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:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The mechanism of G protein-mediated inhibition of an inwardly rectifying K+ current (IIR) in adrenal chromaffin cells was investigated using the whole-cell version of the patch clamp technique. In case of recording with use of ATP-containing patch solution, the IIR was well maintained; otherwise, it ran down within 15 min. This run down was not prevented by replacement with adenylyl-imidodiphosphate, a nonhydrolysable analogue of ATP, but was markedly reduced by the addition to the ATP-free solution of 1 microM calyculin A, a specific inhibitor of serine/threonine phosphatase 1 (PP1) and 2A (PP2A). The addition of alkaline phosphatase to the ATP-containing solution facilitated run down of the current, and application of 100 microM H-7, a general kinase inhibitor, reversibly suppressed IIR. These results taken together suggest that inwardly rectifying K+ channels are under the influence of kinase and phosphatase without external signals. Infusion of nonhydrolysable analogues of GTP, guanosine-5'-O-(3-thiophosphate) (GTP gamma S) or guanylyl-imidodiphosphate, through the pipette produced little inward current at -55 mV, but completely inhibited IIR within approximately 5 or 6 min in all cells tested in the presence of 12 microM Mg2+ inside the cell. In contrast, infusion of aluminum fluoride (AlF) complex, another GTP binding (G) protein activator, consistently produced large inward currents, but did not alter IIR noticeably for 15 min in 17% of the cells tested. In the other cells, the inhibition of IIR developed slowly after long latent periods. This inhibitory potency of AlF was not enhanced by an increase in Mg2+ concentrations. Subtraction of the current-voltage relationship before from that noted during the generation of inward current by AlF complex revealed that the inward current diminished progressively with hyperpolarizations, as is the case with a nonselective cation current (INS) induced by a muscarinic agonist. Thus, AlF complex seems to be potent with the generation of INS, but not with IIR inhibition. The addition of 3 microM calyculin A significantly retarded the IIR inhibition by GTP gamma S, whereas that of 1 microM okadaic acid, another inhibitor of PPI and PP2A, markedly prevented the decline of IIR by AIF complex. Our observations suggest that the low potency of AlF complex in inhibiting IIR may be due to interference with phosphatase activity and that the activation of G protein suppresses IIR, probably by enhancing the apparent activity of phosphatase, which may explain run down of the current.
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PMID:Phosphatase is responsible for run down, and probably G protein-mediated inhibition of inwardly rectifying K+ currents in guinea pig chromaffin cells. 776 18

Inositol polyphosphate 1-phosphatase, inositol monophosphate phosphatase, and fructose 1,6-bisphosphatase share a sequence motif, Asp-Pro-(Ile or Leu)-Asp-(Gly or Ser)-(Thr or Ser), that has been shown by crystallographic and mutagenesis studies to bind metal ions and participate in catalysis. We compared the six alpha-carbon coordinates of this motif from the crystal structures of these three phosphatases and found that they are superimposable with rms deviations ranging from 0.27 to 0.60 A. Remarkably, when these proteins were aligned by this motif a common core structure emerged, defined by five alpha-helices and 11 beta-strands comprising 155 residues having rms deviations ranging from 1.48 to 2.66 A. We used the superimposed structures to align the sequences within the common core, and a distant relationship was observed suggesting a common ancestor. The common core was used to align the sequences of several other proteins that share significant similarity to inositol monophosphate phosphatase, including proteins encoded by fungal qa-X and qutG, bacterial suhB and cysQ (identical to amtA), and yeast met22 (identical to hal2). Evolutionary comparison of the core sequences indicate that five distinct branches exist within this family. These proteins share metal-dependent/Li(+)-sensitive phosphomonoesterase activity, and each predicted tree branch exhibits unique substrate specificity. Thus, these proteins define an ancient structurally conserved family involved in diverse metabolic pathways including inositol signaling, gluconeogenesis, sulfate assimilation, and possibly quinone metabolism. Furthermore, we suggest that this protein family identifies candidate enzymes to account for both the therapeutic and toxic actions of Li+ as it is used in patients treated for manic depressive disease.
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PMID:Definition of a metal-dependent/Li(+)-inhibited phosphomonoesterase protein family based upon a conserved three-dimensional core structure. 776 65

