Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In the presence of Ca2+ and calmodulin (CM), purified smooth muscle myosin light chain kinase (MLCKase) was found to undergo autophosphorylation at a rate that was about 200-fold slower than its catalytic activity. Up to 1.7 mol of phosphate were incorporated per mole of kinase. Lower levels of incorporation could be correlated with the presence of an endogenous protein phosphatase which could be inhibited with okadaic acid or Microcystin-LR. The major autophosphorylation site was identified as Thr-863 or Thr-865 and was located on the 24-kDa C-terminal fragment of the kinase. In addition, there was a relatively low and variable contribution of a Ca/CM-independent autophosphorylation at Ser-814 or Ser-815. The initial autophosphorylation rates and maximal incorporation levels were highest at a molar ratio of 2 MLCKase to 1 CM and were inhibited at higher CM levels. This indicated that binding of one molecule of the kinase apoenzyme by a CM-kinase complex was necessary for the reaction to occur. Kinetic analysis of the autophosphorylation reaction was consistent with this interpretation and indicated a second-order intermolecular process that included MLCKase dimerization or oligomerization. In contrast, the low Ca/CM-independent contribution was of intramolecular type since it did not depend on the kinase concentration. The autophosphorylation appeared to be involved in a relatively slow modification of the oligomeric properties of the kinase leading to a 2-4-fold amplification of the kinase catalytic activity which followed its activation by CM. Oligomerization and dimerization of the kinase was independently demonstrated by light scattering measurements.
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PMID:Calmodulin-dependent autophosphorylation of smooth muscle myosin light chain kinase: intermolecular reaction mechanism via dimerization of the kinase and potentiation of the catalytic activity following activation. 754 20

The endogenous biosynthesis of nitric oxide (NO) is increased during gestation. To begin our investigation of a possible tissue source (or sources), we examined the placenta. We postulated that analogous to the endothelium of blood vessels, the syncytiotrophoblast (STr) cell layer that lines the intervillous blood space of the human placenta would express NO synthase. Our results show that human placental villi express a calcium- and calmodulin-sensitive form of NO synthase, located mainly in the microsomal cell fraction. By in situ hybridization using a riboprobe generated from human endothelial NO synthase cDNA, we observe NO synthase mRNA expression in STr. The STr also shows NADPH-diaphorase staining, indicating the presence of NO synthase, and most likely other flavin-containing enzymes involved in sex steroid metabolism. NO synthase activity was also detected in the villi of a complete mole placenta (which lacks fetal vessels), further supporting a trophoblastic origin. Our findings suggest a previously unrecognized role for STr-derived NO in placental function.
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PMID:Expression of nitric oxide synthase by syncytiotrophoblast in human placental villi. 769 71

Native calponin is able to bind 2 mol of calcium binding protein (CaBP) per mole calponin. This study extends this observation to define the 2 domains of interaction, one of which is near the actin binding site, and the other in the amino-terminal region of calponin. Also, the first evidence for a differentiation in the response of calponin to interaction with caltropin versus calmodulin is demonstrated. The binding of caltropin to cleavage and recombinant fragments of calponin was determined by 3 techniques: tryptophan fluorescence of the fragments, CD measurements to determine secondary structure changes, and analytical ultracentrifugation. In order to delineate the sites of interaction, 3 fragments of calponin have been studied. From a cyanogen bromide cleavage of calponin, residues 2-51 were isolated. This fragment is shown to bind to CaBPs and the affinity for caltropin is slightly higher than that for calmodulin. A carboxyl-terminal truncated mutant of calponin comprising residues 1-228 (CP 1-228) has been produced by recombinant techniques. Analytical ultracentrifugation has shown that CP 1-228, like the parent calponin, is able to bind 2 mol of caltropin per mol of 1-228 in a Ca(2+)-dependent fashion, indicating that there is a second site of interaction between residues 52-228. Temperature denaturation of the carboxyl-terminal truncated fragment compared with whole calponin show that the carboxyl-terminal region does not change the temperature at which calponin melts; however, there is greater residual secondary structure with whole calponin versus the fragment. A second mutant produced through recombinant techniques comprises residues 45-228 and is also able to bind caltropin, thus mapping the location of the second site of interaction to near the actin binding site.
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PMID:Two domains of interaction with calcium binding proteins can be mapped using fragments of calponin. 775 87

