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 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.

Purification of glucosylceramidase by affinity chromatography

1982 ◽  
Vol 60 (11) ◽  
pp. 1025-1031 ◽  
Author(s):  
P. M. Strasberg ◽  
J. A. Lowden ◽  
D. Mahuran
Keyword(s):  
Sodium Dodecyl ◽  
Sodium Taurocholate ◽  
Low Ph ◽  
Affinity Column ◽  
Specific Technique ◽  
Natural Substrate ◽  
Ph Optimum ◽  
Specific Affinity ◽  
Glycol Solution ◽  
Slab Gels

Glucosylceramide:β-glucosidase (glucocerebrosidase, EC 3.2.1.45) has been purified 12 900-fold from human placenta using a specific affinity column. The ligand, glucosyl sphingosine, prepared from glucocerebroside by alkaline hydrolysis, was attached to epoxy-activated Sepharose 6B. The enzyme was applied to the column in citrate–butanol or citrate – ethylene glycol solution at its pH optimum (5.6). No enzyme was bound in the presence of detergent. Glucocerebrosidase was eluted with citrate–taurocholate buffer at low pH or with citrate-taurocholate buffer containing D-gluconolactone at the pH optimum. Citrate–taurocholate solution alone at the pH optimum would not elute the enzyme. The enzyme hydrolyzed both the natural substrate, glucocerebroside, and the artificial substrate, 4-methylumbelliferyl glucopyranoside. Glucocerebrosidase migrated as a single band on 10% sodium dodecyl sulfate–polyacrylamide tube and (or) slab gels, corresponding to a molecular weight of 75 000. It also ran as a single zone of enzyme activity or protein on native gels, composed of 2.2% polyacrylamide – 0.4% agarose containing sodium taurocholate. This is the first reported use of this gel system for the examination of glucocerebrosidase. Overall recovery is 30%. The procedure represents a more rapid and specific technique for purification of glucocerebrosidase than those previously reported.

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.

Immobilization of Chaetomium erraticum dextranase (CED) by adsorption on carboxylated multi walled carbon nanotubes (c-MWCNT)

2021 ◽  
Author(s):  
Yakup Aslan ◽  
Barzan Ismael Ghafour
Keyword(s):  
Storage Conditions ◽  
Reducing Sugar ◽  
Solution Ph ◽  
Multi Walled Carbon Nanotubes ◽  
Activity Yield ◽  
Walled Carbon Nanotubes ◽  
Hydrolysis Of ◽  
Dextran Solution ◽  
Burk Plot

Abstract In this study, CED was immobilized onto c-MWCNT by adsorption. Optimization of immobilization conditions (immobilization buffer's pH and molarity, c-MWCNT amount, and immobilization time) was resulted in 100% immobilization yield and 114.13% activity yield. Further, characterization of FCED and ICED was also studied. After immobilization, the optimum pH shifted from 5.0 to 6.0, while the optimum temperature (55 °C) did not change. Furthermore, kinetic constants for FCED and ICED were also determined using the Lineweaver-Burk plot. The Km value for both FCED and ICED were 54.35 g / L, while Vmax values for FCED and ICED were 2.77 μmol reducing sugar / L.mg.min and 3.19 μmol reducing sugar / L.mg.min, respectively. Moreover, there was no reduction in the initial activity of ICED after 20 consecutive uses and 30 days of storage at optimal storage conditions. Finally, 17.15% and 17.53% of the dextran in 10% dextran solution (pH 6.0) were converted to reduced sugars (IMOs and Glucose) in 12 hours using FCED and ICED, respectively. Consequently, it can be concluded that ICED obtained in this study can be effectively used for industrial production of IMOs and for hydrolysis of dextran.

scholarly journals The Effect of Procoagulative Phospholipids on the Synthetic Substrate Determined Thrombin Generation in Vitro

1977 ◽  
Author(s):  
F. Schulte ◽  
O. Klug ◽  
Ursula Roth
Keyword(s):  
Thrombin Generation ◽  
Chromogenic Substrate ◽  
Chromogenic Substrates ◽  
Platelet Factor ◽  
Synthetic Substrate ◽  
Thrombin Activity ◽  
Vitro Effect ◽  
Hydrolysis Of

The effect of procoagulative phospholipids (Procops) with platelet factor-3 like activity on the generation of thrombin in plasma can be determined in vitro with the aid of synthetic chromogenic substrates. Standard citrated plasma of two manufacturers and from different batches incubated with amounts of 2-10 meg Procops as 1:100 - 1:500 diluted Tachostyptan (micellar Procops in form of a pharmaceutical speciality) was activated. The generated amount of thrombin activity catalyses the hydrolysis of chromogenic substrate to a tripeptide and to p-nitroaniline which was measured kinetically with a spectrophotometer at 405 nm. At optimum concentrations of Procops, activities adequate to 80 (SD ± 7, VK 8. 8%) - 115 i. u. (SD ± 2. 5, VK 2.2%) thrombin per ml plasma were measured depending on the conditions of incubation and activation. Having made the basis of appropriate procedures for incubation and activation the in vitro effect of Procops on the thrombin generation in plasma is reproducibly measurable with the aid of chromogenic substrate.