Phosphorylation of CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) from Saccharomyces cerevisiae protein kinase C was examined. Using pure CTP by synthetase as a substrate, protein kinase C activity was dose- and time-dependent and required calcium, diacylglycerol, and phosphatidylserine for full activation. Protein kinase C activity was also dependent on the concentration of CTP synthetase. Protein kinase C phosphorylated CTP synthetase on serine and threonine residues in vitro whereas the enzyme was primarily phosphorylated on serine residues in vivo. Phosphopeptide mapping analysis of CTP synthetase phosphorylated in vitro and in vivo indicated that the enzyme was phosphorylated on more than one site. Most of the phosphopeptides derived from CTP synthetase phosphorylated in vivo were the same as those derived from CTP synthetase phosphorylated by protein kinase C in vitro. The stoichiometry of the phosphorylation of native CTP synthetase was 0.4 mol of phosphate/mol of enzyme whereas the stoichiometry of the phosphorylation of alkaline phosphatase-treated CTP synthetase was 2.2 mol of phosphate/mol of enzyme. This indicated that CTP synthetase was purified in a phosphorylated state. Phosphorylation of CTP synthetase resulted in a 3-fold activation in enzyme activity whereas alkaline phosphatase treatment of CTP synthetase resulted in a 5-fold decrease in enzyme activity. Overall, the results reported here were consistent with the conclusion that CTP synthetase was regulated by protein kinase C phosphorylation.
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PMID:Phosphorylation of CTP synthetase from Saccharomyces cerevisiae by protein kinase C. 779 79

Human cell lines express two genetically distinct isoforms of DNA topoisomerase (topo II) II: topo II alpha (p170) and topo II beta (p180). We detected a higher molecular weight form with an apparent molecular mass of about 190 kDa in M phase-arrested HeLa cells (Kimura, K., Saijo, M., Ui, M., and Enomoto, T. (1994) J. Biol. Chem. 269, 1173-1176). In this study we confirmed, using anti-topo II alpha and topo II beta monoclonal antibodies, that this higher molecular weight form is topo II beta and consists of doublet bands around 190 kDa. We confirmed that the doublet bands constituted an M phase-specific phenomenon and were not an artifact of the procedure used to accumulate mitotic cells. Digesting the immunoprecipitated materials from mitotic cell extracts with alkaline phosphatase resulted in the disappearance of the doublet bands and the appearance of the 180-kDa band with the concomitant disappearance of 32P label in the region of the doublet bands. Neither heat-inactivated alkaline phosphatase nor phosphodiesterase affected the doublet bands and the 32P label. Topo II beta in interphase cells was also phosphorylated, but the shift in apparent molecular weight was very slight after alkaline phosphatase digestion. Analysis of the labeled phosphoamino acids present in topo II beta from M phase and logarithmically growing cells indicated that phosphorylation occurred mainly on serine and fairly on threonine residues in both topo II beta isoforms. These results indicated that topo II beta is phosphorylated at specific sites in M phase, resulting in the formation of the doublet bands.
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PMID:Identification of the nature of modification that causes the shift of DNA topoisomerase II beta to apparent higher molecular weight forms in the M phase. 792 18

