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:3.4.24.11 (CD10)
9,792 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The hydrophilic GH-binding protein of serum is a derivative of the GH receptor. Little is known how this GH binding protein is released from the receptor which is firmly anchored in the plasma membrane. The IM-9 lymphocytes provide a useful laboratory model for studying this process because they are richly endowed with GH receptors and, under special conditions, are able to shed these receptors during incubation. Incubation of IM-9 cells for 90 min at 30 C did not result in the appearance of significant [125I]hGH binding in conditioned medium as determined with an ultrogel AcA 44 minicolumn. When iodoacetamide, 20 mM, or N-ethylmaleimide, 5 mM, was added during incubation, the conditioned medium bound 20-35% of [125I]human(h)GH. p-Chloromercuriphenyl sulfonic acid was less effective in promoting shedding of GH-binding protein. In contrast, aprotinin, phenylmethylsulfonylfluoride (PMSF), bacitracin, leupeptin, pepstatin, phosphoramidon, or chloroquine did not promote release of GH binding protein and did not affect iodoacetamide-induced release. Release was not inhibited by the addition of serum lacking GH binding protein. GH binding protein release was markedly temperature sensitive and practically ceased at 4 C. GH binding protein incubated with [125I] hGH was cross-linked with disuccinimidyl suberate. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of dithiothreitol the complex migrated with an estimated molecular weight of 100,000 whereas [125I]hGH cross-linked to the membrane-bound GH receptor of the IM-9 cells migrated with an estimated molecular weight of 135,000. The smaller size of the binding protein is consistent with its derivation from the extracellular domain of the GH receptor. Because the release of this GH binding is greatly augmented by iodoacetamide and N-ethylmaleimide, two known sulfhydryl reactive reagents, we suggest that a free sulfhydryl group, either on the GH receptor or on a neighboring protein normally maintains the integrity of the receptor. The loss of this sulfhydryl group destabilizes the receptor and permits a membrane endopeptidase to release the GH binding protein. Cleavage is not dependent on lysosomal action and is not inhibited by protease inhibitors.
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PMID:Release of growth hormone binding protein from IM-9 lymphocytes by endopeptidase is dependent on sulfhydryl group inactivation. 284 6

A nonlysosomal alkaline protease which degrades the oxidatively modified form of Escherichia coli glutamine synthetase has been purified to apparent homogeneity from rat and mouse liver acetone powders. Its molecular weight was determined to be 300,000 by Sephacryl S-300 gel filtration but results of further studies using high pressure liquid chromatography gel filtration suggest a value of 650,000. Examination of the subunit structure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed multiple bands of molecular weights between 22,000 and 34,000. The alkaline protease was inhibited by thiol reagents. Phenylmethylsulfonyl fluoride, aprotinin, leupeptin, antipain, and chymostatin partially inhibited the protease. The inhibition by phenylmethylsulfonyl fluoride was prevented by dithiothreitol, and alpha 1-antitrypsin and soybean trypsin inhibitor did not inhibit. No inhibition was observed with metalloprotease inhibitors. The alkaline protease is active over a broad range of pH with optimum activity for the degradation of oxidized glutamine synthetase around pH 9.0. Its activity is not stimulated by MgATP. A study of the products of insulin B chain degradation demonstrated major cleavage sites at Gln13-Ala14, Leu15-Tyr16, Cys(SO3H)19-Gly20, Gln4-His5, and Leu17-Val18. Based on its endopeptidase activity and its inhibitor specificity, the alkaline protease should be classified as a cysteine proteinase. It appears to be distinct from previously described proteinases and is likely involved in nonlysosomal mechanisms of intracellular protein turnover.
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PMID:Purification of a liver alkaline protease which degrades oxidatively modified glutamine synthetase. Characterization as a high molecular weight cysteine proteinase. 286 41

