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:3.4.16.2 (PCP)
3,761 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A reactive ATP analog, N6-(6-bromoacetamidohexyl)-AMP-PCP, reacted specifically with the ATP inhibitory site of rabbit skeletal muscle phosphofructokinase without affecting the active site. Modification resulted in the incorporation of 1.01 mol of the reagent per mol of enzyme subunit. The modified enzyme was insensitive to allosteric inhibition by ATP and to activation by AMP at pH 7.2, where the native enzyme exhibits allosteric kinetic behavior. These observations demonstrate that we had succeeded in obtaining PFK fixed in the T state. Using the kinetic parameters of this modified enzyme, the kinetic properties of native enzyme can be quantitatively accounted for by the allosteric model of Monod-Wyman-Changeux. Further, the reagent was shown to have reacted with a specific cysteine residue near or at the ATP inhibitory site, and the sequence around the cysteine was determined as Cys-Lys-Asp-Phe-Arg.
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PMID:Analysis of the allosteric properties of rabbit muscle phosphofructokinase by means of affinity labeling with a reactive ATP analog. 16 Apr 16

Dynorphin A-(1-13)-Tyr-Leu-Phe-Asn-Gly-Pro (Dyn Ia; 1-8 nmol) injected intracerebroventricularly in the mouse produces two independent behavioral effects: (1) a norbinaltorphimine (kappa opioid antagonist)-reversible analgesia in the acetic acid-induced writhing test and (2) motor dysfunction characterized by wild running, pop-corn jumping, hindlimb jerking and barrel rolling and antagonized by the irreversible phencyclidine (PCP) and sigma (sigma) receptor antagonist, metaphit and the non-competitive N-methyl-D-aspartate (NMDA) receptor antagonists, dextromethorphan and ketamine. The specific involvement of the PCP receptor in the motor effects of Dyn Ia is supported by the direct competitive interaction of the peptide with the binding of [3H]MK-801 (Ki: 0.63 microM) and [3H]TCP (Ki: 4.6 microM) to mouse brain membrane preparations.
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PMID:Selective involvement of kappa opioid and phencyclidine receptors in the analgesic and motor effects of dynorphin-A-(1-13)-Tyr-Leu-Phe-Asn-Gly-Pro. 135 18

Active gamma subunit of skeletal muscle phosphorylase kinase has been obtained by expression of the rat soleus cDNA in a baculovirus system. The protein exhibited the expected pH 6.8/8.2 activity ratio of 0.6, and its activity was insensitive to Ca2+ addition, indicating that it was free gamma subunit and not a gamma subunit-calmodulin complex. It was stimulated approximately 2-fold by Ca(2+)-calmodulin addition, demonstrating that it had retained high-affinity calmodulin binding. By site-directed mutagenesis, we have examined the role of six of the amino acids that constitute the consensus ATP binding site of the protein kinase, which in the gamma subunit is represented by the sequence 26Gly.Arg.Gly.Val.Ser.Ser.Val.Val33. Changes were evaluated by the kinetic determination of the dissociation constants of gamma-ATP, gamma-ADP, gamma-AMP.PCP, and gamma-phosphorylase and the maximum catalytic activity. The mutants Ser26-gamma, Ser29-gamma, Phe30-gamma, and Gly31-gamma each exhibited an essentially identical dissociation constant for gamma subunit phosphorylase, indicating that these mutations had not caused a global alteration in the protein structure but were limited to changes in the nucleotide binding site domain. Substitution of either Val33 (by Gly) or Gly28 (by Ser), two of the most conserved residues in all protein kinases, resulted in enzyme with marginally detectable activity. In noted contrast, the Ser26 mutant, which substituted the first glycine of the consensus glycine trio motif, and which is also very highly conserved, retained at least 25% of the enzymatic activity. The Gly31 substitution, which restored a glycine to a position characteristic for most protein kinases, had little overall effect upon the maximum rate of catalysis. Restoration of Ser30 to the more typical phenylalanine, which is present in most protein kinases, had minimal effect on catalysis. These data provide the first direct evaluation of the roles that different residues play within this consensus glycine trio/valine motif of the protein kinases, which up to now have only been surmised to be of importance because of their conservation. Two unexpected findings are that for one residue that is very conserved (Gly26) there is some flexibility of substitution not apparent from the evolutionary conservation and that a second quite conserved residue in protein kinases (equivalent to Gly at position 31) does not produce a protein optimized for nucleotide binding.
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PMID:Analysis by mutagenesis of the ATP binding site of the gamma subunit of skeletal muscle phosphorylase kinase expressed using a baculovirus system. 142 Jan 77