The regulation of rat liver S-adenosylmethionine synthetase (AdoMet synthetase), a key enzyme in methionine metabolism, by protein kinase C (PKC) phosphorylation has been studied. Both enzyme forms, tetramer and dimer, are phosphorylated by this kinase in the same residue, Thr-342, of the sequence. Phosphorylation of the dimer leads to its dissociation, with production of a fully-active monomer. The kinetics of the monomer have been studied, and a KmMet of 931.9 microM, a KmATP of 708 microM and a Vmax of 66.8 nmol/min/mg have been calculated. Alkaline phosphatase treatment of both enzyme forms (tetramer and dimer) produces a reduction in their activity with no change in the oligomeric state. On the other hand, PKC phosphorylation of the alkaline phosphatase-treated AdoMet synthetase forms leads to the dissociation of the dimer to produce a monomer. Rephosphorylation occurs again in the same residue, Thr-342, of the sequence. The significance of AdoMet synthetase regulation by PKC phosphorylation is further discussed.
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PMID:Protein kinase C phosphorylation of rat liver S-adenosylmethionine synthetase: dissociation and production of an active monomer. 798 Apr 67

The substrate specificity of furin, a mammalian enzyme involved in the cleavage of many constitutively expressed protein precursors, was studied using substrate phage display. In this method, a multitude of substrate sequences are displayed as fusion proteins on filamentous phage particles and ones that are cleaved can be purified by affinity chromatography. The cleaved phage are propagated and submitted to additional rounds of protease selection to further enrich for good substrates. DNA sequencing of the cleaved phage is used to identify the substrate sequence. After 6 rounds of sorting a substrate phage library comprising 5 randomized amino acids (xxxxx), virtually all clones had an RxxR motif and many had Lys, Arg, or Pro before the second Arg. Nine of the selected sequences were assayed using a substrate-alkaline phosphatase fusion protein system. All were cleaved after the RxxR, and some substrates with Pro or Thr in P2 were also found to be cleaved as efficiently as RxKR or RxRR. To further elaborate surrounding determinants, we constructed 2 secondary libraries (xxRx(K/R)Rx and xxRxPRx). Although no consensus developed for the latter library, many of the sequences in the the former library had the 7-residue motif (L/P)RRF(K/R)RP, suggesting that the furin recognition sequence may extend over more than 4 residues. These studies further clarify the substrate specificity of furin and suggest the substrate phage method may be useful for identifying consensus substrate motifs in other protein processing enzymes.
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PMID:A survey of furin substrate specificity using substrate phage display. 798 14

P1 protamines isolated from ejaculated human, stallion, bull, boar and ram spermatozoa and P2 protamines from human and stallion spermatozoa were subjected, after alkaline phosphatase treatment, to in vitro phosphorylation reactions using cAMP-dependent protein kinase (PKA) and protein kinase C (PKC). All P1 protamines were phosphorylated by PKA, whereas P2 protamines were phosphorylated only by PKC. In addition, human, stallion and boar, but not bull and ram, P1 protamines were phosphorylated by PKC. After phosphoamino acid analysis, the protamines showing positive signals for phosphoserine (P-Ser) were subjected to P-Ser conversion reaction and protein sequencing. Only stallion (St1) and human (HP1) P1 protamines contained P-Ser after PKA phosphorylation, located in the middle region of the molecule, i.e., at Ser29 in St1 and Ser28 in HP1. All other phosphorylated P1 protamines contained only P-Thr, which could not be further localized in the sequence with the present methods. After PKC phosphorylation, the internally located Ser residues in human (ser21) and stallion (Ser29) P1 protamines were phosphorylated and, in boar P1 protamine, only Thr43 was slightly phosphorylated. The N-terminally located Ser residues in P1 protamines, which are known to be phosphorylated in vivo, were not phosphorylated by either kinase, indicating that there must still be other types of protamine kinases in sperm cells responsible for their phosphorylation. Within P2 protamines, HP2 was equally well phosphorylated at all Ser residues in addition to some Thr phosphorylation, whereas, in St2, Ser32 was the main target for PKC phosphorylation in vitro. Collectively, PKC is a good candidate for in vivo phosphorylation of P2 protamines and PKA for phosphorylation of some hydroxyamino acid residues in P1 protamines.
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PMID:P2 protamines are phosphorylated in vitro by protein kinase C, whereas P1 protamines prefer cAMP-dependent protein kinase. A comparative study of five mammalian species. 803 90

