Abstract
Abstract A procedure is presented for the purification of l-tyrosine-2-oxoglutarate aminotransferase from the livers of rats previously treated with the synthetic glucocorticosteroid, triamcinolone (9α-fluoro-16α-hydroxyprednisolone diacetate). A 2500-fold purification over crude liver extract was obtained. The final preparation, which appeared to be over 95% homogeneous by ultracentrifugal and electrophoretic criteria, has twice the specific activity of the most active fraction previously reported. The purified enzyme showed three enzymically active components when examined by acrylamide gel electrophoresis. The major component (approximately 95% of the total protein applied to the gel) migrated more rapidly than the two minor components which appeared to be aggregates of the principal component. The purified enzyme which sediments as a single boundary with an s020,w of 5.3 in the ultra-centrifuge has a molecular weight of 115,000 calculated from sedimentation equilibrium studies. The amino acid composition was determined. The enzyme binds approximately 4 moles of pyridoxal 5'-phosphate per 115,000 g of protein but no cortisol binding was detected.
Highlights
Z-oxoglutarate aminotransferase from the livers of rats previously treated with the synthetic glucocorticosteroid, triamcinolone (9ar-fluoro-16a!-hydroxyprednisolone diacetate)
We report further modifications of the earlier procedures by which we have obtained virtually homogeneous enzyme with a specific activity and a purification factor twice that of the best preparation previously reported [6]
The final product had a specific activity of about 115 units per mg of protein, appeared to be relatively homogeneous, and was estimated to have a molecular weight of 90,000 with 1 mole of pyridoxal-5’-P bound per mole of enzyme
Summary
Materials-Triamcinolone (9ar - fluoro - 16a! - hydroxyprednisolone diacetate) was generously donated by Lederle Laboratories, Pearl River, New York. - hydroxyprednisolone diacetate) was generously donated by Lederle Laboratories, Pearl River, New York. Ammonium sulfate and sucrose (both “Special Enzyme Grade”) and guanidine hydrochloride and urea (both “Ultra Pure”) were purchased from Mann; DEAE-cellulose (Whatman DE52) from Reeve-Angel Company, New York; dithiothreitol and cY-ketoglutaric acid from Calbiochem; L-tyrosine and pyridoxal 5’-phosphate, both A and B grade, from. Sigma; and sodium dodecyl sulfate from Matheson Coleman and. Cellulose acetate strips for paper electrophoresis were obtained from Oxoid Ltd., London, England. (superfine) and G-200 were purchased from Pharmacia. All other reagents were of analytical grade.
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