Catalase: a Tetrameric Enzyme with Four Tightly Bound Molecules of NADPH (Ultrafiltration/Human Erythrocytes/Bovine Liver) HENRY N
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Proc. Natl. Acad. Sci. USA Vol. 81, pp. 4343-4347, July 1984 Biochemistry Catalase: A tetrameric enzyme with four tightly bound molecules of NADPH (ultrafiltration/human erythrocytes/bovine liver) HENRY N. KIRKMAN* AND GIAN F. GAETANIt *Biological Sciences Research Center, University of North Carolina, Chapel Hill, NC 27514; and tlstituto Scientifico di Medicina Interna, University of Genoa, 16132 Genoa, Italy Communicated by Sidney P. Colowick, April 4, 1984 ABSTRACT Catalases (H202:H202 oxidoreductase, EC mined at a wavelength of 240 nm with a recording spectro- 1.11.1.6) from many species are known to be tetramers of photometer (9). Activities of catalase and NADPH diapho- 60,000-dalton subunits, with four heme groups per tetramer. rase (10) (NADPH:methylene blue oxidoreductase, EC Previous authors have determined the amino acid sequence 1.6.99.1) were measured at 25°C. Assays for NADP and and three-dimensional structure of bovine liver catalase. Stud- NADPH were by the cycling method of Lowry and Passon- ies of the regulation of the pentose phosphate pathway led the neau (ref. 11, p. 130) as described previously (12). Binding of present authors to a search for proteins that bind NADP+ and dinucleotides by catalase was quantitated by addition of NADPH in human erythrocytes. An unexpected result of that 0.2-0.4 mg of purified catalase to 6.5 ml of Krebs-Ringer/ search was the finding that a major reservoir of bound 2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid NADPH in human erythrocytes is catalase. Each tetrameric (Tes) buffer, pH 7.4 (12) containing dinucleotide at a final molecule of human or bovine catalase contains four molecules concentration of 5 ,uM. The resulting solutions contained 20- of tightly bound NADPH. The binding sites have the relative 40 molecules of dinucleotide per molecule of catalase. After affinities NADPH > NADH > NADP+ > NAD+. NADPH being allowed to stand on ice for 1 hr, the solution was does not seem to be essential for the enzymic conversion of poured into a CF 25 ultrafiltration cone on a supporting cone H202 to 02 and water but does provide protection of catalase (Amicon) over a 45-ml centrifuge tube. The solution in this against inactivation by H202. assembly was centrifuged for 10-15 min in a swinging-cup rotor at 1000 x g until reduced to a volume of 0.05-0.1 ml. In the presence of catalase (H202:H202 oxidoreductase, EC The volume of concentrate was measured during transfer to 1.11.1.6) hydrogen peroxide is rapidly converted to oxygen a microcentrifuge tube. The cone was washed, by Vortex and water. Catalase was the source of some of the earliest mixing, with two portions of the solution that had passed information about the nature of enzymes. Noting the inhibi- through the cone, the volumes of the washings being such as tion of catalase by cyanide, Warburg suggested in 1923 that to bring the final volume of the concentrate to 0.2 ml when catalase contains iron (1). Chance obtained evidence for an washings were added to the concentrate. The difference in enzyme-substrate complex from studies of the absorption concentration of NADP between ultrafiltrate and the 0.2-ml spectrum of catalase under conditions of rapid flow (2). In sample was regarded as the concentration of bound NADP. 1937, Sumner and Dounce crystallized catalase from bovine The protein assay was with Folin reagent (13). Molecular liver, achieving one of the first successful crystallizations of weights of bovine and human catalase were considered to be an intracellular enzyme (3). The complete amino acid se- 240,000 (7, 14). quence of bovine liver catalase is now known (4), and the Sources of Catalase. Crystalline catalase from bovine liver three-dimensional structure has been determined at resolu- was a product of Boehringer Mannheim. Human catalase tions of 2.5 A for the enzyme from bovine liver (5) and 3.5 A was purified by the method of Morikofer-Zwez et al. (15) but for catalase from Penicillium vitale (6). Catalases from dif- with the following precautions for minimizing contamination ferent sources exhibit similarities in molecular weight, with NADPase from erythrocyte stroma and leukocytes. number of subunits, and types of prosthetic groups (7, 8). Hospital bank blood (500-1000 ml) was filtered through cel- The enzyme is a tetramer with a total molecular weight of lulose (16) for removal of leukocytes and platelets. Washed, approximately 240,000. Each tetrameric molecule contains packed erythrocytes were lysed by addition to 9 vol of 5 ,uM four