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:3.1.6.1 (sulfatase)
3,205 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Metachromatic leukodystrophy and Maroteaux-Lamy syndrome can be diagnosed by assay of leukocyte or fibroblast arylsulfatase A and B activity with the fluorogenic substrate 4-methylumbelliferyl sulfate. The arylsulfatases are extracted into a 27000 x g supernatant by sonication in 0.9% sodium chloride and then separated with CM-32 on columns or in test tubes. In 0.05 M sodium acetate pH 6.0, arylsulfatase A is not absorbed while arylsulfatase B is retained by the resin. The arylsulfatase B is then eluted from the resin with 0.3 M sodium chloride. The arylsulfatase A activity obtained from normal leukocytes and fibroblasts is linear for the initial 10 minutes of the reaction, is stimulated 3-fold by 6 mM lead acetate and inhibited 80% by 0.24 mM silver nitrate. After separation with CM-32, the arylsulfatase B activity is stimulated 3-fold by Triton X-100 (0.1%). Arylsulfatase A but not arylsulfatase B is destroyed by heat (60 degrees). Both leukocyte and fibroblast arylsulfatase A activity was reduced to 11% of control values in metachromatic leukodystrophy. Essentially no arylsulfatase B activity was detected in cells from patients with Maroteaux-Lamy syndrome. Metachromatic leukodystrophy heterozygotes but not Maroteaux-Lamy syndrome heterozygotes can also be distinguished by this method. A heat inactivation technique utilizing the differential thermal stabilities of the two enzymes for diagnosis of patients with Marotezux-Lamy syndrome is also described. The advantages of these 4-methylumbelliferyl sulfate assay procedures over the p-nitrocatechol sulfate method of assay are greater sensitivity, selectivity for the desired enzyme and potential for use in large scale testing.
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PMID:Arylsulfatases A and B in metachromatic leukodystrophy and Maroteaux-Lamy syndrome: studies with 4-methylumelliferyl sulfate. 0 5

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

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

Two methods for the extraction of acrosomal membranes and enzymes from both human and rabbit spermatozoa were compared. Treatment of spermatozoa with hypotonic MgCl2 (0.05 M) solution causes removal of the plasma membrane, vesiculation, disruption and removal of the outer acrosomal membrane posterior to the equatorial segment with accompanying loss of soluble acrosomal material. Subsequent exposure to Hyamine 2389 and Triton X-100 removes acrosomal material bound to the inner acrosomal membrane with concomitant solubilization of this membrane. The MgCl2 extract from rabbit spermatozoa contained a higher yield of hyaluronidase, acrosin, and total proteinase activities, whereas the subsequent detergent extracts contained higher yields of both arylsulfatase A and B activities. By comparison, after 4 minutes of sonication to separate heads and tails, both rabbit and human spermatozoa when viewed by transmission electron microscopy showed alterations of plasma and outer acrosomal membranes with considerable loss of the acrosomal contents. Analysis of acrosomal enzymes indicates the greatest percentage of all the enzymes assayed was located in the extract obtained by sonication in contrast to either the separated head or tail fractions used for further subcellular extraction. Subsequent treatment with Hyamine and Triton yields only minimal amounts of enzyme activity.
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PMID:Extraction of human and rabbit acrosomes: a comparison of sequential and sonication methods. 51 71

Normal reference values of lysosomal enzyme activities (alpha-glucosidase, mannosidase, fucosidase and arylsulfatase-A) were determined in chorionic villi obtained from artificial abortion in the first trimester of normal pregnancies (gestational weeks 6 to 11). Villi were homogenized comparatively either in saline or in Triton X-100 detergent. The alpha-glucosidase, mannosidase and arylsulfatase-A enzyme activities significantly diminished if homogenization was done in saline instead of Triton-X while the difference in fucosidase activity was not significant. Significant correlation was detected between alpha-glucosidase activity and week of gestation. It is suggested that Triton X-100-homogenization should be used for the lysosomal enzyme determinations in chorionic villi because the solubilization of enzymes from the lysosomes is complete in this case than with homogenization in saline.
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PMID:Lysosomal enzyme activities in frozen, non-cultured chorionic villi for prenatal diagnosis of enzymopathies. 136 80

