scholarly journals Hydrolysis of Tay-Sachs Ganglioside by β-Hexosaminidase A of Human Liver and Urine

1973 ◽  
Vol 248 (21) ◽  
pp. 7512-7515 ◽  
Author(s):  
Yu-Teh Li ◽  
Mary Y. Mazzotta ◽  
Chin-Chin Wan ◽  
Roger Orth ◽  
Su-Chen Li
Keyword(s):  
Human Liver ◽  
Hexosaminidase A ◽  
Hydrolysis Of

scholarly journals Interaction of activating protein and surfactants with human liver hexosaminidase A and GM2 ganglioside

1980 ◽  
Vol 185 (3) ◽  
pp. 583-591 ◽  
Author(s):  
Peter Hechtman ◽  
Zarin Kachra
Keyword(s):  
Human Liver ◽  
Anionic Surfactants ◽  
Sodium Taurocholate ◽  
Ionic Surfactants ◽  
Enzymic Hydrolysis ◽  
Gm2 Ganglioside ◽  
Activator Protein ◽  
Hexosaminidase A ◽  
Triton X ◽  
Hydrolysis Of

The effects of surfactants on the human liver hexosaminidase A-catalysed hydrolysis of Gm2 ganglioside were assessed. Some non-ionic surfactants, including Triton X-100 and Cutscum, and some anionic surfactants, including sodium taurocholate, sodium dodecyl sulphate, phosphatidylinositol and N-dodecylsarcosinate, were able to replace the hexosaminidase A-activator protein [Hechtman (1977) Can. J. Biochem.55, 315–324; Hechtman & Leblanc (1977) Biochem. J.167, 693–701) and also stimulated the enzymic hydrolysis of substrate in the presence of saturating concentrations of activator. Other non-ionic surfactants, such as Tween 80, Brij 35 and Nonidet P40, and anionic surfactants, such as phosphatidylethanolamine, did not enhance enzymic hydrolysis of Gm2 ganglioside and inhibited hydrolysis in the presence of activator. The concentration of surfactants at which micelles form was determined by measurements of the minimum surface-tension values of reaction mixtures containing a series of concentrations of surfactant. In the case of Triton X-100, Cutscum, sodium taurocholate, N-dodecylsarcosinate and other surfactants the concentration range at which stimulation of enzymic activity occurs correlates well with the critical micellar concentration. None of the surfactants tested affected the rate of hexosaminidase A-catalysed hydrolysis of 4-methylumbelliferyl N-acetyl-β-d-glucopyranoside. Both activator and surfactants that stimulate hydrolysis of Gm2 ganglioside decrease the Km for Gm2 ganglioside. Inhibitory surfactants are competitive with the activator protein. Evidence for a direct interaction between surfactants and Gm2 ganglioside was obtained by comparing gel-filtration profiles of 3H-labelled GM2 ganglioside in the presence and absence of surfactants. The results are discussed in terms of a model wherein a mixed micelle of surfactant or activator and GM2 ganglioside is the preferred substrate for enzymic hydrolysis.

Substrate binding properties of the human liver hexosaminidase A activator protein

1985 ◽  
Vol 63 (8) ◽  
pp. 830-838 ◽  
Author(s):  
Peter Hechtman ◽  
Claudine Isaacs ◽  
Louise Smith-Jones
Keyword(s):  
Protein Complex ◽  
Human Liver ◽  
Substrate Binding ◽  
Enzyme Activation ◽  
Stable Form ◽  
Ph Optimum ◽  
Substrate Complex ◽  
Activator Protein ◽  
Hexosaminidase A ◽  
Hydrolysis Of

