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Query: UNIPROT:P06889 (Mol)
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We have determined the first genomic structure and characterized the mRNA and protein products of a novel vertebrate gene that encodes a calcium-binding protein with amino acid sequence identity to a protein kinase domain. The elucidation of the complete DNA sequence of this transcription unit and adjacent genomic DNA, Southern blot and polymerase chain reaction analyses of cellular genomic DNA, and examination of mRNA and protein species revealed that the calcium-binding kinase-related protein (KRP)-encoding gene is contained within the gene for a calmodulin-regulated protein kinase, myosin light-chain kinase (MLCK). The KRP gene transcription unit is composed of three exons and a 5'-flanking sequence containing a canonical TATA box motif. The TATA box, the transcription initiation site, and the first 109 nucleotides of the 5' noncoding region of the KRP mRNA correspond to an MLCK gene intron sequence. Both KRP and MLCK are produced in the same adult chicken tissue in relatively high abundance from a single contiguous stretch of genomic DNA and utilize the same reading frame and common exons to produce distinct mRNAs (2.7 and 5.5 kb, respectively) that encode proteins with dissimilar biochemical functions. There appears to be no precedent in vertebrate molecular biology for such a relationship. This may represent a mechanism whereby functional diversity can be achieved within the same vertebrate tissue by use of common exons to produce shuffled domains with identical amino acid sequences in different molecular contexts.
Mol Cell Biol 1992 May
PMID:Structure and expression of a calcium-binding protein gene contained within a calmodulin-regulated protein kinase gene. 137 15

The effect of altering intracellular free Ca2+ on juvenile hormone (JH) and acid synthesis by larval and pupally-committed corpora allata (CA) of fifth stadium Manduca sexta was investigated. Larval CA required extracellular Ca2+ greater than or equal to 0.1 mM for maximal JH synthesis, while JH acid synthesis by glands after pupal commitment was independent of extracellular Ca2+. Free Ca2+ in the hemolymph ranged from 1.4 to 2.1 mM during the fifth stadium. Both calcium ionophores and caffeine, which releases Ca2+ from intracellular stores, inhibited JH synthesis by larval CA but stimulated JH acid synthesis by post-commitment CA. These results suggest that intracellular stores may be the principal source of Ca2+ for the biosynthetic activity of the post-commitment gland. Calcium channel blockers (La3+, Cd2+) and antagonists (verapamil, isradipine and nitrendipine) decreased both JH and JH acid synthesis, indicating the existence of Ca2+ channels in the CA cell membrane. Calmodulin (CaM) antagonists inhibited the activity of both larval and post-commitment CA, suggesting an integral relationship of CaM to the effects of Ca2+ on gland activity. One of these effects is the demonstrated requirement of 0.1 mM extracellular Ca2+ for allatostatin inhibition of JH I synthesis by larval CA.
Mol Cell Endocrinol 1992 Apr
PMID:Manipulation of intracellular calcium affects in vitro juvenile hormone synthesis by larval corpora allata of Manduca sexta. 137 73

In the present study, a relationship between convulsant activity and two cellular events, changes in calmodulin (CaM) concentration and proto-oncogene c-fos expression has been considered. c-fos has been found activated after the administration of the organochlorine insecticide lindane, the Ca2+ channel agonist Bay K, and N-methyl-D-aspartate (NMDA). The administration of the voltage-dependent Ca2+ channel antagonist nifedipine was able to block the expression elicited by lindane. The effect of lindane on c-fos expression could not be blocked by prior administration of MK-801, a non-competitive antagonist of the NMDA receptor. These results suggest a possible role for the voltage-dependent Ca2+ channels in the mechanism of action of lindane. By means of in situ hybridization, the different patterns of c-fos expression after the administration of the mentioned compounds have been described. A possible modification of the levels of CaM has also been investigated. Among all the subcellular fractions considered, only levels of nuclear CaM appeared to be affected after the different treatments. The changes observed seemed to follow a similar pattern to that described for c-fos induction. Calcium entry through these voltage-dependent calcium channels would be the link between membrane depolarizing events and expression of c-fos and/or increase in nuclear CaM.
Brain Res Mol Brain Res 1992 Aug
PMID:Effect of different convulsants on calmodulin levels and proto-oncogene c-fos expression in the central nervous system. 138 76

