INHIBITION AND KINETIC-PROPERTIES OF MEMBRANE-BOUND CARBONIC-ANHYDRASES IN RABBIT SKELETAL-MUSCLES

It was the aim of this study to investigate whether the carbonic anhyd rases associated with the sarcoplasmic reticulum (SR) and sarcolemmal membranes differ in their kinetic and inhibitory properties. To this e nd, sarcolemmal and SR membrane vesicle fractions were prepared from r abbit white and red skeletal muscles, the white muscle sarcolemmal fra ction (WSL), the red muscle sarcolemmal fraction (RSL), the white musc le SR fraction (WSR), and the red muscle SR fraction (RSR), WSL displa yed a specific carbonic anhydrase activity of 22.1 U.ml/mg and RSL of 7.5 U.ml/mg, whereas the SR fractions showed a much lower activity of 0.5 U.ml/mg for WSR and of 2.4 U.ml/mg for RSR. In both SR fractions p hase separation experiments with Triton X-114 demonstrated that the ca rbonic anhydrase activity is due to a membrane-bound enzyme and not du e to a cytosolic isozyme. The kinetic properties of carbonic anhydrase from the four distinct membrane fractions were evaluated by determina tion of the Michaelis constant, K-m, and of the catalytic centre activ ity k(cat). K-m appears to be somewhat lower for SR than for SL, Inhib ition constants of SR and SL carbonic anhydrases were determined apply ing six carbonic anhydrase inhibitors: chlorzolamide, ethoxzolamide, m ethazolamide, benzolamide, and acetazolamide, and also cyanate. The in hibition constants of the SR fractions were significantly different fr om those of the corresponding sarcolemmal fractions, indicating that t he carbonic anhydrase measured in the SR fractions does not originate from contaminating sarcolemmal membrane vesicles, but appears to repre sent a distinct carbonic anhydrase associated with the SR membrane. (C ) 1998 Academic Press.