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Proc Natl Acad Sci U S A. Jan 15, 1992; 89(2): 490–494.
PMCID: PMC48264

Purification and characterization of a mammalian myosin I.

Abstract

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.

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  • Korn ED, Hammer JA., 3rd Myosin I. Curr Opin Cell Biol. 1990 Feb;2(1):57–61. [PubMed]
  • Pollard TD, Doberstein SK, Zot HG. Myosin-I. Annu Rev Physiol. 1991;53:653–681. [PubMed]
  • Fukui Y, Lynch TJ, Brzeska H, Korn ED. Myosin I is located at the leading edges of locomoting Dictyostelium amoebae. Nature. 1989 Sep 28;341(6240):328–331. [PubMed]
  • Miyata H, Bowers B, Korn ED. Plasma membrane association of Acanthamoeba myosin I. J Cell Biol. 1989 Oct;109(4 Pt 1):1519–1528. [PMC free article] [PubMed]
  • Mooseker MS, Conzelman KA, Coleman TR, Heuser JE, Sheetz MP. Characterization of intestinal microvillar membrane disks: detergent-resistant membrane sheets enriched in associated brush border myosin I (110K-calmodulin). J Cell Biol. 1989 Sep;109(3):1153–1161. [PMC free article] [PubMed]
  • Adams RJ, Pollard TD. Binding of myosin I to membrane lipids. Nature. 1989 Aug 17;340(6234):565–568. [PubMed]
  • Hayden SM, Wolenski JS, Mooseker MS. Binding of brush border myosin I to phospholipid vesicles. J Cell Biol. 1990 Aug;111(2):443–451. [PMC free article] [PubMed]
  • Spudich JA. In pursuit of myosin function. Cell Regul. 1989 Nov;1(1):1–11. [PMC free article] [PubMed]
  • Harrington WF, Rodgers ME. Myosin. Annu Rev Biochem. 1984;53:35–73. [PubMed]
  • Hoshimaru M, Nakanishi S. Identification of a new type of mammalian myosin heavy chain by molecular cloning. Overlap of its mRNA with preprotachykinin B mRNA. J Biol Chem. 1987 Oct 25;262(30):14625–14632. [PubMed]
  • Titus MA, Warrick HM, Spudich JA. Multiple actin-based motor genes in Dictyostelium. Cell Regul. 1989 Nov;1(1):55–63. [PMC free article] [PubMed]
  • Pollard TD, Korn ED. Acanthamoeba myosin. I. Isolation from Acanthamoeba castellanii of an enzyme similar to muscle myosin. J Biol Chem. 1973 Jul 10;248(13):4682–4690. [PubMed]
  • Côté GP, Albanesi JP, Ueno T, Hammer JA, 3rd, Korn ED. Purification from Dictyostelium discoideum of a low-molecular-weight myosin that resembles myosin I from Acanthamoeba castellanii. J Biol Chem. 1985 Apr 25;260(8):4543–4546. [PubMed]
  • Matsudaira PT, Burgess DR. Identification and organization of the components in the isolated microvillus cytoskeleton. J Cell Biol. 1979 Dec;83(3):667–673. [PMC free article] [PubMed]
  • Maruta H, Korn ED. Acanthamoeba cofactor protein is a heavy chain kinase required for actin activation of the Mg2+-ATPase activity of Acanthamoeba myosin I. J Biol Chem. 1977 Dec 10;252(23):8329–8332. [PubMed]
  • Maruta H, Gadasi H, Collins JH, Korn ED. The isolated heavy chain of an Acanthamoeba myosin contains full enzymatic activity. J Biol Chem. 1978 Sep 25;253(18):6297–6300. [PubMed]
  • Hammer JA, 3rd, Albanesi JP, Korn ED. Purification and characterization of a myosin I heavy chain kinase from Acanthamoeba castellanii. J Biol Chem. 1983 Aug 25;258(16):10168–10175. [PubMed]
  • Maruta H, Gadasi H, Collins JH, Korn ED. Multiple forms of Acanthamoeba myosin I. J Biol Chem. 1979 May 10;254(9):3624–3630. [PubMed]
  • Coluccio LM, Bretscher A. Calcium-regulated cooperative binding of the microvillar 110K-calmodulin complex to F-actin: formation of decorated filaments. J Cell Biol. 1987 Jul;105(1):325–333. [PMC free article] [PubMed]
  • Swanljung-Collins H, Collins JH. Ca2+ stimulates the Mg2(+)-ATPase activity of brush border myosin I with three or four calmodulin light chains but inhibits with less than two bound. J Biol Chem. 1991 Jan 15;266(2):1312–1319. [PubMed]
