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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)

The purpose of the present study was to evaluate the properties of beta-glucuronidase (EC 3.2.1.31) in human synovial fluid. It was shown to have a pH requirement of 5.0 and a KM value of about 8.0 - 10(-3) M using phenolphthalein beta-glucuronide as the substrate. At low substrate concentration an endogenous inhibitor is demonstrable. The inhibition is of the competitive type and is removed by proteolytic digestion of synovial fluid, whereas hyaluronidase digestion and addition either of Triton X-100 or of various salts to the assay mixture, are ineffective. The possibility that the inhibitor is a protein from serum is discussed.
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PMID:Properties of beta-glucuronidase activity in human synovial fluid. 0 Nov 63

The optimal reaction conditions and kinetic properties of eleven leukocyte acid hydrolases determined with the use of fluorigenic derivatives of 4-methyl-umbelliferone are described. The enzymes studied were acid phosphatase, aryl sulfatase, alpha- and beta-glucosidase, alpha- and beta-galactosidase, alpha-mannosidase, N-acetyl-beta-glucosaminidase, N-acetyl-beta-galactosaminidase, beta-glucuronidase and alpha-fucosidase. More than 90% of the activity of each enzyme was released into a 27,000 X g supernatant by a double sonication procedure employing 0.9% sodium chloride and 0.1% Triton X-100. The Km values obtained were similar to those previously reported for chromogenic subtrates. A single Km value could not be derived for beta-galactosidase because its double reciprocal plot was not linear. All enzymes could be measured with less than 10 mug of protein within 15 min. Activators and inhibitors studied included the chloride salts of Na+, K+, Zn2+, Ca2+, Mg2+, Hg2+, and Fe2+ as well as p-chloromercuriphenysulfonate, glutathione, BAL, EDTA, EGTA, Triton X-100 and sodium taurocholate. The reaction conditions described in this report can be used for the diagnosis of various lysosomal storage diseases and should facilitate the development of automated procedures for the analysis of these eleven enzyme activities with small quantities of blood.
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PMID:Human leukocyte acid hydrolases: characterization of eleven lysosomal enzymes and study of reaction conditions for their automated analysis. 0 26

The relative activities of uridine diphosphoglucuronyltransferase (UDPGT) and beta-glucuronidase (betaG) were measured during perinatal development of hepatic and extrahepatic tissues to determine the balance between glucuronidation and deglucuronidation reactions at different developmental stages. Liver, lung, kidney, intestine, and placenta were studied in guinea pigs and rabbits. In general, betaG activities exceeded those of UDPGT in fetal tissues, whereas the converse was evident in adults. There were significant species and age differences in the onset of betaG and UDPGT activities and the occurrence of developmental peaks. A dramatic betaG developmental peak was observed in fetal guinea pig intestine and newborn rabbit intestine. Both microsomal and lysosomal betaG exhibited similar developmental patterns in all tissues tested. Hepatic nonsteroid UDPGT activities were higher at parturition than in adult animals, whereas no such developmental peak occurred for steroid UDPGT. Triton X-100 activated fetal UDPGT in vitro by approximately the same factor as it did for adult UDPGT.
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PMID:Glucuronidation and deglucuronidation reactions in hepatic and extrahepatic tissues during perinatal development. 1 27