heme groups in which the iron is in the ferric state. NADP+. The preparation was allowed to stand at 0°C for 10 We now report that bovine and human catalase also con- min with occasional mixing. After centrifugation at 16,000 x tain four tightly bound molecules of NADPH. This reduced g for 20 min, supernatant fluid was collected for the two dinucleotide is not essential for activity of catalase. Instead, steps (15) for ion-exchange chromatography. Catalase was NADPH decreases the susceptibility of catalase to inactiva- further purified by chromatography in Krebs-Ringer/Tes tion when the enzyme is exposed to low concentrations of its buffer/1 mM EDTA/5 ,uM NADP+ on a 2.6 x 96 cm column toxic substate, H202. Purified samples of human and bovine of Sephadex G-200 (Pharmacia). The catalase fraction from catalase were found to bind and release NADPH in a manner the Sephadex column was washed free of excess NADP+ by suggesting that catalase may also function as a regulatory several ultrafiltrations and dilutions in 0.01 M sodium phos- protein, releasing NADP+ when the cell is under peroxida- phate buffer, pH 6.0, adjusted to a protein concentration of 5 tive stress. This release would augment removal of H202 by mg/ml in the buffer, then passed through a 1 x 5 cm column the glutathione reductase-glutathione peroxidase mecha- of type 3 agarose-hexane-NADP+ affinity resin from P-L nisms. Biochemicals. After a wash with 6 ml of the phosphate buff- er, catalase activity was eluted with 5 ml of 0.01 M sodium MATERIALS AND METHODS phosphate buffer/100 ,uM NADP+. The catalase had a spe- Assays. Activity of catalase was expressed as the first-or- cific activity (3.8 x 107 M-1 sec-1), matching that reported der kinetic constant for disappearance of H202, as deter- for pure preparations (15). Human catalase from both labora- The publication costs of this article were defrayed in part by page charge Abbreviations: NADP, nicotinamide adenine dinucleotide phos- payment. This article must therefore be hereby marked "advertisement" phate in the oxidized form (NADP+) or reduced form (NADPH); in accordance with 18 U.S.C. §1734 solely to indicate this fact. Tes, 2-{[tris(hydroxymethyl)methyl]aminokethanesulfonic acid. 4343 Downloaded by guest on September 30, 2021 4344 Biochemistry: Kirkman and Gaetani Proc. NatL Acad Sci. USA 81 (1984) tories moved essentially as a single band on electrophoresis in NaDodSO4/7.5% acrylamide gels. Labeled NAD and NADP. Nicotinamide [U-_4C]adenine di- nucleotide ([14C]NAD+) was obtained as an aqueous solu- tion from Amersham. NAD' kinase (Sigma) with an enzy- mic specific activity of 10-20 nmol min-1 mg-' was dis- E E solved in 0.1 M Tris'HCl buffer, pH 7.5. After being brought E to dryness with a stream of air at 250C, 10 ,uCi (35 nmol; 1 Ci = 0: c] c 37 GBq) of [14C]NAD' was dissolved with 173 Al of water 0 and mixed with 5 /O of 0.5 M MgCl2, 30 kJ of 10 mM ATP at 0 z 0~ pH 7.5, 16.5 pil of 10 mM NAD', and 25 Al of a 10 mg/ml 0 solution of NAD' kinase. Enzymic determination (ref. 11, p. 17) of NAD+ and NADP+ at the end of a 36-hr incubation at 37°C revealed that all NAD+ had become NADP+. After the addition of 1.0 ml of ethanol, the mixture was brought to Elution volume, ml dryness with a stream of air at 25°C, then dissolved in 1.0 ml of water. The preparation was centrifuged at 1000 x g for 10 FIG. 1. Distribution of ["C]NADP after chromatography of a min; the supernatant fluid was evaporated to dryness; and hemolysate of normal erythrocytes in Sephadex G-200. Through a the powder was dissolved in 0.25 ml of water. For 2.6 x 96 cm column of the Sephadex was passed one bed volume of studies of Krebs-Ringer/Tes buffer containing, in the following final concen- binding of [14C]NAD+ or [14C]NADP+ by purified catalase, trations: 5 mM 2-mercaptoethanol, 1 mM EDTA, and 5 ttM ["C]- labeled and unlabeled dinucleotide were added to Krebs- NADP+ (0.4 ,pCi/,4mol). The addition of 0.017 ACi (0.25 nmol) of Ringer/Tes buffer to a final concentration of 5 ,tM with a [14C]NADP+ to 12 ml of stroma-free hemolysate brought the specific specific activity of 0.5 uCi/,umol. activity of the NADP in the hemolysate also to 0.4 ,tCi/,umol. Incu- [14C]NADH was generated on the day of use by the addi- bation of the hemolysate for 30 min at 0C allowed the conversion of tion of 45 ,ug (15 units) of crystalline alcohol dehydrogenase [14C]NADP+ to [14C]NADPH by the intrinsic dehydrogenases and to 3 ml of Krebs-Ringer/Tes/1 M ethanol/80 ,uM [14C]NAD+ substrates of the pentose phosphate pathway. The 12 ml of hemoly- (0.5 followed by incubation at 25°C sate was mixed with 3 ml of 0.75 M NaCl, then passed down the ,uCi/,umol), for 30 min, column at a rate of 8 ml/hr.