The free heavy chain of vertebrate skeletal myosin subfragment 1 shows full enzymatic activity.

Sivaramakrishnan, M; Burke, M. Desmond

doi:10.1016/s0021-9258(19)68313-4

Cited by 128 publications

(23 citation statements)

References 29 publications

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“…In addition to its ability to split ATE it binds actin in a calciumsensitive manner, and decoration of actin filaments with ll0K-calmodulin complex resembles by electron microscopy that which occurs after incubation of actin with myosin fragments. We now observe that the enzymatic activity resides on the ll0-kD heavy chain; the calmodulin chains, as has been found for the light chains of skeletal muscle myosin (Wagner and Giniger, 1981;Sivaramakrishnan and Burke, 1982) and Acanthamoeba myosin (Maruta et al, 19"/8), are not essential.…”

Section: Discussionsupporting

confidence: 74%

Mapping of the microvillar 110K-calmodulin complex: calmodulin-associated or -free fragments of the 110-kD polypeptide bind F-actin and retain ATPase activity

Coluccio

Bretscher

1988

The Journal of Cell Biology

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Abstract. The ll0K-calmodulin complex isolated from intestinal microvilli is an ATPase consisting of one polypeptide chain of 110 kD in association with three to four calmodulin molecules. This complex is presumably the link between the actin filaments in the microvillar core and the surrounding cell membrane. To study its structural regions, we have partially cleaved the ll0K-caimodulin complex with ~t-chymotrypsin; calmodulin remains essentially intact under the conditions used. As determined by 125I-calmodulin overlays, ion exchange chromatography, and actinbinding assays, a 90-kD digest fragment generated in EGTA remains associated with calmodulin. The 90K-calmodulin complex binds actin in an ATP-reversible manner and decorates actin filaments with an arrowhead appearance similar to that found after incubation of F-actin with the parent complex; binding occurs in either calcium-or EGTA-containing buffers. ATPase activity of the 90-kD digest closely resembles the parent complex. In calcium a digest mixture containing fragments of 78 kD, a group of three at m40 kD, and a 32-kD fragment (78-kD digest mixture) is generated with a-chymotrypsin at a longer incubation time; no association of these fragments with calmodulin is observed. Time courses of digestions and cyanogen bromide cleavage indicate that the 78-kD fragment derives from the 90-kD peptide. The 78-kD mixture can also hydrolyze ATE Furthermore, removal of the calmodulin by ion exchange chromatography from this 78-kD mixture had no effect on the ATPase activity of the digest, indicating that the ATPase activity resides on the ll0-kD polypeptide. The 78 kD, two of the three fragments at *40 kD, and the 32-kD fragments associate with F-actin in an ATP-reversible manner. Electron microscopy of actin filaments after incubation with the 78-kD digest mixture reveals coated filaments, although the prominent arrowhead appearance characteristic of the parent complex is not observed. These data indicate that calmodulin is not required either for the ATPase activity or the ATP-reversible binding of the ll0K-calmodulin complex to F-actin. In addition, since all the fragments that bind F-actin do so in an ATP-reversible manner, the sites required for F-actin binding and ATP reversibility likely reside nearby.

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Section: Discussionsupporting

confidence: 74%

Mapping of the microvillar 110K-calmodulin complex: calmodulin-associated or -free fragments of the 110-kD polypeptide bind F-actin and retain ATPase activity

Coluccio

Bretscher

1988

The Journal of Cell Biology

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“…myosin-nucleotide tertiary complex. This diagram does not conflict with the previous reports showing that myosin alkali light chains are not essential for the ATP hydrolysis of S-l ATPase (Wagner & Giniger, 1981;Sivaramakrishnan & Burke, 1982).…”

Section: Discussionsupporting

confidence: 67%

Binding of the Amino-Terminal Region of Myosin Alkali 1 Light Chain to Actin and Its Effect on Actin-Myosin Interaction

Hayashibara¹,

Miyanishi²

1994

Biochemistry

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The role of the amino-terminal region of myosin alkali 1 light chain (A1) in the interaction between actin and myosin subfragment-1 (S-1) was explored. Papain digestion of skeletal myosin filaments produced S-1 whose A1 was found to lose the basic 13 amino-terminal amino acid residues (A1'). We obtained three types of papain S-1 isoenzymes differing in their alkali light chain content: recombined papain S-1 (A1), papain S-1 (A1'), and papain S-1 (A2). Both the maximum turnover rate (Vmax) and the dissociation constant (Km) for actin-activated papain S-1 (A1') ATPase activity were similar to those for papain S-1 (A2) and remarkably larger than those for recombined papain S-1 (A1). The 13 amino-terminal residue peptide of A1 (N-pep) was isolated and characterized. 1H-NMR spectroscopy suggested that the N-pep was relatively immobilized in the presence of actin filaments. A cross-linking study suggested that N-pep binds to actin. The addition of N-pep to acto-S-1 (A1) made Km and Vmax for the actin-activated ATPase activity close to those for S-1 (A2). Removal of the trimethyl group from the N-pep suppressed the above effect on the actin-S-1 interaction. Our findings suggest that the amino-terminal region of A1 binds to the actin molecule to affect the mechanism of actin-activated S-1 ATPase.