The human liver hexosaminidase A activator protein has been shown to bind to the substrate GM2 ganglioside by cosedimentation in sucrose density gradients. Among other proteins tested only serum albumin forms a GM2 ganglioside – protein complex. Both activator protein and albumin bind to the monomeric form of GM2 ganglioside and not to the micellar form of the substrate. The GM2 ganglioside – activator protein complex can be recovered in a stable form. Storage at various temperatures or incubation with monosaccharides or with detergent does not result in dissociation of the complex. GM2 ganglioside in the activator–substrate complex is exchangeable with exogenous GM2 ganglioside. Hexosaminidase A, prepared from human liver, hydrolyzes GM2 ganglioside in the activator–substrate complex as efficiently as GM2 ganglioside supplied exogenously. The activator – Gm2 ganglioside complex forms at pH 3.0 and exhibits an optimum similar to the pH optimum of hexosaminidase A catalyzed hydrolysis of GM2 ganglioside in the presence of the activator; however, the ability of the activator to stimulate enzymic hydrolysis of substrate is rapidly lost after heating at 75 °C, whereas its ability to bind substrate is increased. The sphingolipids cerebroside sulfate and sphingomyelin show little or no binding to the hexosaminidase A activator protein nor do they inhibit activation of hexosaminidase A catalyzed hydrolysis of GM2 ganglioside. By contrast GM1 ganglioside inhibits both substrate binding and enzyme activation.

scholarly journals Purification and properties of the hexosaminidase A-activating protein from human liver

1977 ◽  
Vol 167 (3) ◽  
pp. 693-701 ◽  
Author(s):  
Peter Hechtman ◽  
Dorothy LeBlanc
Keyword(s):  
Human Liver ◽  
Gel Filtration ◽  
Disc Electrophoresis ◽  
Exchange Chromatography ◽  
Hydrolase Activity ◽  
Gm2 Ganglioside ◽  
Purification And Properties ◽  
Hexosaminidase A ◽  
Hydrolysis Of

Human liver extracts contain an activating protein which is required for hexosaminidase A-catalysed hydrolysis of the N-acetylgalactosaminyl linkage of GM2 ganglioside [N-acetylgalactosaminyl-(N-acetylneuraminyl) galactosylglucosylceramide]. A partially purified preparation of human liver hexosaminidase A that is substantially free of GM2 ganglioside hydrolase activity is used to assay the activating protein. The proceudres of heat and alcohol denaturation, ion-exchange chromatography and gel filtration were used to purify the activating protein over 100-fold from crude human liver extracts. When the purified activating protein is analysed by polyacrylamide-gel disc electrophoresis, two closely migrating protein bands are seen. When purified activating protein is used to reconstitute the GM2 ganglioside hydrolase activity, the rate of reaction is proportional to the amount of hexosaminidase A used. The activation is specific for GM2 ganglioside and and hexosaminidase A. The activating protein did not stimulate hydrolysis of asialo-GM2 ganglioside by either hexosaminidase A or B. Hexosaminidase B did not catalyse hydrolysis of GM2 ganglioside with or without the activator. Kinetic experiments suggest the presence of an enzyme–activator complex. The dissociation constant of this complex is decreased when higher concentrations of substrate are used, suggesting the formation of a ternary complex between enzyme, activator and substrate. Determination of the molecular weight of the activating protein by gel-filtration and sedimentation-velocity methods gave values of 36000 and 39000 respectively.

scholarly journals Identification of Catalposide Metabolites in Human Liver and Intestinal Preparations and Characterization of the Relevant Sulfotransferase, UDP-glucuronosyltransferase, and Carboxylesterase Enzymes

Pharmaceutics ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 355 ◽  
Author(s):  
Deok-Kyu Hwang ◽  
Ju-Hyun Kim ◽  
Yongho Shin ◽  
Won-Gu Choi ◽  
Sunjoo Kim ◽  
...  
Keyword(s):  
Human Liver ◽  
Individual Variability ◽  
Hydroxybenzoic Acid ◽  
Catalpa Ovata ◽  
Anti Oxidant ◽  
Extrahepatic Tissues ◽  
Udp Glucuronosyltransferase ◽  
Hydrolysis Of

