Hen ovomucoid-agarose: A new conjugate for the isolation by affinity chromatography of UDP-galactose: Glycoprotein galactosyl-transferase.
A new conjugate for the affinity chromatography of UDP-galactose:glycoprotein galactosyltransferase has been synthesized by coupling hen ovomucoid, a ligand similar to the acceptor substrate, to agarose.The hen ovomucoid-Sepharose conjugate binds galactosyl transferase more tightly that other acceptor-Sepharose conjugates.The new adsorbent gives comparable yields and purifications with those obtained by ligands similar to the nucleotide moiety of the substrate and to the "specifier" protein, α-lactalbumin.The soluble galactosyltransferase from rat ventral prostate is effectively removed from the high speed supernatant by an ovomucoid-Sepharose column. The enzyme can be eluted with buffer containing EDTA andN-acetylglucosamine in a high yield (75-80%) and in a purified form (4000-fold purification). The stability of ovomucoid to heat and to high concentrations of urea and its inhibition of some proteases makes the conjugate easy to operate with an quite useful even with rather crude preparations.
Istituto di Chimica Biologica, Università di Bologna, Via Irnerio 48, 40126, Bologna, Italy.
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Tadolini
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Hakim