We investigated the ability of human T cells to be directed to lyse murine and human tumor targets by antibodies (Ab) to the T11-E rosette (CD2) receptor. We found that the human cytotoxic T lymphocyte clone TBI-6, which is specific for the Epstein-Barr virus-transformed cell line, CM-EBV, could be directed to lyse the Fc receptor-positive murine tumor P388D1, by the combination of anti-T11(2) plus anti-T11(3) Ab. This activation and lysis was demonstrable only with an Fc receptor expressing tumor target and only with those Ab or with anti-T3 (CD3) Ab but not with other anti-T11 Ab or other Ab directed against surface structures on the clone. We therefore constructed heterodimeric Ab consisting of anti-T11(2) or anti-T11(3) Ab and the J5 anti-common acute lymphoblastic leukemic antigen (anti-CALLA) Ab. The purity and retained functional properties of the dimers were demonstrated by sodium dodecyl sulfate-polyacrylamide gels, fluorescence-activated cell sorter analysis on relevant cells, and by the ability of these conjugates to activate human peripheral blood lymphocytes to proliferate. These heterodimeric Ab conjugates were shown to be able to direct the lysis of CALLA+ targets by TBI-6. The specificity of this lysis was demonstrated by the inability of these heterodimers to direct the lysis of CALLA- targets by the cytotoxic T lymphocyte clone, and by the ability of excess free J5, but not an irrelevant Ab of the same isotype, to block this type of lysis. The potential clinical significance of these reagents is discussed.
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PMID:Human T cells can be directed to lyse tumor targets through the alternative activation/T11-E rosette receptor pathway. 289 67

A novel proteinase, termed human T cell-associated serine proteinase (HuTSP), has been highly purified from a human CD8+ T lymphocyte clone. By using a panel of chromogenic model peptide substrates the enzyme was found to specifically recognize L-arginine and to cleave Tos-Gly-Pro-Arg-nitroanilide with high efficiency at a pH optimum of 10.5-11. Exposure to class-specific proteinase inhibitors revealed that HuTSP is a serine endopeptidase. The enzyme has a mol. mass of approximately 50 kDa (non-reduced) and of approximately 25-30 kDa (reduced) when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggesting HuTSP to be a disulfide-linked dimer. The enzyme is shown to be inducible by lectin in both CD4+ and CD8+ lymphocytes. Moreover, HuTSP was detected in a number of independent CD4+ and CD8+ T cell clones and was found to be released from effector cells upon ligand binding to the CD3-T cell receptor complex.
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PMID:A novel serine proteinase (HuTSP) isolated from a cloned human CD8+ cytolytic T cell line is expressed and secreted by activated CD4+ and CD8+ lymphocytes. 296 May 46

The expression of common acute lymphoblastic leukemia antigen (CALLA) on a human neuroblastoma cell line, SJ-N-CG, was demonstrated by indirect membrane immunofluorescence, complement-dependent cytotoxicity, and quantitative absorption, using two monoclonal antibodies (J-5 and BA-3) directed against CALLA. Immunoprecipitation of solubilized 125I-labeled membrane proteins from SJ-N-CG cells with J-5 antibody revealed a protein with a molecular weight of 100,000 as determined on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Morphological differentiation of SJ-N-CG cells could be induced in the presence of 2.0 mM dibutyryl adenosine 3'-5'-cyclic monophosphoric acid for 10 days of culture. Changes in cell surface membrane antigens associated with morphological differentiation were studied by indirect immunofluorescence and complement-dependent cytotoxicity using a panel of seven monoclonal antibodies. Increases in the antigens recognized by BA-2 (detecting leukemia-associated antigen), anti-Thy-1, and antibody 390 (Thy-1 antigen) were found in "differentiated cells," while those detected by BA-1 (B-cell-associated antigen) and J-5 (CALLA) were unchanged. In contrast, the antitransferrin receptor defined by B3/25 was inhibited, and expression of B7/21-defined la-like antigen was not induced. Kinetic studies on antigenic alterations showed that the expression of BA-2-defined antigen rose on Day 2 and remained at the same level until Day 10. The expression of CALLA was not changed from Days 2 to 10. The augmentation of Thy-1 antigen was noted on Day 4 and reached the maximum on Day 10. These results show that dibutyryl adenosine 3'-5'-cyclic monophosphoric acid is capable of inducing phenotypic changes in SJ-N-CG cells. The changes of expression of some antigens on exposure of cells to dibutyryl adenosine 3'-5'-cyclic monophosphoric acid may enable us to have a greater understanding of the differentiation of neuroblastoma to a more mature ganglioneuroblastoma phenotype.
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PMID:Altered expression of cell surface membrane antigens in a common acute lymphoblastic leukemia-associated antigen-expressing neuroblastoma cell line (SJ-N-CG) with morphological differentiation. 298 Nov 59