In soluble fractions prepared from rat liver homogenates, L-penicillamine hydantoin appeared to be, on the basis of SH consumption measurements, a substrate for glutathione peroxidase but not transferase reactions. When glutathione is incubated with rat liver soluble proteins in the presence of penicillamine hydantoin, formation of oxidized glutathione is inhibited. Calculations from Lineweaver-Burk plots point out that inhibition by L-penicillamine hydantoin of the peroxide-dependent oxidations of glutathione is mixed, since both apparent Km and Vmax values are modified. Preincubation of rat liver soluble proteins with L-penicillamine hydantoin led to a progressive inactivation of glutathione peroxidase. The kinetics of this inactivation process with respect to time and inactivator concentration were studied. Inclusion in the preincubation mixture of SH-containing molecules such as dithiothreitol, L-cysteine or glutathione protected the enzyme against inactivation. However, none of these molecules and neither hydantoin, Triton X-100, phenol, nor dialysis could reverse the enzyme from inactivated to activated form. Mitochondrial glutathione peroxidase was inhibited and inactivated by L-penicillamine hydantoin to the same extent as its cytosolic counterpart. Modifications by penicillamine hydantoin of various subcellular markers enzymes (lactate dehydrogenase, N-acetyl beta-glucosaminidase, arylsulfatase C, butyryl-CoA dehydrogenase, lauryl-CoA and glycolate oxidases) were of weak amplitude consisting of either inhibition, inactivation or stimulation.
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PMID:Effect of L-penicillamine hydantoin, an analogue of glutathione, on rat liver glutathione peroxidase, reductase and transferase reactions. 156 76

Steroid sulfatase was purified approximately 170-fold from normal human placental microsomes and properties of the enzyme were investigated. The major steps in the purification procedure included solubilization with Triton X-100, column chromatofocusing, and hydrophobic interaction chromatography on phenylsepharose CL-4B. The purified sulfatase showed a molecular weight of 500-600 kDa on HPLC gel filtration, whereas the enzyme migrated as a molecular mass of 73 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isoelectric point of steroid sulfatase was estimated to be 6.7 by isoelectric focusing in polyacrylamide gel in the presence of 2% Triton X-100. The addition of phosphatidylcholine did not enhance the enzyme activity in the placental microsomes obtained from two patients with placental sulfatase deficiency (PSD) after solubilization and chromatofocusing. This result indicates that PSD is the result of a defect in the enzyme rather than a defect in the membrane-enzyme structure. Amino acid analysis revealed that the purified human placental sulfatase did not contain cysteine residue. The Km and Vmax values of the steroid sulfatase for dehydroepiandrosterone sulfate (DHA-S) were 7.8 microM and 0.56 nmol/min, while those for estrone sulfate (E1-S) were 50.6 microM and 0.33 nmol/min, respectively. The results of the kinetic study suggest the substrate specificity of the purified enzyme, but further studies should be done with different substrates and inhibitors.
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PMID:Purification and properties of steroid sulfatase from human placenta. 160 23

The effect of progesterone and nine synthetic progestogens on the activity rate of microsome estrone sulfatase obtained from human breast carcinoma tissues was studied. The progestogens were classified into three groups: group I with a strict inhibitor effect: demegestone and chlormadinone acetate; group II with a strict activator effect: medroxyprogesterone acetate, quingestanol acetate, lynestrenol and progesterone and group III with a nonsignificant effect: dydrogesterone, promegestone, norgestrel and danazol. Demegestone was the most potent inhibitor and medroxyprogesterone acetate and quingestanol acetate had the highest activator effect. The effect of Triton X-100, a nonionic detergent, was also tested. This detergent consistently increased the microsome estrone sulfatase activity. A comparison was made between the effects of demegestone, medroxyprogesterone acetate and danazol on estrone sulfatase activity measured with or without Triton X-100 in the incubation medium. The presence of the detergent modified the progestogen action. Our results suggest that synthetic progestogens can influence the estrone sulfatase activity measured in human breast carcinoma tissues. However, the effect of progestogens was dependent on experimental conditions. Progestogens such as demegestone and chlormadinone acetate which inhibited estrone sulfatase activity in intact preparations, can reduce the intracellular production of biological active estrogen via the sulfatase pathway.
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PMID:In vitro effect of synthetic progestogens on estrone sulfatase activity in human breast carcinoma. 175 97

Estrone sulfate sulfohydrolase (estrogen sulfatase) activity was solubilized by treatment with Triton X-100 from 105,000 g pellets of guinea pig uterus, testis and brain, as well as from rat liver and human placenta. The solubilized forms were subjected to chromatofocusing in the fast protein liquid chromatography (FPLC) system and on conventional columns packed in our laboratory. The guinea pig tissue pattern was complex. Uterus showed peaks of activity with apparent pI's of 9.11 and 7.6; testis contained 3 peaks with pI's of 9.18, 8.7 and 7.5; brain possessed peaks with pI's of 9.28 and 8.6. In each case the major activity peak was that with pI greater than 9. Rat liver activity chromatofocused as a single peak of apparent pI = 6.87 and the human placental enzyme also showed a single, though broad, peak, of pI = 6.57. This suggests not only that the guinea pig enzyme(s) differs markedly from those of rat liver and human placenta, but that there may be qualitative differences between the forms in the three guinea pig tissues. Chromatofocusing behaviour was not independent of the specific exchange resins and ampholytes utilized. The recovered enzyme activity was fairly stable and it seems that chromatofocusing could be a useful step in purification of the guinea pig enzyme(s), particularly the main form possessing a pI greater than 9.
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PMID:Chromatofocusing of mammalian estrone sulfate sulfohydrolase activity. 346 42


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