Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Plasma membranes have been prepared from porcine thyroid glands using sucrose gradients. The fractions having a density in sucrose of 1.18 g/ml mainly contained plasma membranes and were moderately contaminated with other subcellular components as shown by marker enzyme data. Purified plasma membranes incubated in the presence of [32-P]gamma ATP incorporated 32-P. Kinetics of incorporation of 32-P into endogenous substrates studied in various buffers and with increasing ATP concentration suggest a phosphodephosphorylating system related to cAMP-dependent protein kinase and phosphoprotein phosphatase activities. The two enzymatic activities associated with plasma membranes have been demonstrated using exogenous substrates. cAMP increases and fluoride ions decrease the extent of membrane phosphorylation. The specific activity of protein kinase was 10-12 times higher than in the initial homogenate and was only slightly enhanced in the presence of 0.5% Nonidet as compared to microsomal fraction. cAMP binding to membrane proteins was 3 times higher than to the other particulate fractions. TSH present in the incubating medium or added after 5 min of 32-P labelling induced a rapid stimulation of endogenous phosphorylation followed by a rapid decrease. Phosphorylated membrane substrates were analyzed: high voltage paper electrophoresis after partial hydrolysis indicated that [32-P]phosphate is incorporated into serine and threonine residues as o-phosphate derivatives. SDS-polyacrylamide gel electrophoresis showed several 32--labelled fractions. When enhanced by cAMP, no specific phosphorylation of protein components was observed.
Mol Cell Endocrinol 1975 May
PMID:Phosphorylation of purified thyroid plasma membranes incubated with [32-P]ATP. 16 13

A heat-and acid-stable protein inhibitor of phosphorylase phosphatase is present in a highly purified preparation of protein inhibitor of cyclic AMP-dependent protein kinase from rabbit skeletal muscle. Although these two inhibitors have strikingly similar properties to each other, such as sensitivity to trypsin and behavior on gel permeation chromatography, they can be separated by polyacrylamide disc gel electrophoresis. This indicates that the phosphatase-inhibitory and kinase-inhibitory activities reside with different protein species. The inhibition of both the enzymes is not altered by incubating the inhibitor preparation with a general phosphoprotein phosphatase, with phosvitin kinase, or with cyclic AMP-dependent protein kinase. Inhibition of phosphorylase phosphatase is of a non-competitive type supporting the idea that the phosphatase inhibitor is not an alternative substrate for the enzyme. Inhibition of phosphatase activity is selective in that it does no occur when phosphorylated histone or phosphorylated protamine are used as substrates.
Mol Cell Biochem 1977 Apr 12
PMID:Protein inhibitors of phosphorylase phosphatase and cyclic AMP-dependent protein kinase from rabbit skeleta muscle. 19 98

By using an assay specific for detection of calcineurin, a Ca2+/calmodulin-dependent phosphoprotein phosphatase, this enzyme was purified approximately 5,000-fold from extracts of the yeast Saccharomyces cerevisiae. Cna1p and Cna2p, the products of two yeast genes encoding the catalytic (A) subunits of calcineurin, were major constituents of the purified fraction. A third prominent component of apparent molecular mass 16 kDa displayed several properties, including ability to bind 45Ca2+, that are characteristic of the regulatory (B) subunit of mammalian calcineurin and was recognized by an antiserum raised against bovine calcineurin. These antibodies were used to isolate the structural gene (CNB1) encoding this protein from a yeast expression library in the vector lambda gt11. The nucleotide sequence of CNB1 predicted a polypeptide similar in length and highly related in amino acid sequence (56% identity) to the mammalian calcineurin B subunit. Like its counterpart in higher cells, yeast Cnb1p was myristoylated at its N terminus. Mutants lacking Cnb1p, or all three calcineurin subunits (Cna1p, Cna2p, and Cnb1p), were viable. Extracts of cnb1 delta mutants contained no detectable calcineurin activity, even though Cna1p and Cna2p were present at normal levels, suggesting that the B subunit is required for full enzymatic activity in vitro. As was observed previously for MATa cna1 cna2 double mutants, MATa cnb1 mutants were defective in their ability to recover from alpha-factor-induced growth arrest. Thus, the B subunit also is required for the function of calcineurin in promoting adaptation of haploid yeast cells to pheromone in vivo.
Mol Cell Biol 1992 Aug
PMID:Regulatory subunit (CNB1 gene product) of yeast Ca2+/calmodulin-dependent phosphoprotein phosphatases is required for adaptation to pheromone. 132 37

