Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:O95477 (membrane-bound)
 29,236 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A neutral endoprotease was isolated from porcine antral mucosa and purified to homogeneity as examined by SDS-polyacrylamide gel electrophoresis (PAGE). Throughout the purification, t-butyloxycarbonyl-Arg-Val-Arg-Arg-4- methylcoumaryl-7-amide (MCA) was used as a substrate, which was found to be hydrolyzed specifically by the enzyme at the Arg-Arg bond. Unexpectedly, however, the enzyme was also found to hydrolyze vasoactive intestinal polypeptide (VIP) fairly specifically and more efficiently when various neuropeptides and related peptides were examined as substrates. It could degrade VIP by cleaving three peptide bonds not containing an arginine residue(s) with Km = 7.7 x 10(-6) M and kcat/Km = 7.4 x 10(6) M-1 s-1 (at pH 7.6 in the presence of 0.1% Lubrol PX), whereas only secretin, substance P, and a few others were hydrolyzed at much slower rates among the various peptides examined. Both activities toward the MCA substrate and VIP behaved in parallel throughout the purification procedures and showed essentially the same pH optimum and susceptibility toward various inhibitors and detergents. Therefore, both activities are thought to be due to the same enzyme. This endoprotease required 0.001% or a higher concentration of a detergent such as Lubrol PX or Triton X-100 for its maximal activity. Its optimum pH was about 7.5 and the molecular weight was estimated to be approximately 37,000 by SDS-PAGE. This enzyme was strongly inhibited by serine protease inhibitors such as diisopropyl-fluorophosphate and phenylmethanesulfonyl fluoride. It was also inhibited by p-chloromercuribenzoic acid, but not by some other cysteine protease inhibitors. Therefore, the enzyme appears to be most likely a kind of serine protease although its possibility as a cysteine protease cannot be completely excluded. Analysis of its cleavage specificity toward various oligopeptides indicated the possibility that the protease might recognize a specific amino acid sequence(s) and/or conformation in the vicinity of the cleavage site of the target peptide. Various characteristics of the endoprotease suggest that it is a novel membrane-bound neuropeptide-degrading endoprotease fairly specific for VIP.
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PMID:Purification and characterization of a vasoactive intestinal polypeptide-degrading endoprotease from porcine antral mucosal membranes. 771 70

Urokinase plasminogen activator (uPA) is a serine protease involved in cancer invasion and metastasis. uPA mediates its action while attached to a membrane-bound receptor (uPAR). In this investigation we show that uPAR levels correlate with uPA levels in human breast cancers. uPAR levels, however, do not correlate with other components of the plasminogen activator system such as tissue-type plasminogen activator (t-PA), PAI-I or PAI-2. In addition, uPAR levels showed no correlation with tumor size, axillary-node status or estrogen-receptor status. On the basis of an optimum cut-off point, patients with breast cancers containing high levels of uPAR had a worse prognosis than patients with low levels of the receptor. However, as a prognostic marker in breast cancer, uPAR was not as strong as uPA. Our results are consistent with data from model systems suggesting that both uPA and uPAR are necessary for metastasis.
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PMID:Urokinase plasminogen activator and urokinase plasminogen activator receptor in breast cancer. 776 29

The action of non-detergent sulphobetaines (NDSBs) as new mild agents for protein purification is described. The solubilization effects of non-detergent sulphobetaines are shown in different examples; all obtained under non-denaturing conditions: (1) microsomal proteins extraction; (2) recovery after dialysis of nuclear proteins; (3) reduction of precipitation in isoelectric focusing experiments under non-denaturing conditions; and (4) purification of a membrane-bound serine protease from Plasmodium falciparum involved in erythrocyte invasion by malaria merozoites. The absence of a significant denaturation effect induced by NDSBs is demonstrated by tests on beta-galactosidase and alkaline phosphatase. A simple NDSB synthesis and some possible explanations of the action of NDSBs are also presented.
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PMID:Non-detergent sulphobetaines: a new class of mild solubilization agents for protein purification. 782 51