A synthetic tris-sulfotyrosyl dodecapeptide (TRDIY(S)ETDY(S)Y(S)RK-amide), whose primary sequence is identical to the 1142-1153 sequence of the insulin proreceptor, inhibited insulin receptor dephosphorylation in solubilized membranes, and digitonin-permeabilized cells derived from Chinese hamster ovary (CHO) cells expressing high levels of human insulin receptors (CHO/HIRc). It also inhibited the dephosphorylation of a synthetic tyrosine phosphorylated substrate by recombinant PTP-1B, a protein tyrosine phosphatase (PTPase), indicating that it acted via interaction with PTPase(s). A N-stearyl derivative of the peptide caused an approximately 4.5-fold increase in insulin-stimulated receptor autophosphorylaction in intact CHO/HIRc cells. The peptide displayed specificity toward tyrosine-class phosphatases only, as it had no effect on the activities of the serine/threonine phosphatases PP-1 and PP-2A, or alkaline phosphatase. The tyrosine sulfate ester bonds of the peptide were stable when incubated with PTP-1B (1 h, 30 degrees C). These data suggest that the sulfotyrosyl peptide functions as a nonhydrolyzable phosphotyrosyl peptide analogue capable of direct interaction with PTPase catalytic domain.
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PMID:A synthetic tris-sulfotyrosyl dodecapeptide analogue of the insulin receptor 1146-kinase domain inhibits tyrosine dephosphorylation of the insulin receptor in situ. 808 98

Urinary trypsin inhibitor is a glycoprotein with a structure in which two Kunitz-type inhibitory domains are linked in a row. We isolated two genes encoding the 70 amino acid sequence from the 78th amino acid (Thr) to the C-terminal and the 68 amino acid sequence from the 80th (Ala) to the C-terminal of human urinary trypsin inhibitor, both which correspond to the second Kunitz-type inhibitory domain, and then constructed expression plasmids by ligating it to the E. coli alkaline phosphatase signal peptide gene. These plasmids under the control of the tryptophan promoter expressed the second domain in E. coli strain JE5505 which lacks the membrane lipoprotein. The recombinant second domain purified from the culture supernatant of the transformant inhibited trypsin, plasmin, leukocyte elastase and chymotrypsin which are known to be inhibited by urinary trypsin inhibitor. In addition it inhibited blood coagulation factor Xa and plasma kallikrein in a concentration dependent and competitive manner, and significantly prolonged the plasma-based activated partial thromboplastin time (APTT). The truncated natural counterpart obtained by a limited degradation of human urinary trypsin inhibitor also revealed the identical inhibitory activities.
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PMID:Novel factor Xa and plasma kallikrein inhibitory-activities of the second Kunitz-type inhibitory domain of urinary trypsin inhibitor. 819 13

The correlation between sequence homology and catalytic importance of a specific amino acid in the E. coli and Liver/Kidney/Bone (L/K/B) human alkaline phosphatase was investigated. For this reason Ala-161 in the E. coli enzyme was substituted with a threonine residue via site-specific mutagenesis. The homologous amino acid in the L/K/B alkaline phosphatase sequence has been shown to cause inactivation of the enzyme. In E. coli alkaline phosphatase the Ala-161-->Thr substitution results in a mutant enzyme with virtually unchanged catalytic properties when compared to the wild-type enzyme. Our results show that Ala-161 in the E. coli alkaline phosphatase does not have an important catalytic role. The results suggest that the three-dimensional topology of the L/K/B alkaline phosphatase may be different from that observed for the enzyme from E. coli.
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PMID:The Ala-161-->Thr substitution in Escherichia coli alkaline phosphatase does not result in loss of enzymatic activity although the homologous mutation in humans causes hypophosphatasia. 832 35


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