Using cytostatic factor metaphase II-arrested extracts as a model system, we show that protein phosphatase 1 is regulated during early embryonic cell cycles in Xenopus. Phosphatase 1 activity peaks during interphase and decreases shortly before the onset of mitosis. A second peak of activity appears in mitosis at about the same time that cdc2 becomes active. If extracts are inhibited in S-phase with aphidicolin, then phosphatase 1 activity remains high. The activity of phosphatase 1 appears to determine the timing of exit from S-phase and entry into M-phase; inhibition of phosphatase 1 by the specific inhibitor, inhibitor 2 (Inh-2), causes premature entry into mitosis, whereas exogenously added phosphatase 1 lengthens the interphase period. Analysis of DNA synthesis in extracts treated with Inh-2, but lacking the A- and B-type cyclins, shows that phosphatase 1 is also required for the process of DNA replication. These data indicate that phosphatase 1 is a component of the signaling pathway that ensures that M-phase is not initiated until DNA synthesis is complete.
Mol Biol Cell 1992 Jun
PMID:Multiple roles for protein phosphatase 1 in regulating the Xenopus early embryonic cell cycle. 132 52

Type 1 protein phosphatases (PP-1) comprise a group of widely distributed enzymes that specifically dephosphorylate serine and threonine residues of certain phosphoproteins. They all contain an isoform of the same catalytic subunit, which has an extremely conserved primary structure. One of the properties of PP-1 that allows one to distinguish them from other serine/threonine protein phosphatases is their sensitivity to inhibition by two proteins, termed inhibitor 1 and inhibitor 2, or modulator. The latter protein can also form a 1:1 complex with the catalytic subunit that slowly inactivates upon incubation. This complex is reactivated in vitro by incubation with MgATP and protein kinase FA/GSK-3. In the cell the type 1 catalytic subunit is associated with noncatalytic subunits that determine the activity, the substrate specificity, and the subcellular location of the phosphatase. PP-1 plays an essential role in glycogen metabolism, calcium transport, muscle contraction, intracellular transport, protein synthesis, and cell division. The activity of PP-1 is regulated by hormones like insulin, glucagon, alpha- and beta-adrenergic agonists, glucocorticoids, and thyroid hormones.
Crit Rev Biochem Mol Biol 1992
PMID:The structure, role, and regulation of type 1 protein phosphatases. 135 Feb 40

Sulfation is an important pathway in the metabolism of many hormones and drugs. Human liver contains at least three well characterized sulfotransferase (ST) enzymes, i.e., dehydroepiandrosterone (DHEA) ST and two forms of phenol sulfotransferase (PST). Our goal was to purify, to obtain partial amino acid sequence for, and to clone and express cDNA for human liver DHEA ST. Polymerase chain reaction primers were designed on the basis of homology among rat liver hydroxysteroid ST, rat liver PST, and bovine estrogen ST. These primers amplified a unique sequence from human liver cDNA, and this polymerase chain reaction product was used to screen a human liver cDNA library. Two clones were isolated that contained identical open reading frames, of 855 nucleotides, that encoded a protein of 285 amino acids. The deduced amino acid sequence of the encoded protein included two separate 27- and 23-amino acid sequences that were identical to those obtained by microsequencing of proteolytic fragments from purified human liver DHEA ST. Translation, in a rabbit reticulocyte lysate system, of mRNA transcribed in vitro from the two cDNA clones resulted in a 35-kDa translation product that comigrated with purified human liver DHEA ST during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This translation product also catalyzed the sulfation of DHEA but not the sulfation of model substrates for the two forms of PST found in human liver. The two cDNA clones were also used to create expression constructs with the eukaryotic expression vector P91023(B), and these constructs were used to transfect COS-1 cells. The transfected cells expressed a high level of DHEA ST activity, and this enzyme activity displayed a pattern of inhibition by the ST inhibitor 2,6-dichloro-4-nitrophenol identical to that of human liver DHEA ST. Cloning of cDNA for this important human sulfate-conjugating enzyme will enhance understanding of the relationship between DHEA ST and other human liver STs, as well as ST enzymes in other species.
Mol Pharmacol 1992 May
PMID:Human liver dehydroepiandrosterone sulfotransferase: molecular cloning and expression of cDNA. 158 21