A major histopathological hallmark of Alzheimer's disease (AD) is the presence of amyloid deposits in the parenchyma of the amygdala, hippocampus, and neocortex. The principal component of amyloid is the beta-amyloid protein (A beta), a 39-43 amino acid peptide composed of a portion of the transmembrane domain and the extracellular domain of the amyloid precursor protein (APP). APP occurs as several A beta-containing isoforms of 695, 751, and 770 amino acids, with the latter two APP containing a domain that shares structural and functional homologies with Kunitz serine protease inhibitors. In cultured cells, APP mature through the constitutive secretory pathway, and some cell surface-bound APP are cleaved by an enzyme, designated as alpha-secretase, within the A beta domain, an event that precludes A beta amyloidogenesis. Several studies have delineated two additional pathways of APP processing: first, an endosomal/lysosomal pathway generates a complex set of APP-related membrane-bound fragments, some of which contain the entire A beta sequence; and, second, by mechanisms which are not fully understood, A beta 1-40 is secreted into the conditioned medium in vitro and is present in cerebrospinal fluid in vivo. The intracellular sites of enzymes responsible for proteolytic cleavage at the NH2 and COOH termini of A beta, termed gamma- and beta-secretase, respectively, have not been identified. Finally, recent molecular genetic investigations have identified a variety of mutations in APP that segregate with early-onset familial AD and with hereditary cerebral hemorrhage with amyloid, Dutch type (HCHWA-D). Several of these mutations appear to influence APP processing and result in the production of higher levels or longer A beta-related peptides that are inherently more fibrillogenic. Although a variety of lines of evidence implicates APP/A beta in AD, the mechanisms by which A beta influences the biology and vulnerability of neural cells are not fully understood but are very active areas of investigation. This review focuses on the present state of our understanding of APP and A beta in the context of AD.
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PMID:Role of the beta-amyloid protein in Alzheimer's disease. 789 5

The human fibroblast activation protein alpha (FAP alpha) is a M(r) 95,000 cell surface antigen selectively expressed in reactive stromal fibroblasts of epithelial cancers, granulation tissue of healing wounds, and malignant cells of bone and soft tissue sarcomas. Normal adult tissues are generally FAP alpha-, but some fetal mesenchymal tissues transiently express the molecule. Because of its restricted normal tissue distribution and abundant expression in the stroma of over 90% of breast, colorectal, and lung carcinomas, FAP alpha is under clinical evaluation as a target for immunodetection and immunotherapy of epithelial cancers. In the present study, we have isolated a full-length cDNA for FAP alpha through expression cloning in COS-1 cells. The FAP alpha cDNA codes for a type II integral membrane protein with a large extracellular domain, transmembrane segment, and short cytoplasmic tail. FAP alpha shows 48% amino acid sequence identity to the T-cell activation antigen CD26, a membrane-bound protein with dipeptidyl peptidase IV (DPPIV) activity; however, unlike FAP alpha, CD26 is widely expressed in normal tissues. Three catalytic domains shared by DPPIV homologues in different species and by other serine proteases are conserved in FAP alpha. Immunochemical analysis of COS-1 cells coexpressing FAP alpha and CD26 revealed that the two molecules form heteromeric cell surface complexes, suggesting that a previously identified FAP alpha-associated M(r) 105,000 protein of cultured fibroblasts and growth factor-stimulated melanocytes, FAP beta, is identical to CD26. In vivo coexpression of FAP alpha and CD26 is found in reactive fibroblasts of healing wounds but not in tumor stromal fibroblasts or sarcomas (FAP alpha +/CD26-). The putative serine protease activity of FAP alpha and its in vivo induction pattern may indicate a role for this molecule in the control of fibroblast growth or epithelial-mesenchymal interactions during development, tissue repair, and epithelial carcinogenesis.
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PMID:Molecular cloning of fibroblast activation protein alpha, a member of the serine protease family selectively expressed in stromal fibroblasts of epithelial cancers. 791 Dec 42

