Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Myosin light chain kinase, which is located primarily in the soluble fraction of bovine myocardium, has been isolated and purified approximately 1200-fold with 16% yield by a three-step procedure. The approximate content of soluble myosin light chain kinase in heart is calculated to be 0.63 microM. The isolated kinase is active only as a ternary complex consisting of the kinase, calmodulin, and Ca2+; the apparent Kd for calmodulin is 1.3 nM. The enzyme also exhibits a requirement for Mg2+ ions. Myosin light chain kinase is a monomeric enzyme with Mr = 85,000. The enzyme exhibits a Km for ATP of 175 microM, and a K0.5 for the regulatory light chain of cardiac myosin of 21 microM. The optimum pH is 8.1. Kinase activity is specific for the regulatory light chain of myosin. The specific activity of the isolated enzyme (30 nmol 32P/min/mg of protein) is considerably less than and corresponding values reported for the skeletal and smooth muscle light chain kinases. This is probably due to proteolysis during extraction of the myocardium, a phenomenon which has, as yet, proven impossible to eliminate. In contrast to the smooth muscle enzyme (Adelstein, R.S., Conti, M.A., Hathaway, D.R., and Klee, C.B. (1978) J. Biol. Chem. 253, 8347-8350), the cardiac kinase is not phosphorylated by the catalytic subunit of cAMP-dependent protein kinase.
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PMID:Purification and characterization of bovine cardiac calmodulin-dependent myosin light chain kinase. 50 Jul 1

Phosphorylation of beta-connectin (titin 2), an elastic protein of chicken breast muscle, occurred in the presence of [gamma-32P] ATP, 0.2 mM CaCl2 and 25 mM phosphate buffer, pH 7.0. Addition of 3 mM MgCl2 did not affect the phosphorylation. However, Ca2+ ions were required for the phosphorylation and EGTA inhibited it even if MgCl2 were present. Myosin light chain kinase (gizzard MLCK), cAMP dependent protein kinase (A kinase), and protein kinase C (C kinase) did not phosphorylate beta-connectin in vitro under optimal conditions. Thus it appears that beta-connectin, possibly containing a domain homologous with MLCK, has an autophosphorylating action.
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PMID:Autophosphorylation of beta-connectin (titin 2) in vitro. 154 1

We examined the extranuclear effects of thyroid hormones on human platelets. Pretreatment with DL-thyroxine or DL-triiodothyronine inhibited collagen-induced aggregation, in a dose-dependent manner, but other derivatives of thyroid hormone had no significant effects. In contrast to collagen, 12-O-tetradecanoylphorbol-13-acetate-induced aggregation was not affected by thyroid hormones at the same concentration range. Thyroxine also inhibited the release of [14C] serotonin from collagen-stimulated platelets, with a marked reduction in the phosphorylation of 20,000-dalton protein. Thyroxine and triiodothyronine had inhibitory effects on myosin light chain kinase purified from human platelets and inhibited more markedly the myosin light chain kinase than protein kinase C (Ca2+/phospholipid-dependent enzyme) and cAMP-dependent protein kinase. In addition, L-thyroxine behaved as a competitive inhibitor of myosin light chain kinase toward calmodulin, and the Ki value was calculated to be 2.6 microM. To determine whether or not thyroxine directly binds myosin light chain kinase, we prepared an affinity column, using L-thyroxine as the ligand. Myosin light chain kinase was selectively bound to the column while calmodulin passed through. We also designed a procedure for the purification of myosin light chain kinase from human platelets, using L-thyroxine-affinity chromatography. A markedly increased purification was thus achieved, and DEAE-cellulose and L-thyroxine-affinity chromatography were made feasible. These results suggest that thyroxine can serve as a pharmacological tool for elucidating the biological significance of myosin light chain kinase-mediated reactions and is a pertinent ligand which can be used to purify myosin light chain kinase from platelets as a substitute for calmodulin.
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PMID:Thyroid hormones inhibit platelet function and myosin light chain kinase. 272 89

Myosin light chain kinase activity in the placental region of the rabbit myometrium on day 28 of gestation was 4.7 +/- 0.1 (mean +/- S.E.M.) nmol/min per mg protein, which was significantly higher than that (3.6 +/- 0.1 nmol/min per mg protein) in the non-placental region. The amount of calmodulin in the placental region was 4.2 +/- 0.1 micrograms/mg protein, which was significantly higher than that (3.2 +/- 0.1 micrograms/mg protein) in the non-placental region. In contrast, cyclic AMP-dependent protein kinase activities showed no difference between the two regions. These findings suggest that calcium- and calmodulin-dependent protein phosphorylation is activated mainly in the placental region, and uterine contractions can occur more strongly in this part than in the non-placental region. Such enzymatic phenomena may be related to the mechanism whereby the placenta separates from the myometrium after delivery of the fetus.
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PMID:Myosin light chain kinase and cyclic AMP-dependent protein kinase activities and calmodulin levels in placental and non-placental regions of the rabbit myometrium. 300 78