Rabphilin-3A is a putative target protein for Rab3A small GTP-binding protein which is implicated in neurotransmitter release. We have examined here whether Rabphilin-3A is phosphorylated by calmodulin-dependent protein kinase II (CaMKII). Recombinant Rabphilin-3A was phosphorylated by CaMKII purified from rat brain. Two moles of phosphate were maximally incorporated into one mole of Rabphilin-3A. The phosphorylation sites were Ser34, Thr205, Thr209, and Thr537. These results suggest that the CaMKII-catalyzed phosphorylation of Rabphilin-3A may modulate neurotransmitter release.
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PMID:Phosphorylation of Rabphilin-3A by calmodulin-dependent protein kinase II. 781 Dec 64

In the present investigation, we observed that lead in vitro activates calmodulin at lower concentrations, and the maximum activation was observed at 30 microM concentration. In vivo lead exposure (50 mg/kg body weight, intragastrically) for a period of 8 weeks also stimulated the activity of calmodulin by 45%. The addition of trifluoperazine resulted in partially inhibiting the lead-stimulated calmodulin activity, whereas the calcium-stimulated calmodulin activity was completely inhibited by trifluoperazine. Studies with purified calmodulin from the brain of control and lead-treated animals indicate that approximately 4 mole of calcium was present bound/mole of calmodulin in control animals and this fraction was reduced in lead-treated animals to approximately 3 mole of calcium/mole of calmodulin. Lead distribution revealed that approximately 68% of the total lead present was bound to calmodulin and the remaining 32% present was bound to non-calmodulin binding sites following lead exposure. These results indicate that in vivo lead exposure is able to displace and mimic the action of calcium and this may constitute a molecular mechanism of lead neurotoxicity.
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PMID:Effect of lead on the biological activity of calmodulin in rat brain. 799 80

Phosphorylation of D-glyceraldehyde-3-phosphate dehydrogenase (GPDH) by Ca2+/phospholipid- and Ca2+/calmodulin-dependent protein kinases was shown to take place in rabbit skeletal muscle and brain extracts. The kinases could be "picked up" from the extract, using GPDH immobilized on CNBr-activated Sepharose 4B as an affinity adsorbent. Washing of the column with GPDH solutions resulted in elution of the protein kinases; the same effect was observed when anti-GPDH antibodies were used. The most effective elution took place under the conditions favouring the dissociation of the immobilized GPDH into dimers. Based on these findings, a method for purification of Ca2+/calmodulin-dependent protein kinase has been elaborated, which includes chromatography on phenyl-Sepharose to separate the kinase from GPDH. The susceptibility of GPDH to phosphorylation by tissue protein kinases was confirmed by analyses of GPDH preparations purified from rabbit muscle for endogenous phosphate content: 0.7-1.5 moles of covalently bound phosphate were found per mole of the enzyme.
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PMID:[Phosphorylation of glyceraldehyde-3-phosphate dehydrogenase]. 838 8

The function of the uterine smooth muscle in gestation and parturition is affected by a variety of hormones and biomolecules, some of which alter the intracellular levels of cAMP and Ca2+. Since the activity of smooth muscle MLCK has been shown to be modulated by phosphorylation, the effect of this modification of pregnant sheep myometrium (psm) MLCK by the catalytic subunit of cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) was studied. In contrast to other smooth muscle MLCK reported, PKA incorporates 2.0-2.2 moles phosphate into a mole of psm MLCK both in the presence and absence of Ca(2+)-calmodulin. Modification of serine residues inhibited the activity of the enzyme. PKC also incorporated 2.0-2.1 moles of phosphate per mole psmMLCK under both conditions but had no effect on the MLCK activity. Sequential phosphorylation by PKC and PKA incorporated 3.8-4.1 moles phosphate suggesting that the amino acid residues modified by the two kinases are different. Phosphoamino acid analysis of the MLCK revealed that PKC phosphorylated serine and threonine residues. The double reciprocal plots of the enzyme activity and calmodulin concentrations showed that the Vmax of the reaction is not altered by phosphorylation by PKA but the calmodulin concentration require for half-maximal activation is increased about 4-fold. Only 10 out of 17 monoclonal antibodies to various regions of the turkey gizzard MLCK cross-reacted with psmMLCK suggesting structural differences between these enzymes. Comparison of the deduced amino acid sequence of the cDNA encoding the C-terminal half of the psmMLCK molecule showed that while cgMLCK and psmMLCK are highly homologous, a number of nonconservative substitutions are present, particularly near the PKA phosphorylation site B (S828).
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PMID:Phosphorylation and partial sequence of pregnant sheep myometrium myosin light chain kinase. 856 50