Transforming growth factor-alpha (TGF-alpha) is synthesized and transported to the cell surface as a membrane-anchored precursor (proTGF-alpha) that is converted to the soluble form by proteolytic cleavage. ProTGF-alpha cleavage is activated in response to tumor-promoting phorbol ester or to calcium ionophore. Mechanism(s) controlling conversion of the membrane-anchored precursor to the soluble TGF-alpha are unknown, and the responsible protease has not been identified. Using the fluorogenic substrate succinyl-Ala-Ala-Ala-4-methylcoumaryl-7-amide (Suc-Ala-Ala-Ala-MCA), containing the sequence similar to the cleavage sites for proTGF-alpha processing, we identified a putative candidate proTGF-alpha-converting enzyme in the membrane fractions of Chinese hamster ovary (CHO) cells. The enzyme activity was about 5-6-fold increased by exposure of the cells with phorbol ester or with calcium ionophore. The enzyme activity was released from the cells by trypsin added exogenously; hence, the activity probably locates on the outer surface of the cell membrane. The enzyme was partially purified from membranes of phorbol ester-treated CHO cells. The molecular mass of the enzyme was estimated to be about 84 kDa by gel filtration analysis. Inhibitor profile of the enzyme, including inhibition by serine protease inhibitors and no inhibition by elastatinal, coincides with that previously reported for proTGF-alpha cleavage activity on CHO cells. These findings suggest that the enzyme is a processing protease involved in the cleavage of proTGF-alpha and that induction of proTGF-alpha conversion by phorbol ester or by calcium ionophore can be attributed to activation of the processing protease. In the cell-free reconstitution system, the membrane-bound Suc-Ala-Ala-Ala-MCA-hydrolyzing enzyme from CHO cells was also activated by phorbol ester in the presence of the soluble fraction, ATP, and vanadate. Requirement of vanadate, an inhibitor for protein phosphatases, for activation of the enzyme in this system suggests that phosphorylation-dephosphorylation probably plays a key role in the regulated cleavage of proTGF-alpha.
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PMID:Phorbol ester-induced activation of a membrane-bound candidate pro-transforming growth factor-alpha processing enzyme. 805 Nov 25

Coagulation factor Va is an essential cofactor which combines with the serine protease factor Xa on a phospholipid surface to form the prothrombinase complex. In the present study, the structure of factor Va interacting with lipid surfaces containing phosphatidylserine was studied by electron microscopy. Two-dimensional crystals of factor Va were obtained on planar lipid films under quasi-physiological conditions. The two-dimensional projected structure of factor Va was calculated at a resolution of 2 nm, revealing dimers of factor Va arranged on the surface lattice with the symmetry of the plane group p2. Average unit cell dimensions are a = 14.4 nm, b = 8.8 nm, gamma = 107 degrees. Each factor Va molecule presents two distinct domains of protein density consisting of one small domain, of 3 nm in diameter, connected to a larger domain of about 6 nm x 4.5 nm. The projected structure of factor Va covers an area equivalent to about fifty phospholipid molecules. In addition, edge-on views of factor Va molecules bound to liposomes reveal a globular structure connected through a thin stem to the liposome surface. A three-dimensional model of membrane-bound factor Va is proposed.
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PMID:Structure of membrane-bound human factor Va. 808 90

epsilon receptor modulating protein (epsilon RMP) was identified and purified in our previous studies as a murine T cell-derived soluble 17-kDa chymotryptic serine protease which suppresses avidity of binding between IgE and CD23 (low affinity Fc receptor for IgE) without decreasing the quantitative expression of the CD23 molecule. Some, but not all, of the other known soluble serine proteases showed epsilon RMP-like CD23-modulating activities. Further studies indicated that epsilon RMP exists not only as a soluble protein but also as a 36-kDa T-cell surface form. Both soluble and membrane-bound epsilon RMP can induce purified splenic B cells to secrete IgE in the presence of IL-4 even without lipopolysaccharide (LPS). In this study, therefore, we have tested effects of several known serine proteases on Ig production in vitro and have found that: (i) coculture of splenic B cells in the presence of LPS and IL-4 with serine proteases which have epsilon RMP-like substrate specificity, such as kallikrein and alpha-chymotrypsin, results in a significant increase of IgG1 and a slight increase of IgE secretion at low concentrations, and significant suppression at high concentrations in an isotype-selective manner; and (ii) the effects of these proteases are blocked by phenylmethylsulfonyl fluoride but not by indomethacin, suggesting that serine protease activity but not prostaglandin E2 is involved. The biological significance of the possible involvement of serine proteases on Ig class switching is discussed.
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PMID:Biphasic effect of kallikrein on IgE and IgG1 syntheses by LPS/IL-4-stimulated B cells. 842 28

