Inhibition of rabbit muscle creatine kinase by iodomethane [proceedings].

carbonic anhydrase III with an estimated purity of greater than 95%, as judged by electrophoresis in sodium dodecyl sulphate/polyacrylamide gels. Ion-exchange chromatography and salt fractionation were used to purify the bovine carbonic anhydrase 111. Bovine muscle was homogenized and adjusted t o 40% saturation with (NH4)*S04. The supernatant was applied t o a DEAE-cellulose column (2.5cm x 25cm) at pH8.7 in 0.01 M-Tris and elution was with the same buffer. The enzyme was then applied to a CM-celluiose column ( I .5cmx IlOcm) in 0.01 M-sodium phosphate, pH6.6. The active fractions were eluted with a linear gradient of NaCl ( 1 5 0 m l , 0 . 3 ~ 0 . 2 ~ N a c I , in 0.1 M-sodium phosphate, pH6.6). The resulting preparation was pure, as judged by electrophoresis in sodium dodecyl sulphate/polyacrylamide gels. (Both human and bovine carbonic anhydrase Ill had mol.wts. of 27000-28000.) The kinetic properties of these two carbonic anhydrase I11 isoenzymes were similar, but in sharp contrast with those of carbonic anhydrase I and 11, thus the human carbonic anhydrase 111 gave 5.0% of the COz hydration rate of isoenzyme CAI under identical assay conditions and about 1 % of the esterase activity of carbonic anhydrase I (2.5 nmol/min per mg of enzyme) measured with p-nitrophenyl acetate as substrate (Tashian, 1969). The bovine carbonic anhydrase 111 gave about 30% of the COz hydrase activity of carbonic anhydrase I and the esterase activity was undetectable under conditions similar to those described for the human enzyme. Inhibition kinetics with acetazolamide showed that the bovine carbonic anhydrase I11 was only weakly inhibited in comparison with isoenzymes CAI and CAII. For example, the inhibition constant (Iso; M I ) for bovine isoenzyme CAI was 2 . 0 ~ lo’, and 1.1 x lo7 for isoenzyme CAII, whereas for bovine isoenzyme CAI11 the value was 2 4 0 0 ~ 10’. Determinations of energy of activation gave values of 1029 and 714kJ/mol for bovine isoenzymes CAII and CAI respectively and 395 kJ/mol for isoenzyme CAIII. These results indicate that isoenzyme CAIII, which is predominant in red skeletal muscle, has unique properties. It has very low COz hydrase and esterolytic activity relative to the other carbonic anhydrase isoenzymes and is poorly inhibited by sulphonamides. However, the CAIII enzyme protein is present a t a concentration of about 1 mg/g wet weight of muscle, and the true catalytic function of the enzyme remains a paradox. Preliminary sequence analyses (R. E. Tashian, unpublished work) show a number of regions of homology for CAI, CAI1 and CAIII confirming the common evolutionary origin of these isoenzymes.