Human polymorphonuclear neutrophils (PMN) respond to ATP with an elevation in intracellular calcium and a marked enhancement of O2-production in response to stimulation by the chemotactic peptide N'-formyl-Met-Leu-Phe (FMLP). These pertussis toxin-sensitive pathways appear to be mediated by a nucleotide receptor(s) on the surface of human PMN. In the current study, we have examined the binding to intact human PMN of the ATP analog, adenosine 5'-O-(3-thio[35S] triphosphate) [( 35S]ATP gamma S). On the basis of Scatchard analysis, the binding of [35S]ATP gamma S involves at least two sites, one of high and one of low affinity. In the presence of sodium thiophosphate, a compound which did not affect intracellular increases in calcium induced by ATP or N'-formyl-Met-Leu-Phe, a significant fraction of the [35S]ATP gamma S binding was eliminated. This reduction involved both high and low affinity binding of [35S]ATP gamma S and was related to a reduction in numbers of binding sites. The Kd values for the high affinity binding site were unaffected by the presence of sodium thiophosphate, although the low affinity Kd values were numerically increased by 2-fold. In the presence of thiophosphate, [35S]ATP gamma S binding was specific, saturable, and reversible, and was related to a single class of high affinity (Kd = 36 +/- 19 nM) binding sites (184 +/- 144 sites/cell), together with a second class of low affinity (Kd = 1110 +/- 503 nM) binding sites (13,562 +/- 6,851 sites/cells). Competitive binding experiments, based on the ability of nucleotides and ATP analogs to block [35S]ATP gamma S binding to PMN, revealed a rank order of ATP gamma S greater than ATP greater than 2-MeS-ATP = 8-Bromo ATP greater than ADP = ITP greater than AMP-PCP = GTP much greater than CTP. A comparison between the ability of nucleotides to compete with [35S]ATP gamma S binding and their ability to induce a biologic response (elevation of intracellular calcium) revealed a close correlation (r2 = 0.83). These findings support the possibility of a common nucleotide PMN receptor functionally linked to a cellular response which involves increases in intracellular calcium.
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PMID:Adenosine-5'-O-(3-thiotriphosphate) binding to human neutrophils. Evidence for a common nucleotide receptor. 165 77

Bradykinin (BK) (Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) was degraded by rat brain synaptic membranes at a rate comparable to that found for Met-enkephalin, but approximately 40 times the rate for vasopressin and oxytocin. The catabolic pathway for BK and its metabolites was elucidated through the use of high performance liquid chromatography for metabolite identification and peptidase inhibitors for blocking specific cleavage sites. BK was hydrolyzed at three sites: at the -Phe5-Ser6- bond by metalloendopeptidase 24.15, at the -Pro7-Phe8- bond by an apparently novel peptidyl dipeptidase, and at the -Phe8-Arg9 bond by a carboxypeptidase B-like enzyme. Each enzyme contributed about equally to BK degradation under the assay conditions used. Some of the resulting metabolites were further hydrolyzed: BK(1-8) to BK(1-7) + Phe by a DFP inhibitable prolyl carboxypeptidase-like enzyme, BK(1-8) to BK(1-5) + BK(6-8) by metalloendopeptidase 24.15, BK(1-7) slowly to BK(1-5) by a second peptidyl dipeptidase which was captopril inhibited, and Phe-Arg to Phe + Arg by a bestatin-inhibited dipeptidase. A number of properties of the individual enzymes were determined including sensitivity to a variety of peptidase inhibitors. These results provide a starting point for investigating the potential physiological role of each enzyme in BK function in the brain.
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PMID:Degradation of bradykinin and its metabolites by rat brain synaptic membranes. 260 54

