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.

The β-glucosidases of porcine kidney

1977 ◽  
Vol 55 (2) ◽  
pp. 140-145 ◽  
Author(s):  
Julian N. Kanfer ◽  
Richard A. Mumford ◽  
Srinivasa S. Raghavan
Keyword(s):  
Sodium Taurocholate ◽  
Hydrolytic Activity ◽  
Acidic Ph ◽  
Porcine Kidney ◽  
Ph Optimum ◽  
Pig Kidney ◽  
Competitive Inhibitors ◽  
Triton X ◽  
Hydrolysis Of ◽  
Estradiol 17Β

Some of the properties of a partially purified particle bound and soluble β-glucosidase (EC 3.2.1.21) from pig kidney were compared. The soluble β-glucosidase (1) hydrolyzed 4-methylumbelliferyl-β-D-glucoside (4-MU-β-D-glucoside) 17α-estradiol 3β-glucoside, 17α-estradiol 17β-glucoside, and salicin, but not glucosylceramide, (2) possessed a broad pH optimum (5.5–7.0), (3) had an isoelectric point of 4.9, and (4) was inhibited by Triton X-100. Several compounds were found to be competitive inhibitors of its hydrolytic activity, gluconolactam and estrone β-glucoside being the most effective. In contrast, a particulate β-glucosidase purified from the same tissue (1) had an acidic pH optimum (5.0), (2) was stimulated by sodium taurocholate and 'Gaucher's factor' for the hydrolysis of both 4-MU-β-glucoside and glucosylceramide, and (3) was capable of catalyzing a transglucosylation reaction employing 4-MU-β-D-glucoside or glucosylceramide as the glucosyl donor, and [l4C]ceramide as acceptor.

Characterization of an activating factor required for hydrolysis of Gm2 ganglioside catalyzed by hexosaminidase A

1977 ◽  
Vol 55 (4) ◽  
pp. 315-324 ◽  
Author(s):  
Peter Hechtman
Keyword(s):  
Enzyme Activity ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Hydrolase Activity ◽  
Gm2 Ganglioside ◽  
Heat Stable ◽  
Hexosaminidase A ◽  
Heat Stable Protein ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of

Separation of the hexosaminidase A (EC 3.2.1.52) and B isozymes of human liver by ion-exchange chromatography results in recovery of greater than 80% of the activity in crude extracts when synthetic substrates are used to monitor enzyme activity. Only 15% of hexosaminidase activity toward the N-acetylgalactosaminyl (N-acetylneuraminyl) galactosyl glucosylceramide (Gm2 ganglioside) substrate is recovered and all of this activity is associated with the hexosaminidase A fraction.The low level of Gm2 ganglioside hydrolase activity in the hexosaminidase A fraction could be enhanced by coincubation with column fractions which contain hexosaminidase B. The activating factor, which has been partially purified by gel filtration, is a heat-stable protein with a molecular weight of 36 000 and is without enzyme activity toward hexosaminidase substrates.Highly purified hexosaminidase A or crude hexosaminidase A recovered after gel filtration on Sephadex G-100 has no Gm2 ganglioside hydrolase activity. The Gm2 ganglioside hydrolase activity of these hexosaminidase A preparations can be completely restored by addition of activating factor. The activating factor does not affect the rate of hydrolysis of synthetic substrate or asialo Gm2 ganglioside catalyzed by hexosaminidase A.

scholarly journals In Cellulo Examination of a Beta-Alpha Hybrid Construct of Beta-Hexosaminidase A Subunits, Reported to Interact with the GM2 Activator Protein and Hydrolyze GM2 Ganglioside

2021 ◽  
Author(s):  
Warren W. Wakarchuk ◽  
Incilay Sinici ◽  
Sayuri Yonekawa ◽  
Ilona Tkachyova ◽  
Steven J. Gray ◽  
...  
Keyword(s):  
In Vitro Assays ◽  
Variant Form ◽  
Gm2 Ganglioside ◽  
Activator Protein ◽  
Hexosaminidase A ◽  
Hybrid Construct ◽  
Gm2 Activator ◽  
Gm2 Activator Protein