The thrombin receptor (ThrR) is a membrane-bound, G-protein-coupled receptor for the serine protease thrombin. This receptor is expressed in a wide variety of cells and tissues, and elicits a range of physiological responses associated with tissue injury, inflammation, and wound repair. To achieve a better understanding of the physiological role of the ThrR, we have employed homologous recombination to create mice with a disrupted ThrR gene. Following heterozygous (+/-) intercrosses, a total of 351 surviving offspring were genotyped. Only 7% of these offspring were identified as homozygous (-/-) for the disrupted allele, indicating a profound effect on embryonic development. Paradoxically, adult ThrR-/- mice appeared to be normal by anatomical and histological analysis, including their platelet number and function. Similarly, ThrR deficiency had no detectable effect in adult ThrR-/- mice on basal heart rate, arterial blood pressure, vasomotor responses to angiotensin II and acetycholine, and coagulation parameters, even though the ThrR is expressed in many cardiovascular tissue types. In addition, the loss of ThrR function in the peripheral vasculature of adult ThrR-/- mice was confirmed by the absence of various standard hemodynamic effects of the ThrR-activating peptides SFLLRN-NH2 and TFLLRNPNDK-NH2. Our results indicate that ThrR deficiency has a strong impact on fetal development; however. ThrR-/- mice that proceed to full development display surprisingly little change in phenotype compared to the wild-type.
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PMID:Biological consequences of thrombin receptor deficiency in mice. 897 1

We have investigated the effect of UVC irradiation on "TGF alpha ase" activity using both intact HeLa cells and isolated membrane fragments with an assay based on the previously described nonapeptide substrate method [Brown et al. (1992): J Cell Biochem 48:411-423]. This method allows recognition of cleavage at the scissile bond cognate with that of the TGF alpha N-terminal cleavage site from its membrane-bound precursor. The level of ectoendopeptidase (including "TGF alpha ase") activity observed on intact cells was lower than that of ectoaminopeptidases. Addition of foetal bovine serum (FBS) enhanced aminopeptidase and dipeptidyl peptidase activity but inhibited "TGF alpha ase" activity, while phorbol 12-myristate 13-acetate (PMA) had no significant effect on the ectopeptidases monitored, expect for "TGF alpha ase," which was also inhibited, in contradistinction to their effects in other cell systems. Sublethal UVC irradiation (10 Jm-2) of the cultures resulted in activation of the ectoaminopeptidase and ectoendopeptidases which was maximal 16 and 20-24 h after irradiation, respectively. The addition of FBS to these irradiated cells appeared to reduce the increase in endopeptidase products, due in part to increased aminopeptidase activity but also to the direct inhibitory effect of FBS on the "TGF alpha ase." The activation of these proteases by UVC, even at high fluences (500 Jm-2), was not observed within the first 30 min after the cells were irradiated. Purified plasma membrane fragments were prepared from suspension cultures of HeLa cells and displayed high levels of "TGF alpha ase" activity. The rate of "TGF alpha ase" activity using 140 nM peptide substrate (P9) was 5.6 pmol/min/mg membrane protein, which was elevated to 13.7 pmol/min/mg membrane protein, 20 h after the cells had been irradiated with 10 Jm-2 UVC. Inhibition studies indicate that the plasma membrane "TGF alpha ase is a metalloenzyme as it was inhibited by EDTA, EGTA, and 1,10-phenanthroline but not by elastase or serine protease inhibitors. "TGF alpha ase" activity on intact cells was shown to be inhibited by 1,10-phenanthroline, which further supports this suggestion. Treatment of the membranes with Triton X-100 resulted in a loss of "TGF alpha ase" activity, raising the possibility that this enzyme may require a cofactor to be fully functional. We show that in purified membrane preparations of HeLa cells there is evidence for the presence of a "TGF alpha ase" which can be activated by UV irradiation, which differs from the putative "TGF alpha ase" described in various other cell lines, and which does not seem dependent on protein kinase C (PKC) activity.
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PMID:UVC activation of the HeLa cell membrane "TGF alpha ase," a metalloenzyme. 905 93


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