Interaction between pyridine adenine dinucleotides and bovine liver catalase: a chromatographic and spectral study.
Two different fractions were present in crystalline bovine liver catalase, and could be resolved using dye-ligand affinity chromatography with Red-A Matrex gel containing Procion HE 3B. The major part (alpha) was not adsorbed on this gel. The second fraction (beta) was firmly adsorbed to the gel, and could be eluted either by high salt or by NADPH in the micromolar range. Elution of catalase beta was also obtained with NADH, NADP+, and ADP at higher concentration. Fractions alpha and beta displayed no detectable difference in specific activity, stability to heat, and light absorption data. It is suggested that the difference in behavior between alpha and beta is related to the binding of NADPH to the mammalian catalase [H. N. Kirkman and G. F. Gaetani (1984) Proc. Natl. Acad. Sci. USA 81, 4343-4347], and that the beta fraction corresponds to the enzyme molecules that have at least one free site for NADPH binding. Modifications of catalase molecules in the presence of dithioerythritol (DTE) were examined using light absorption and EPR data. Thiol induced changes that corresponded to the formation of catalase complex II. They were partially reversed by NADPH at very low level, and the dinucleotide appeared to be oxidized in this process. DTE-treated bovine catalase was totally adsorbed on the Red-A Matrex columns, and could be eluted as fraction beta. Similar spectral changes in the presence of DTE and NADPH were displayed by a bacterial catalase from Proteus mirabilis. This enzyme was also able to oxidize NADPH, but was not adsorbed by Red-A Matrex. This work suggests that dye-affinity chromatography provides a very convenient tool for isolating dinucleotide-depleted catalase from bovine liver, facilitating further study of the physiological function of this cofactor within the enzyme.
D000327:Adsorption; D000818:Animals; D001234:Aspergillus niger / Q000201:enzymology; D002374:Catalase / Q000378:metabolism*; D002417:Cattle; D002846:Chromatography, Affinity; D004226:Dithioerythritol / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D008099:Liver / Q000201:enzymology*; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism* / Q000494:pharmacology; D010084:Oxidation-Reduction; D011513:Proteus mirabilis / Q000201:enzymology; D013053:Spectrophotometry
D009243:NAD; D009249:NADP; D004226:Dithioerythritol; D002374:Catalase