Saccharomyces cerevisiae genomic clones that encode calmodulin-binding proteins were isolated by screening a lambda gt11 expression library using 125I-labeled calmodulin as probe. Among the cloned yeast genes, we found two closely related genes (CMP1 and CMP2) that encode proteins homologous to the catalytic subunit of phosphoprotein phosphatase. The presumed CMP1 protein (62,999 Da) and CMP2 protein (68,496 Da) contain a 23 amino acid sequence very similar to those identified as calmodulin-binding sites in many calmodulin-regulated proteins. The yeast genes encode proteins especially homologous to the catalytic subunit of mammalian phosphoprotein phosphatase type 2B (calcineurin). The products of the CMP1 and CMP2 genes were identified by immunoblot analysis of cell extracts as proteins of 62,000 and 64,000 Da, respectively. Gene disruption experiments demonstrated that elimination of either or both of these genes had no effect on cell viability, indicating that these genes are not essential for normal cell growth.
Mol Gen Genet 1991 May
PMID:The Saccharomyces cerevisiae genes (CMP1 and CMP2) encoding calmodulin-binding proteins homologous to the catalytic subunit of mammalian protein phosphatase 2B. 164 87

The crystal structure of subtilisin BPN' complexed with a proteinaceous inhibitor SSI (Streptomyces subtilisin inhibitor) was refined at 1.8 A resolution to an R-factor of 0.177 with a root-mean-square deviation from ideal bond lengths of 0.014 A. The work finally established that the SSI-subtilisin complex is a Michaelis complex with a distance between the O gamma of active Ser221 and the carbonyl carbon of the scissile peptide bond being an intermediate value between a covalent bond and a van der Waals' contact, 2.7 A. This feature, as well as the geometry of the catalytic triad and the oxyanion hole, is coincident with that found in other highly refined crystal structures of the complex of subtilisin Novo, subtilisin Carlsberg, bovine trypsin or Streptomyces griseus protease B with their proteinaceous inhibitors. The enzyme-inhibitor beta-sheet interaction is composed of two separate parts: that between the P1-P3 residues of SSI and the 125-127 chain segment (the "S1-3 site") of subtilisin and that between the P4-P6 residues of SSI and th 102-104 chain segment (the "S4-6 site") of subtilisin. The latter beta-interaction is unique to subtilisin. In contrast, the beta-sheet interaction previously found in the complex of subtilisin Novo and chymotrypsin inhibitor 2 or in the complex of subtilisin Carlsberg and Eglin C is distinct from the present complex in that the two types of beta-interactions are not separate. As for the flexibility of the molecules comprising the present complex, the following observations were made by comparing the B-factors for free and complexed SSI and comparing those for free and complexed subtilisin BPN'. The rigidification of the component molecules upon complex formation occurs in a very localized region: in SSI, the "primary" and "secondary" contact regions and the flanking region; in subtilisin BPN', the S1-3 and S4-6 sites and the flanking region.
J Mol Biol 1991 Sep 05
PMID:Refined crystal structure of the complex of subtilisin BPN' and Streptomyces subtilisin inhibitor at 1.8 A resolution. 192 Apr 11