Nitric oxide synthases (NOSs) are proposed to generate NO and citrulline from L-arginine in two steps: initial N-hydroxylation to generate Nomega-hydroxyarginine (NOHA) followed by a three-electron oxidation of the hydroxylated nitrogen to form products. Both steps consume NADPH and may involve heme iron-based activation of O2. Studies done under multiple-turnover conditions suggest that 0.5 mol of NADPH is consumed to convert 1 mol of NOHA to products, implying that one electron from NADPH may be sufficient. To test this, we studied NOHA oxidation under single-turnover conditions using neuronal NOS (nNOS), whose heme iron reduction requires bound calmodulin. The heme iron in calmodulin-bound nNOS was reduced with excess NADPH under anaerobic conditions, calmodulin was then dissociated from nNOS to prevent subsequent heme iron reduction, NOHA was added, and the reaction initiated by exposure to air. Spectra obtained at each step were consistent with buildup of NOHA-bound ferrous nNOS prior to air exposure. Reactions containing graded amounts of nNOS produced L-citrulline in linear relation (1.2 +/- 0.1 mol of citrulline per mole of nNOS). Nitrite and nitrate also accumulated as NO-derived products. Control reactions that contained L-arginine instead of NOHA, no enzyme, or ferric nNOS did not generate products. Thus supplying a single electron from NADPH to the heme iron permits nNOS to catalyze one full round of citrulline and NO synthesis from NOHA upon exposure to O2. These data provide a molecular explanation for the NADPH requirement in the second step of the biosynthetic reaction, implicate ferrous-dioxy nNOS as a critical reactant in that step, and eliminate a number of possible alternative catalytic mechanisms or products.
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PMID:Analysis of neuronal NO synthase under single-turnover conditions: conversion of Nomega-hydroxyarginine to nitric oxide and citrulline. 931 70

Membrane binding of the myristoylated alanine-rich C kinase substrate (MARCKS) requires both its myristate chain and basic "effector" region. Previous studies with a peptide corresponding to the effector region, MARCKS-(151-175), showed that the 13 basic residues interact electrostatically with acidic lipids and that the 5 hydrophobic phenylalanine residues penetrate the polar head group region of the bilayer. Here we describe the kinetics of the membrane binding of fluorescent (acrylodan-labeled) peptides measured with a stopped-flow technique. Even though the peptide penetrates the polar head group region, the association of MARCKS-(151-175) with membranes is extremely rapid; association occurs with a diffusion-limited association rate constant. For example, kon = 10(11) M-1 s-1 for the peptide binding to 100-nm diameter phospholipid vesicles. As expected theoretically, kon is independent of factors that affect the molar partition coefficient, such as the mole fraction of acidic lipid in the vesicle and the salt concentration. The dissociation rate constant (koff) is approximately 10 s-1 (lifetime = 0.1 s) for vesicles with 10% acidic lipid in 100 mM KCl. Ca2+-calmodulin (Ca2+.CaM) decreases markedly the lifetime of the peptide on vesicles, e.g. from 0.1 to 0.01 s in the presence of 5 micrM Ca2+.CaM. Our results suggest that Ca2+.CaM collides with the membrane-bound MARCKS-(151-175) peptide and pulls the peptide off rapidly. We discuss the biological implications of this switch mechanism, speculating that an increase in the level of Ca2+-calmodulin could rapidly release phosphatidylinositol 4, 5-bisphosphate that previous work has suggested is sequestered in lateral domains formed by MARCKS and MARCKS-(151-175).
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PMID:Kinetics of interaction of the myristoylated alanine-rich C kinase substrate, membranes, and calmodulin. 934 Nov 59

Gustin, a zinc-metalloprotein constituting about 3% of human parotid saliva protein was previously isolated and characterized as a single polypeptide chain of 37kDa with one mole of zinc tightly bound to the protein. It exhibited biological activity activating calmodulin dependent bovine brain cAMP phosphodiesterase and was decreased in saliva of patients with loss of taste in whom taste buds showed a specific pathological morphology. Determination of its primary structure by amino acid sequence revealed it was identical with carbonic anhydrase (CA) [EC 4.2.1.1] VI and had two N-linked glycosylation sites. Analysis by reverse phase HPLC and SDS-PAGE before and after deglycosylation confirmed a single peak with molecular weight of the purified protein being 37kDa, the deglycosylated protein, 33kDa. N-linked carbohydrate chains contained N-acetyl glucosamine, galactose, mannose, and fucose interior to di, tri and tetra sialyated termini. By isoelectric focusing five increasingly acidic pI values were determined consistent with addition of sialic acid as the terminal carbohydrate residue on the N-linked glycoforms of the protein. Gustin was found to exhibit CA activity but was inhibited by known CA inhibitors in a different manner than CA I or II. These findings, consistent with analysis of previous investigators, indicate that parotid saliva gustin is CA VI.
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PMID:Gustin from human parotid saliva is carbonic anhydrase VI. 978 98


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