Heat Stability of the Bile Salt-Stimulated Lipase in Human Milk Fortified with Sodium Taurocholate1

1988 ◽  
Vol 51 (4) ◽  
pp. 310-313 ◽  
Author(s):  
H. L. PAN ◽  
C. W. DILL ◽  
E. S. ALFORD ◽  
S. L. DILL ◽  
C. A. BAILEY ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Human Milk ◽  
Bile Salt ◽  
Lipase Activity ◽  
Sodium Taurocholate ◽  
Heat Stability ◽  
Heat Inactivation ◽  
Temperature Differential ◽  
Heat Stable ◽  
Control Samples

Time-temperature relationships for heat-inactivation of the bile salt-stimulated lipase activity were compared in whole human milk and in the same product fortified to 9 mM/ml with sodium taurocholate. Heat treatments were varied from 45 to 70°C for times ranging from 15s to 40 min. Enzyme activity was more heat stable in human milk fortified with taurocholate than in control samples. The temperature required for the onset of heat inactivation at 30-min holding time was increased from 45°C for control samples to 60°C following addition of taurocholate. A temperature differential of approximately 12°C was required in the fortified milks to produce inactivation equivalent to that observed in the control milks over the heating range studied.

MECHANISM OF ACTIVATION OF HOG PANCREATIC LIPASE BY SODIUM TAUROCHOLATE

1963 ◽  
Vol 41 (1) ◽  
pp. 719-730 ◽  
Author(s):  
Paul J. Fritz ◽  
Paul Melius
Keyword(s):  
Activation Energy ◽  
Enzymatic Hydrolysis ◽  
Pancreatic Lipase ◽  
Sodium Taurocholate ◽  
Enzyme Complex ◽  
Temperature Dependent ◽  
Fast Reaction ◽  
Presence And Absence ◽  
Hydrolysis Of

A new preparation of hog pancreatic lipase is described which is evidently the most stable one of its kind. From the composition of the reaction mixtures after enzymatic hydrolysis with and without sodium taurocholate, it appears that the hydrolysis of triglyceride to diglyceride is facilitated and the hydrolysis of diglyceride to monoglyceride is depressed in the presence of taurocholate. The differences in the curves showing the rates of hydrolysis of triolein, monoolein, tributyrin, and monobutyrin in the presence and absence of taurocholate also indicate that the taurocholate acts to split the diglyceride–enzyme complex and thus increases the action of the enzyme on the triglyceride ester. The hydrolysis of the triglyceride is a fast reaction whereas the di- and mono-glycerides are hydrolyzed at much slower rates. The activation energy for the lipolysis of the triolein and tributyrin has been calculated in the presence and absence of taurocholate. This was possible because in spite of earlier reports that the lipolysis of triolein was not temperature dependent between 10 and 40 degrees, we found these reactions to be temperature dependent. The lesser activating effect at the highest taurocholate concentrations indicates this is not a simple emulsifying effect.

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.

Effects of Pulsed Electric Fields on Inactivation Kinetics of Listeria innocua

1999 ◽  
Vol 65 (12) ◽  
pp. 5364-5371 ◽  
Author(s):  
Patrick C. Wouters ◽  
Nicole Dutreux ◽  
Jan P. P. M. Smelt ◽  
Huub L. M. Lelieveld
Keyword(s):  
Electric Field ◽  
Physiological State ◽  
Pulse Length ◽  
Listeria Innocua ◽  
Heat Inactivation ◽  
Inactivation Kinetics ◽  
Inlet Temperature ◽  
Similar Time ◽  
Process Factors ◽  
Kinetics Of

ABSTRACT The effects of pulsed electric field (PEF) treatment and processing factors on the inactivation kinetics of Listeria innocuaNCTC 11289 were investigated by using a pilot plant PEF unit with a flow rate of 200 liters/h. The electric field strength, pulse length, number of pulses, and inlet temperature were the most significant process factors influencing the inactivation kinetics. Product factors (pH and conductivity) also influenced the inactivation kinetics. In phosphate buffer at pH 4.0 and 0.5 S/m at 40°C, a 3.0-V/μm PEF treatment at an inlet temperature of 40°C resulted in ≥6.3 log inactivation of strain NCTC 11289 at 49.5°C. A synergistic effect between temperature and PEF inactivation was also observed. The inactivation obtained with PEF was compared to the inactivation obtained with heat. We found that heat inactivation was less effective than PEF inactivation under similar time and temperature conditions.L. innocua cells which were incubated for a prolonged time in the stationary phase were more resistant to the PEF treatment, indicating that the physiological state of the microorganism plays a role in inactivation by PEF. Sublethal injury of cells was observed after PEF treatment, and the injury was more severe when the level of treatment was increased. Overall, our results indicate that it may be possible to use PEF in future applications in order to produce safe products.

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