In this paper we report that while 55% of the total post-proline dipeptidyl-aminopeptidase activity in guinea-pig brain is associated with the soluble fraction of the cells, the remaining activity is widely distributed throughout the particulate fractions. A significant portion of this particulate activity is, however, associated with a synaptosomal membrane fraction. The specific activity of this enzyme rose as the synaptosomal membrane fraction was prepared from a synaptosomal fraction and had previously risen at the synaptosomal fraction was prepared from a postmitochondrial pellet. The synaptosomal membrane post-proline dipeptidyl-aminopeptidase was released from the membrane by treatment with Triton X-100 and partially purified by chromatography on Sephadex G-200. By contrast with the soluble enzyme the partially purified solubilised synaptosomal membrane post-proline dipeptidyl-aminopeptidase was not inhibited by 1.0 mM p-chloromercuribenzoate, 1.0 mM N-ethylmaleimide or 0.5 mM puromycin but was inhibited by 0.5 mM bacitracin. The partially purified solubilised enzyme was capable of releasing His-Pro from His-Pro-Val, His-Pro-Leu, His-Pro-Phe and His-Pro-Tyr and of releasing Gly-Pro from Gly-Pro-Ala but could not release Arg-Pro from Arg-Pro-Pro or from Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg (bradykinin). It was also unable to release Pro-Pro from Pro-Pro-Gly or Glp-Pro from Glp-Pro-Ser-Lys-Asp-Ala-Phe-Ile-Gly-Leu-MetNH2 (eledoisin). Using [Pro-3H]thyroliberin we show that the membrane-bound enzyme converts His-ProNH2, produced by the action of the synaptosomal membrane pyroglutamate aminopeptidase, to His-Pro thus competing with the spontaneous cyclisation of His-ProNH2 to His-Pro diketopiperazine. Purified preparations of synaptosomal membrane pyroglutamate aminopeptidase were used to generate His-ProNH2, which could then be converted to His-Pro by the presence of the partially purified synaptosomal membrane post-proline dipeptidyl-aminopeptidase. This preparation was free of contaminating post-proline cleaving endopeptidase, carboxypeptidase P, aminopeptidase P, prolyl carboxypeptidase or proline dipeptidase.
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PMID:Post-proline dipeptidyl-aminopeptidase from synaptosomal membranes of guinea-pig brain. A possible role for this activity in the hydrolysis of His-ProNH2, arising from the action of synaptosomal membrane pyroglutamate aminopeptidase on thyroliberin. 286 1

