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:3.2.1.31 (beta-glucuronidase)
7,680 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A chemotactic peptide CHO.Met.Leu.Phe.OH has been synthesized classically using the mixed anhydride procedure. The formyl group was introduced by coupling formic acid in the presence of dicyclohexylcarbodiimide to the partially protected triptide. The final product was obtained by treatment of the intermediate CHO.Met.Leu.Phe.OBzl with hydrogen fluoride. The ED50 of the peptide in the Boyden chamber assay was 7 x 10(-11) M; in the lysozyme release assay 2.4 x 10(-10) M and in the beta-glucuronidase release assay 2.6 x 10(-10) M. In a radioreceptor assay the ID50 of the peptide was 3.3 x 10(-10) M.
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PMID:Synthesis of Nalpha-formyl-Met-Leu-Phe-OH: an inducer of chemotaxis in peritoneal polymorphonuclear neutrophils. 42 7

24 di-, tri-, and tetrapeptides have been synthesized as a start of a systematic study of the structural requirements for chemotactic activity and lysosomal enzyme-releasing ability in rabbit neutrophils. All but two of them are N-formyl methionyl peptides. Using the method of Zigmond and Hirsch (10), two representative peptides, F-Met-Leu-Phe and F-Met-Met-Met, were shown to stimulate directed, as well as, random locomotion; thus, they were truly chemotactic. The various peptides showed a wide spread in activity. F-Met-Leu-Phe, the most active peptide studied, had an ED50 for induced migration of 7 X 10(-11) M and for lysozyme and beta-glucuronidase release of 2.4 X 10(-10) M and 2.6 X 10(-10) M, respectively; the least active, Met-Leu-Glu was 26 million times less active in these respects. The relation of activity to structure is exceedingly specific, very small changes in structure making large changes in activity. Moreover, this specificity exhibits a definite regularity and pattern; the activity of a given peptide depends not only on its constituent amino acids but on the position of the amino acid in the peptide chain. Most striking in this last regards is the high activity conferred by phenylalanine when it is in the carboxyl terminal position of a tripeptide, whereas, as the second amino acid from the NH2 terminal end whether in a tripeptide or a dipeptide, it contributes no more to the activity than other amino acids with hydrophobic side chains such as leucine or methionine. The high activity and the specificity and nature of the structural requirements strongly suggest that the primary interaction of peptide and neutrophil leading to either chemotaxis or lysosomal enzyme release is a binding of the peptide with a stereospecific receptor on the neutrophil surface. Whether all chemotactic factors act through the same receptor is not known. An essentially exact correlation exists between the concentrations of the various synthetic peptides required to induce migration and their ability to induce release of lysozyme or beta-glucuronidase. This implies that these two neutrophil functions are triggered by teh same primary interaction; possibly, the binding of the peptides to the same putative receptor. A higher concentration of a given peptide is required to stimulate lysosomal enzyme release than a corresponding migratory response. A slightly but significantly higher concentration of peptide is required to induce beta-glucuronidase secretion than lysozyme release.
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PMID:The structure-activity relations of synthetic peptides as chemotactic factors and inducers of lysosomal secretion for neutrophils. 126 85

The radiographic contrast agent sodium diatrizoate (DTR) reportedly inhibits f-Met-Leu-Phe-induced chemotaxis in human neutrophils. DTR is also an ingredient of Ficoll-Paque, a density centrifugation medium widely used to purify human polymorphonuclear leukocytes (PMNs). Exposure of PMNs to DTR during preparation had no detrimental effect on subsequent binding characteristics of tritiated f-Met-Leu-Phe, probably owing to a rapid dissociation of DTR from the PMN receptors. DTR competed directly with f-Met-Leu-Phe for receptor binding, but was 160- and 640-fold less potent than phenylbutazone and 1,2-diphenyl-4-[3-(1-naphthyl)-propyl]-3,5-pyrazolidinedione (DPN; an analog of phenylbutazone), respectively. Iohexol and the methylamide of DTR did not compete with [3H]f-Met-Leu-Phe in receptor binding, supporting the existence of a definite interaction between iodinated aromatic molecules and the f-Met-Leu-Phe receptor. DTR did not inhibit prostaglandin synthesis, as did DPN. Both drugs inhibited chemotactic peptide-induced release of superoxide anion in a concentration-dependent manner, and were relatively selective for f-Met-Leu-Phe, as opposed to C5a. Both drugs at 10 microM interfered non-selectively with chemotactic peptide-induced beta-glucuronidase release from PMNs. Available non-peptide antagonists of f-Met-Leu-Phe exhibited other pharmacodynamic properties that could make them unsuitable for future in vivo studies designed to probe the physiological role of the receptor.
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PMID:Comparison of two classes of non-peptide drugs as antagonists of neutrophil receptors for f-Met-Leu-Phe. Pyrazolons and iodinated radiographic contrast agents. 131 83