The synthetic thioether phospholipid BM 41.440 (1-S-hexadecyl-2-methoxymethyl-rac-glycero-3-phosphocholine) was found to inhibit protein kinase C (PKC) activity competitively with respect to phosphatidylserine, with an apparent Ki value of about 6.4 microM. The agent also inhibited the enzyme activated by diolein or 12-O-tetradecanoylphorbol-13-acetate (TPA), without affecting binding of [3H]phorbol-12,13-dibutyrate to the enzyme. Myosin light chain kinase and cAMP-dependent protein kinase were not inhibited by BM 41.440, indicating a specificity of the action of the agent. BM 41.440 partly blocked the TPA-induced depletion of soluble PKC in HL60 and KG-1 cells (responsive to the differentiating effect of TPA) but not in K562 cells (resistant to the TPA effect). The thioether inhibited the phosphatidylserine/Ca2+-dependent phosphorylation of several common proteins in the solubilized homogenates of HL60 and KG-1 cells, and that of apparently distinct proteins in the preparation of K562 cells. The TPA-induced differentiation of HL60 and KG-1 cells was inhibited by BM 41.440 at a concentration inhibitory to PKC, but differentiation of HL60 cells promoted by dimethyl sulfoxide, retinoic acid, and 1,25-dihydroxyvitamin D3, on the other hand, was not affected. The present data suggested that PKC inhibition might partly account for the antineoplastic effect of BM 41.440, and that the agent could be useful in studying involvements of the PKC system in cellular processes.
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PMID:Effects of thioether phospholipid BM 41.440 on protein kinase C and phorbol ester-induced differentiation of human leukemia HL60 and KG-1 cells. 318 76

To investigate the intracellular physiology of cerebral arterial vasospasm after subarachnoid hemorrhage (SAH), we measured the activity of three protein kinases related to vascular smooth muscle function. Myosin light chain kinase (MLCK), cyclic adenosine monophosphate (cAMP)-dependent protein kinase and cyclic guanosine monophosphate (cGMP)-dependent protein kinase activity was determined in canine basilar arteries using the SAH model created by two intracisternal injections of autologous blood. Moderate chronic cerebral vasospasm was confirmed angiographically 7 days after the first injection of blood, with a mean decrease in the diameter of the basilar artery to 61.2 +/- 2.3% (SE) of preinjection controls. Enzymatic analysis of basilar arteries excised from control dogs and from SAH model dogs 2 to 10 days after an intracisternal injection of blood demonstrated detectable MLCK and cyclic nucleotide-dependent protein kinase activity. The MLCK activity was approximately 3 times higher than the cyclic nucleotide-dependent protein kinase activity. There was no significant difference in basilar artery protein kinase activity between control animals and SAH model animals at any time for up to 10 days after the intracisternal injection of blood. As MLCK is involved with smooth muscle contraction and cAMP- and cGMP-dependent protein kinases are involved in smooth muscle relaxation, our results suggest that the metabolic machinery of vascular smooth muscle in the cerebral arterial wall is intact after SAH and that prolonged vasospasm represents an appropriate physiological response by the vascular smooth muscle to external stimuli. If this is correct, the successful pharmacological manipulation of vasospasm after SAH is more likely than if profound disruption had occurred, and further investigative efforts in this area should be encouraged.
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PMID:Protein kinase activity in canine basilar arteries after subarachnoid hemorrhage. 341 63

The proteolytic susceptibility of chicken gizzard myosin light chain kinase, a calmodulin-dependent enzyme, has been utilized to define the relative location of the catalytic and regulatory domains of the enzyme. Myosin light chain kinase isolated from this source exhibits a Mr of 130,000 and is extremely sensitive to trypsin at 24 degrees C; however, the molecule is divided into susceptible and resistant domains such that proteolysis proceeds rapidly and at multiple sites in the sensitive regions even at 4 degrees C while the rest of the molecule remains relatively resistant to digestion. One of these sensitive areas is the calmodulin-binding domain. On the other hand, Staphylococcus aureus V8 protease digestion generates a calmodulin-binding fragment (Mr = 70,000) that retains Ca2+/calmodulin-dependent enzymatic activity and both of the phosphorylation sites recognized by cAMP-dependent protein kinase. In contrast, treatment with chymotrypsin produces a 95,000 Mr calmodulin-binding fragment that contains only the calmodulin-modulated phosphorylation site. Sequential proteolytic digestion studies demonstrated that the chymotryptic cleavage site responsible for the generation of this 95,000 Mr peptide is within 3,000 Mr of the V8 protease site which produces the 70,000 Mr fragment. Moreover, the non-calmodulin-modulated phosphorylation site must exist in this 3,000 Mr region. A calmodulin-Sepharose affinity adsorption protocol was developed for the digestion and used to isolate both the 70,000 and 95,000 Mr fragments for further study. Taken together, our results are compatible with a model for chicken gizzard myosin light chain kinase in which there is no overlap between the active site, the calmodulin-binding region, and the two sites phosphorylated by cAMP-dependent protein kinase with regard to their relative position in the primary sequence of the molecule.
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PMID:Functional domains of chicken gizzard myosin light chain kinase. 383 92

