Purification of γ-glutamyltransferase of rat kidney by affinity chromatography using concanavalin a conjugated with sepharose 4B

Takahashi, Shizuko; Pollack, J D; Seifter, Sam

doi:10.1016/0005-2795(74)90156-1

Cited by 19 publications

(3 citation statements)

References 5 publications

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“…The earliest preparations of y-glutamyltransferase employed detergent extracts of microsomal fractions and yielded a very-high-molecular-weight form still associated with membrane components and soluble with difficulty in the absence of detergents (Revel & Ball, 1959;Connell & Adamson, 1970;Orlowski & Meister, 1970;Takahashi et al, 1974). The enzyme obtained by papain treatment of microsomal fractions, in contrast, is freely soluble in water and more readily purified.…”

Section: Discussionmentioning

confidence: 99%

“…The enzyme was obtained from the kidneys of adult male Sprague-Dawley rats by established procedures. A microsomal pellet was treated with papain (Curthoys & Kuhlenschmidt, 1975), and the solubilized enzyme was purified by passage through columns of Sephadex G-200 and concanavalin A-Sepharose 4B (Takahashi et al, 1974; Hughey & Curthoys, 1976). The activity of the enzyme was assayed in a total volume of 1 ml containing 2.5 mmy-glutamic acid p-nitroanilide, 20 mM-glycylglycine and enzyme sample in 50mM-2-amino-2-methylpropane-1,3-diol, pH 8.0, at 200C, by observing the rate of increase of absorption at 410nm (Orlowski & Meister, 1965; Szasz, 1969).…”

Section: Experimental Materials and Methodsmentioning

confidence: 99%

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Active-site amino acid residues in γ-glutamyltransferase and the nature of the γ-glutamyl-enzyme bond

Elce

1980

Biochemical Journal

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Active-site residues in rat kidney gamma-glutamyltransferase (EC 2.3.2.2) were investigated by means of chemical modification. 1. In the presence of maleate, the activity was inhibited by phenylmethanesulphonyl fluoride, and the inhibition was not reversed by beta-mercaptoethanol, suggesting that a serine residue is close to the active site, but is shielded except in the presence of maleate. 2. Treatment of the enzyme with N-acetylimidazole modified an amino group, exposed a previously inaccessible cysteine residue and inhibited hydrolysis of the gamma-glutamyl-enzyme intermediate, but not its formation. 3. After reaction of the enzyme successively with N-acetylimidazole and with non-radioactive iodoacetamide/serine/borate, two active-site residues reacted with iodo[(14)C]acetamide. One of these possessed a carboxy group, which formed a [(14)C]glycollamide ester, and the other was cysteine, shown by isolation of S-[(14)C]carboxymethylcysteine after acid hydrolysis. When N-acetylimidazole treatment was omitted, only the carboxy group reacted with iodo[(14)C]acetamide. 4. Isolation of the gamma-[(14)C]glutamyl-enzyme intermediate was made easier by prior treatment of the enzyme with N-acetylimidazole. The gamma-glutamyl-enzyme bond was stable to performic acid, and to hydroxylamine/urea at pH10, but was hydrolysed slowly at pH12, indicating attachment of the gamma-[(14)C]glutamyl group in amide linkage to an amino group on the enzyme. Proteolysis of the gamma-[(14)C]glutamyl-enzyme after performic acid oxidation gave rise to a small acidic radioactive peptide that was resistant to further proteolysis and was not identical with gamma-glutamyl-epsilon-lysine. 5. A scheme for the catalytic mechanism is proposed.

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Section: Discussionmentioning

confidence: 99%

Section: Experimental Materials and Methodsmentioning

confidence: 99%

Active-site amino acid residues in γ-glutamyltransferase and the nature of the γ-glutamyl-enzyme bond

Elce

1980

Biochemical Journal

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“…It is bound to Sepharose 4B by the CNBr method of Axen et al (1967). Concanavalin A-Sepharose has been used to purify several glycoproteins: a-foetoprotein (Caron et al, 1973), verylow-density lipoprotein (Shore & Shore, 1973) and more recently y-glutamyltransferase (EC 2.3.2.2) (Takahashi et al, 1974). Human alkaline phosphatases isolated from placenta, liver, bone and kidney are glycoproteins that contain sialic acid residues (Ghosh, 1969;Robinson & Pierce, 1964) and more specifically the enzyme as isolated from placenta contains glucose, galactose, mannose and fucose (Ghosh et al, 1974).…”

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confidence: 99%

Affinity purification and some molecular properties of human liver alkaline phosphatase

Trepanier

Seargeant

Stinson

1976

Biochemical Journal

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Alkaline phosphatase from human liver was purified to homogeneity. The purification procedure included solubilization with butanol, fractionation with acetone, and chromatography on concanavalin A-Sepharose, DEAE-cellulose, Sephadex G-200 and DEAE-Sephadex. Purity was established by standard and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The isoelectric point of the protein was determined to be 4.0. Sephadex-gel filtration gave a mol.wt. of 146000, although a higher value was obtained in the presence of 100mM-NaC1. The subunit mol.wt. 76700, was determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Neuraminidase treatment resulted in two enzyme-activity bands on isoelectric-focused gels with isoelectric points of 6.6 and 6.8. The desialylated enzyme gave only one protein band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with a subunit molecular weight indistinguishable from that of the non-neuraminidase-treated protein. The desialylated enzyme was more readily denatured by sodium dodecyl sulphate in the presence of mercaptoethanol than was the native enzyme.

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Membrane-Bound γ-Glutamyl Transpeptidase

Meister

Tate

Ross

1976

The Enzymes of Biological Membranes

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Purification of γ-glutamyltransferase of rat kidney by affinity chromatography using concanavalin a conjugated with sepharose 4B

Cited by 19 publications

References 5 publications

Active-site amino acid residues in γ-glutamyltransferase and the nature of the γ-glutamyl-enzyme bond

Active-site amino acid residues in γ-glutamyltransferase and the nature of the γ-glutamyl-enzyme bond

Affinity purification and some molecular properties of human liver alkaline phosphatase

Membrane-Bound γ-Glutamyl Transpeptidase

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