scholarly journals Isolation of the native form of chicken gizzard myosin light-chain kinase

1984 ◽  
Vol 218 (3) ◽  
pp. 863-870 ◽  
Author(s):  
P K Ngai ◽  
C A Carruthers ◽  
M P Walsh
Keyword(s):  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Binding Protein ◽  
Cross Reactivity ◽  
Native Form ◽  
Chicken Gizzard ◽  
Calmodulin Binding ◽  
Chymotryptic Peptide ◽  
Dependent Protein

A simple and rapid procedure for the purification of the native form of chicken gizzard myosin light-chain kinase (Mr 136000) is described which eliminates problems of proteolysis previously encountered. During this procedure, a calmodulin-binding protein of Mr 141000, which previously co-purified with the myosin light-chain kinase, is removed and shown to be a distinct protein on the basis of lack of kinase activity, different chymotryptic peptide maps, lack of cross-reactivity with a monoclonal antibody to turkey gizzard myosin light-chain kinase, and lack of phosphorylation by the purified catalytic subunit of cyclic AMP-dependent protein kinase. This Mr-141000 calmodulin-binding protein is identified as caldesmon on the basis of Ca2+-dependent interaction with calmodulin, subunit Mr, Ca2+-independent interaction with skeletal-muscle F-actin, Ca2+-dependent competition between calmodulin and F-actin for caldesmon, and tissue content.

Calcium-dependent mechanisms of regulation of smooth muscle contraction

1991 ◽  
Vol 69 (12) ◽  
pp. 771-800 ◽  
Author(s):  
Michael P. Walsh
Keyword(s):  
Smooth Muscle ◽  
Muscle Contraction ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Binding Protein ◽  
Smooth Muscle Contraction ◽  
Light Chains ◽  
Contractile State ◽  
Calmodulin Binding

The contractile state of smooth muscle is regulated primarily by the sarcoplasmic (cytosolic) free Ca2+ concentration. A variety of stimuli that induce smooth muscle contraction (e.g., membrane depolarization, α-adrenergic and muscarinic agonists) trigger an increase in sarcoplasmic free [Ca2+] from resting levels of 120–270 to 500–700 nM. At the elevated [Ca2+], Ca2+ binds to calmodulin, the ubiquitous and multifunctional Ca2+-binding protein. The interaction of Ca2+ with CaM induces a conformational change in the Ca2+-binding protein with exposure of a site(s) of interaction with target proteins, the most important of which in the context of smooth muscle contraction is the enzyme myosin light chain kinase. The interaction of calmodulin with myosin light chain kinase results in activation of the kinase that catalyzes phosphorylation of myosin at serine-19 of each of the two 20-kDa light chains (native myosin is a hexamer composed of two heavy chains (230 kDa each) and two pairs of light chains (one pair of 20 kDa each and the other pair of 17 kDa each)). This simple phosphorylation reaction triggers cycling of myosin cross-bridges along actin filaments and the development of force. Relaxation of the muscle follows removal of Ca2+ from the sarcoplasm, whereupon calmodulin dissociates from myosin light chain kinase regenerating the inactive kinase; myosin is dephosphorylated by myosin light chain phosphatase(s), whereupon it dissociates and remains detached from the actin filament and the muscle relaxes. A substantial body of evidence has been accumulated in support of this central role of myosin phosphorylation–dephosphorylation in the regulation of smooth muscle contraction. However, a wide range of physiological and biochemical studies supports the existence of additional, secondary Ca2+-dependent mechanisms that can modulate or fine-tune the contractile state of the smooth muscle cell. Three such mechanisms have emerged: (i) the actin-, tropomyosin-, and calmodulin-binding protein, calponin; (ii) the actin-, myosin-, tropomyosin-, and calmodulin-binding protein, caldesmon; and (iii) the Ca2+- and phospholipid-dependent protein kinase (protein kinase C).Key words: smooth muscle, Ca2+, myosin phosphorylation, regulation of contraction.

scholarly journals Inhibition by melittin of phospholipid-sensitive and calmodulin-sensitive Ca2+ -dependent protein kinases

1982 ◽  
Vol 202 (1) ◽  
pp. 217-224 ◽  
Author(s):  
N Katoh ◽  
R L Raynor ◽  
B C Wise ◽  
R C Schatzman ◽  
R S Turner ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Dependent Protein Kinase ◽  
Calmodulin Binding ◽  
Substrate Protein ◽  
Dependent Protein ◽  
A Site