The nucleotide dependence of the Ca(2+)-ATPase purified from cardiac sarcolemma by calmodulin-affinity chromatography was investigated for preparations either in the basal state or activated by three procedures: (i) addition of calmodulin; (ii) addition of phosphatidylserine and (iii) controlled proteolysis. Upon activation, the maximal velocity of ATP hydrolysis increases by a factor of 4-5, while the curves of ATP dependence of ATP hydrolysis change from hyperbolic to biphasic, revealing the presence of two Kmapp for ATP. A tight coupling between Ca2+ and ATP binding sites was also observed. At high ATP concentration, the ATPase activity of the basal state shows a complex dependence on Ca2+ concentration, increasing sharply at millimolar Ca2+. Our results indicate that this increase in ATPase activity is paralleled by the appearance of a second, low affinity Kmapp for ATP. When only the high affinity site for ATP is occupied the ATPase activity of the basal state displays a simple, hyperbolic dependence on the Ca2+ concentration. In addition, increasing Ca2+ concentration appears to decrease the ATP binding at the low affinity site of the enzyme. The effect of ADP on ATP hydrolysis was also examined. The finding that ADP is a potent inhibitor of the purified Ca(2+)-ATPase from heart suggests that the stimulatory action of ADP observed in cardiac sarcolemmal vesicles is not an intrinsic property of the enzyme.
J Mol Cell Cardiol 1992 Mar
PMID:Regulation of the nucleotide dependence of the cardiac sarcolemma Ca(2+)-ATPase. 138 33

The presence of actin has been determined in mammalian spermatozoa. However, its function in these cells is still almost unknown. Only in boar spermatozoa has evidence for F-actin and a possible function for it been presented. In this work, actin distribution and F-actin were determined in uncapacitated, capacitated, and acrosomal-reacted guinea pig spermatozoa, by means of monoclonal and polyclonal antibodies, using an indirect immunoperoxidase technique, and by the use of rhodamine-phalloidin. With the last probe we found filamentous actin in these cells. By both techniques, actin was detected in the acrosome and in the entire tail. In some cells with acrosomal reaction, actin was also detected in the equatorial and in the postacrosomal regions. SDS-PAGE and Western blots immunostained with monoclonal and polyclonal anti-actin antibodies confirmed the presence of actin in extracts of guinea pig spermatozoa. Actin was also detected in preparations of Percoll-purified spermatozoa. We have communicated that guinea pig spermatozoa show a change on calmodulin location during the acrosome reaction. They present it first in the equatorial region and later in the postacrosomal region. To determine if F-actin participates in this calmodulin translocation, we studied the effect of cytochalasin D. It was found that the number of cells with calmodulin in the equatorial region increased in the presence of cytochalasin D while the number of cells with calmodulin in the postacrosomal region decreased. We also found that after cytochalasin D treatment acrosome loss was increased and sperm motility was slightly inhibited. Our results suggest that actin participate in calmodulin translocation to the postacrosomal region during acrosome reaction, in maintaining the acrosome structure, and perhaps also in sperm motility.
Mol Reprod Dev 1992 Oct
PMID:F-actin in guinea pig spermatozoa: its role in calmodulin translocation during acrosome reaction. 141 86

Ca2+ release from skeletal sarcoplasmic reticulum (SR) could be regulated by at least three mechanisms: 1) Ca2+, 2) calmodulin, and 3) Ca2+/calmodulin-dependent phosphorylation. Bell-shaped Ca(2+)-dependence of Ca2+ release from both actively- and passively-loaded SR vesicles suggest that opening and closing of the Ca2+ release channel could be regulated by [Ca2+o]. The time- and concentration-dependent inhibition of Ca2+ release from skeletal SR by calmodulin was also studied using passively-Ca2+ loaded SR vesicles. Up to 50% of Ca2+ release was inhibited by calmodulin (0.01-0.5 microM); this inhibition required 5-15 min preincubation time. The hypothesis that Ca2+/calmodulin-dependent phosphorylation of a 60 kDa protein regulates Ca2+ release from skeletal SR was tested by stopped-flow fluorometry using passively-Ca2+-loaded SR vesicles. Approximately 80% of the initial rates of Ca(2+)-induced Ca2+ release was inhibited by the phosphorylation within 2 min of incubation of the SR with Mg-ATP and calmodulin. We identified two types of 60 kDa phosphoproteins in the rabbit skeletal SR, which was distinguished by solubility of the protein in CHAPS. The CHAPS-soluble 60 kDa phosphoprotein was purified by column chromatography on DEAE-Sephacel, heparin-agarose, and hydroxylapatite. Analyses of the purified protein indicate that the CHAPS-soluble 60 kDa protein is an isoform of phosphoglucomutase (PGM). cDNAs encoding isoforms of PGM were cloned and sequenced using synthetic oligonucleotides. Two types of PGM isoforms (Type I and Type II) were identified. The translated amino acid sequences show that Type II isoform is SR-form.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Biochem 1992 Sep 08
PMID:Regulation of Ca2+ release from sarcoplasmic reticulum in skeletal muscles. 146 Dec 55