Catalposide, an active component of Veronica species such as Catalpa ovata and Pseudolysimachion lingifolium, exhibits anti-inflammatory, antinociceptic, anti-oxidant, hepatoprotective, and cytostatic activities. We characterized the in vitro metabolic pathways of catalposide to predict its pharmacokinetics. Catalposide was metabolized to catalposide sulfate (M1), 4-hydroxybenzoic acid (M2), 4-hydroxybenzoic acid glucuronide (M3), and catalposide glucuronide (M4) by human hepatocytes, liver S9 fractions, and intestinal microsomes. M1 formation from catalposide was catalyzed by sulfotransferases (SULTs) 1C4, SULT1A1*1, SULT1A1*2, and SULT1E1. Catalposide glucuronidation to M4 was catalyzed by gastrointestine-specific UDP-glucuronosyltransferases (UGTs) 1A8 and UGT1A10; M4 was not detected after incubation of catalposide with human liver preparations. Hydrolysis of catalposide to M2 was catalyzed by carboxylesterases (CESs) 1 and 2, and M2 was further metabolized to M3 by UGT1A6 and UGT1A9 enzymes. Catalposide was also metabolized in extrahepatic tissues; genetic polymorphisms of the carboxylesterase (CES), UDP-glucuronosyltransferase (UGT), and sulfotransferase (SULT) enzymes responsible for catalposide metabolism may cause inter-individual variability in terms of catalposide pharmacokinetics.

scholarly journals Organic pyrophosphates as substrates for human alkaline phosphatases

1967 ◽  
Vol 105 (3) ◽  
pp. 1307-1312 ◽  
Author(s):  
R. Helen Eaton ◽  
D W Moss
Keyword(s):  
Gel Electrophoresis ◽  
Human Liver ◽  
Inorganic Phosphate ◽  
Starch Gel Electrophoresis ◽  
Alkaline Phosphatases ◽  
Starch Gel ◽  
Pyrophosphatase Activity ◽  
Small Intestinal ◽  
Hydrolysis Of ◽  
Single Enzyme

1. Purified human liver and small-intestinal alkaline orthophosphatases release inorganic phosphate at appreciable rates from a variety of organic pyrophosphate substrates. 2. The pyrophosphatase action is inhibited by Mg2+ ions at concentrations that activate the hydrolysis of orthophosphate substrates by these enzymes. 3. The results of mixed-substrate experiments, denaturation studies with heat or urea and starch-gel electrophoresis suggest that both orthophosphatase and pyrophosphatase activities are, in each preparation, properties of a single enzyme. 4. Intestinal phosphatase shows greater pyrophosphatase activity relative to orthophosphatase than the liver enzyme.

scholarly journals N-Acetyl-β-d-hexosaminidase component A. Different forms in human tissues and fluids

1973 ◽  
Vol 135 (3) ◽  
pp. 457-462 ◽  
Author(s):  
J. U. Ikonne ◽  
R. B. Ellis
Keyword(s):  
Salt Concentration ◽  
Human Liver ◽  
Deae Cellulose ◽  
Minor Component ◽  
Human Tissues ◽  
Serum Enzyme ◽  
Hexosaminidase A ◽  
Minor Form ◽  
A Minor ◽  
Cellulose Column

1. Hexosaminidase A of human serum was resolved into two components, a minor form with properties identical with those of the single hexosaminidase A component of human liver, and a major form with significantly different properties. 2. The major serum hexosaminidase A form was eluted from a DEAE-cellulose column at a lower salt concentration than that required to elute the liver form. 3. A multiple-pass technique was used to elute the major serum enzyme A from a Sephadex G-150 column before that of liver enzyme A. 4. Clostridium perfringens neuraminidase converted the major component of serum hexosaminidase A into a form that was held less tightly by DEAE-cellulose, but the minor component of the A enzyme of serum, and the A enzyme of liver were not affected. 5. The hexosaminidase A from tears was similar to the A enzyme from serum, whereas those from several human tissues and from urine and lymph were similar to the liver form. 6. The A enzyme from serum may be derived from the A enzyme from liver by glycosylation before secretion.

