The Dissociation and Reconstitution of Aldolase^*

Abstract

Rabbit muscle aldolase, which exists in solution as essentially globular, compact, highly organized particles of molecular weight 1.42 X 106, was readily dissociated into three polypeptide chains apparently without the rupture of covalent bonds. This dissociation was effected by treatment with any of the reagents, urea, HC1, acetic acid, or sodium dodecyl sulfate. Detailed studies of the subunits generated in 4 m urea solutions at pH 5.5 and in acid solutions (pH 2) showed that the polypeptide chains were markedly disorganized as coil-like particles of weight-average molecular weight 0.46 X 105. Upon dissociation and denaturation the reduced viscosity increased from 4.0 ml/g for the native enzyme to 23 ml/g at pH 2 and 18 ml/g in 4 m urea solutions. The optical rotation, [a]d, changed from -23°for the native protein to -62°and -83°for the aciddissociated and urea-dissociated subunits respectively. Similarly there was a marked change in the Drude constant, Ac, from 283 mp to 238 mp and 223 m^i. Accompanying the change in the conformation of the polypeptide chains was a blue shift in the absorption spectrum with AmaI decreasing from 279.7 mp for the native protein to 277.0 mp for the subunits. Analyses of the shift by difference spectra revealed contributions from chromophoric groups of tyrosine, tryptophan, and phenylalanine due to the altered environment of these chromophores in the dissociated chains as compared to the intact enzyme. Titration of the sulfhydryl groups showed that the 16 groups which are "masked" in the native enzyme reacted readily in the dissociated subunits.No enzymic activity could be detected in the subunits in the presence of urea. However, activity was regained with yields of 65% when the dissociating agents (hydrogen ions or urea) were removed by dilution or dialysis. Reconstitution experiments were performed under different conditions; pH 5.5 and concentrations greater than about 50 /il/ml were found to be optimal for the restoration of activity. Kinetic studies showed that extensive annealing was unnecessary, for enzymic activity was regained in only a few minutes. The reconstituted protein was characterized by its reduced viscosity, sedimentation coefficient, molecular weight, optical rotatory dispersion, absorption spectrum, titratable sulfhydryl groups, and specific activity. In all respects the reconstituted macromolecules were virtually identical to the native enzyme.

Posttranslational Folding of α1-Inhibitor 3

“…However, the fact that the sedimentation rate seemed to increase stepwise with time of chase makes this possibility unlikely. The sedimentation rate of the ␣ form was similar to that of aldolase (data not shown), which has a sedimentation coefficient of 7.4 S (Stellwagen and Schachman, 1962); that of secreted ␣ 1 I 3 is 8.6 S (Esnard and Gauthier, 1980).…”

Intermolecular Bonding of the Antibiotic Diumycin

“…The methyl ester and acetate (10 M acetate/ 1 M diumycin) of diumycin aggregate in aqueous buffer with molecular weights of 32,000 and 38,000 daltons, respectively (Table I). The stability of the 32,000-dalton aggregate under these conditions indicates that ionic forces (noncovalent interactions between polar groups), including salt linkages (14,15) and hydrogen bonds (16,17), contribute only slightly to stabilize the 32,000-dalton aggregate. Some involvement of ionic forces is indicated by the molecular weight of 17,000 daltons in 5 M NaC14.02 M phosphate buffer (pH 6.85) and the higher concentration of the 65,000-dalton aggregate than the 32,000-dalton aggregate at pH 2.2 and 4.05 (Table I) (Table I); (b) the inability of a lipidless diumycin to aggregate ( Table I); and (c) the increasing efficacy of alcohols as disaggregating agents as the length of their hydrocarbon portion increases (Table I).…”

“…Vinylpyridine-vinylpyrrolidone copolymers-synthesis 0 Equilibrium dialysis-copolymer-p-toluene sulfonic acid sodium interaction Binding sites-copolymer-p-toluene sulfonate ions Urea effect-alkyl copolymer binding sulfonate ions UV spectrophotometry-identity During the past 3 decades, several interactions have been reported which involved binding of drugs by plasma proteins; notable examples are penicillins (I), sulfonamides (2-5), methyl orange (6-7), and shortand long-chain fatty acids (8)(9)(10)(11)(12)(13)(14). In connection with these binding studies, it has been reported that the interactions primarily take place through ionic forces, but a further contribution to the stability of proteindrug complex is made by the hydrophobic part of a drug molecule.…”

Kinetics of Refolding and Reactivation of Rabbit‐Muscle Aldolase after Acid Dissociation

“…Enzymically active intermediates formed during reactivation are selectively labilized in the presence of 2.3 M urea [4,24] or at elevated temperature [25,26]. This observation in connection with the concentration dependence and sigmoidicity of the reactivation kinetics after treatment with 2.3 M urea [4] suggest precursors of the final tetrameric product of refolding to be active.…”