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Properties of caldesmon isolated from chicken gizzard.

ABSTRACT: Chicken gizzard smooth muscle contains two major calmodulin-binding proteins: caldesmon (11.1 microM; Mr 141 000) and myosin light-chain kinase (4.6 microM; Mr 136 000), both of which are associated with the contractile apparatus. The amino acid composition of caldesmon is distinct from that of myosin light-chain kinase and is characterized by a very high glutamic acid content (25.5%), high contents of lysine (13.6%) and arginine (10.3%), and a low aromatic amino acid content (2.4%). Caldesmon lacked myosin light-chain kinase and phosphatase activities and did not compete with either myosin light-chain kinase or cyclic nucleotide phosphodiesterase (both calmodulin-dependent enzymes) for available calmodulin, suggesting that calmodulin may have distinct binding sites for caldesmon on the one hand and myosin light-chain kinase and cyclic nucleotide phosphodiesterase on the other. Consistent with the lack of effect of caldesmon on myosin phosphorylation, caldesmon did not affect the assembly or disassembly of myosin filaments in vitro. As previously shown [Ngai & Walsh (1984) J. Biol. Chem. 259, 13656-13659], caldesmon can be reversibly phosphorylated. The phosphorylation and dephosphorylation of caldesmon were further characterized and the Ca2+/calmodulin-dependent caldesmon kinase was purified; kinase activity correlated with a protein of subunit Mr 93 000. Caldesmon was not a substrate of myosin light-chain kinase or phosphorylase kinase, both calmodulin-activated protein kinases.

SUBMITTER: Ngai PK

PROVIDER: S-EPMC1152673 | biostudies-other | 1985 Sep

REPOSITORIES: biostudies-other

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Project description:Caldesmon, an actin- and calmodulin-binding protein of smooth muscle, is a protein serine/threonine kinase capable of Ca2+/calmodulin-dependent autophosphorylation [Scott-Woo & Walsh (1988) Biochem. J. 252, 463-472]. Phosphorylation nullifies the inhibitory effect of caldesmon on the actin-activated Mg2+-ATPase activity of smooth-muscle myosin [Ngai & Walsh (1987) Biochem. J. 244, 417-425]. We have characterized the kinase activity of caldesmon of chicken gizzard smooth muscle. Autophosphorylation requires Ca2+/calmodulin, but is unaffected by other second messengers (Ca2+/phospholipid/diacylglycerol, cyclic AMP or cyclic GMP), and is inhibited by the calmodulin antagonists chlorpromazine and compound 48/80, with 50% inhibition at 39.8 microM and 12.0 ng/ml respectively. Half-maximal activation of autophosphorylation occurs at 60-80 nM-Ca2+ and 0.14 microM-calmodulin, and maximal activity at 0.14-0.18 microM-Ca2+ and 1 microM-calmodulin; activation is gradually lost at higher Ca2+ and calmodulin concentrations. Autophosphorylation is pH-dependent, with maximal activity over the range pH 7-9, and requires free Mg2+ in addition to the MgATP2- substrate. The Km for ATP is 15.6 +/- 4.1 microM (mean +/- S.D., n = 4), and kinase activity is inhibited by increasing ionic strength [half-maximal inhibition at I = 0.094 +/- 0.009 M (mean +/- S.D., n = 4)]. Autophosphorylation does not affect the rate of hydrolysis of caldesmon (free or bound to calmodulin) by alpha-chymotrypsin. However, a slight difference in peptides generated from phospho- and dephospho-forms of caldesmon is observed. The binding of phospho- or dephospho-caldesmon to F-actin protects the protein against chymotryptic digestion, but does not alter the pattern of peptide generation. Characterization of proteolytic fragments of caldesmon generated by alpha-chymotrypsin and Staphylococcus aureus V8 protease enables localization of the phosphorylation sites and the kinase active site within the caldesmon molecule.

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Properties of caldesmon isolated from chicken gizzard.

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