Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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We have isolated a cDNA clone for the human slow-twitch muscle isoform myosin light-chain 1slow-a (MLC1sa) from a skeletal muscle library and for the human nonmuscle isoform myosin light-chain 3nonmuscle (MLC3nm) from a fibroblast library. The nucleotide sequence of both isoforms was determined, and isoform-specific probes were constructed. In addition, MLC1sa was subsequently isolated from the fibroblast library. MLC1sa and MLC3nm were found to be very closely related to each other and distant from all other myosin light-chain isoforms so far described. We concluded that MLC1sa arose by duplication of MLC3nm rather than from any other isoform. A comparison was made between all human myosin light chains described to date and a model proposed for the evolution of this multigene family. A comparison between human and chicken myosin light-chain isoforms showed that human isoforms are more similar to their chicken counterparts than to human MLC1sa. The expression of MLC1sa and MLC3nm was studied in humans, rabbits, mice, and rats. MLC1sa was detected at the onset of both human and murine myogenesis in vitro. With development, MLC1sa may be replaced by the other slow-twitch muscle isoform, 1sb, in slow-twitch skeletal muscle, but the proportion of MLC1sa to 1sb expression varies between different species. MLC1sa was detected in nonmuscle cells in humans, mice, and rats. MLC3nm was the major nonmuscle alkaline myosin light chain in all species tested, but its pattern of expression in nonmuscle tissues was not identical to that of beta- or gamma-actin. We have shown that in the human, as in the chicken, one exon is spliced out of the MLC3nm transcript in smooth muscle to give an alternative product. We concluded that all alkali myosin light-chain isoforms may be functionally different.
Mol Cell Biol 1990 Mar
PMID:Characterization of human myosin light chains 1sa and 3nm: implications for isoform evolution and function. 230 59

The formation of human myotubes in culture is accompanied by the induction of developmentally regulated, muscle-specific genes. We have studied the expression of human myosin light chain proteins and mRNAs during myogenesis in culture, in particular the skeletal embryonic myosin light chain 1 (MC1emb), which is indistinguishable from MLC1 of adult atrial cardiac muscle (MLC1A) as has been shown for rodent and bovine MLC1emb. We have identified distinct MLC1emb/MLC1A mRNAs in cultured human skeletal muscle cells that differ in their 5' and 3' untranslated regions but contain identical protein-coding regions. The alternative 3' untranslated region is detectable also in RNA of human atria. The different MLC1emb RNAs are likely to be encoded by one gene. It appears that the two MLC1emb 5' untranslated regions of the human gene are specific for man. In the mouse, only one 5' untranslated region of the MLC1emb gene has been detected.
J Mol Biol 1990 Feb 05
PMID:Heterogenic mRNAs with an identical protein-coding region of the human embryonic myosin alkali light chain in skeletal muscle cells. 230 63

KT5926, (8R*,9S*,11S*)-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl-14-n-propoxy-2,3 ,9, 10-tetrahydro-8,11-epoxy, 1H,8H, 11H-2,7b,11a-triazadibenzo[a,g]cycloocta[cde] trinden-1-one, was found to be a potent and selective inhibitor of myosin light chain kinase. The compound inhibited both Ca2+/calmodulin-dependent and -independent smooth muscle myosin light chain kinases to a similar extent. The inhibition was not affected by the concentration of calmodulin. Kinetic analyses showed that the mode of inhibition was of the competitive type with respect to ATP (Ki, 18 nM) and of the noncompetitive type with respect to myosin light chain (Ki, 12 nM). These results indicated that KT5926 directly interacted with the enzyme at the catalytic site. KT5926 also inhibited other protein kinases, but with relatively high Ki values; the values for protein kinase C, cAMP-dependent protein kinase, and cGMP-dependent protein kinase were 723, 1200, and 158 nM, respectively. Ca2(+)-ATPase, Na+/K(+)-ATPase, hexokinase, and 5'-nucleotidase were not inhibited by KT5926 at less than 10 microM. The effect of KT5926 on serotonin secretion and protein phosphorylation induced by platelet-activating factor or phorbol ester was examined in rabbit platelets. KT5926 inhibited the phosphorylation of a 20-kDa protein but had no effect on the phosphorylation of a 40-kDa protein, thereby indicating that the compound exerts its selective inhibition of myosin light chain kinase in intact cells. The compound inhibited serotonin secretion induced by platelet-activating factor, but its potency was significantly less than that of K-252a, (8R*,9S*,11S*)-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9, 10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b, 11a-triazadibenzo[a,g]cycloocta [cde]trinden-1-one, which inhibited the phosphorylation of both the 20-kDa protein and the 40-kDa protein. Phorbol ester-induced secretion was not suppressed by KT5926. These results provide the evidence that both the 20-kDa protein phosphorylation by myosin light chain kinase and the 40-kDa protein phosphorylation by protein kinase C substantially contribute to the secretion response in platelets.
Mol Pharmacol 1990 Apr
PMID:KT5926, a potent and selective inhibitor of myosin light chain kinase. 232 35

