Forms Of Β-galactosidase Research Articles

Does pH 6 β-galactosidase activity indicate cell senescence?

Apparently two forms of β-galactosidase (β-GAL) in cells or tissue sections can be detected by enzyme histochemical staining (X-GAL). Using a sensitive and specific HPLC method we have determined the pH dependent activity of β-GAL in cell lines of lung carcinoma (A549), colon carcinoma (Caco2-TC7), promyelocytic leukemia (HL60), hepatoma (HepG2) and human liver homogenates. The HPLC method has been validated and the influence of pH and substrate concentration was studied. There was a good linear correlation between HPLC and quantitative enzyme histochemistry (pH 4.5: r=0.985; pH 6.0: r=0.967). Both, pH 4.5 β-GAL and pH 6 β-GAL could be demonstrated in all biological material tested and pH 6 β-GAL activity was always lower (25–50%) than pH 4.5 activity. In Caco2-TC7 cells both activities increased by a factor of 10 from day 3 to day 17 after seeding. In addition, since the β-GAL activity decreased with increase in pH both in human liver homogenates (independent of the age of the donor) as well as in tumor cell lysates in a similar fashion we believe that the activity at pH 6 can hardly be considered as an exclusive ‘senescence marker’. In addition, the more sensitive HPLC method could demonstrate activity in cells that showed negative reaction with X-GAL.

Stabilization effect of polyvinyl alcohol on horseradish peroxidase, glucose oxidase, ?-galactosidase and alkaline phosphatase

The stabilization effect of different molecular weights of polyvinyl alcohol (PVA) upon solutions of commonly used analytical enzymes was studied. It was found that various PVA conditions could stabilize the activity of horseradish peroxidase and glucose oxidase but not β-galactosidase or alkaline phosphatase. It is expected that PVA can stabilize conjugate forms of glucose oxidase as well as horseradish peroxidase (as previously shown) but not the conjugate forms of β-galactosidase or alkaline phosphatase.

PURIFICATION AND CHARACTERIZATION OF β-GALACTOSIDASE OF `FUJI' APPLES

β-Galactosidase was purified from apple, Malus domestica Borkh, cv. Fuji by gel filtration, CM cellulose ion exchange chromatography, and characterizied by means of several biochemical methods. One form of β-Galactosidase was detected and the Km and Vmax values were calculated to be 0.035 and 0.036 mM with para-nitrophenyl-β-D-galactoside lmM/15min., respectively. The β-galactosidase was active between pH 3 and 7 with the optimum pH of about 4-5. The stable temperature for β-galactosidase was lower than 45°C with 30°C optimum. The β-galactosidase activities were inhibited by Ag*. EDTA and SDS.

Characterization of β-galactosidases from the germinating seeds of Vigna sinensis

Four forms of β-galactosidase from the germinating seeds of Vigna sinensis were separated and partially purified by ammonium sulphate precipitation, ion exchange chromatography (DE-52) and gel filtration to more than 50% purity as judged by PAGE. The pH and temperature optima, stability, M r , kinetic parameters and energy of activation of each enzyme have been determined. The four forms differed in their M, s and ionic charges.

Acid Glycosidase Isoenzymes and Activities in Developing Hulls and Seeds of Pisum sativum L.

Summary The changes in β-glucosidase (EC 3.2.1.21), β-galactosidase (EC 3.2.1.23), α-mannosidase (EC 3.2.1.24) and α-glucosidase (EC 3.2.1.20) activities that occur during development of the pea seed have been measured. The highest tissue specific activity of β-glucosidase is found in the seedcoats, whereas the highest specific activities of the other enzymes occur in the cotyledons. Glycosidase isoenzymes were separated by ammonium sulphate fractionation followed by anion exchange chromatography and gel filtration. The α-mannosidase and β-galactosidase activities of the cotyledons are attributed mainly to single forms of each specific enzyme of MW 70,000 and 46,000 daltons respectively. By comparison, a wider spectrum of isoenzymes was observed in the hull and seedcoats. For seedcoats, two forms of β-glucosidase, three forms of β-galactosidase and four forms of α-mannosidase were distinguished. The main form of β-glucosidase from the seedcoats, isoenzyme 2, had a MW estimated to be 65,000 daltons.

MULTIPLE FORMS OF β -GALACTOSIDASE AND THE CHANGES IN ITS PATTERN DURING DEVELOPMENT OF DICTYOSTELIUM DISCOIDEUM.

Four forms of β-galactosidase (EC 3.2.1.23) were separated from cell extracts of Dictyostelium discoideum by polyacrylamide gel electrophoresis. Changes in the pattern of multiple forms of this enzyme during differentiation and dedifferentiation were studied. The band closest to the anode (Band 1) existed throughout development, but the other three bands appeared and disappeared at certain stages. One of those bands was specific for the vegetative stage (Band 3), and another for the morphogenetic stages (Band 2). The last one predominantly appeared during dedifferentiation of disaggregated slug cells (Band 4), although it was slightly detected during culmination. During the process of dedifferentiation, the increase in activity of Band 4 coincided with the decrease in Band 2. After the completion of dedifferentiation, Band 4 also disappeared, only Band 1 remaining. These multiple forms were not only electrophoretically separable but different in their sensitivities to various inhibitors. All forms of the enzyme were localized in subcellular particles, probably in lysosomes and phagosomes, and the relative activities in these two fractions varied during development.

β-Galactosidase, β-glucosidase and N-acetyl- β-glucosaminidase in human kidney

The levels of N-acetyl- β-glucosaminidase, β-galactosidase and β-glucosidase activity in normal human kidney were determined fluorimetrically. All three glycosidases were studied by ion exchange chromatography and gel electrophoresis. N-acetyl- β-glucosamin idase exists in two forms, acidic and basic, closely resembling those in human spleen. There are also two forms of β-galactosidase, one of which appears to be identical with the single β-glucosidase component. The same enzymes were assayed in urine from normal subjects. In contrast to the kidney, the urinary level of β-galactosidase activity is greater than the N-acetyl- β-glucosaminidase, which occurs almost entirely in the acidic form. Only one form of β-galactosidase could be found and β-glucosidase could not be detected. The β-galactosidase and β-glucosidase activities in human kidney and urine have characteristics similar to the corresponding enzymes in the rat. The possible use of the glycosidases for the detection of kidney damage is discussed.