Subcellular distribution and characterization of porcine kidney catalase.
Subcellular distribution of catalase for porcine kidney was analyzed by differential centrifugation of kidney homogenate. The specific activity of catalase was the highest in light mitochondrial fraction, followed by mitochondrial, cytosolic, nuclear, and microsomal fractions. However, about a half of the total activity was found in supernatant (cytosol) fraction and the other half was mainly associated with both mitochondrial and light mitochondrial fractions. Osmotic shock using hypotonic solution, 50 mm sodium phosphate buffer (pH 7.0) was found to be most effective for solubilization of particulate-bound catalase. Both particulate and cytosol catalases from porcine kidney were purified by ammonium sulfate fractionation followed by DEAE-cellulose, CM-cellulose, and Sephadex G-100 column chromatographies. The purified enzymes showed two distinct bands, one major and the other minor, on disc gel electrophoresis. Both particulate and cytosol enzymes showed identical molecular weights estimated from disc gel electrophoresis; that of the major component was 219,000 corresponding to native molecule and that of the minor one 421,000. A similar value, 210,000, was also obtained for the major component by gel filtration on a Bio Gel A-1.5 m column. It was inferred that the minor component was formed by dimerization of native molecule caused by formation of disulfide cross-links due to oxidation of SH groups in protein moiety. The particulate and cytosol catalases showed essentially identical molecular characteristics, although a slight difference was detected in stability in guanidine-HCl solution. The effect of NaCl on enzyme activity and optical properties of catalase were also measured.
D000818:Animals; D002374:Catalase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology; D007668:Kidney / Q000201:enzymology*; D012965:Sodium Chloride / Q000494:pharmacology; D012995:Solubility; D013347:Subcellular Fractions / Q000201:enzymology; D013552:Swine
D012965:Sodium Chloride; D002374:Catalase