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)

This paper describes the glycosylation sites of kappa-casein component P-5 from bovine mature milk. A short glycopeptide was prepared from kappa-casein component P-5 containing two carbohydrate chains by pronase P digestion, followed by gel filtration and ion exchange chromatographies. The glycopeptide obtained corresponded to residues 128-141 (Gly-Glu-Pro-Thr-Ser-Thr-Pro-Thr-Thr-Glu-Ala-Val-Glu-Ser) of kappa-casein A from the results of analyses with chemical and enzymatic procedures. The effect of alkaline borohydride treatment indicated the presence of serine as well as threonine as the binding site of carbohydrate moieties. From the facts of Edman degradation and carboxypeptidase P hydrolysis of glycopeptide treated with alkali, the carbohydrate moieties were considered to be attached to threonine residue No. 133 and serine residue No. 141.
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PMID:Presence of O-glycosidic linkage through serine residue in kappa-casein component from bovine mature milk. 679 4

The activity of prolylcarboxypeptidase (PCP), or angiotensinase C, was measured in lung tissues, leukocytes, and cultured human cells using Cbz-Pro-[14C]Ala as a substrate. A lysosomal fraction of homogenized rat or human lung contained most of the PCP activity in that tissue. Polymorphonuclear neutrophils, macrophages, and lymphocytes isolated from human blood had PCP activity. Fibroblasts cultured from human tissues had the highest activity (0.56-1.15 mumol/h per 10(6) cells), more than endothelial cells cultured from human pulmonary arteries. PCP of cultured human fibroblasts was similar to the human renal enzyme because it was resistant to moderate heating and was not inhibited by p-chloromercuriphenyl sulfonic acid. These properties and the substrate specificity distinguish PCP from cathepsin A, which is also in fibroblasts. Antibody to human renal PCP reacted with fibroblast PCP in immunofluorescence, indicating common antigenic determinants. Hydrocortisone changed PCP activity in fibroblasts in parallel with changes in beta-glucuronidase activity and cell-protein concentration; the activity was depressed at low concentration of the hormone. PCP activity was also found in synovial fluid from arthritic joints and in fibroblasts from the synovium. That PCP is found in both inflammatory exudates and in cells that appear at sites of inflammation indicates that, in addition to inactivating angiotensins, this enzyme may have a role in inflammation.
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PMID:Prolylcarboxypeptidase (angiotensinase C) in human lung and cultured cells. 745 50

Lysine 318 in the conserved sequence SXXXGXGKS of bacteriophage T7 gene 4A' protein was mutated to an alanine to understand the effect of this substitution on the helicase and primase activities. The dTTPase activity of 4A'/K318A mutant protein was much lower than that of 4A', and both Km and kcat values were affected. The Km of the mutant protein was 3-5-fold higher, and the kcat was about 100-fold lower, than that of 4A'. The mutation did not affect the ability of 4A'/K318A to assemble into hexamers or bind DNA in the presence of MgdTTP. Interestingly, the mutant protein does not bind DNA in the presence of MgdTMP-PCP. The reduced dTTPase activity, however, decreased the helicase activity of the mutant protein to an undetectable level, whereas its primase activity was only 1.5-2.5-fold lower. When 4A'/K318A mutant protein was mixed with 4A', heterooligomers were formed and the helicase and the DNA-dependent dTTPase activities of 4A' were inhibited, but the DNA-independent activity actually increased. The extent of decrease in activities upon heterooligomer formation depended both on the length of time 4A' and 4A'/K318A proteins were incubated and on the concentration of the mutant protein. In addition, the decrease in the dTTPase activity was observed only when the two proteins were incubated in the absence of MgdTTP and DNA, conditions under which both proteins form unstable hexamers. Even though 4A'/K318A does not bind a 30-mer DNA in the presence of MgdTMP-PCP, heterooligomers were capable of binding DNA with the same stoichiometry as 4A'. Protein-DNA cross-linking experiments with (dT)30 and poly(5-BrdU) showed that DNA interacts with five and perhaps all six subunits of 4A'. Therefore, unless heterooligomer restores the ability of the mutant protein to bind DNA in the presence of MgdTMP-PCP, these results suggest that the DNA can bind 4A' by interacting with a few subunits. However, a fully active hexamer is required for both the helicase and the single-stranded M13 DNA-dependent dTTPase activities.
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PMID:The K318A mutant of bacteriophage T7 DNA primase-helicase protein is deficient in helicase but not primase activity and inhibits primase-helicase protein wild-type activities by heterooligomer formation. 801 49