scholarly journals Purification and Cloning of a Broad Substrate Specificity Human Liver Carboxylesterase That Catalyzes the Hydrolysis of Cocaine and Heroin

1997 ◽  
Vol 272 (23) ◽  
pp. 14769-14775 ◽  
Author(s):  
Evgenia V. Pindel ◽  
Natalia Y. Kedishvili ◽  
Trent L. Abraham ◽  
Monica R. Brzezinski ◽  
Jing Zhang ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Human Liver ◽  
Broad Substrate Specificity ◽  
Hydrolysis Of

Phosphoester specificity of purified human liver alkaline phosphatase

1979 ◽  
Vol 57 (7) ◽  
pp. 1000-1007 ◽  
Author(s):  
L. E. Seargeant ◽  
R. A. Stinson
Keyword(s):  
Alkaline Phosphatase ◽  
Atpase Activity ◽  
Human Liver ◽  
Magnesium Ions ◽  
Calcium Dependent ◽  
Ph Values ◽  
Regulator Protein ◽  
Calcium And Magnesium ◽  
Hydrolysis Of ◽  
Calcium And Magnesium Ions

Kinetic parameters for the hydrolysis of a number of physiologically important phosphoesters by purified human liver alkaline phosphatase have been determined. The enzyme was studied at pH values of 7.0 to 10.0. The affinity of the enzyme for the compounds was determined by competition experiments and by their direct employment as substrates. Phosphodiesters and phosphonates were not hydrolysed but the latter were inhibitors. Calcium and magnesium ions inhibited the hydrolysis of ATP and PP1 and evidence is presented to show that the metal complexes of these substrates are not hydrolysed by alkaline phosphatase. A calcium-stimulated ATPase activity could not be demonstrated for the purified enzyme or the enzyme in the presence of a calcium-dependent regulator protein. Nevertheless, the influence of magnesium and calcium ions on the ATPase activity of alkaline phosphatase means that precautions must be taken when assaying for Ca2+-ATPase in the presence of alkaline phosphatase.The low substrate Km values and the hydrolysis which occurs at pH 7.4 mean that the enzyme could have a significant phosphohydrolytic role. However, liver cell phosphate concentrations, if accessible to the enzyme, are sufficient to strongly inhibit this activity.

scholarly journals Characterization of proteins structurally related to human N-acetyl-β-d-glucosaminidase

1978 ◽  
Vol 173 (1) ◽  
pp. 191-196 ◽  
Author(s):  
M Carroll
Keyword(s):  
Molecular Weight ◽  
Human Liver ◽  
Deae Cellulose ◽  
Low Molecular Weight ◽  
Enzyme Preparations ◽  
Molecular Weights ◽  
Functional Relationships ◽  
Hexosaminidase A ◽  
Cellulose Column

Those proteins of human liver that cross-reacted with antibodies raised to apparently homogenous hexosamindases A and B were detected by immunodiffusion. Cross-reacting proteins with high molecular weights (greater than 2000000) and intermediate molecular weights (70000–200000) were present both in the unadsorbed fraction and in the 0.05–0.2M-NaCl eluate obtained by DEAE-cellulose chromatography at pH7.0. The unadsorbed fraction also contained a cross-reacting protein of low molecular weight (10000–70000). The possible structural and functional relationships between hexosaminidase and the cross-reacting proteins are discussed. An apparently cross-reacting protein present in the 0.05M-NaCl eluate from the DEAE-cellulose column was serologically unrelated to hexosaminidase, but it gave a reaction of immunological identify with one of the apparently cross-reacting proteins having the charge and size characteristics of hexosaminidase A. It is suggested that immunochemical methods may provide criteria for the homogeneity of enzyme preparations superior to those of conventional methods.

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