The hydrolysis in lysosomes of GM2 ganglioside to GM3 ganglioside requires the correct synthesis, intracellular assembly and transport of three separate gene products; i.e., the alpha and beta subunits of heterodimeric beta-hexosaminidase A, E.C. # 3.2.1.52 (encoded by the HEXA and HEXB genes, respectively), and the GM2-activator protein (GM2AP, encoded by the GM2A gene). Mutations in any one of these genes can result in one of three neurodegenerative diseases collectively known as GM2 gangliosidosis (HEXA, Tay-Sachs disease, MIM # 272800; HEXB, Sandhoff disease, MIM # 268800; and GM2A, AB-variant form, MIM # 272750). Elements of both of the hexosaminidase A subunits are needed to productively interact with the GM2 ganglioside-GM2AP complex in the lysosome. Some of these elements have been predicted from the crystal structures of hexosaminidase and the activator. Recently a hybrid of the two subunits has been constructed and reported to be capable of forming homodimers that can perform this reaction in vivo, which could greatly simplify vector-mediated gene transfer approaches for Tay-Sachs or Sandhoff diseases. A cDNA encoding a hybrid hexosaminidase subunit capable of dimerizing and hydrolyzing GM2 ganglioside could be incorporated into a single vector, whereas packaging both subunits of hexosaminidase A into vectors, such as adeno-associated virus, would be impractical due to size constraints. In this report we examine the previously published hybrid construct (H1) and a new more extensive hybrid (H2), with our documented in cellulo (live cell- based) assay utilizing a fluorescent GM2 ganglioside derivative. Unfortunately when Tay-Sachs cells were transfected with either the H1 or H2 hybrid construct and then were fed the GM2 derivative, no significant increase in its turnover was detected. In vitro assays with the isolated H1 or H2 homodimers confirmed that neither was capable of human GM2AP-dependent hydrolysis of GM2 ganglioside.

Enzymic hydrolysis of sphingolipids. Hydrolysis of ceramide glucoside by an enzyme from ox brain

1966 ◽  
Vol 101 (3) ◽  
pp. 687-691 ◽  
Author(s):  
G Shimon
Keyword(s):  
Palmitic Acid ◽  
Acetate Buffer ◽  
Enzymic Hydrolysis ◽  
Ionic Detergent ◽  
Anionic Detergents ◽  
Beta Glucosidase ◽  
Triton X ◽  
Hydrolysis Of

Ceramide glucoside (1-O-glucosido-2-N-acyl-sphingosine) was hydrolysed to ceramide (N-acyl-sphingosine) and glucose by beta-glucosidase from ox brain. The reaction was stimulated by the non-ionic detergent, Triton X-100, or by the anionic detergents, cholate or taurocholate. It was not reversible, had optimum pH5.0 (with acetate buffer) or 5.6 (with pyridine buffer), had K(m) 1.8x10(-4)m and was inhibited by delta-gluconolactone and sphingosine, but not by ceramide or palmitic acid.

scholarly journals A protein activator for the enzymic hydrolysis of GM2 ganglioside.

1981 ◽  
Vol 256 (12) ◽  
pp. 6234-6240 ◽  
Author(s):  
S.C. Li ◽  
Y. Hirabayashi ◽  
Y.T. Li
Keyword(s):  
Enzymic Hydrolysis ◽  
Gm2 Ganglioside ◽  
Protein Activator ◽  
Hydrolysis Of

scholarly journals Isolation and characterization of an activator protein for the hydrolysis of ganglioside GM2 from the roe of striped mullet (Mugil cephalus)

1989 ◽  
Vol 260 (3) ◽  
pp. 777-783 ◽  
Author(s):  
R DeGasperi ◽  
Y T Li ◽  
S C Li
Keyword(s):  
Mugil Cephalus ◽  
Striped Mullet ◽  
Activator Protein ◽  
Isolation And Characterization ◽  
Mammalian Tissues ◽  
Hexosaminidase A ◽  
Ammonium Sulphate Fractionation ◽  
Gm2 Activator ◽  
Hydrolysis Of

After the revelation of the presence of ganglioside GM2 as the major ganglioside in the roe of striped mullet, Mugil cephalus [Li, Hirabayashi, DeGasperi, Yu, Ariga, Koerner & Li (1984) J. Biol. Chem. 259, 8980-8985], we have continued to investigate the catabolism of GM2 in this tissue. We have found that mullet roe contains a specific activator protein which stimulates the hydrolysis of GM2 carried out by the beta-hexosaminidase isolated from the same tissue. This activator has been purified by using conventional procedures including ammonium sulphate fractionation and chromatography on Sepharose 6B, DEAE-Sephadex A-50, octyl-Sepharose and Matrex Gel Blue A columns. This activator protein is also able to stimulate the hydrolysis of GM2 carried out by human beta-hexosaminidase A. Unlike human GM2-activator, the roe activator protein does not stimulate the hydrolysis of GgOse3Cer or GbOse4Cer. The molecular mass (18 kDa) of the roe activator protein was found to be similar to that of human GM2-activator; however, the pI (pH 4.1) was found to be lower than that of human GM2-activator. This is the first report on the presence of a GM2-activator protein in a source other than mammalian tissues.