Intracerebroventricular injection of the D-forms of alanine (Ala; 2-200 micrograms/rat) and serine (Ser; 20-2000 micrograms/rat) caused a dose-dependent inhibition of the ability of 10 mg/kg of phencyclidine (PCP; given i.p.) to increase automatically quantitated locomotor counts and cumulated scores of locomotion, stereotypy and ataxia for 90 min after PCP administration. D-Ala and D-Ser were found to be more potent than the corresponding L-isomers in attenuating the PCP-induction of these behavioral abnormalities. Although L-, but not D-Ser, at moderate doses (400 micrograms/rat) produced a slight decrease in cumulative ataxia scores after a 10-mg/kg PCP administration, D-, but not L-Ser, reduced the behavioral scores at large doses (more than 1000 micrograms/rat). Similarly, bilateral i.c.v. infusion of D-Ala (140 micrograms/rat) reduced the increasing effects of a lower dose of PCP (5 mg/kg i.p.) on locomotion, stereotypy and ataxia scores, whereas the L-form of Ala (140 micrograms/rat) lacked the inhibitory influence. The stereo-selectivity of the antagonism by Ala and Ser of PCP-induced abnormal behavior parallels that of the potencies of these amino acids as agonists for the strychnine-insensitive glycine site linked to the N-methyl-D-aspartate type excitatory amino acid receptor. Furthermore, the decreasing effects of D-Ala (200 micrograms/rat i.c.v.) and D-Ser (2000 micrograms/rat i.c.v.) on PCP-induced hyperactivity were antagonized by i.c.v. application of 5,7-dichlorokynurenate and 7-chlorokynurenate which are selective antagonists of the glycine modulatory site.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Stereoselective antagonism by enantiomers of alanine and serine of phencyclidine-induced hyperactivity, stereotypy and ataxia in the rat. 801 48

1. We have investigated the effects of a schizophrenomimetic drug phencyclidine (PCP) and N-methyl-D-aspartate (NMDA)-related agents alone or in combination on dopamine metabolism in the medial prefrontal cortex and striatum of the rats by measuring the tissue concentrations of dopamine and its metabolite, 3,4-dihyroxyphenylacetic acid (DOPAC), and the rate of dopamine disappearance (dopamine utilization) after its synthesis inhibition. 2. Systemic injection of PCP and selective, non-competitive, NMDA antagonists caused an increase of both tissue concentrations of DOPAC and dopamine utilization in the prefrontal cortex but not in the striatum. The PCP-induced augmentation of cortical dopamine metabolism was not influenced by selective lesion of ascending noradrenergic neurones. 3. Intra-prefrontal cortical infusion of PCP or selective competitive or non-competitive antagonists of the NMDA receptor mimicked the ability of systemic PCP injection to enhance DOPAC levels and dopamine utilization in the prefrontal cortex. However, an NMDA antagonist injected into the cell body area of the mesocortical dopaminergic neurones failed to affect dopamine metabolism in the prefrontal cortex. 4. The increasing effects of PCP and selective NMDA antagonists on cortical dopamine utilization were not additive, although a dopamine receptor antagonist, haloperidol, still accelerated the disappearance of dopamine, even in the presence of PCP. 5. Intra-cortical or intra-ventricular infusion of NMDA or D-alanine but not L-alanine, attenuated the ability of systemic PCP administration to facilitate prefrontal dopamine utilization. 6. These data suggest that PCP might activate prefrontal cortical dopaminergic neurones, at least in part, by blocking the NMDA receptor in the prefrontal cortex which participates in a tonic inhibitory control of the mesoprefrontal dopaminergic projections.
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PMID:Characterization of the phencyclidine-induced increase in prefrontal cortical dopamine metabolism in the rat. 964 56

