Simple and unique purification by size-exclusion chromatography for an oligomeric enzyme, rat liver cytosolic acetyl-coenzyme A hydrolase
Introduction
This review covers original papers on the purification of a cytosolic acetyl-coenzyme A (CoA) hydrolase (CACH, EC 3.1.2.1), which produces acetate and CoA from acetyl-CoA. The enzyme had escaped previous detection presumably due to its extreme cold lability until its first identification in rat liver in 1980 [1]. At room temperature the enzyme is usually present as active oligomeric forms, but at low temperature, they dissociate into an inactive monomer [2], [3]. Because of its extreme cold lability, the enzyme had to be purified at room temperature (RT) [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12] that caused a great loss of the enzyme activity, presumably due to denaturation of the enzyme protein by oxidation and degradation by contaminating proteases. Recently, the cold-inactivated monomeric form of the enzyme was found to be reconstituted to an active oligomeric form at 37 °C in the presence of a very low concentration of Triton X-100 [13]. The new finding allowed us to purify the cold-labile enzyme in the cold environment (0–4 °C) and further to develop an excellent purifying strategy using successive gel-filtration steps targeted for different quaternary structures of the enzyme.
Section snippets
Oligomeric properties of CACH
CACH had been detected in rat liver (∼20 μmol/min per g wet mass of liver at 30 °C) and kidney (5% of the activity in liver cytosol per mg protein) [1], [2]. We purified the enzyme [1] and demonstrated that at RT it is usually present as active forms: homodimer (Mr 135 000) and homotetramer (Mr 240 000) whose Km values for acetyl-CoA are 170 μM and 60 μM, respectively [2], [3]. The active oligomeric forms are stable at RT or in 1.3 M sucrose, but at 0 °C in 0.3 M sucrose, they rapidly dissociate
Effect of nucleotides, acetyl-CoA, phosphate, pyrophosphate, proteins, peptides or sucrose on rewarming
It was found, with the purified CACH, that rewarming under appropriate conditions partially reversed the cold-induced inactivation and dissociation of the enzyme: at a low protein concentration of 14 μg/ml, simple rewarming only partially restored the enzyme activity (less than 3% of the original activity), while at a higher concentration of 370 μg/ml, the reactivation by warming was greater (about 90% of the initial enzyme activity) [3]. Warming at 37 °C appeared to be optimal for reactivation;
Novel purification method of the cold-labile CACH in the cold environment
With the aid of 0.01% Triton X-100, an effective stabilizer as well as a reactivator of the cold-inactivated CACH, we have developed a novel purification method of cold-labile CACH in the cold environment. The reconstitution of the monomeric to oligomeric form of the enzyme in the presence of Triton X-100 further allowed us to adopt an excellent purifying strategy using two successive size-exclusion chromatography steps, first for the monomeric and second for the oligomeric form of the enzyme.
Conclusions
We previously demonstrated that CACH is an allosteric enzyme regulated by ATP (activator) and ADP (inhibitor) [1], [2]. Physiological observations in rat liver have revealed that CACH activity greatly increases in opposite metabolic states: in enhanced fatty acid oxidation and in heightened fatty acid synthesis [1], [4]. Further, this enzyme was markedly induced by CPIB [5], a hypolipidemic drug or a peroxisome proliferator, which enhances rat liver mitochondrial and peroxisomal β-oxidation [24]
Acknowledgments
This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan and by a grant from the Vitamin Society of Japan.
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