Isoelectric focusing was used to study the multiple forms of acid phosphatase, arylsulfatase, beta-glucuronidase and beta-N-acetylhexosaminidase in lysosomes isolated from rat kidney. The isoelectric points of the main protein and hydrolase peaks were 1-1.5 units lower when electrofocusing was done in a pH 3-10 gradient than in a pH 10-3 gradient, apparently because the lysosomal constituents aggregated strongly at their isoelectric points and tended to settle somewhat in the gradient due to gravity. In the extended pH gradient the acidic form of each hydrolase occurred as asingle, relatively discrete peak. However, when pooled acidic fractions were refocused in a restricted pH gradient (pH 6-3 or 3-5) multiple acidic enzyme and protein components were resolved with isoelectric points between 2.7 and 5.1. When autolysis was minimized by extracting lysosomal fractions at alkaline pH (0.2% Triton X-100, 0.1%p-nitrophenyloxamic acid, 0.1 M glycine buffer, pH9) and including 0.1%p-NITROPHENYLOXAMIC ACID, AN INHIBITOR OF LYSOSOMAL NEURAMINIDASE AND CATHEPSIN D, in the pH gradient, arylsulfatase, beta-glucuronidase and beta-N-acetylhexosaminidase occurred in two forms, an acidic form with an isoelectric point of about 4.4, and a basic form with an isoelectric point close to 6.2, 6.7 and 8.0, respectively. Acid phosphatase occurred in three forms with isoelectric points of 4.1, 5.6 and 7.4. When some autolytic digestion was permitted by extracting lysosomal fractions in an acidic medium (0.2% Triton X-100, 0.1 M sodium acetate buffer, pH 5.2) AT 0-4DEGREES C and omitting p-nitrophenyloxamic acid from the gradient, the acidic form of beta-glucuronidase and the intermediate form of acid phosphatase were lost, the isoelectric points of the acidic forms of acid phosphatase, arylsulfatase and beta-N-acetylhexosaminidase were increased 0.6-1.2 units, and the isoelectric point of the basic forms of acid phosphatase, arylsulfatase and beta-glucuronidase was increased 0.5 unit. When lysosomal extracts were incubated with bacterial neuraminidase before electrofocusing, the acidic forms of acid phosphatase, arylsulfatase and beta-glucuronidase were largely lost, the isoelectric point of the acidic form of beta-N-acetylhexosaminidase was increased from 4.5 to 6.4, and the isoelectric points of the basic forms of all four hydrolases were increased 0.5-1.5 units. Autoincubation of lysosomal extracts in vitro at pH 5.2 PRODUCED SIMILAR, THOUGH LESS MARKED, effects. cont'd
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PMID:Isoelectric-focusing behavior of acid hydrolases in rat kidney lysosomes. Effects of the pH gradient, autolysis and neuraminidase. 23 55

Isoelectric focusing was used to investigate the multiple forms of acid phosphatase, arylsulfatase, beta-glucuronidase, beta-galactosidase and beta-N-acetylhexosaminidase in the following, previously characterized subcellular fractions from rat kidney: a special rough microsomal fraction, enriched up to 9-fold over the homogenate in acid hydrolases; a smooth microsomal fraction; a Golgi membrane fraction enriched about 2.5-fold in acid hydrolases and 10- to 20-fold in several glycosyl transferases; and a lysosomal fraction enriched up to 25-fold in acid hydrolases. The electro-focusing behavior of the hydrolases in these fractions was markedly sensitive to the autolytic changes that occur under acidic conditions, even at 4 degrees C. Autolysis was minimized by extracting fractions in an alkaline medium (0.2% Triton X-100, 0.1 M sodium glycinate buffer, pH 10, 0.1 % p-nitrophenyloxamic acid) and adding p-nitrophenyloxamic acid (0.1 %), AN INHIBITOR OF LYSOSOMAL NEURAMINIDASE AND cathepsin D, to the pH gradient. The enzymes in the lysosomal fraction displayed a characteristic bimodal or trimodal distribution. Arylsulfatase, beta-glucuronidase and beta-N-acetylhexosaminidase occurred in an acidic form with an isoelectric point of 4.4, and a basic form with an isoelectric point of 6.2, 6.7 and 8.0, respectively. Acid phosphatase and beta-galactosidase occurred in an acidic, intermediate and basic form with isoelectric points of about 4. 1, 5.6 and 7.4, respectively. In the special rough microsomal fraction these enzymes were mostly in a basic form with isoelectric points between 7.5 and 9; these were 1-2 units higher than the corresponding basic forms in the lysosomal fraction. Treatment of extracts of the rough microsomal fraction with bacterial neuraminidase raised the isoelectric points of all five hydrolases by 1-2.5 units, indicating the presence of some N-acetylneuraminic acid residues in these basic glycoenzymes. The hydrolases in the Golgi fraction were largely in an acidic form with isoelectric points similar to or lower than those of the corresponding acidic components in the lysosomal fraction. The hydrolases in the smooth microsomal fraction showed isoelectric-focusing patterns intermediate between those in the rough microsomal and the Golgi fractions. These findings support the following scheme for the synthesis, transport and packaging of the lysosomal enzymes. Each hydrolase is synthesized in a restricted portion of the r
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PMID:Changes in electronegativity of lysosomal hydrolases during intracellular transport. An isoelectric-focusing study in subcellular fractions of rat kidney. 23 56