The hepatitis C virus NS3 gene encodes a RNA helicase with several sequence motifs conserved among the members of the DExH box protein family. The contributions of the sequence motifs to enzyme activity were assessed in this study by substitution of alanine for the Lys in the ATP binding motif GxGK (referred to as K1236A mutation), or for the Asp in the DExH motif (D1316A), or for the Arg in the middle of the QRxGRxGR motif known for RNA binding (R1490A). Histidine-tagged recombinant proteins of Mr 54,000 were expressed in Escherichia coli and purified by chromatography on nickel agarose. All three mutants were severely defective in ATPase and RNA helicase activities, but loss of the ATPase activity was not dependent on polynucleotide cofactors. With the exception of R1490A mutant, a stable complex was formed between dsRNA substrates and recombinant proteins, indicating that the arginine-rich motif is required for efficient RNA binding. Complex formation was not affected by omission of ATP or substitution by a non-hydrolyzable analog AMP-PCP, suggesting that neither binding nor hydrolysis of ATP is required for RNA binding. Moreover, the K1236A mutant which was defective in binding ATP exhibited an unusually strong affinity for RNA duplex. These results suggest that the conserved motifs cooperatively constitute a large functional domain rather than act as individual domains with strictly independent functions, and that alteration of one motif affects functions of other motifs in a mutually interactive fashion.
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PMID:Functional interactions between conserved motifs of the hepatitis C virus RNA helicase protein NS3. 1049 48

Bacterial phosphoribulokinases (PRKs) are octameric members of the adenylate kinase family of enzymes. The enzyme is allosterically activated by NADH and allosterically inhibited by AMP. We have determined the crystal structure of PRK from Rhodobacter sphaeroides bound to the ATP analogue AMP-PCP to a resolution of 2.6 A. The structure reveals that the ATP analogue does not bind to the canonical ATP site found in adenylate kinase family members. Rather, the AMP-PCP binds in two different orientations at the interface of three of the monomers in the octamer. This interface was previously characterized as having an unusually large number of arginine residues. Of the five arginine residues that are near the bound nucleotide, one (Arg 221) is highly conserved in both prokaryotic and eukaryotic (nonallosterically regulated) PRKs, two (Arg 234 and Arg 257) are on a second subunit and conserved in only prokaryotic PRKs, and two (Arg 30 and Arg 31) are on a third subunit with only one of them (Arg 31) conserved in prokaryotic PRKs. Each of these arginine residues was converted by site-directed mutagenesis to alanine. Fluorescence binding data suggest that none of these arginines are involved in active site ATP binding and that Arg 234 and Arg 257 on the second subunit are directly involved in NADH binding, while the other arginines have a minimal effect on NADH binding. While the wild-type enzyme exhibits low maximal activity and hyperbolic kinetics with respect to ATP in the absence of NADH and high maximal activity and sigmoidal kinetics in the presence of NADH, the R31A mutant exhibits identical hyperbolic kinetics with respect to ATP in the presence or absence of NADH. Thus, the transmission of allosteric information from one subunit to another is conducted through a single path that includes NADH and Arg 31.
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PMID:Identification of the allosteric regulatory site in bacterial phosphoribulokinase. 1056 98

We determined the effect of nucleotides and protein kinase A (PKA) on the Ca(2+)-dependent gating of the cloned intermediate conductance, Ca(2+)-dependent K(+) channel, hIK1. In Xenopus oocytes, during two-electrode voltage-clamp, forskolin plus isobutylmethylxanthine induced a Ca(2+)-dependent increase in hIK1 activity. In excised inside-out patches, addition of ATP induced a Ca(2+)-dependent increase in hIK1 activity (NP(o)). In contrast, neither nonhydrolyzable (AMP-PNP, AMP-PCP) nor hydrolyzable ATP analogs (GTP, CTP, UTP, and ITP) activated hIK1. The ATP-dependent activation of hIK1 required Mg(2+) and was reversed by either exogenous alkaline phosphatase or the PKA inhibitor PKI(5-24). The Ca(2+) dependence of hIK1 activation was best fit with a stimulatory constant (K(s)) of 350 nM and a Hill coefficient (n) of 2.3. ATP increased NP(o) at [Ca(2+)] >100 nM while having no effect on K(s) or n. Mutation of the single PKA consensus phosphorylation site at serine 334 to alanine (S334A) had no effect on the PKA-dependent activation during either two-electrode voltage-clamp or in excised inside-out patches. When expressed in HEK293 cells, ATP activated hIK1 in a Mg(2+)-dependent fashion, being reversed by alkaline phosphatase. Neither PKI(5-24) nor CaMKII(281-309) or PKC(19-31) affected the ATP-dependent activation. Northern blot analysis revealed hIK1 expression in the T84 colonic cell line. Endogenous hIK1 was activated by ATP in a Mg(2+)- and PKI(5-24)-dependent fashion and was reversed by alkaline phosphatase, whereas CaMKII(281-309) and PKC(19-31) had no effect on the ATP-dependent activation. The Ca(2+)-dependent activation (K(s) and n) was unaffected by ATP. In conclusion, hIK1 is activated by a membrane delimited PKA when endogenously expressed. Although the oocyte expression system recapitulates this regulation, expression in HEK293 cells does not. The effect of PKA on hIK1 gating is Ca(2+)-dependent and occurs via an increase in NP(o) without an effect on either Ca(2+) affinity or apparent cooperativity.
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PMID:Kinase-dependent regulation of the intermediate conductance, calcium-dependent potassium channel, hIK1. 1061 55