We have used a continuous spectrofluorimetric method to analyse the role of cytosolic free Ca2+ ([Ca2+]i) in the lysosomal enzyme release from the azurophilic granules in human neutrophils stimulated with f-Met-Leu-Phe (fMLP) in the presence of cytochalasin B. Measurements were performed with the beta-glucuronidase substrate 4-methylumbelliferyl-beta-D-glucuronide. We found that the transient rise in [Ca2+]i induced by fMLP is a necessary signal to obtain maximal degranulation. When this Ca2+ transient is prevented by the Ca2+ chelator BAPTA, degranulation can still be induced by a stimulated Ca2+ influx, albeit to a lower extent. We also studied the degranulation process in the neutrophils of a patient with a generalized chemotactic defect. Release of beta-glucuronidase from the patient's neutrophils could not be induced despite the occurrence of a normal Ca2+ response and normal degranulation of specific granules. We conclude that, besides an increase in [Ca2+]i, an additional signal is required for the fusion of azurophilic granules with the plasma membrane in human neutrophils.
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PMID:Release of azurophilic granule contents in fMLP-stimulated neutrophils requires two activation signals, one of which is a rise in cytosolic free Ca2+. 178 9

Coordinate secretion of two prohormone/proneuropeptide processing enzymes [pro-opiomelanocortin converting enzyme (PCE) and an aminopeptidase B-like enzyme (APBE)] and alpha-melanotropin (alpha-MSH) from bovine intermediate lobe pituitary cells was studied. Stimulation of secretion with 8-bromo-cyclic AMP produced significant increases in levels of immunoreactive alpha-MSH, PCE, and APBE. Treatment of cells with the dopaminergic agonist 2-bromo-alpha-ergocryptine resulted in significant decreases in secretion of alpha-MSH, PCE, and APBE. In neither case were there significant changes in levels of cytosolic lactic dehydrogenase or lysosomal beta-glucuronidase in the medium. The secreted PCE activity was shown to process frog and mouse pro-opiomelanocortin primarily to 23,000-Mr corticotropin (ACTH), 13,000-Mr ACTH, beta-lipotropin, a beta-endorphin-like peptide, and beta-endorphin, products comparable to those synthesized by the mouse and frog intermediate lobe in situ. The secreted enzymatic activity had a pH optimum between 4.0 and 5.0, was strongly inhibited by pepstatin A, and had an inhibitor profile similar to the purified bovine intermediate lobe PCE. The secreted APBE activity cleaved Argo-[Met]-enkephalin to [Met]-enkephalin and had a pH optimum and inhibitor profile similar to that previously reported for an activity from purified secretory vesicle fractions of bovine intermediate and neural lobes. The coordinate regulated secretion of alpha-MSH and enzyme activities (PCE and APBE) strongly indicates their colocalization in the same secretory vesicle compartment within the cell. The characteristics of the two enzymes secreted in the medium paralleled those seen in the tissue and further support their role in pro-opiomelanocortin processing in vivo.
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PMID:Regulated secretion of pro-opiomelanocortin converting enzyme and an aminopeptidase B-like enzyme from dispersed bovine intermediate lobe pituitary cells. 254 Feb 80