scholarly journals Hydrolysis of ceramide trihexoside by a specific α-galactosidase from human liver

1973 ◽  
Vol 133 (1) ◽  
pp. 1-10 ◽  
Author(s):  
Mae Wan Ho
Keyword(s):  
Human Liver ◽  
Sodium Taurocholate ◽  
Optimum Shape ◽  
Heat Inactivation ◽  
Inactivation Kinetics ◽  
Natural Substrate ◽  
Synthetic Substrate ◽  
Ceramide Trihexoside ◽  
Hydrolysis Of ◽  
Burk Plot

1. Partially purified ceramide trihexoside α-galactosidase from human liver was studied by using ceramide trihexoside specifically tritiated in the terminal galactose. 2. The hydrolysis of ceramide trihexoside was absolutely dependent on a mixture of sodium taurocholate and Triton X-100 and was markedly inhibited by human serum albumin and by NaCl. 3. The Lineweaver–Burk plot for ceramide trihexoside hydrolysis was upward curving. Ceramide lactoside inhibited hydrolysis of all concentrations of ceramide trihexoside. Ceramide digalactoside stimulated hydrolysis of low concentrations of ceramide trihexoside, but inhibited hydrolysis of high concentrations of the lipid. 4. α-Galactosidase activity assayed with the synthetic substrate 4-methylumbelliferyl α-d-galactopyranoside fractionated together with activity assayed with the natural substrate ceramide trihexoside. Both activities had identical heat-inactivation kinetics. 5. Characteristics of the hydrolysis of the synthetic substrate differed considerably from those of the natural substrate, including pH optimum, shape of the Lineweaver–Burk plot, and differential effects of inhibitors and activators. Mutual inhibition of hydrolysis between the synthetic and natural substrates was predominantly non-competitive. 6. These results are discussed in the light of special problems involved in the hydrolysis of lipids in an aqueous milieu.

scholarly journals Non-enzymic hydrolysis of bilirubin mono- and diglucuronide to unconjugated bilirubin in model and native bile systems. Potential role in the formation of gallstones

1987 ◽  
Vol 242 (2) ◽  
pp. 323-329 ◽  
Author(s):  
W Spivak ◽  
D DiVenuto ◽  
W Yuey
Keyword(s):  
Guinea Pig ◽  
Sodium Taurocholate ◽  
Gallstone Formation ◽  
Unconjugated Bilirubin ◽  
Bile Pigment ◽  
Bile Pigments ◽  
Enzymic Hydrolysis ◽  
Pigment Gallstones ◽  
Model Bile ◽  
Hydrolysis Of

Pigment gallstones contain considerable amounts of unconjugated bilirubin (UCB) in the form of calcium bilirubinate and/or bilirubin polymers. Since more than 98% of bile pigments are excreted as conjugates of bilirubin, the source of this UCB needs to be identified. By using a rapid h.p.l.c. method, we compared the non-enzymic hydrolysis of bilirubin monoglucuronide (BMG) and bilirubin diglucuronide (BDG) to UCB in model bile and in native guinea-pig bile. Model biles containing 50 microM solutions of pure BMG and BDG were individually incubated in 25 mM-sodium taurocholate (NaTC) and 0.4 M-imidazole/5 mM-ascorbate buffer (TC-BUF) at 37 degrees C. Over an 8 h period, BMG hydrolysis produced 4-6 times more UCB than BDG hydrolysis. At pH 7.4, 25% of the BMG was converted into UCB, whereas only 4.5% of BDG was converted into UCB. Hydrolysis rates for both BMG and BDG followed the pH order 7.8 greater than 7.6 approximately equal to 7.4 greater than 7.1 Incubation with Ca2+ (6.2 mM) at pH 7.4 in TC-BUF resulted in precipitated bile pigment which, at 100 X magnification, appeared similar to precipitates seen in the bile of patients with pigment gallstones. At pH 7.4, lecithin (crude phosphatidylcholine) (4.2 mM) was a potent inhibitor of hydrolysis of BMG and BDG. The addition of a concentration of cholesterol equimolar with that of lecithin eliminated this inhibitory effect. Guinea-pig gallbladder bile incubated with glucaro-1,4-lactone (an inhibitor of beta-glucuronidase) underwent hydrolysis similar to the model bile systems. The non-enzymic hydrolysis of bile pigments, especially BMG, may be an important mechanism of bile-pigment precipitation and, ultimately, of gallstone formation.

Sign in / Sign up

Export Citation Format

Share Document