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“…In smooth muscle, invertebrate muscle and vertebrate nonmuscle myosin, myosin light chain 2 (MLC2) ~ regulates the actin-activated ATPase of myosin in response to the calcium concentration of the cell (for reviews see Kamm and Stull, 1985;Citi and Kendrick-Jones, 1988). The role of MLC1 in myosin function is less clear (see Wagner and Giniger, 1981;Sivaramakrishnan and Burke, 1982). However, isoforms of MLC1 have been shown to be associated with different shortening velocities of skeletal muscle (Sweeney et al, 1988).The interactions of the subunits of myosin are not precisely defined, but both biochemical and electron microscopic studies suggest that MLCs bind near the junction of the subfragments 1 and 2 ($1 and $2).…”

mentioning

confidence: 99%

Identification of sequences necessary for the association of cardiac myosin subunits.

McNally

Bravo-Zehnder

Leinwand

1991

The Journal of Cell Biology

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Abstract. To begin to understand the nature of myosin subunit assembly, we determined the region of a vertebrate sarcomeric myosin heavy chain required for binding of light chain 1. We coexpressed in Escherichia coli segments of the rat alpha cardiac myosin heavy chain which spanned the carboxyl terminus of subfragment 1 and the amino terminus of subfragment 2 with a full-length rat cardiac myosin light chain 1. A 16 amino acid region of the myosin heavy chain (residues 792-808) was shown to be required for myosin light chain 1 binding in an immunoprecipitation assay.M YosiN is an asymmetric hexamer comprised of two heavy chains (210 kD) and two pairs of light chains (18-25 kD). In smooth muscle, invertebrate muscle and vertebrate nonmuscle myosin, myosin light chain 2 (MLC2) ~ regulates the actin-activated ATPase of myosin in response to the calcium concentration of the cell (for reviews see Kamm and Stull, 1985;Citi and Kendrick-Jones, 1988). The role of MLC1 in myosin function is less clear (see Wagner and Giniger, 1981;Sivaramakrishnan and Burke, 1982). However, isoforms of MLC1 have been shown to be associated with different shortening velocities of skeletal muscle (Sweeney et al., 1988).The interactions of the subunits of myosin are not precisely defined, but both biochemical and electron microscopic studies suggest that MLCs bind near the junction of the subfragments 1 and 2 ($1 and $2). Scallop myosin reacted with anti-light chain antibodies demonstrates MLC2 binding near the head-rod junction (Flicker et al., 1983). Similar positions were noted for vertebrate MLC2, with MLC1 binding distally to MLC2, in the S1 head (Tokunaga et al., 1987;Katoh and Lowey, 1989). Proteolytic peptides of myosin heavy chain (MHC) from the carboxyl terminus of the S1 head bind MLC (Szentkiralyi, 1984;Mitchell et al., 1986). Burke et al. (1983) also implicated the carboxyl terminus of the S1 head by abolishing MLC1 binding with proteolytic digestion of a 3-kD segment from the S1-$2 junction. Sellers and Harvey (1984) used gel overlay to assign the light chain binding sites to 26 kD at the carboxyl terminus of smooth muscle myosin $1. However, Okamoto et al. (1986) have shown that MLC1 binds near the site of ATP binding on smooth muscle myosin. In the scallop it has been shown that exchange of MLC1 with exogenously added MLC1 can occur only in the absence of MLC2 (Ashiba and Szent-Gyorgyi, 1985). This finding, coupled with the observation that the 1. Abbreviations used in this Paper: MHC, myosin heavy chain; MLC, myosin light chain; S1 and $2, subfragment 1 and 2, respectively. sulfhydryl groups of scallop MLC1 are only available for labeling in the absence of MLC2, suggests that MLC1 lies between MLC2 and MHC (Walliman and Szent-Gyorgyi, 1981;Hardwicke et al., 1982).The use of recombinant DNA methods to produce myosin segments that have precisely defined sequences would be of great use in mapping the interactions of myosin subunits. A portion of the Caenorhabditis elegans unc54 MHC gene corresponding to S1 was expressed in Es...

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The free heavy chain of vertebrate skeletal myosin subfragment 1 shows full enzymatic activity.

Cited by 128 publications

References 29 publications

Mapping of the microvillar 110K-calmodulin complex: calmodulin-associated or -free fragments of the 110-kD polypeptide bind F-actin and retain ATPase activity

Mapping of the microvillar 110K-calmodulin complex: calmodulin-associated or -free fragments of the 110-kD polypeptide bind F-actin and retain ATPase activity

Binding of the Amino-Terminal Region of Myosin Alkali 1 Light Chain to Actin and Its Effect on Actin-Myosin Interaction

Identification of sequences necessary for the association of cardiac myosin subunits.

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