Transcarboxylase: IX. PARAMETERS EFFECTING DISSOCIATION AND REASSOCIATION OF THE ENZYME

Abstract

Abstract Transcarboxylase (E.C.2.1.3.1), a high molecular weight biotin enzyme, dissociates in a complex fashion to inactive subunits. The rate of dissociation is increased by increasing pH and temperature and decreased by increasing concentration of protein and polyvalent anions. The active species exhibits two sedimenting forms of s20,w ∼ 16 and ∼18 S when sedimented at speeds of 50,000 rpm or above. At the same speeds, the subunits formed show s20,w values of ∼12 S and ∼6S. Dissociation in the presence of 20% glycerol is slower and leads to subunits of similar sedimentation coefficients but they behave differently on reassociation. When dissociation occurs at pH 10 a species of s20,w ∼5 S and a smaller component are formed. Small components also arise in urea, dodecyl sulfate or after succinylation. Transcarboxylase which has been inactivated by dissociation can be reassociated with concomitant reactivation. Three methods have been successful: (a) adjustment to pH 5 with acetate buffer, (b) addition of a high concentration of phosphate buffer, and (c) transfer to dilute phosphate buffer at pH 6.8. The latter method has succeeded only with subunits formed in the presence of glycerol and leads to an active species with approximately the same sedimentation coefficient as the native enzyme. Reassociation by any of the methods, with subunits formed in the absence of glycerol leads to an active species of s20,w ∼ 25 S. A 1.3 S biotin-carboxyl carrier subunit is formed during the dissociation but it is not observed in the Schlieren patterns of dissociated enzyme because of its low concentration. By use of transcarboxylase labeled with tritiated biotin and by sucrose density gradient separation of subunits it has been shown that the reassociation does involve the use of the 1.3 S biotin-carboxyl carrier subunit. In addition to inactivation by dissociation, transcarboxylase undergoes a second type of inactivation which occurs when the purified enzyme is stored at -20°. No changes in the sedimentation coefficient or distribution of sedimenting species occur during this type of inactivation. This partially inactive enzyme is reactivated by incubation with 1.5 µ of (NH4)2SO4 at 25°. The enzyme appears to be pressure sensitive. Although it exhibits two forms, ∼16 and ∼18 S, when sedimented at 50,000 rpm, at speeds of 30,000 or 40,000 rpm only one species is observed of s20,w ∼17 S.