Purified preparations of human polymorphonuclear leucocytes contain a protein kinase in the cytosol which is stimulated by cyclic AMP and cyclic IMP but not by other cyclic nucleotides. The holoenzyme had a molecular weight of 66000 estimated by gel filtration; when it was incubated with histone or cyclic AMP, it dissociated into two smaller subunits of molecular weight 45000 and 30000; the former remained cyclic AMP-sensitive, whereas the latter had become independent of added cyclic AMP. By means of substrate-affinity chromatography on histone-Sepharose 4B, cyclic [3H5AMP-binding activity (regulatory or R subunit) could be resolved into two peaks of enzyme activity, one again independent of added cyclic AMP, with a molecular weight of 30000 (catalytic or C subunit). Also by means of substrate-affinity chromatography it was possible to resolve 'specific' polymorphonuclear leukocyte histone phosphatases from 'non-specific' phosphomonesterases capable of dephosphorylating histone previously phosphorylated by the protein kinase. Specific histone phosphatase displayed greatest affinity for histone-Sepharose 4B, followed by acid p-nitrophenyl phosphatase, and the unretained acid beta-glucerophosphatase. Polymorphonuclear leucocyte histone phosphatase, purified approx. 40-fold, was further resolved from the other phosphatases by gel filtration on Sephadex G-150 from which it was eluted with apparent molecular weights of 45000 and 18700. The apparent Km values for dephosphorylation of histone are 4.3 X 10-6M and 3.6 X 10-6M. Most (69%) of cytoplasmic histone phosphatase was found in the cell sap, whereas 20% remained tightly associated with polymorphonuclear leucocyte lysosomes from which it could not be solubilized by treatments (Triton X-100, freeze-thawing) that released approx. 70% of lysosomal beta-glucuronidase or acid phosphatases. Although both soluble and particulate enzymes required 5-10 mM-Mn2 for maximal activation, and showed a pH maximum of 6.5-7.0, only the particulate enzyme was partly inhibited by ammonium molybdate. Polymorphonuclear leucocyte histone phosphatases were neither inhibited nor stimulated by those cyclic nucleotides that greatly stimulate the protein kinase of the same subcellular fraction
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PMID:Protein kinase and phosphatases from human polymorphonuclear leucoytes. 23 86

A binding protein with apparent specificity for beta-glucuronidase has been partially purified from a Triton X-100 extract of rat liver microsomes by affinity chromatography on glucuronidase-Sepharose 2B. It appears that once removed from the membrane, this binding protein self-aggregates to form large macromolecular complexes. With the use of polyacrylamide gel electrophoretic and sucrose density gradient ultracentrifugation assays to monitor the conversion of glucuronidase tetramer to a very high molecular weight complex, it was shown that the binding activity is heatlabile and protease-sensitive. However, binding activity is not influenced by salts, carbohydrates, other proteins or glycoproteins, or by extensive periodate oxidation of beta-glucuronidase, nor does binding occur with any other protein tested. The binding protein does not discriminate against any form of beta-glucuronidase from any rat organ tested. However, the binding protein does show organ localization, being present in the liver and kidney but not the spleen. The possible relationship of this binding protein to egasyn, a membrane protein which stabilizes beta-glucuronidase in mouse liver endoplasmic reticulum, is discussed.
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PMID:Demonstration of a rat liver microsomal binding protein specific for beta-glucuronidase. 43 56

