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)

Antibodies raised in rabbits to detergent-solubilized pig kidney microvillar proteins have been used to investigate the membrane hydrolases by crossed immunoelectrophoresis. Eight enzymes were detected by specific staining methods: aminopeptidase M, dipeptidylpeptidase IV, neutral endopeptidase, aminopeptidase A, carboxypeptidase P, gamma-glutamyltransferase, trehalase and phosphodiesterase I. The mobility of all these enzymes, with the exception of trehalase and neutral endopeptidase, was increased by treatment of the detergent-solubilized preparation with papain. The difference between the detergent and proteinase forms of these enzymes is attributed to the removal of a small, non-antigenic peptide to which detergent is bound in significant quantities. This interpretation was further supported by experiments in which the microvillus fraction was labelled with an intramembrane photolabelling reagent, 1-azido-4-[125I]iodobenzene. After photolysis, the radioactivity in the membrane could be solubilized by detergent treatment but not by papain treatment. Radioautography after crossed charge-shift immunoelectrophoresis showed a good correlation between charge-shift (signifying the presence of detergent bound to a hydrophobic domain) and the presence of the label.
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PMID:Proteins of the kidney microvillar membrane. Immunoelectrophoretic analysis of the membrane hydrolases: identification and resolution of the detergent- and proteinase-solubilized forms. 48 90

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 interaction of phencyclidine (PCP) with its specific receptor sites in the central nervous system has been further characterized. Kinetic association and dissociation rate constants of 2.9 X 10(6) M-1 and 4.8 X 10(-1) min-1 were determined, yielding a kinetic KD of 1.6 X 10(-7) M, in agreement with the KD previously determined at equilibrium. Permissible separation time of 13 s was calculated from the kinetic data, well above the actual separation time of less than 10 s in the rapid filtration assay. Presoaking of filters in 0.01% poly-L-lysine eliminated displacable [3H]PCP adsorption to filter material. Binding data obtained via centrifugation assays was identical to that obtained with the rapid filtration method. Stereospecificity of the PCP receptor was demonstrated by the finding that (+)-ketamine is four-fold more potent than (-)-ketamine in displacing specifically bound [3H]PCP. Several proteolytic enzymes including trypsin, papain and thermolysin potently inactivated PCP receptors. Detailed regional distribution studies showed highest density of PCP receptors in subicular cortex and hippocampus, intermediate levels in hypothalamus, striatum, frontal cortex and cerebellum, lower levels in brainstem and spinal cord, and negligible levels in corpus callosum, a white-matter control area. Benzomorphan opiates with PCP-like behavioral effects interact with the PCP receptor. These data support the pharmacological relevance of the PCP receptor site as demonstrated by the rapid filtration method.
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PMID:Specific binding of [3H]phencyclidine in rat central nervous tissue: further characterization and technical considerations. 629 64

The purpose of these studies was to explore the use of phencyclidine (PCP)-specific high affinity antibodies as a possible treatment for phencyclidine toxicity. High affinity (Kd = 1.8 nM) anti-PCP monoclonal Fab fragments were purified from papain digested anti-PCP immunoglobulin produced in mouse ascites. Control animals (n = 5) received an i.v. bolus dose of 1 mg/kg of PCP, along with a tracer dose of 250 microCi of [3H]PCP. Fab-treated rats (n = 5) also received this PCP dose, but at 2 hr after dosing (when PCP distribution was complete) they received an equimolar dose of anti-PCP Fab (50 mg). Within 5 min after the anti-PCP Fab administration, serum [3H]PCP concentrations increased approximately 60- to 100-fold. Fab treatment caused the [3H]PCP volume of distribution at steady state to decrease from 12.6 +/- 3.0 liters/kg (mean +/- S.D.) in control animals to 0.6 +/- 0.2 liters/kg in the Fab-treated animals (about 5% of control values). Systemic clearance changed from 66.3 +/- 16.9 to 6.8 +/- 2.8 ml/min/kg (about 10% of control values). Because both volume of distribution and systemic clearance decreased to a similar degree, the terminal elimination half-life did not change significantly (3.9 hr in controls vs. 4.9 hr in treated animals, harmonic means). Renal clearance decreased from 1.8 +/- 0.6 to 0.62 +/- 0.17 ml/min/kg after Fab treatment. The anti-PCP Fab caused the percentage of PCP recovered in urine to increase from 2.5 +/- 0.5 to 10.3 +/- 4.7%.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Anti-phencyclidine monoclonal Fab fragments markedly alter phencyclidine pharmacokinetics in rats. 801 50