The potent opiate radioligands [3H]etorphine, [3H]ethylketocyclazocine (EKC), and [3H]naloxone, bound specifically and saturably to a single class of membrane-binding sites in rat neurointermediate lobe (NIL), with Kd values of 3.7, 24, and 51 nM, respectively. In the hypothalamus (Ht), [3H]etorphine bound to specific and saturable sites with a Kd of 2.9 nM. Binding-inhibition studies with [3H]etorphine and unlabeled etorphine-HCl as well as [3H]EKC and unlabeled EKC, revealed high and low affinity binding sites in rat Ht and NIL as well as in the neural lobe of the bovine pituitary gland. [3H]naloxone also bound specifically to two classes of sites in Ht membranes, but to only a single class of low affinity sites in NIL membranes. Specific binding represented 80-90% of total [3H]etorphine binding, about 75% of total [3H]EKC binding, and 45-55% of total [3H]naloxone binding at 22 C in NIL and Ht, respectively. Relative binding potencies derived from Ki values for binding-inhibition studies of [3H]etorphine with opioid peptides and opiates were: NIL, etorphine-HCl greater than dynorphin A greater than naloxone-HCl greater than dynorphin-(1-9) greater than beta-endorphin much greater than alpha-neoendorphin approximately (Leu5)enkephalin approximately DAGO (Tyr-D-Ala-Gly-NMe-Phe-Gly-ol); Ht, etorphine HCl greater than naloxone-HCl greater than beta-endorphin greater than dynorphin A much greater than DAGO greater than morphiceptin much greater than (Leu5)enkephalin. Specific [3H]etorphine binding was also demonstrable after preincubation of NIL membranes with DAGO and (Leu5)enkephalin and after preincubation of Ht membranes with morphiceptin and (Leu5)enkephalin; such binding could be displaced by nonradioactive dynorphin A. In addition, [3H]etorphine binding to bovine neural lobe was displaceable by naloxone-HCl, with an ED50 of 43 nM. Specific ligands for sigma-opiate receptors, such as (+)SKF 10,047 (N-allylnorcyclazocine), phencyclidine (PCP), and (-)cyclazocine, displaced specifically bound [3H]etorphine and [3H]EKC from NIL membranes only at high (micromolar) concentrations. However, specific [3H]PCP sites were of higher affinity in NIL and Ht membranes, with similar Kd values of 102 and 190 nM respectively, and different concentrations (0.15 and 1.32 pmol/mg protein, respectively). These data have revealed several differences in the opiate-binding properties of rat Ht and NIL membranes.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Opiate receptor subtypes in the rat hypothalamus and neurointermediate lobe. 303 71

Fumarases in the mitochondrial and cytosolic fractions of rat liver were separately purified and crystallized. These two fumarases were not distinguishable in physicochemical, catalytic, or immunochemical properties. The sequences of seven amino acids in the C-terminal portions of the two fumarases were shown using carboxypeptidase P to be identical, i.e.-Val-Asp-Glu-Thr-Ala-Leu-Lys-. The amino acid sequence of the N-terminal portion of the mitochondrial fumarase was determined by the Edman method as Ala-Gln-Gln-Asn-Phe-Glu-Ile-Pro-Asp-, but that of the cytosolic fumarase could not be determined by the Edman method, since the N-terminal amino acid was blocked. The N-terminal amino acid of the cytosolic fumarase was identified as N-acetyl-alanine by analysis of the acidic amino acid produced by digestion of the enzyme protein with pronase E, carboxypeptidase A and B. Then the sequence of five amino acids in the N-terminal portion was determined by analyzing the acidic peptide obtained by limited proteolysis of the enzyme protein with carboxypeptidase A as Ac-Ala-Ser-Gln-Asn-Ser-. Peptide mapping of the tryptic peptides obtained from the mitochondrial and cytosolic fumarases showed no difference in the amino acid sequences of the two except in their N-terminal portions. The turnover rates of the mitochondrial and cytosolic fumarases were determined by injecting L-[U-14C]leucine into rat and following the decay of specific radioactivity incorporated into immunoprecipitates from the partially purified enzyme. The half-life of the cytosolic fumarase was estimated as 4.8 days from the decay curve of its specific radioactivity. The decay curve of the specific radioactivity of the mitochondrial fumarase, obtained after a single injection of L-[U-14]leucine, was quite unusual: its specific radioactivity remained constant for about 7 days after pulse labeling, and then decreased exponentially with a half-life of 9.7 days. Similar amounts of cytosolic and mitochondrial fumarase were found in the livers of the rat, mouse, rabbit, dog, chicken, snake, frog, and carp, respectively. Similar subcellular distributions of the enzyme were also found in the kidney, heart, and skeletal muscle of rats, and in hepatoma cells (AH-109A). However, in rat brain no fumarase activity was detected in the cytosolic fraction. Two putative precursor polypeptides of rat liver fumarase were synthesized when rat liver RNA was translated in vitro in a rabbit reticulocyte lysate system.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Mechanism of synthesis and localization of mitochondrial and cytosolic fumarases in rat liver. 381 85