Yersiniabactin (Ybt) synthetase is a three-subunit, 17-domain [7 domains in high molecular weight protein (HMWP)2, 9 in HMWP1, and 1 in YbtE] enzyme producing the virulence-conferring siderophore yersiniabactin in Yersinia pestis. The 350-kDa HMWP1 subunit contains a polyketide synthase module (KS-AT-MT(2)-KR-ACP) and a nonribosomal peptide synthetase module (Cy(3)-MT(3)-PCP(3)-TE). The full-length HMWP1 was heterologously overexpressed in Escherichia coli and purified to near homogeneity. The purified HMWP1 showed thioesterase activity toward acyl-CoAs, such as acetyl-CoA, benzoyl-CoA, and malonyl-CoA, with saturation kinetics and relative catalytic efficiencies of 172:50:1. A chain-releasing thioesterase (TE) activity is ascribed to the C-terminal TE domain, and this was substantiated by the fact that acyl-N-acetylcysteamines were hydrolyzed by the didomain PCP(3)-TE fragment of HMWP1. However, PCP(3)-TE failed to hydrolyze any of the acyl-CoAs, suggesting the TE domain does not recognize CoA moiety, thus the acyl-CoA hydrolysis by HMWP1 must involve other domains. Ser-to-Ala mutants in each of the AT, ACP, PCP(3), and TE domains reduced hydrolysis rates of the two fastest substrates, acetyl-CoA and benzoyl-CoA, by more than two orders of magnitude. Thus, the acyl-CoA hydrolysis activity requires 4 of the 9 domains of HMWP1, and it is consistent with autoacylation of the AT domain active site serine and subsequent passage of the itinerant acyl chain from AT to ACP to PCP(3) to the TE domain, a cascade of four sequential acyl-enzyme intermediates, for hydrolytic turnover. This could represent an editing pathway for this polyketide synthase/nonribosomal peptide synthetase assembly line.
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PMID:Acyl-CoA hydrolysis by the high molecular weight protein 1 subunit of yersiniabactin synthetase: mutational evidence for a cascade of four acyl-enzyme intermediates during hydrolytic editing. 1110 85

Dipeptidyl peptidase II (DPP II; EC 3.4.14.2) from rat kidney was purified to a specific activity of 65.4 micromol/min per mg of protein for Lys-Ala-beta-naphthylamide. The N-terminal and partial amino acid sequences of the enzyme were determined. The peptide sequences were used to identify expressed sequence tag (EST) clones. By using the cDNA fragment of one of the EST clones as a probe, we isolated a cDNA clone with 1710 bp encoding DPP II from a rat kidney cDNA library. The cDNA of rat DPP II contained an open reading frame of 1500 bp, coding for a protein of 500 amino acids. The first 10 residues of the purified enzyme matched the deduced protein sequence starting with residue 37, suggesting the presence of a signal peptide. The mature enzyme (464 residues) had a calculated molecular mass of 51400 Da, which was lower than the value (about 60000 Da) determined by SDS/PAGE; and the deduced amino acid sequence showed six potential N-glycosylation sites. The deduced amino acid sequence of rat DPP II shared high similarity with quiescent-cell proline dipeptidase (78% identity) and prolyl carboxypeptidase (38% identity) and bore the putative catalytic triad (Ser, Asp, His) conserved in serine peptidase families. We transiently transfected COS-7 cells with pcDNA3.1 containing the cloned cDNA and obtained the overexpression of an immunoreactive protein (of about 60000 Da). The transfected cells showed Lys-Ala-methylcoumarinamide-hydrolysing activity that was 50 times higher than the control cells.
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PMID:Cloning and functional expression of rat kidney dipeptidyl peptidase II. 1113 92


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