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)

Through a series of kinetic studies involving the inactivation effects of diisopropylfluorophosphate, an affinity label that modifies the active site serine residue involved in the mechanism of action, it has been firmly established that carboxypeptidase P (CPP) requires a serine residue for catalytic activity. The essential kinetic parameters were determined to be 1.33 mM for the apparent dissociation constant with a limiting half-life of inactivation of 20.1 min. Structural elucidation of the primary amino acid sequence surrounding the essential serine, and comparing that with the reactive site of carboxypeptidase Y (CPY), revealed a significant degree of homology at the active site between these two enzymes. These regions, however, were quite divergent from other known serine proteases, leading to the speculation that these serine exopeptidases may comprise a unique family in the overall classification of serine proteases. It was established that CPY could be inactivated with either of the classic histidine affinity labels tosylphenylalanylchloromethyl ketone (TPCK) or carbobenzoxyphenylalanylchloromethyl ketone (ZPCK) with Ki's of 1.2 and 12.8 microM, respectively. This is in marked contrast to CPP, which was unaffected by saturating levels of the known histidine affinity labels, TPCK, tosyllysylchloromethyl ketone, or ZPCK. This point may be a significant element in differentiating specificity among these two serine proteases. Further investigation into the structural nature of CPP revealed that it is a glycoprotein with a single site of carbohydrate attachment. In addition, the carbohydrate moiety itself appears to contribute 1217 Da to the overall molecular weight and it is characterized as an asparagine linked high mannose type. This is significantly different from CPY with its four sites of carbohydrate attachment contributing approximately 17% to its molecular weight.
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PMID:Structural determination of the essential serine and glycosylation sites of carboxypeptidase P. 157 19

Recent advances in glycobiology have greatly stimulated carbohydrate research; however, improving techniques for identification and isolation of specific glycosylation sites in protein structure analysis remains a challenge. We report here a practical approach utilizing a membrane staining technique on Problott, a PVDF-type membrane, to screen glycoproteins and glycopeptides derived from enzymatic digests of glycoproteins. To improve the detection sensitivity, an amplified staining technique using biotinylated lectins, avidin, and biotinylated peroxidase was employed. In addition, we describe a micro-batch affinity binding procedure to isolate glycopeptides from complex glycoprotein enzymatic digests. These protocols allow us to start with a subnanomole quantity of glycoprotein and locate the glycosylation sites; isolate glycopeptides in a homogeneous form; and perform amino acid composition, amino acid sequence, and mass analyses on the isolated glycopeptides. The characterization of glycosylation site of a model glycoprotein, carboxypeptidase P, of which the structure is still largely unknown, has been investigated.
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PMID:A general approach for characterizing glycosylation sites of glycoproteins. 172 19

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

In healthy male subjects (n = 12) phencyclidine (PCP) free fraction was 22.0 +/- 2.8 % (mean +/- SD). In male patients with mild to moderate alcoholic liver disease (n = 16) free fraction (23.0 +/- 3.4%) was of the same order as in healthy subjects although age and the concentrations of albumin, bilirubin, and high-density lipoproteins were different (P less than 0.05). Free fraction (76.2 /+- 0.06%) in fatty acid free human serum albumin (HSA, 4.4 gm/dl) was far greater than in plasma. Both the increased binding of PCP in plasma over HSA and the lack of a difference in PCP binding between normals and patients was associated with alpha 1-acid glycoprotein (alpha 1-AGP). This protein is an acute-phase reactant that binds cationic drugs and rises nonspecifically in a variety of diseases. Free fraction of PCP in alpha 1-AGP (75 mg/dl) was 36.4 +/- 1.7%. Half of the variance in PCP binding can be accounted for (r = 0.67, P less than 0.01) from percentage of free PCP = 39.24 - 2.18 (albumin) - 0.094 (alpha 1-AGP). Male rats (n = 14, weight = 251 +/- 7 gm) were alternatively assigned to pretreatment with either saline or alpha 1-AGP (11.6 mg) by cardiac puncture. PCP brain concentrations were reduced (11%, P less than 0.05) in the protein-treated group 5 min after cardiac 3H-PCP (0.17 mg) administration, demonstrating that increased plasma protein binding can reduce free drug concentration during the distribution phase and, thereby, the rate and extent of drug distribution.
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PMID:Plasma protein binding of phencyclidine. 705 8

