyacrylamide gel pattern and in their steady-state kinetic properties. The Vmax of the actin-activated MgATPase activity varied between 0.8 and 2.5 s-1, and the KATPase (the apparent dissociation constant derived from a double-reciprocal plot of the MgATPase activity) was about 1-2 microM. This low value for the apparent dissociation constant was similar to the dissociation constant of HMM for actin directly measured under similar conditions and is about 40 times lower than that determined with avian smooth muscle HMM. The KATPase of the cytoplasmic HMM was only slightly increased when the ionic strength was raised from 12 to 112 mM.
Sellers
J R
JR
Laboratory of Molecular Cardiology, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892.
Soboeiro
M S
MS
Faust
K
K
Bengur
A R
AR
Harvey
E V
EV
D000199:Actins / Q000378:metabolism; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism; D007700:Kinetics; D015879:Myosin Subfragments / Q000302:isolation & purification* / Q000378:metabolism; D009218:Myosins / Q000302:isolation & purification* / Q000378:metabolism; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000302:isolation & purification* / Q000378:metabolism
D000199:Actins; D015879:Myosin Subfragments; D010446:Peptide Fragments; D017301:Ca(2+) Mg(2+)-ATPase; D009218:Myosins