ABSTRACT
Kinetics of thermal aggregation of model protein substrates (glycogen phosphorylase b from rabbit skeletal muscle and yeast alcohol dehydrogenase) were investigated under heat stress conditions (41-48 degrees C) in the presence of macrophage migration inhibitory factor (MIF), a heat-stable hydrophobic protein (12.5 kD). Anti-chaperone MIF activity found by turbidimetry manifests itself in significantly accelerated protein aggregation and increased limiting value of apparent optical absorption at 360 nm and t --> infinity in the sub-stoichiometric range of MIF concentrations. The aggregation kinetics is shown to have cooperative character. Possible reversibility of aggregation after removal of denaturing conditions was demonstrated using alcohol dehydrogenase aggregation at a temperature close to the physiological level (41.5 degrees C). This reversibility is caused by solubility of aggregates and stabilization of oligomeric structure of the substrate as a result of MIF binding to the partially denatured protein. The data suggest that in spite of distinct anti-chaperone effect, the chaperone-like activity of MIF can be observed in the case of heat stress removal and restoration of the system to normal conditions.
Subject(s)
Alcohol Dehydrogenase/metabolism , Glycogen Phosphorylase, Muscle Form/metabolism , Hot Temperature , Macrophage Migration-Inhibitory Factors/chemistry , Oxidative Stress , Alcohol Dehydrogenase/chemistry , Animals , Brain Chemistry , Cattle , Electrophoresis, Polyacrylamide Gel , Glycogen Phosphorylase, Muscle Form/chemistry , Heat-Shock Proteins/chemistry , Heat-Shock Proteins/metabolism , Kinetics , Macrophage Migration-Inhibitory Factors/isolation & purification , Macrophage Migration-Inhibitory Factors/physiology , Muscle, Skeletal/enzymology , Rabbits , Saccharomyces cerevisiae/enzymologyABSTRACT
The purification of macrophage migration inhibitory factor (MIF) from bovine brain cytosol and its partial characterization are reported. A rapid and relatively simple method for MIF isolation was developed based mainly on size-exclusion chromatography on Toyopearl TSK polymer having a tendency to adsorb MIF as compared to elution of other proteins with similar molecular weights. The method gives a high yield of MIF (0.1 mg homogenous protein per g wet tissue). The retardation is conveniently utilized to achieve good separations of MIF from other proteins of similar molecular weights. The isolated protein was identified as MIF by SDS-electrophoresis, immunoblotting, sequencing of the N-terminal amino acid residues, and also by determination of keto-enol tautomerase activity that is characteristic of MIF with p-hydroxyphenylpyruvic acid as a substrate.