Myosin light chain kinase plays a central role in the regulation of smooth muscle contraction. The activity of this enzyme is controlled by protein-protein interaction (the Ca2+-dependent binding of calmodulin) and by phosphorylation catalyzed by cAMP-dependent protein kinase. The effects of these two regulatory mechanisms on the conformation of myosin light chain kinase and the locations of the phosphorylation sites, the calmodulin-binding site, and the active site have been probed by limited proteolysis. Phosphorylated and nonphosphorylated myosin light chain kinases were subjected to limited digestion by four proteases having different peptide bond specificities (trypsin, chymotrypsin, Staphylococcus aureus V8 protease, and thrombin), both in the presence and in the absence of bound calmodulin. The digests were compared in terms of gel electrophoretic pattern, distribution of phosphorylation sites, and Ca2+ dependence of kinase activity. A 24 500-dalton chymotryptic peptide containing both sites of phosphorylation was purified and tentatively identified as the amino-terminal peptide. The following conclusions can be drawn: neither phosphorylation nor calmodulin binding induces dramatic changes in the conformation of the kinase; the kinase contains two regions that are particularly susceptible to proteolytic cleavage, one located approximately 25 000 daltons from the amino terminus and the other near the center of the molecule; the two phosphorylation sites are located within 24 500 (probably 17 500) daltons of the amino terminus; the active site is located close to the center of the molecule; the calmodulin-binding site is located in the amino-terminal half of the molecule, between the sites of phosphorylation and the active site, and this region is very susceptible to cleavage by trypsin.
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PMID:Limited proteolysis of smooth muscle myosin light chain kinase. 384 33

Antiestrogens (tamoxifen, clomiphene and nafoxidine) were found to inhibit phospholipid/Ca2+-dependent protein kinase (PL/Ca-PK, or protein kinase C), whereas estrogens (estradiol and diethylstilbesterol) and the weakly estrogenic chlorotrianisene were inactive. Kinetic analysis indicated that the antiestrogens inhibited PL/Ca-PK competitively with respect to phosphatidylserine (Ki = 16-27 microM), but non-competitively with Ca2+ (Ki = 14-30 microM). Tamoxifen, but not diethylstilbesterol, also inhibited the phospholipid/Ca2+-dependent phosphorylation of various endogenous proteins from the total, solubilized fraction of the rat brain and ovary. Myosin light chain kinase, a calmodulin/Ca2+-dependent class of protein kinase, was similarly inhibited by tamoxifen; the drug, however, was without effect on cyclic AMP-dependent and cyclic GMP-dependent protein kinases. It is suggested that PL/Ca-PK, by virtue of the hydrophobic interactions required for the enzyme activation, may represent a potential site of action for the lipophilic antiestrogens, in addition to the commonly recognized intracellular estrogen receptors.
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PMID:Effect of tamoxifen, a nonsteroidal antiestrogen, on phospholipid/calcium-dependent protein kinase and phosphorylation of its endogenous substrate proteins from the rat brain and ovary. 384 Mar 75

Myosin light chain kinase from smooth muscle has been shown to be phosphorylated by cyclic AMP-dependent protein kinase, which leads to a decrease in the affinity of the kinase for Ca2+ . calmodulin and, hence, a decrease in enzymatic activity. This event has been proposed as a mechanism for the relaxation of smooth muscle in response to increased intracellular concentrations of cyclic AMP. The ratio of myosin light chain kinase activities measured in the presence of 4 microM or 100 microM Ca2+, at 1 microM calmodulin, permits evaluation of such a change in the calmodulin activation properties of myosin light chain kinase. This activity ratio was decreased by phosphorylation of either purified bovine tracheal smooth muscle myosin light chain kinase, or the endogenous myosin light chain kinase in a homogenate of tracheal smooth muscle, with the addition of the catalytic subunit of cyclic AMP-dependent protein kinase. The ratio was unchanged, however, by activation of the endogenous cyclic AMP-dependent protein kinase in homogenates of tracheal smooth muscle by the addition of cyclic AMP. Incubation of tracheal smooth muscle with isoproterenol, at a concentration sufficient to relax the muscle and to increase phosphorylase a formation, had no effect upon the activity ratio. Incubation of tracheal smooth muscle for 2 hr in the presence of carbachol resulted in a transient increase and then a decrease in myosin light chain phosphate content to control values with no decrease in isometric force. The addition of isoproterenol at 2 hr still resulted in relaxation. These findings are inconsistent with a role of myosin light chain kinase phosphorylation in mediating relaxation of tracheal smooth muscle by beta-adrenergic agonists. Cyclic AMP-dependent effects on cytoplasmic calcium concentrations may be more important in mediating relaxation.
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PMID:The role of myosin light chain kinase phosphorylation in beta-adrenergic relaxation of tracheal smooth muscle. 613 4


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