A monoclonal antibody (2C5) raised against rat liver lysosomal membranes was used to identify a 78-kD glycoprotein that is present in the membranes of both endosomes and lysosomes and, therefore, is designated endolyn-78. In cultures of rat hepatoma (Fu5C8) and kidney cells (NRK), this glycoprotein could not be labeled with [35S]methionine or with [32P]inorganic phosphate but was easily labeled with [35S]cysteine and [3H]mannose. Pulse-chase experiments and determinations of endoglycosidase H (endo H) sensitivity showed that endolyn-78 is derived from a precursor of Mr 58-62 kD that is processed to the mature form with a t1/2 of 15-30 min. The protein has a 22-kD polypeptide backbone that is detected after a brief pulse in tunicamycin-treated cells. During a chase in the presence of the drug, this is converted into an O-glycosylated product of 46 kD that despite the absence of N-linked oligosaccharides is effectively transferred to lysosomes. This demonstrates that the delivery of endolyn-78 to this organelle is not mediated by the mannose-6-phosphate receptor (MPR). Immunocytochemical experiments showed that endolyn-78 is present in the limiting membranes and the interior membranous structures of morphologically identifiable secondary lysosomes that contain the lysosomal hydrolase beta-glucuronidase, lack the MPR, and could not be labeled with alpha-2-macroglobulin at 18.5 degrees C, a temperature which prevents appearance of endocytosed markers in lysosomes. Endolyn-78 was present at low levels in the plasma membrane and in peripheral tubular endosomes, but was prominent in morphologically diverse components of the endosomal compartment (vacuolar endosomes and various types of multivesicular bodies) which acquired alpha-2-macroglobulin at 18.5 degrees C, and frequently contained substantial levels of the MPR and variable levels of beta-glucuronidase. On the other hand, the MPR was very rarely found in endolyn-containing structures that were not labeled with alpha-2-macroglobulin at the low temperature. Thus, the process of lysosomal maturation appears to involve the progressive delivery of lysosomal enzymes to various types of endosomes that may have already received some of the lysosomal membrane proteins. Although endolyn-78 would be one of the proteins added early to endosomes, other lysosomal membrane proteins may be added only to multivesicular endosomes that represent very advanced stages of maturation.
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PMID:Endolyn-78, a membrane glycoprotein present in morphologically diverse components of the endosomal and lysosomal compartments: implications for lysosome biogenesis. 265 37

The susceptibility of a number of human neutrophil granule enzymes to oxidative inactivation was investigated. Addition of H2O2 to the cell-free medium from stimulated neutrophils resulted in inactivation of all enzymes tested. This was inhibited by azide and methionine, indicating that inactivation was due to myeloperoxidase-derived oxidants. Lysozyme was more than 50% inactivated by one addition of 100 nmol of H2O2/ml, whereas myeloperoxidase, beta-glucuronidase, gelatinase and collagenase were almost completely inactivated by three additions. Cathepsin G was slightly less susceptible, whereas elastase was extremely resistant to oxidative attack. Myeloperoxidase-dependent enzyme inactivation may be a means whereby the neutrophil can terminate the activity of its granule enzymes and control the release of degradative enzymes into the tissues.
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PMID:Myeloperoxidase-dependent oxidative inactivation of neutrophil neutral proteinases and microbicidal enzymes. 282 16

Protein I, the major outer membrane protein of Neisseria gonorrhoeae, is a voltage-dependent anion channel which can translocate from the gonococcus into human cells. Since granule exocytosis from neutrophils is regulated by ion fluxes, we examined the effect of protein I on neutrophil activation. Pretreatment with protein I (250 nM) impaired degranulation from neutrophils: beta-glucuronidase release decreased to 27 +/- 6% S.E. of cells treated with N-f-Met-Leu-Phe (fMLP, 0.1 microM) and to 13 +/- 4% of cells treated with leukotriene B4 (LTB4, 0.1 microM); lysozyme release decreased to 52 +/- 17% of fMLP-treated cells and 22 +/- 9% of LTB4-treated cells. Morphometric analysis was consistent: control neutrophils increased their surface membrane after fMLP (43.3 +/- 5.6 microns relative perimeter versus 71.4 +/- 3.7 microns) while protein I-treated neutrophils did not (29.4 +/- 2 (S.E.) microns relative perimeter versus 34 +/- 4 microns). Enzyme release after exposure to phorbol myristate acetate was not affected (lysozyme: 86 +/- 27% of control). Cell/cell aggregation in response to fMLP was inhibited by treatment with protein I. However, generation of O2 was not affected. Protein I altered the surface membrane potential (Oxonol V): protein I evoked a transient membrane hyperpolarization which was not inhibited by furosemide. After exposure to fMLP, protein I-treated neutrophils underwent a furosemide-sensitive hyperpolarization rather than the usual depolarization. Protein I did not alter increments in [Ca]i (Fura-2) stimulated by fMLP (460 +/- 99 nM (S.E.) versus 377 +/- 44 nM) nor decrements in [pH]i (7.22 +/- 0.04 S.E. versus 7.22 +/- 0.02, bis-(carboxy-ethyl)carboxyfluorescein). The results suggest that degranulation and O2 generation have separate ionic requirements and that protein I interrupts the activation sequence proximal to activation of protein kinase C.
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PMID:Protein I, a translocatable ion channel from Neisseria gonorrhoeae, selectively inhibits exocytosis from human neutrophils without inhibiting O2- generation. 282 69