The RNA polymerase gene of murine coronavirus MHV-JHM encodes a polyprotein of greater than 750 kDa. This polyprotein is proposed to be processed by two papain-like cysteine proteinases, PCP-1 and PCP-2, and a poliovirus 3C-like proteinase domain, 3C-pro, to generate protein products. The amino-terminal product of the MHV polymerase polyprotein, p28, is generated by cleavage of the polyprotein by PCP-1. To identify the viral products downstream of p28, we generated a fusion-protein specific antiserum directed against the region adjacent to p28 and used the antiserum to detect virus-specific proteins from MHV-JHM infected cells. When this antiserum was used to immunoprecipitate radiolabeled proteins from MHV-JHM infected cell lysates, virus-specific proteins of 72 and 65 kDa were detected. Furthermore, pulse and chase experiments demonstrated that p72 is likely a precursor to the mature protein product, p65. To investigate which viral proteinase may be responsible for generating p72 and p65, we expressed the 5'-region of the MHV-JHM RNA polymerase gene including the two papain-like cysteine proteinase domains in an in vitro transcription/translation system and analyzed the translation products for proteolytic processing. We also cloned and expressed the 72 kDa region immediately downstream from p28, and tested the ability of in vitro translated PCP-1 and PCP-2 to cleave p72 to p65 in trans. Our results indicate that neither viral proteinase domain PCP-1 nor PCP-2 is capable of cleavage of p72 to produce p65 in vitro. The role of MHV proteinases in the processing of p72 and p65 is discussed.
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PMID:Identification of the polymerase polyprotein products p72 and p65 of the murine coronavirus MHV-JHM. 889 45

Expression of the coronaviral gene 1 polyproteins, pp 1a and pp 1ab, involves a series of proteolytic events that are mediated by virus-encoded proteinases similar to cellular papain-like cysteine-proteinases and the 3C-like proteinases of picornaviruses. In this study, we have characterized, in vitro, the human coronavirus HCV 229E papain-like cysteine-proteinase PCP 1. We show that PCP 1 is able to mediate cleavage of an aminoterminal polypeptide, p9, from in vitro translation products representing the aminoproximal region of pp 1a/pp 1ab. Mutagenesis studies support the prediction of Cys1054 and His1278 as the catalytic amino acids of the HCV 229E PCP 1, since mutation of these residues abolishes the proteolytic activity of the enzyme.
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PMID:Characterization of a papain-like cysteine-proteinase encoded by gene 1 of the human coronavirus HCV 229E. 978 76

ORF 1a of Beet yellows closterovirus (BYV) encodes the domains of the papain-like proteinase (PCP), methyltransferase (MT) and RNA helicase. BYV cDNA inserts encoding the PCP-MT region were cloned in pGEX vectors next to the glutathione S-transferase gene (GST). In a 'double tag' construct, the GST-PCP-MT cDNA was flanked by the 3'-terminal six histidine triplets. Following expression in E. coli, the fusion proteins were specifically self-cleaved into the GST-PCP and MT fragments. MT-His(6) was purified on Ni-NTA agarose and its N-terminal sequence determined by Edman degradation as GVEEEA, thus providing direct evidence for the Gly(588)/Gly(589) bond cleavage. The GST-PCP fragment purified on glutathione S-agarose was used as an immunogen to produce anti-PCP monoclonal antibodies (mAbs). On Western blots of proteins from virus-infected Tetragonia expansa, the mAbs recognized the 66 kDa protein. Immunogold labelling of BYV-infected tissue clearly indicated association of the PCP with the BYV-induced membranous vesicle aggregates, structures related to closterovirus replication.
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PMID:Processing and subcellular localization of the leader papain-like proteinase of Beet yellows closterovirus. 1286 60