Rat liver cytosol has low hydrolytic activity against [3H]methylcasein at neutrality, but activity increases greatly on addition of various compounds such as poly-L-lysine, N-ethylmaleimide, and sodium dodecyl sulfate, suggesting that it contains latent proteolytic activity. The latent enzyme was found to be stabilized in the presence of 20% glycerol and to be activated by addition of poly-L-lysine. The latent enzyme was purified from a crude extract of rat liver to apparent homogeneity in the presence of 20% glycerol by conventional chromatographic techniques. The purified enzyme showed endoproteolytic activity toward various proteins when it was activated by the compounds listed above. It preferentially degraded N-substituted tripeptide substrates with a basic amino acid at the carboxyl terminus, as well as peptides containing neutral hydrophobic amino acids. It did not require activation for these peptidase activities, in contrast to its activity toward large proteins. Interestingly, a proteinase and a trypsin-like and a chymotrypsin-like peptidase activity could not be separated by customary chromatographic methods but were distinguishable by their sensitivities to various inhibitors, activators, and covalent modifiers, suggesting that the enzyme has three distinct active sites within a single protein. The enzyme seems to be a seryl endopeptidase showing maximal activity at neutral and weakly alkaline pH values. Thus, the enzyme is a unique protease with latent multifunctional catalytic sites. The distribution of the protease in soluble extracts of various rat tissues and cells was examined quantitatively by an enzyme immunoassay. The enzyme level was highest in liver and also in spleen, stomach, lung, small intestine, and kidney, but was low in heart, diaphragm, skeletal muscle, brain, and skin. The concentrations of enzyme in some established cell lines including hepatoma and rat kidney cells were comparable to that in normal liver hepatocytes. The enzyme was found mainly in the cytosol fraction, although a small amount was associated with microsomal membranes, suggesting that it is an extralysosomal protease. Immunohistochemical staining of the liver and skeletal muscles showed that the protease is distributed diffusely in panlobular hepatocytes with slight centrilobar predominance and is present in Kupffer cells, vascular endothelial cells, and bile duct epithelial cells in the liver and also diffusely in the intermyofibrillar spaces and vascular endothelial cells in skeletal muscle. The quantitative data obtained in the present study indicate the presence of the protease in the cytosol fraction of all rat tissues.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:A high molecular weight protease in the cytosol of rat liver. I. Purification, enzymological properties, and tissue distribution. 309 25

A monoclonal antibody to enkephalinase (membrane metalloendopeptidase E.C. 3.4.24.11) raised by immunizing mice with rabbit renal cortical cells (mAb 85A2) was used to assess the tissue distribution of the enzyme in adult rats. When incubated with enkephalinase purified from renal brush border or with solubilized brush border proteins in the presence of antimouse IgG, the mAb specifically precipitates active enkephalinase as assessed by thiorphan inhibitable hydrolysis of [3H]-(D.Ala2, Leu5)]enkephalin. Immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis studies of radiolabeled brush border proteins indicate that mAb 85A2 binds specifically an antigen with an apparent molecular weight of 90 kilodaltons, corresponding to published values for enkephalinase. This antibody was used to localize enkephalinase by autoradiographic and immunofluorescence techniques. Immunohistochemical data show intense expression of this ubiquitous enzyme in brain, kidney, thyroid, parts of intestine, lung, seminal vesicle, and prostate. Specific areas of the brain contain more enkephalinase than others. Similarly, specific intense localization demonstrated in pulmonary alveolar cells and along the brush borders of the gut and the pars recta of the renal proximal tubule indicates that only select cell populations and/or cell compartments express this enzyme.
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PMID:Distribution of enkephalinase (membrane metalloendopeptidase, E.C. 3.4.24.11) in rat organs. Detection using a monoclonal antibody. 327 60

Intact human neutrophils hydrolyzed N-formyl-Met-Leu-[3H]Phe (fMLP) and released Leu-[3H]Phe, cleaving 45-50% of the peptide within 20 min at 37 degrees C. The dipeptide after its release was then hydrolyzed to free amino acids by a dipeptidase (EC 3.4.13.11). This activity, present in plasma membrane-enriched fractions of neutrophil lysates, was also inhibited over 90% by phosphoramidon, an inhibitor of neutral endopeptidase (NEP, EC 3.4.24.11). Dithiothreitol and EDTA inhibited the activity to a comparable degree, suggesting the requirement for a heavy metal cofactor. Bestatin and amastatin, inhibitors of aminopeptidases (but not human kidney NEP), did not inhibit the rate of fMLP degradation but prevented the production of free phenylalanine and enhanced the accumulation of Leu-Phe. Of other inhibitors, alpha 1-antitrypsin and alpha 2-macroglobulin slightly enhanced the rate of fMLP hydrolysis by neutrophils, and others tested were ineffective. Rabbit antiserum to homogeneous human kidney NEP reacted specifically with a 100-kDa protein present in sodium dodecyl sulfate-solubilized neutrophils. The Mr of this protein was slightly larger than that of the kidney enzyme in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antiserum incubated with intact cells specifically inhibited the degradation of fMLP over 70%. First, we confirm that NEP present on the plasma membrane cleaves fMLP at the Met-Leu bond; then the dipeptide Leu-Phe is cleaved by a dipeptidase. Finally, inhibition of NEP completely blocks fMLP-mediated chemotaxis. Thus, the enzyme may play an important role in modulating chemotactic responses.
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PMID:Function of neutral endopeptidase on the cell membrane of human neutrophils. 328 36

