Preparation and Studies on the Characterization of Sheep Brain Glutamine Synthetase

Robert A. Ronzio, W. Bruce Rowe, Sherwin Wilk, Alton Meister

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48 Scopus citations

Abstract

Glutamine synthetase from sheep brain has been isolated by a new procedure, which gives much higher yields of the enzyme than have been obtained previously. The new procedure may be conveniently carried out in the laboratory and may be adapted to a 20-fold scale-up for use in a pilot plant. The amino acid composition of the enzyme has been determined. A single N-terminal amino acid, arginine, was found. Close to 65 peptides were obtained after digestion of the enzyme with trypsin and about 16 peptides were found after treatment of the enzyme with cyanogen bromide. These findings are consistent with the view that the eight subunits of the enzyme are identical. The data indicate that 12-14 of the 15 half-cystine residues/enzyme subunit found on amino acid analysis are cysteine residues. Titration with 5,5′-dithiobis( 2-nitrobenzoate) indicates that one sulfhydryl group per subunit reacts readily with this reagent and that the other enzyme sulfhydryl groups react more slowly and are presumably buried. The enzyme is inactivated by treatment with N-ethylmaleimide and protected from such inactivation by ATP+ Mg2+.

Original languageEnglish
Pages (from-to)2670-2674
Number of pages5
JournalBiochemistry
Volume8
Issue number6
DOIs
StatePublished - 1 Jun 1969
Externally publishedYes

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