A novel immunobiologically active fraction was prepared from a phenol-water extract of Prevotella intermedia ATCC 25611 by Sephadex G-100 column chromatography. The fraction consisted mainly of carbohydrate and protein and was devoid of fatty acid. The fraction showed high-molecular-weight bands (10,000 to 12,000) on deoxycholate polyacrylamide gel electrophoresis (DOC-PAGE) and was scarcely active in a Limulus test. We designated the fraction Prevotella glycoprotein (PGP). The PGP fraction showed strong mitogenicity on splenocytes and cytokine-inducing activities on peritoneal macrophages from both C3H/HeJ and C3H/HeN mice, and it stimulated human gingival fibroblasts to produce cytokines. The activities of the PGP fraction were resistant to heat inactivation (100 degrees C for 1 h) and protease treatments and were scarcely inhibited by polymyxin B. In contrast, the purified lipopolysaccharide fraction (LPS-PCP) extracted from the same bacterium with a phenol-chloroform-petroleum ether mixture, which showed strong Limulus activity and a single low-molecular-weight band (approximately 3,000) on DOC-PAGE, lacked the activities on splenocytes and macrophages from C3H/HeJ mice and human gingival fibroblasts. The activities of the LPS-PCP fraction on cells from C3H/HeN mice were completely inhibited by polymyxin B. The LPS extracted from the same bacterium with hot phenol-water (LPS-PW) exhibited the properties of both the PGP fraction and the LPS-PCP fraction. These findings suggest that the unique bioactivities of the LPS-PW fraction of oral black-pigmented bacteria reported to date, which differed from those of the classical endotoxin, were derived from the PGP fraction and not from the LPS itself.
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PMID:A novel component different from endotoxin extracted from Prevotella intermedia ATCC 25611 activates lymphoid cells from C3H/HeJ mice and gingival fibroblasts from humans. 935 30

Self-incompatibility (SI) in Brassica species is controlled by a single polymorphic locus (S) with multiple specificities. Two stigmatically expressed genes that have been cloned from this region encode the S locus glycoprotein (SLG) and S receptor kinase (SRK). Both appear to be essential for the operation of SI. It is believed that rejection of incompatible pollen grains is effected by recognition events between an as yet unidentified S locus-encoded pollen coating-borne protein and the SLG/SRK. We previously identified a small pollen coat protein PCP7 (renamed here PCP-A1, for pollen coat protein, class A, 1) that binds with high affinity to SLGs irrespective of S genotype. Here, we report the cloning of PCP-A1 from Brassica oleracea and demonstrate that it is unlinked to the S locus. In situ localization of PCP-A1 transcripts revealed that they accumulate specifically in pollen at the late binucleate/trinucleate stage of development rather than in the tapetum, which previously was taken to be the principal source of the pollen coat. PCP-A1 is characterized by the presence of a structurally important motif consisting of eight cysteine residues shared by the plant defensins. Based on the presence of this motif and other data, homology modeling has been used to produce a putative structure for PCP-A1. Protein-protein interaction analyses demonstrate that SLG exists in monomeric and dimeric forms, both of which bind PCP-A1. Evidence is also presented for the existence of putative membrane-associated PCP-A1 binding proteins in stigmatic tissue.
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PMID:PCP-A1, a defensin-like Brassica pollen coat protein that binds the S locus glycoprotein, is the product of gametophytic gene expression. 970 33

In Brassica, two self-incompatibility genes, encoding SLG (S locus glycoprotein) and SRK (S-receptor kinase), are located at the S locus and expressed in the stigma. Recent molecular analysis has revealed that the S locus is highly polymorphic and contains several genes, i.e., SLG, SRK, the as-yet-unidentified pollen S gene(s), and other linked genes. In the present study, we searched for expressed sequences in a 76-kb SLG/SRK region of the S(9) haplotype of Brassica campestris (syn. rapa) and identified 10 genes in addition to the four previously identified (SLG(9), SRK(9), SAE1, and SLL2) in this haplotype. This gene density (1 gene/5.4 kb) suggests that the S locus is embedded in a gene-rich region of the genome. The average G + C content in this region is 32.6%. An En/Spm-type transposon-like element was found downstream of SLG(9). Among the genes we identified that had not previously been found to be linked to the S locus were genes encoding a small cysteine-rich protein, a J-domain protein, and an antisilencing protein (ASF1) homologue. The small cysteine-rich protein was similar to a pollen coat protein, named PCP-A1, which had previously been shown to bind SLG.
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PMID:Genomic organization of the S locus: Identification and characterization of genes in SLG/SRK region of S(9) haplotype of Brassica campestris (syn. rapa). 1047 21