A multicatalytic endopeptidase (ME) with three distinct activities, chymotrypsin-like, cucumisin-like, and trypsin-like, occurred in all rat tissues examined with highest activities in kidney, testes, liver, and spleen; they were assayed with benzyloxycarbonyl-Gly-Gly-Leu-p-nitroanilide (Z-Gly-Gly-Leu-pNA), benzyloxycarbonyl-Leu-Leu-Glu-2-naphthylamide (Z-Leu-Leu-Glu-2NA), and benzyloxycarbonyl-Gly-Gly-Arg-2-naphthylamide (Z-Gly-Gly-Arg-2NA), respectively. All three activities were recovered from a single protein band on a polyacrylamide gel after electrophoresis of purified human kidney ME. The native enzyme had a Mr of 650,000, and it consisted of about 5,135 amino acid residues. After denaturation and electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gels kidney ME dissociated into several low Mr components ranging from 23,000 to 33,000. Kidney ME had a pH optimum of 7.6-8.1 with Z-Gly-Gly-Leu-pNA, 7.3 with Z-Leu-Leu-Glu-2NA, and 9.8 with Z-Gly-Gly-Arg-2NA. SDS enhanced chymotrypsin- and cucumisin-like activities by two to three times whereas trypsin-like activity was not enhanced. The specificity constant (kappa cat/Km) of human kidney ME for Z-Gly-Gly-Leu-pNA was 6.7 X 10(3) M-1 S-1; Z-Gly-Gly-Leu-2NA was not hydrolyzed. The specificity constant for Z-Leu-Leu-Glu-2NA was similar to, and for Z-Gly-Gly-Arg-2NA was one half of that for Z-Gly-Gly-Leu-pNA. ME cleaves only the Phe5-Ser6 bond of bradykinin (BK); however, all three ME activities were inhibited by BK. Strong inhibition of ME by albumin suggests that ME is involved in cleavage of larger polypeptides. Antipain and leupeptin almost completely inactivated the trypsin-like activity whereas they had no significant effect on the other two activities. ME is not a metal-loenzyme nor is the serine residue essential for its activities; however, thiol groups are involved. Na+ and K+ inhibited all ME activities. Trypsin-like activity was more sensitive to divalent cations than the other two.
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PMID:A multicatalytic high-molecular-weight neutral endopeptidase from human kidney. 331 Sep 3

In addition to its ability to hydrolyze acetylcholine, purified eel acetylcholinesterase possesses a trypsin-like endopeptidase activity. The tryptic activity is associated with a serine residue at a site that is distinct from the esteratic site. To label both the esteratic and tryptic sites, the enzyme was incubated with the serine hydrolase inhibitor [3H]diisopropyl fluorophosphate. This compound labelled the protein in a biphasic manner, with both slow and rapid labelling kinetics. The time course of the rapid phase was similar to the time course of inactivation of the esteratic activity. The time course of the slow phase was similar to the time course of inactivation of the tryptic activity. Labelling of the nonesteratic site was inhibited by the trypsin inhibitor N alpha-p-tosyl-L-lysine chloromethyl ketone. The total number of sites labelled by [3H]diisopropyl fluorophosphate on eel acetylcholinesterase was 2.6 mol/280,000 g protein, whereas the number of tryptic sites was less (0.52 mol/280,000 g). The results suggest that a subpopulation of acetylcholinesterase molecules may possess tryptic activity. Extensive chromatography of the purified enzyme by ion-exchange and gel filtration failed to separate the labelled tryptic component from acetylcholinesterase. On sodium dodecyl sulfate-polyacrylamide gels, the labelled tryptic component comigrated with a polypeptide of 50,000 molecular weight, which is a major proteolytic digestion product derived from the intact acetylcholinesterase monomer. Because of its localization in many noncholinergic peptide-containing cells, acetylcholinesterase could act as a neuropeptide processing enzyme in these cells.
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PMID:Identification of a trypsin-like site associated with acetylcholinesterase by affinity labelling with [3H]diisopropyl fluorophosphate. 337 13


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