  • Conzelman KA, Mooseker MS. The 110-kD protein-calmodulin complex of the intestinal microvillus is an actin-activated MgATPase. J Cell Biol. 1987 Jul;105(1):313–324. [PMC free article] [PubMed]
  • Collins K, Sellers JR, Matsudaira P. Calmodulin dissociation regulates brush border myosin I (110-kD-calmodulin) mechanochemical activity in vitro. J Cell Biol. 1990 Apr;110(4):1137–1147. [PMC free article] [PubMed]
  • Mooseker MS, Coleman TR. The 110-kD protein-calmodulin complex of the intestinal microvillus (brush border myosin I) is a mechanoenzyme. J Cell Biol. 1989 Jun;108(6):2395–2400. [PMC free article] [PubMed]
  • Garcia A, Coudrier E, Carboni J, Anderson J, Vandekerkhove J, Mooseker M, Louvard D, Arpin M. Partial deduced sequence of the 110-kD-calmodulin complex of the avian intestinal microvillus shows that this mechanoenzyme is a member of the myosin I family. J Cell Biol. 1989 Dec;109(6 Pt 1):2895–2903. [PMC free article] [PubMed]
  • Bikle DD, Munson S, Mancianti ML. Limited tissue distribution of the intestinal brush border myosin I protein. Gastroenterology. 1991 Feb;100(2):395–402. [PubMed]
  • Scholey JM, Smith RC, Drenckhahn D, Groschel-Stewart U, Kendrick-Jones J. Thymus myosin. Isolation and characterization of myosin from calf thymus and thymic lymphocytes, and studies on the effect of phosphorylation of its Mr = 20,000 light chain. J Biol Chem. 1982 Jul 10;257(13):7737–7745. [PubMed]
  • Barylko B, Tooth P, Kendrick-Jones J. Proteolytic fragmentation of brain myosin and localisation of the heavy-chain phosphorylation site. Eur J Biochem. 1986 Jul 15;158(2):271–282. [PubMed]
  • Watterson DM, Harrelson WG, Jr, Keller PM, Sharief F, Vanaman TC. Structural similarities between the Ca2+-dependent regulatory proteins of 3':5'-cyclic nucleotide phosphodiesterase and actomyosin ATPase. J Biol Chem. 1976 Aug 10;251(15):4501–4513. [PubMed]
  • Strzelecka-Golaszewska H, Jakubiak M, Drabikowski W. Changes in the state of actin during superprecipitation of actomyosin. Eur J Biochem. 1975 Jun 16;55(1):221–230. [PubMed]
  • Glenney JR, Jr, Weber K. Detection of calmodulin-binding polypeptides separated in SDS-polyacrylamide gels by a sensitive [125I]calmodulin gel overlay assay. Methods Enzymol. 1983;102:204–210. [PubMed]
  • Maruta H, Korn ED. Direct photoaffinity labeling by nucleotides of the apparent catalytic site on the heavy chains of smooth muscle and Acanthamoeba myosins. J Biol Chem. 1981 Jan 10;256(1):499–502. [PubMed]
  • Korn ED, Collins JH, Maruta H. Myosins from Acanthamoeba castellanii. Methods Enzymol. 1982;85(Pt B):357–363. [PubMed]
  • Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. [PubMed]
  • Carboni JM, Conzelman KA, Adams RA, Kaiser DA, Pollard TD, Mooseker MS. Structural and immunological characterization of the myosin-like 110-kD subunit of the intestinal microvillar 110K-calmodulin complex: evidence for discrete myosin head and calmodulin-binding domains. J Cell Biol. 1988 Nov;107(5):1749–1757. [PMC free article] [PubMed]
  • Swanljung-Collins H, Collins JH. Rapid, high-yield purification of intestinal brush border myosin I. Methods Enzymol. 1991;196:3–11. [PubMed]
  • Coluccio LM, Bretscher A. Mapping of the microvillar 110K-calmodulin complex: calmodulin-associated or -free fragments of the 110-kD polypeptide bind F-actin and retain ATPase activity. J Cell Biol. 1988 Feb;106(2):367–373. [PMC free article] [PubMed]
  • Korn ED, Hammer JA., 3rd Myosins of nonmuscle cells. Annu Rev Biophys Biophys Chem. 1988;17:23–45. [PubMed]

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