We used an antibody specific for Dictyostelium discoideum myosin to screen a lambda gt11 cDNA expression library to obtain cDNA clones which encode the Dictyostelium essential myosin light chain (EMLC). The amino acid sequence predicted from the sequence of the cDNA clone showed 31.5% identity with the amino acid sequence of the chicken EMLC. Comparisons of the Dictyostelium EMLC, a nonmuscle cell type, with EMLC sequences from similar MLCs of skeletal- and smooth-muscle origin, showed distinct regions of homology. Much of the observed homology was localized to regions corresponding to consensus Ca2+-binding of E-F hand domains. Southern blot analysis suggested that the Dictyostelium genome contains a single gene encoding the EMLC. Examination of the pattern of EMLC mRNA expression showed that a significant increase in EMLC message levels occurred during the first few hours of development, coinciding with increased actin expression and immediately preceding the period of maximal chemotactic activity.
Mol Cell Biol 1988 Feb
PMID:Dictyostelium discoideum myosin: isolation and characterization of cDNAs encoding the essential light chain. 245 Nov 26

Phosphorylation of the regulatory light chains (RMLC) of nonmuscle myosin can increase the actin-activated ATPase activity and filament formation. Little is known about these regulatory mechanisms and how the RMLC are involved in ATP hydrolysis. To better characterize the nonmuscle RMLC, we isolated cDNAs encoding the Dictyostelium RMLC. Using an antibody specific for the RMLC, we screened a lambda gt11 expression library and obtained a 200-base-pair clone that encoded a portion of the RMLC. The remainder of the sequence was obtained from two clones identified by DNA hybridization, using the 200-base-pair cDNA. The composite RMLC cDNA was 645 nucleotides long. It contained 60 base pairs of 5' untranslated, 483 bases of coding, and 102 base pairs of 3' untranslated sequence. The amino acid sequence predicted an 18,300-dalton protein that shares 42% amino acid identity with Dictyostelium calmodulin and 30% identity with the chicken skeletal myosin RMLC. This sequence contained three regions that were similar to the E-F hand calcium-binding domains found in calmodulin, troponin C, and other myosin light chains. A sequence similar to the phosphorylation sequence found in chicken gizzard and skeletal myosin light chains was found at the amino terminus. Genomic Southern blot analysis suggested that the Dictyostelium genome contains a single gene encoding the RMLC. Analysis of RMLC expression patterns during Dictyostelium development indicated that accumulation of this mRNA increases just before aggregation and again during culmination. This pattern is similar to that obtained for the Dictyostelium essential myosin light chain and suggests that expression of the two light chains is coordinated during development.
Mol Cell Biol 1989 Jul
PMID:Dictyostelium discoideum myosin: isolation and characterization of cDNAs encoding the regulatory light chain. 255 Jul 95