We have evaluated a PCR technique using primers based on Pneumocystis carinii major surface glycoprotein (MSG) genes, a multicopy gene family, for utility in detection of P. carinii in BAL and oropharyngeal samples obtained from immunosuppressed patients. These primers were able to detect P. carinii DNA in as little as 16 fg of genomic DNA. PCR using MSG primers detected P. carinii DNA in 7 smear-positive BAL samples (100% sensitivity), and found no P. carinii DNA in 12 smear-negative BAL samples (100% specificity). Mitochondrial ribosomal RNA (mrRNA) primers, commonly used in PCR studies of PCP, detected P. carinii in six of seven positive samples (85.7% sensitivity) and none of 12 were negative samples (100% specificity). Diagnosis of PCP by amplification of 81 oropharyngeal samples using MSG primers had a 50% sensitivity (4/8) and 96% specificity (70/73). PCR with mrRNA primers was 37.5% sensitive (3/8) and 100% specific (73/73). All three false-positive MSG results showed a very low intensity on Southern hybridization. PCR using MSG gene primers should prove valuable in the diagnosis of PCP.
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PMID:Development of a PCR assay for diagnosis of Pneumocystis carinii pneumonia based on amplification of the multicopy major surface glycoprotein gene family. 1052 78

Many flowering plants possess self-incompatibility (SI) systems that prevent inbreeding. In Brassica, SI is controlled by a single polymorphic locus, the S locus. Two highly polymorphic S locus genes, SLG (S locus glycoprotein) and SRK (S receptor kinase), have been identified, both of which are expressed predominantly in the stigmatic papillar cell. We have shown recently that SRK is the determinant of the S haplotype specificity of the stigma. SRK is thought to serve as a receptor for a pollen ligand, which presumably is encoded by another polymorphic gene at the S locus. We previously have identified an S locus gene, SP11 (S locus protein 11), of the S(9) haplotype of Brassica campestris and proposed that it potentially encodes the pollen ligand. SP11 is a novel member of the PCP (pollen coat protein) family of proteins, some members of which have been shown to interact with SLG. In this work, we identified the SP11 gene from three additional S haplotypes and further characterized the gene. We found that (i) SP11 showed an S haplotype-specific sequence polymorphism; (ii) SP11 was located in the immediate flanking region of the SRK gene of the four S haplotypes examined; (iii) SP11 was expressed in the tapetum of the anther, a site consistent with sporophytic control of Brassica SI; and (iv) recombinant SP11 of the S(9) haplotype applied to papillar cells of S(9) stigmas, but not of S(8) stigmas, elicited SI response, resulting in inhibition of hydration of cross-pollen. All these results taken together strongly suggest that SP11 is the pollen S determinant in SI.
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PMID:The pollen determinant of self-incompatibility in Brassica campestris. 1067 56

Poria cocos is an important Oriental medical fungus with multiple functionalities, yet its bioactive substances and the mechanisms involved have not been fully characterized. A novel immunomodulatory protein (P. cocos immunomodulatory protein; PCP) was purified from the dried sclerotium of P. cocos (Schw.) Wolf using DE-52 cellulose and gel filtration chromatography. Chromatography and electrophoresis results indicated that the native PCP (35.6 kDa) is a disulfide-linked heterodimeric glycoprotein consisting of 14.3 and 21.3 kDa subunits with N- and O-glycosylation. PCP was capable of stimulating RAW 264.7 macrophages in vitro through the induction of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1beta) as well as the regulation of nuclear factor-kappa B (NF-kappaB)-related gene expression. In primary mouse macrophages, PCP directly activated peritoneal cavity macrophages to induce Toll-like receptor 4 (TLR4)-mediated myeloid differentiation factor 88 (MyD88)-dependent signaling. This study demonstrated the cell surface interactions of PCP with TLR4 and the capacity of PCP for TLR4 tyrosine phosphorylation. Results obtained with peritoneal macrophages from TLR4-deficient C57BL/10ScN mice revealed that PCP-induced activation and PCP cell surface binding were significantly attenuated. Moreover, enzymatic deglycosylation decreased PCP-mediated responses, indicating that the glycosylated portion of PCP was a key factor in PCP signaling through TLR4 in peritoneal macrophages. These findings suggest that PCP is a new potential immune stimulator within P. cocos and that TLR4 is primarily responsible for PCP signaling in murine macrophages.
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PMID:A novel immunomodulatory protein from Poria cocos induces Toll-like receptor 4-dependent activation within mouse peritoneal macrophages. 1954 79


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