Purification and characterization of a mammalian myosin I.
Myosin I, an actin-dependent force-generating enzyme, has been purified from three mammalian sources: bovine adrenal medulla, adrenal cortex, and brain. The purification procedure includes extraction of tissue with ATP at low ionic strength and coprecipitation with actin, followed by gel filtration on Sepharose 4B, anion-exchange chromatography on Q Sepharose, and affinity chromatography on ATP-agarose. Mammalian myosin I molecules are composed of a heavy chain of 116 kDa and multiple low molecular weight polypeptides identified as calmodulin. The structural and enzymatic properties of adrenal medulla myosin I were further characterized. This enzyme exhibits high K+,EDTA- and Ca(2+)-ATPase specific activities (about 0.2 mumol.min-1 per mg of protein), whereas the Mg(2+)-ATPase activity is very low (1-3 nmol.min-1.mg-1). The Mg(2+)-ATPase of medulla myosin I is activated by F-actin in a Ca(2+)-dependent manner: activity is stimulated 40-fold in the presence of EGTA and 90-fold in the presence of 10 microM Ca2+. Two structural domains of the myosin I heavy chain were identified. A 74-kDa chymotryptic fragment contains the catalytic site, while a 36-kDa polypeptide contains the calmodulin-binding sites. These results indicate that mammalian myosin I is more closely related to myosin I from the avian intestinal brush border than to the enzymes isolated from the protozoans Acanthamoeba and Dictyostelium.
Barylko
B
B
University of Texas Southwestern Medical Center, Department of Pharmacology, Dallas 75235.
Wagner
M C
MC
Reizes
O
O
Albanesi
J P
JP
D000199:Actins / Q000378:metabolism; D000302:Adrenal Cortex / Q000201:enzymology*; D000313:Adrenal Medulla / Q000201:enzymology*; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002850:Chromatography, Gel; D004789:Enzyme Activation; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009218:Myosins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry
D000199:Actins; D010455:Peptides; D009218:Myosins