Carboxypeptidase P has been purified by immunoaffinity chromatography from pig kidneys. A single-step assay with Z-Pro-Met (where Z represents benzyloxycarbonyl) as substrate was used, methionine being determined by using L-amino acid oxidase and horseradish peroxidase. The enzyme constitutes about 1.5% of the kidney microvillar proteins. Triton X-100-solubilized and papain-released forms of the enzyme were isolated. The former had an apparent subunit Mr of 135 000, and the latter form contained two polypeptide chains of Mr 128 000 and 95 000. The undenatured forms were dimeric proteins. In common with other microvillar hydrolases, carboxypeptidase P was a glycoprotein and each subunit contained one Zn atom. MnCl2 (1 mM) in the assay was necessary for maximum activity; in its absence, 0.5 mM-ZnSO4 produced a limited activation, but was inhibitory at higher concentrations. The Km for Z-Pro-Met, in the presence of MnCl2, was 4.1 mM, and the kcat. for freshly prepared enzyme was 1230 min-1. The enzyme lost activity during storage at -20 degrees C. In a limited survey of peptides, hydrolysis was observed only with substrates containing a proline, alanine or glycine residue in the P1 position, and these included angiotensins II and III. The best substrate in this series was Val-Ala-Ala-Phe.
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PMID:Proteins of the kidney microvillar membrane. Purification and properties of carboxypeptidase P from pig kidneys. 403 59

The translocation of the mRNA in relation to the ribosome during peptide synthesis represents an example for a mechanochemical reaction in which the chemical bond energy of GTP is transformed into coordinated motion. We demonstrate here that translocation can be explained simply by binding equilibria between the tRNA, the mRNA, and their binding sites on the ribosome. The presence of two cognate tRNAs shifts the association constant for the 70 S ribosome . AUGU3 complex from 6.8 x 10(5) to 2.2 x 10(8) M-1. The elongation factor G and GTP or guanosine-5'-(beta,gamma-methylene)triphosphate GMP-PCP) displace the methionine tRNAs which can be formylated (tRNAfMet) from the quaternary complex 70 S . AUGU3 . tRNAfMet . tRNAPhe. Only the ternary complex Phe-tRNAPhe . elongation factor Tu . GMP-PCP shows an absolute preference for the aminoacyl-tRNA binding site (A site) (K a = 6.6 x 10(6) M-1). AcPhe-tRNAPhe, (N alpha-acetylphenylalanyl-tRNA) an analogue of a peptidyl-tRNA exhibits a 20-fold higher affinity to the peptidyl-tRNA binding site (P site) (K a = 3.5 x 10(6) M-1) as against the A site (K a = 1.8 x 10(6) M-1) at 8 mM Mg2+. Compared to aminoacyl-tRNA and tRNA, peptidyl-tRNA shows a 3- to 15-fold higher affinity toward complementary oligonucleotides both in the binary complex and in the presence of 70 S ribosomes (UUCA . AcPhe-tRNAPhe: K a = 1.9 x 10(5) M-1), UUCA . tRNAPhe:K a = 3.2 x 10(4) M-1). This indicates a stabilization of the peptidyl-tRNA . mRNA complex during translocation. Our data support a concept of mRNA translocation in which the removal of the deacylated tRNA from the P site requires GTP energy and a peptidyl-tRNA . mRNA complex diffuses from its low affinity site (A) to its high affinity binding site (P).
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PMID:Mechanism of translocation. Binding equilibria between the ribosome, mRNA analogues, and cognate tRNAs. 703 57


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