Interrelationship of hexosaminidases A and B: conformation of the common and the unique subunit theory.

Srivastava, Satish K.; Wiktorowicz, John E.; Awasthi, Yogesh C.

doi:10.1073/pnas.73.8.2833

Cited by 36 publications

(28 citation statements)

References 31 publications

(42 reference statements)

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“…2). When both these peaks are pooled separately and subjected individually to re-isoelectrofocusing on a thin-layer bed of Sephadex G-75 by the method described by Srivastava et al (1976), they appear as single peaks corresponding to the pI of the parent fractions. During several isoelectrofocusing experiments the pl values of GSH S-transferase co (major component) and ,v (minor component) were found to be about 4.6 and 5.4 respectively.…”

Section: Resultsmentioning

confidence: 99%

Interrelationship between anionic and cationic forms of glutathione S-transferases of human liver

Awasthi¹,

Dao²,

Saneto³

1980

Biochemical Journal

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199

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Human liver glutathione S-transferases (GSH S-transferases) were fractionated into cationic and anionic proteins. During fractionation with (NH4)2SO4 the anionic GSH S-transferases are concentrated in the 65%-saturated-(NH4)2SO4 fraction, whereas the cationic GSH S-transferases separate in the 80%-saturated-(NH4)2SO4 fraction. From the 65%-saturated-(NH4)2SO4 fraction two new anionic GSH S-transferases, ct and y/, were purified to homogeneity by using ion-exchange chromatography on DEAEcellulose, Sephadex G-200 gel filtration, affinity chromatography on GSH bound to epoxy-activated Sepharose and isoelectric focusing. By a similar procedure, cationic GSH S-transferases were purified from the 80%-saturated-(NH4)2SO4 fraction. Isoelectric points of GSH S-transferases co and ,v are 4.6 and 5.4 respectively. GSH S-transferase co is the major anionic GSH S-transferase of human liver, whereas GSH S-transferase yv is present only in traces. The subunit mol.wt. of GSH S-transferase co is about 22 500, whereas that of cationic GSH S-transferases is about 24 500. Kinetic and structural properties as well as the amino acid composition of GSH S-transferase co are described. The antibodies raised against cationic GSH S-transferases cross-react with GSH S-transferase co. There are significant differences between the catalytic properties of GSH S-transferase co and the cationic GSH S-transferases. GSH peroxidase II activity is displayed by all five cationic GSH S-transferases, whereas both anionic GSH S-transferases do not display this activity.

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Section: Resultsmentioning

confidence: 99%

Interrelationship between anionic and cationic forms of glutathione S-transferases of human liver

Awasthi¹,

Dao²,

Saneto³

1980

Biochemical Journal

Self Cite

199

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“…The enzyme assay with H202 as substrate was performed by the method of Beutler (1971). Polyacrylamide-disc-gel electrophoresis was carried out by the method of Davis (1964) and thin-layer isoelectrofocusing on Sephadex G-75 bed was performed by the method of Srivastava et al (1976). Protein was determined by the method of Lowry et al (1951).…”

Section: Methodsmentioning

confidence: 99%

Purification and properties of glutathione peroxidase from human placenta

Awasthi¹,

Dao²,

Lal³

et al. 1979

Biochemical Journal

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103

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Glutathione peroxidase (glutathione-H2O2 oxidoreductase; EC 1.11.1.9) was purified to homogeneity from human placenta by using (NH4)2SO4 precipitation, ion-exchange chromatography, Sephadex gel filtration and preparative polyacrylamide-disc-gel electrophoresis. Glutathione peroxidase from human placenta is a tetramer, having 4g-atoms of selenium/mol of protein. The molecular weight of the enzyme is about 85 000 with a subunit size of about 22000. Kinetic properties of the enzyme are described. On incubation with cyanide, glutathione peroxidase is completely and irreversibly inactivated and selenium is released as a low-molecular-weight fragment. Reduced glutathione, f,-mercaptoethanol and dithiothreitol protect the enzyme from inactivation by cyanide and the release of selenium. Properties of human placental glutathione peroxidase are similar to those of isoenzyme A reported earlier by us from human erythrocytes. The presence of isoenzyme B, reported earlier by us in human erythrocytes, was not detected in placenta. Also selenium-independent glutathione peroxidase (isoenzyme II), which is specific for cumene hydroperoxide, was not present in human placenta.

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“…21 -231 but the active sites for these respective two substrates might be distinct [24, [8,[27][28][29] seem to be identical to form P characterized in the serum of pregnant women [30 - [34,351 or alternatively identical to Hex B [36, 371. Hex S [38,39], a minor acidic form (PI about 4.5) present in normal liver [35] and in Sandhoff disease tissues [36,401 hydrolyses substrate containing both the P-GlcNAc and @-GalNAc linkages.…”

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confidence: 99%

Molecular Forms of β‐N‐Acetylhexosaminidase in Epstein‐Barr Virus‐Transformed Lymphoid Cell Lines from Normal Subjects and Patients with Tay‐Sachs Disease

Salvayre

Maret

Nègre

et al. 1983

European Journal of Biochemistry

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In whole leukocytes and in lymphocytes from normal subjects, the percentage activity of heat‐stable β‐N‐acetylhexosaminidase (30 ± 5% and 45 ± 5%, respectively) was higher than in the transformed lymphoid cell line (19 ± 3%). In Tay‐Sachs transformed cells as well as non‐transformed lymphocytes, β‐N‐acetylhexosaminidase was almost completely heat‐stable (95–98%). In the transformed cells from normal subjects, the β‐N‐acetylhexosaminidase B (Hex B) activity (5% of total) was significantly lower than in blood lymphocytes (average 25–30% of total activity), whereas Hex A and Hex I were similar in the either cell type. Blood lymphocytes and lymphoid cell lines established from a Tay‐Sachs patient lacked heat‐labile Hex A and expressed high heat‐stable Hex I and Hex B activities (3–6‐fold). After neuraminidase treatment, Hex A peak sharpened while Hex I peaks switched to higher pI than normal Hex I, in the region of Hex B. PreHex A/S pI was not affected. Hydrolytic properties using the both substrates (4‐methylumbelliferyl‐2‐acetamido‐2‐deoxy‐β‐d‐glucopyranoside and 4‐methylumbelliferyl‐2‐acetamido‐2‐deoxy‐β‐d‐galactopyranoside) of each molecular form were similar in transformed and non‐transformed cells. Data derived from the use of a mixture of substrates were consistent with the model which proposes a common active site for either substrate in the case of preHex A, Hex B and Hex I, but not for Hex A.Thus Epstein‐Barr virus‐transformed lymphoid cell lines represent an accurate model system for studies on Tay‐Sachs disease.

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Interrelationship of hexosaminidases A and B: conformation of the common and the unique subunit theory.

Cited by 36 publications

References 31 publications

Interrelationship between anionic and cationic forms of glutathione S-transferases of human liver

Interrelationship between anionic and cationic forms of glutathione S-transferases of human liver

Purification and properties of glutathione peroxidase from human placenta

Molecular Forms of β‐N‐Acetylhexosaminidase in Epstein‐Barr Virus‐Transformed Lymphoid Cell Lines from Normal Subjects and Patients with Tay‐Sachs Disease

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