The structural organization of the chromosomal gene for human parvalbumin was determined mostly by sequencing exons and intron exon junctions of a 7500 base-pair (bp) long genomic clone derived from a chromosome 22-specific gene library. Four exons coding for 100 from a total of 109 amino acids were detected in this clone and 472 bp of the 5'-flanking region were sequenced. The region corresponding to the C-terminal amino acids 101 to 109 of human parvalbumin was determined by sequencing a cDNA fragment derived from human brain mRNA after amplification by the polymerase chain reaction. The first intron is placed 7 bp upstream from the ATG translation start signal, whereas all other splice sites divide putative Ca2+-binding domains. All intron positions coincide exactly with those reported for the rat parvalbumin gene. The 5' mRNA leader sequence has a similarity of 57%, the coding region of 91% and the 3' non-coding region of 83% to the corresponding rat sequences. Only nine conservative amino acid replacements were observed between human and rat parvalbumins. The predicted secondary structures for human, rat, mouse and rabbit parvalbumins are very similar, indicating a strong structural relationship among mammalian parvalbumins. Several elements with potential transcription regulatory activities were found in the region immediately 5' to the transcription start site including a TATA box (TATATA) and a CAAT box (CCAAAAT). Several regions in the putative promoter are strongly conserved between the human and rat parvalbumin genes. One of these with a length of 32 bp is identical with the rat counterpart and has a high degree of homology to a promoter region in the myosin light chain 3F gene, which is expressed in fast contracting/relaxing muscle fibers (anaerobic/type IIb), the cell type that also exhibits highest levels of parvalbumin expression. The human parvalbumin mRNA contains the putative polyadenylation signal AATAAA 13 nucleotides upstream from the polyadenylation site. A 700-nucleotide long parvalbumin mRNA is synthesized at low levels in the human cerebellum as well as in the neuroblastoma cell line SK-N-BE.
J Mol Biol 1989 Dec 05
PMID:Parvalbumin genes from human and rat are identical in intron/exon organization and contain highly homologous regulatory elements and coding sequences. 261 29

The thyroid hormones have direct effects on vascular smooth muscle and are potent vasorelaxants. In the present study, the effects of d- and l-thyroxine (d-T4 and l-T4), 3,5,3'-triiodo-d-thyronine (d-T3), and 3,5,3'-triiodo-l-thyronine (l-T3) on the isolated mesenteric artery of the rabbit and superprecipitation of actomyosin from bovine aorta were examined. These thyroid hormones dose dependently relaxed vascular strips previously contracted with 50 mM KCl in the presence of phentolamine (1 microM), propranolol (1 microM), and atropine (0.3 microM), and the order of the inhibitory potency was l-T4 greater than d-T4 greater than l-T3 greater than d-T3 for the contraction. Pretreatment with l-T4 (10 and 30 microM) inhibited the contractile response concomitant with the inhibition of the 20,000-Da myosin light chain phosphorylation, without significant suppression of the increase in La3+-resistant 45Ca influx and uptake (5 and 30 min) induced by 50 mM KCl, suggesting that the inhibitory effect of l-T4 may not be primarily related to Ca2+ entry through the voltage-dependent Ca2+ channel. The l-T4 (10 and 30 microM) showed noncompetitive antagonism against the Ca2+-induced contraction in the high K+-depolarized vascular strips. Superprecipitation of actomyosin was inhibited by the addition of l-T4, in a dose-dependent manner, and calmodulin (1 microgram/ml) partly reversed the inhibitory effect of l-T4. Thyroid hormones were found to inhibit Ca2+/calmodulin-dependent smooth muscle myosin light chain kinase, and the Ki value for l-T4 was 2.5 microM. Although the concentrations of l-T4 used in this study are high, relative to circulating physiological levels, thyroid hormones act directly at the blood vessel wall to cause inhibition of the contractile process in vascular smooth muscle in vitro. Modulation of the 20,000-Da myosin light chain phosphorylation via the inhibition of myosin light chain kinase activity may at least in part contribute to the inhibitory effect of l-T4.
Mol Pharmacol 1989 Jun
PMID:Thyroid hormones directly interact with vascular smooth muscle strips. 273 94

We have found that phosphorylation of the 18,000 mol. wt protein in rat basophilic leukemia cells (RBL-2H3 cells) is enhanced by stimulation by an antigen. This phenomenon was also observed when cells were treated with phorbol myristate (TPA) and a calcium ionophor, A23187. The phosphorylated 18,000 mol. wt protein was mainly located in the membrane fraction. It was identified as one of the myosin light chains as follows: (1) the mol. wt of one of the major myosin light chains of RBL-2H3 cells was 18,000; (2) more than half of the phosphorylated 18,000 mol. wt protein was recovered in an actomyosin fraction; (3) this phosphorylated 18,000 mol. wt protein was immunoprecipitated with anti-myosin antibody. Since the presence of Ca2+ in the cell culture medium was essential for the phosphorylation of the 18,000 mol. wt protein and, since trifluoperazine (a potent inhibitor of calmodulin as well as of the degranulation process of RBL-2H3 cells) inhibited the reaction, the phosphorylation may be catalyzed by a Ca2+-calmodulin-dependent process, most likely by myosin light chain kinase. These results, together with our previous observation [Teshima et al. Molec Immun. 23, 279-284 (1986)], suggest that simultaneous phosphorylation of the 18,000 mol. wt myosin light chain and a 36,000 mol. wt membranous protein is a prerequisite for the degranulation of RBL-2H3 cells.
Mol Immunol 1989 Jul
PMID:Enhancement of the phosphorylation of membrane bound myosin light chain by antigen stimulation in rat basophilic leukemia cells. 277 87