We have determined and refined the crystal structure of a recombinant calmodulin at 1.7 A resolution. The structure was determined by molecular replacement, using the 2.2 A published native bovine brain structure as the starting model. The final crystallographic R-factor, using 14,469 reflections in the 10.0 to 1.7 A range with structure factors exceeding 0.5 sigma, is 0.216. Bond lengths and bond angle distances have root-mean-square deviations from ideal values of 0.009 A and 0.032 A, respectively. The final model consists of 1279 non-hydrogen atoms, including four calcium ions, 1130 protein atoms, including three Asp118 side-chain atoms in double conformation, 139 water molecules and one ethanol molecule. The electron densities for residues 1 to 4 and 148 of calmodulin are poorly defined, and not included in our model, except for main-chain atoms of residue 4. The calmodulin structure from our crystals is very similar to the earlier 2.2 A structure described by Babu and coworkers with a root-mean-square deviation of 0.36 A. Calmodulin remains a dumb-bell-shaped molecule, with similar lobes and connected by a central alpha-helix. Each lobe contains three alpha-helices and two Ca2+ binding EF hand loops, with a short antiparallel beta-sheet between adjacent EF hand loops and one non-EF hand loop. There are some differences in the structure of the central helix. The crystal packing is extensively studied, and facile crystal growth along the z-axis of the triclinic crystals is explained. Herein, we describe hydrogen bonding in the various secondary structure elements and hydration of calmodulin.
J Mol Biol 1992 Dec 20
PMID:Calmodulin structure refined at 1.7 A resolution. 147 85

The single gene encoding calmodulin in the eukaryotic microorganism Dictyostelium discoideum was cloned and sequenced. The gene was found to contain three introns, one lying immediately after the translation initiation codon. The deduced amino acid sequence indicated that Dictyostelium calmodulin contains 19 amino acid differences from vertebrate calmodulin, including extensions at both termini. Northern blot analysis showed that similar levels of calmodulin mRNA are present throughout growth and development of wild-type cells. A complete copy of the calmodulin cDNA was prepared, and an 87-base pair fragment complementary to the 5'-end of the calmodulin mRNA was subcloned into the Dictyostelium transformation vector pVEII, such that expression of the antisense transcript was driven by the discoidin I gamma promoter. Transformed cells were selected and maintained at low cell density, a condition resulting in minimal activity of the discoidin I promoter. High level expression was induced by allowing the transformants to reach high cell density or by growing them in the presence of medium conditioned by high density cells. Under these conditions, in which calmodulin mRNA and protein levels were reduced about twofold, the calmodulin antisense transformants lost the ability to complete cytokinesis. A contractile ring formed and constricted, but the midbody linking daughter cells failed to break. The resulting cell population contained multinucleated cells and networks of cells connected by cytoplasmic bridges. Normal cell division was restored when the cells were diluted to low density. These observations have identified a new point at which calmodulin may regulate cell cleavage.
Mol Biol Cell 1992 Dec
PMID:Inducible expression of calmodulin antisense RNA in Dictyostelium cells inhibits the completion of cytokinesis. 149 36

In situ hybridization and northern/slot blot analyses were used to quantify the expression of calcyclin (2A9, 5B10), osteopontin (opn, secreted phosphoprotein, 2ar) and calmodulin mRNAs in mouse tissues that support pregnancy. High-to-moderate levels of the mRNAs of all three genes were detected at discrete locations in the uterus, decidua and placenta as a function of gestation time. Calmodulin expression was constant in these tissues; calcyclin mRNA was high during early pregnancy and declined after day 8-9 of gestation; and opn mRNA was undetectable before day 7, with maximal levels on days 9-12 in each of these tissues. Calcyclin, but not opn, expression was also observed in the chorioamnion after day 12. Calcyclin was expressed throughout the decidua on day 8 but became restricted to the primary (antimesometrial) decidual zone and decidua lateralis on day 9, and the decidua capsularis after day 9. By contrast, opn mRNA was localized on day 9 to the mesometrial triangle, which contains a large population of granulated metrial gland cells, and to the decidua basalis. These two genes may serve as markers for the two types of decidual tissue. We suggest that one function of OPN, which may be an indicator of cells in the decidua that have a bone marrow genealogy, is to mediate the flux of calcium from the maternal circulation to the developing embryo.
Mol Reprod Dev 1992 Aug
PMID:Regulated temporal and spatial expression of the calcium-binding proteins calcyclin and OPN (osteopontin) in mouse tissues during pregnancy. 149 79

A codon-based approach to estimating the number of variable sites in a protein is presented. When first and second positions of codons are assumed to be replacement positions, a capture-recapture model can be used to estimate the number of variable codons from every pair of homologous and aligned sequences. The capture-recapture estimate is compared to a maximum likelihood estimate of the number of variable codons and to previous approaches that estimate the number of variable sites (not codons) in a sequence. Computer simulations are presented that show under which circumstances the capture-recapture estimate can be used to correct biases in distance matrices. Analysis of published sequences of two genes, calmodulin and serum albumin, shows that distance corrections that employ a capture-recapture estimate of the number of variable sites may be considerably different from corrections that assume that the number of variable sites is equal to the total number of positions in the sequence.
J Mol Evol 1992 Sep
PMID:Estimating the fraction of invariable codons with a capture-recapture method. 151 92


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