Glucuronidase present in lysosomes of mouse liver occurs as the free tetramer, whereas glucuronidase present in endoplasmic reticulum occurs in macromolecular complexes containing one to four molecules of the protein egasyn. Earlier genetic and biochemical studies suggest that these complexes, or M forms, function to stabilize the membrane binding of glucoronidase. The detergent Triton X-100 extracts glucuronidase-egasyn complexes intact and they dissociate in the presence of the detergent deoxycholate or upon heating. We have now purfied egasyn by releasing it from antiglucuronidase immunoprecipitates of M forms under relatively mild conditions, such as treatment with deoxycholate or heating at 50 degrees. Isolated egasyn is a glycoprotein of molecular weight about 64,000 and is not unusually hydrophobic in amino acid composition. Monospecific antibody to egasyn was raised. This antibody showed no cross-reactivity with purified beta-glucuronidase and antibody to glucuronidase failed to react with purified egasyn; however, both antibodies bound to egasyn-glucuronidase complexes. A procedure for the radioimmunoassay of egasyn was developed utilizing egasyn labeled with iodine 125. Most of the antigenic sites of egasyn in homogenates of normal liver are masked after extraction with Triton X-100 and only become immunoreactive after exposure to deoxycholate. After unmasking, mouse liver proved to contain about 56 mug of egasyn/g, nearly all of which is localized to the microsomal fraction. Of this total only about 10% was complexed with glucuronidase, suggesting theat the bulk of the egasyn present may be complexed with other proteins. Mice of the inbred strain YBR, which carry the EgO mutation resulting in the absence of microsomal glucuronidase, lacked immunoreactive egasyn, suggesting that the primary defect in this strain lies in the unavailabililty of agasyn to form complexes. There is now considerable evidence in support of the concept that the microsomal forms of glucuronidase exist in membranes complexed with egasyn and that formation of these complexes is required for maintenance of glucuronidase in membranes. Egasyn may represent one of a class of membrane anchor proteins that each stabilize the membrane binding of a charcteristic set of proteins.
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PMID:Isolation, characterization, and radioimmunoassay of murine egasyn, a protein stabilizing glucuronidase membrane binding. 82 34

Non-latent (free) activities of two lysosomal enzymes (acid phosphatase and beta-glucuronidase) and urea production were measured in purified rat liver parenchymal cells incubated in the presence and absence of insulin. Non-latent enzyme activity was measured by including 0.25M sucrose in the assay mixtures to provide osmotic protection to the lysosomes. Total enzyme activity was estimated with Triton X-100 in the homogenates. Insulin was found to inhibit ureogenesis and to reduce non-latent lysosomal enzyme activity in the hepatocytes in vitro. Our data support the idea that insulin inhibits autophagy in rat liver parenchymal cells. Such an effect of insulin may also explain the inhibitory action of insulin on urea production in the rat liver.
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PMID:Influence of insulin on lysosomal activity and urea production in isolated parenchymal cells from rat liver. 103 39

1. Structure-linked latency, a trait for most lysosome hydrolase activities, is customarily ascribed to the permeability-barrier function performed by the particle-limiting membrane, which shields enzyme sites from externally added substrates. 2. The influence of various substrate concentrations on the reaction rate has been measured for both free (non-latent) and total (completely unmasked by Triton X-100) hydrolase activities in rat liver cell-free preparations. The substrates were: beta-glycerophosphate, phenolphthalein mono-beta-glucuronide. p-nitrophenyl N-acetyl-beta-D-glucosaminide and p-nitrophenyl beta-D-galactopyranoside. The ratio (free activity/total activity) X 100 is called fractional free activity at any given substrate concentration. 3. The fractional free activity of beta-glucuronidase and beta-N-acetylglucosaminidase were clearly independent of substrate concentration, over the range examined, in both homogenates and lysosome-rich fractions. The fractional free activity of acid phosphatase appeared to be either unaffected (homogenate) or even depressed (lysosome-rich fraction) by increasing the beta-glycerophosphate concentration. The fractional free activity of beta-galactosidase consistently showed a non-linear increase with increasing substrate concentration in both homogenates and lysosome-rich fractions. 4. Procedures such as treatment with digitonin, hypo-osmotic shock and acid autolysis, although effective in causing varying degrees of resolution of the latency of lysosome hydrolase activities, were unable to modify appreciably the pattern of dependence or independence of their fractional free activities on substrate concentration, as compared with that exhibited by control preparations. Ouabain did not affect the free beta-N-acetylglucosaminidase activity of liver homogenates at all. 5. Preincubation of control preparations with beta-glycerophosphate or p-nitrophenyl beta-galactoside did not result in any significant stimulation of the free hydrolytic activity toward these substrates. 6. The results consistently support the view that the membrane of "intact" lysosomes is virtually impermeable to all the substrates tested, except for p-nitrophenyl beta-galactoside, for which the evidence is contradictory. Moreover the progressive unmasking of the hydrolase activities produced by these procedures in vitro reflects the increasing proportion of enzyme sites that are fully accessible to their substrates rather than a graded increase in the permeability of the lysosomal membrane.
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PMID:Structural equivalents of latency for lysosome hydrolases. 104 Dec 36


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