The enhanced phosphorylations via cAMP, Ca2+ mobilization, and diacyl glycerol formation via the activation of the respective kinases is now classical. The decreased phosphorylation via inhibition of adenylate cyclase via the alpha adrenergic receptor is also becoming understood. What the insulin studies on the control of glycogen synthesis have taught us is that the rate limiting enzyme glycogen synthase is regulated by multiple covalent phosphorylation in an elegant but complex manner. The overall pattern of dephosphorylation is influenced by effecting both phosphatase and kinase activities in a set of interrelated mechanisms. In the presence of glucose, in muscle, fat, and liver under physiological conditions G-6-P acts as a signal to stimulate the phosphatase. An additional stimulation could occur via a novel insulin phosphatase stimulatory mediator. The phosphatase is also stimulated by at least three covalent mechanisms involving altered phosphorylation state. In one there is a decreased phosphorylation of the phosphatase inhibitor 1 potentially related to decreased cAMP-dependent protein kinase activity. In the second, there is decreased phosphorylation of the deinhibitor also potentially related to decreased cAMP-dependent protein kinase phosphorylation. In the third, an increased activity of casein kinase 2 could activate the ATP-Mg dependent phosphatase by an increased phosphorylation of phosphatase inhibitor 2 (modulatory subunit). In the liver, allosteric control of the phosphatase by G-6-P and nucleotides is of great importance. Insulin also stimulates the phosphatase in long-term experiments via increased protein synthesis. It is clear that future work will be required to determine which species of the various classes of phosphatases are regulated in short-term and long-term regulation by insulin. In terms of kinases, the effects of insulin to inactivate and desensitize the cAMP-dependent protein kinase are established. The molecular mechanisms of this effect remain to be worked out. The enhanced activity of MAP and S-6 kinase would appear to be part of a cascade of reactions perhaps originating in the autophosphorylation and activation of the insulin receptor tyrosine kinase. The mechanism of the short-term activation of casein kinase 2 remains to be elucidated. A cAMP-dependent protein kinase inhibitory mediator, which also inhibits adenylate cyclase is an important element in the regulation of kinase and adenylate cyclase activity by insulin. Its physiological significance must be established in the future, in terms of its control of glycogen synthase activation by insulin. Clearly this kinase inhibitor as well as the phosphatase stimulator are potential regulators of glycogen synthase activity by insulin.
Adv Enzymol Relat Areas Mol Biol 1990
PMID:Insulin and the stimulation of glycogen synthesis. The road from glycogen structure to glycogen synthase to cyclic AMP-dependent protein kinase to insulin mediators. 215 10

We present a calculation of the relative changes in binding free energy between the complex of ribonuclease T1 (RNase Tr) with its inhibitor 2'-guanosine monophosphate (2'GMP) and that of RNase T1-2'-adenosine monophosphate (2'AMP) by means of a thermodynamic perturbation method implemented with molecular dynamics. Using the available crystal structure of the RNase T1-2'GMP complex, the structure of the RNase T1-2'AMP complex was obtained as a final structure of the perturbation calculation. The calculated difference in the free energy of binding (delta delta Gbind) was 2.76 kcal/mol. This compares well with the experimental value of 3.07 kcal/mol. The encouraging agreement in delta delta Gbind suggests that the interactions of inhibitors with the enzyme are reasonably represented. Energy component analyses of the two complexes reveal that the active site of RNase T1 electrostatically stabilizes the binding of 2'GMP more than that of 2'AMP by 44 kcal/mol, while the van der Waals' interactions are similar in the two complexes. The analyses suggest that the mutation from Glu46 to Gln may lead to a preference of RNase T1 for adenine in contrast to the guanine preference of the wild-type enzyme. Although the molecular dynamics equilibration moves the atoms of the RNase T1-2'GMP system about 0.9 A from their X-ray positions and the mutation of the G to A in the active site increases the deviation from the X-ray structure, the mutation of the A back to G reduces the deviation. This and the agreement found for delta delta Gbind suggest that the molecular dynamics/free energy perturbation method will be useful for both energetic and structural analysis of protein-ligand interactions.
J Mol Biol 1990 Mar 05
PMID:Calculation of the relative binding free energy of 2'GMP and 2'AMP to ribonuclease T1 using molecular dynamics/free energy perturbation approaches. 215 20


1 2 3 4 5 6 7 8 9 10 Next >>