There is general agreement now that elevation of intracellular calcium ion concentration [( Ca2+]i) mediates the physiological actions of a number of hormones, neurotransmitters and other pharmacological agents. In smooth muscle one way by which these agents elevate [Ca2+]i and induce contraction involves formation of inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DG) from the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2). Our initial observation that the muscarinic-stimulated breakdown of PIP2 into IP3 and DG in the iris smooth muscle is a primary event that could couple activated receptors to contraction is now supported by the following: 1. In the iris sphincter contractions by carbachol (CCh) indicated close correlations, using different concentrations of CCh, atropine- and pirenzepine antagonists, time course, temperature and Ca2+, between the stimulated hydrolysis of PIP2, myosin light chain (MLC) phosphorylation and muscle contraction. Further, studies on the relationships between receptor occupancy of muscarinic acetylcholine receptors in this tissue, measured by [3H]QNB binding, and the CCh-stimulated PIP2 hydrolysis, MLC phosphorylation and contraction revealed that all of these responses are coupled to the M2 receptor subtype. 2. NaF, which activates G proteins by promoting the dissociation of their subunits, produced a concentration-dependent activation of phospholipase C, measured as IP3 accumulation, and contraction in the iris sphincter muscle. 3. In permeabilized smooth muscle fibers, IP3 has been shown to release Ca2+ from the SR and induce contraction. 4. In the iris sphincter, as well as in other smooth muscles, phorbol 12,13-dibutyrate, which mimics the action of DG, induced MLC phosphorylation and contraction in a dose- and time-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Biochem
PMID:Polyphosphoinositides, generation of second messengers, myosin light chain phosphorylation and contraction in rabbit iris sphincter smooth muscle. 284 9

Troponin has been prepared from the asynchronous flight muscle of Lethocerus (water bug) taking special care to prevent proteolysis. The regulatory complex contained tropomyosin and troponin components. The troponin components were Tn-C (18,000 Mr), Tn-T (apparent Mr 53,000) and a heavy component, Tn-H (apparent Mr 80,000). The troponin was tightly bound to tropomyosin and could not be dissociated from it in non-denaturing conditions. A complex of Tn-T, Tn-H and tropomyosin inhibited actomyosin ATPase activity and the inhibition was relieved by Tn-C from vertebrate striated muscle in the presence of Ca2+. However, unlike vertebrate Tn-I, Tn-H by itself was not inhibitory. Monoclonal antibodies were obtained to Tn-T and Tn-H. Antibody to Tn-T was used to screen an expression library of Drosophila cDNA cloned in lambda phage. The sequence of cDNA coding for the protein was determined and hence the amino acid sequence. The Drosophila protein has a sequence similar to that of vertebrate skeletal and cardiac Tn-T. The sequence extends beyond the carboxyl end of the vertebrate sequences, and the last 40 residues are acidic. Part of the sequence of Drosophila Tn-T is homologous to the carboxyl end of the Drosophila myosin light chain MLC-2 and one anti-Tn-T antibody cross-reacted with the light chain. Lethocerus Tn-H is related to the large tropomyosins of Drosophila flight muscle, for which the amino acid sequence is known, since antibodies that recognize this component also recognize the large tropomyosins. Tn-H is easily digested by calpain, suggesting that part of the molecule has an extended configuration. Electron micrographs of negatively stained specimens showed that Lethocerus thin filaments have projections at about 39 nm intervals, which are not seen on thin filaments from vertebrate striated muscle and are probably due to the relatively large troponin complex. Decoration of the thin filaments with myosin subfragment-1 in rigor conditions appeared not to be affected by the troponin. The troponin of asynchronous flight muscle lacks the Tn-I component of vertebrate striated muscle. Tn-H occurs only in the flight muscle and may be involved in the activation of this muscle by stretch.
J Mol Biol 1988 Dec 05
PMID:Troponin of asynchronous flight muscle. 285 58


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