Acid hydrolases were isolated from the lysosome fraction of beta-galactosidase-deficient human fibroblasts and from the mannose 6-phosphate containing medium in which they were grown. Nearly half of the total beta-hexosaminidase and beta-glucuronidase from both sources bound to Ricin specifically. Lysosomal beta-hexosaminidase, metabolically labelled with [35S]-methionine, was also fractionated on Ricin-agarose. SDS-PAGE of immunoprecipitates from Ricin-binding and non-binding fractions revealed approximately equivalent amounts of cross-reacting material at the appropriate MW. We interpret these results to mean that acid hydrolases which are segregated to lysosomes are exposed to trans-Golgi processing enzymes to about the same extent as enzymes which are secreted, and that segregation by the Man 6-P receptor occurs after transit through the trans-Golgi compartment.
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PMID:Ricin-binding properties of acid hydrolases from isolated lysosomes implies prior processing by terminal transferases of the trans-Golgi apparatus. 293 47

Endocytosis of acid hydrolases via the cell surface mannose 6-phosphate (Man 6-P) receptor results in the delivery of the enzymes to lysosomes. To examine the fate of the ligand-associated phosphorylated high mannose oligosaccharides, we have analyzed the asparagine-linked oligosaccharides attached to beta-glucuronidase after uptake and processing by Man 6-P receptor-positive mouse L cells. beta-Glucuronidase, double-labeled with [2-3H]mannose and [35S]methionine, was isolated from the growth medium of mouse P388D1 cells. 80% of the [3H]mannose associated with the secreted enzyme was recovered as high mannose-type oligosaccharides, and 24-37% of these units were phosphorylated. Three species of phosphorylated oligosaccharides were identified; high mannose-type units containing either one or two phosphomonoesters, and hybrid-type units containing one phosphomonoester and one sialic acid residue. After endocytosis by the L cells, the beta-glucuronidase molecules migrated faster on an SDS gel, suggesting that the enzymes had been processed within lysosomes. Examination of the cell-associated beta-glucuronidase molecules indicated that: (a) the percentage of phosphorylated oligosaccharides remained comparable to the input form of the enzyme, even after a 24-h chase period, (b) the presence of a single species of phosphorylated oligosaccharide that contained one phosphomonoester, and (c) the positioning of the phosphate within the intracellular monophosphorylated species was comparable to the positioning of the phosphate within the two phosphomonoester species originally secreted by the P388D1 cells. Therefore, the internalized beta-glucuronidase molecules undergo a limited dephosphorylation; oligosaccharides containing two phosphomonoesters are converted to monophosphorylated species, but the one phosphomonoester forms are conserved. A comparison of the phosphorylated oligosaccharides recovered from ligands internalized by the L cells at 37 degrees and 20 degrees C indicated that: (a) molecules internalized at 20 degrees C retain a higher percentage of phosphorylated structures; and (b) at both temperatures the predominant phosphorylated oligosaccharide contains a single phosphomonoester group. The results indicate that the Man 6-P recognition marker persists after endocytosis and delivery to lysosomes and support the possibility that the limited dephosphorylation of the oligosaccharides may occur en route to these organelles.
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PMID:Mannose 6-phosphate receptor-mediated endocytosis of acid hydrolases: internalization of beta-glucuronidase is accompanied by a limited dephosphorylation. 294 1


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