Effects of melittin, an amphipathic polypeptide, on various species of protein kinases were investigated. It was found that melittin inhibited the newly identified phospholipid-sensitive Ca2+-dependent protein kinase (from heart, brain, spleen and neutrophils) and the cardiac myosin light-chain kinase, a calmodulin-sensitive Ca2+-dependent enzyme. In contrast, melittin had little or no effect on either the holoenzymes of the cardiac cyclic AMP-dependent and cyclic GMP-dependent protein kinases or the catalytic subunit of the former. Kinetic analysis indicated that melittin inhibited phospholipid-sensitive Ca2+-dependent protein kinase non-competitively with respect to ATP (Ki = 1.3 microM); although exhibiting complex kinetics, its inhibition of the enzyme was overcome by phosphatidylserine (a phospholipid cofactor), but not by protein substrate (histone H1) or Ca2+. On the other hand, melittin inhibited myosin light-chain kinase non-competitively with respect to ATP (Ki = 1.4 microM) or Ca2+ (Ki = 1.9 microM), and competitively with respect to calmodulin (Ki = 0.08 microM); although exhibiting complex kinetics, its inhibition of the enzyme was reversed by myosin light chains (substrate protein). The present findings indicate the presence of functionally important hydrophobic or hydrophilic loci on the Ca2+-dependent protein kinases, but not on the cyclic nucleotide-dependent class of protein kinase, with which melittin can interact. Moreover, the kinetic data suggest that melittin inhibited myosin light-chain kinase by interacting with a site on the enzyme the same as, or proximal to, the calmodulin-binding site, thus interfering with the formation of active enzyme-calmodulin-Ca2+ complex.

scholarly journals Calcium-independent contraction in lysed cell models of teleost retinal cones: activation by unregulated myosin light chain kinase or high magnesium and loss of cAMP inhibition.

1987 ◽  
Vol 105 (1) ◽  
pp. 397-402 ◽  
Author(s):  
B Burnside ◽  
N Ackland
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Myosin Phosphorylation ◽  
Calmodulin Binding ◽  
Dependent Protein ◽  
Myosin Interaction ◽  
High Concentrations

The retinal cones of teleost fish contract at dawn and elongate at dusk. We have previously reported that we can selectively induce detergent-lysed models of cones to undergo either reactivated contraction or reactivated elongation, with rates and morphology comparable to those observed in vivo. Reactivated contraction is ATP dependent, activated by Ca2+, and inhibited by cAMP. In addition, reactivated cone contraction exhibits several properties that suggest that myosin phosphorylation plays a role in mediating Ca2+-activation (Porrello, K., and B. Burnside, 1984, J. Cell Biol., 98:2230-2238). We report here that lysed cone models can be induced to contract in the absence of Ca2+ by incubation with trypsin-digested, unregulated myosin light chain kinase (MLCK) obtained from smooth muscle. This observation provides further evidence that MLCK plays a role in regulating cone contraction. We also report here that lysed cone models can be induced to contract in the absence of Ca2+ by incubation with high concentrations of MgCl2 (10-20 mM). Mg2+-induced reactivated contraction is supported by inosine triphosphate (ITP) just as well as by ATP. Because ITP will not serve as a substrate for MLCK, this finding suggests that Mg2+-activation of contraction does not require myosin phosphorylation. Although Ca2+-induced contraction is completely blocked by cAMP at concentrations less than 10 microM, cAMP has no effect on cone contraction activated by unregulated MLCK or by high Mg2+ in the absence of Ca2+. Because trypsin digestion of MLCK cleaves off not only the Ca2+/calmodulin-binding site but also the site phosphorylated by cAMP-dependent protein kinase, and because Mg2+ activation of cone contraction circumvents MLCK action altogether, both these observations would be expected if cAMP inhibits reactivated cone contraction by catalyzing the phosphorylation of MLCK and thus reducing its affinity for Ca2+, as has been described for smooth muscle. Together our results suggest that in lysed cone models, myosin phosphorylation is sufficient for activating cone contraction, even in the absence of other Ca2+-mediated events, that cAMP inhibition of contraction is mediated by cAMP-dependent phosphorylation of MLCK, and that 10-20 mM Mg2+ can activate actin-myosin interaction to produce contraction in the absence of myosin phosphorylation.

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