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 anhydrases associated with the sarcoplasmic reticulum (SR) and sarcolemmal membranes differ in their kinetic and inhibitory properties. To this end, sarcolemmal and SR membrane vesicle fractions were prepared from rabbit white and red skeletal muscles, the white muscle sarcolemmal fraction (WSL), the red muscle sarcolemmal fraction (RSL), the white muscle SR fraction (WSR), and the red muscle SR fraction (RSR). WSL displayed 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 phase separation experiments with Triton X-114 demonstrated that the carbonic anhydrase activity is due to a membrane-bound enzyme and not due to a cytosolic isozyme. The kinetic properties of carbonic anhydrase from the four distinct membane fractions were evaluated by determination of the Michaelis constant, Km, and of the catalytic centre activity kcat. Km appears to be somewhat lower for SR than for SL. Inhibition constants of SR and SL carbonic anhydrases were determined applying six carbonic anhydrase inhibitors: chlorzolamide, ethoxzolamide, methazolamide, benzolamide, and acetazolamide, and also cyanate. The inhibition constants of the SR fractions were significantly different from those of the corresponding sarcolemmal fractions, indicating that the carbonic anhydrase measured in the SR fractions does not originate from contaminating sarcolemmal membrane vesicles, but appears to represent a distinct carbonic anhydrase associated with the SR membrane.
Zentrum Physiologie, Medizinische Hochschule Hannover, Carl Neuberg Strasse 1, Hannover, 30623, Germany.
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D000818:Animals; D002257:Carbonic Anhydrase Inhibitors / Q000494:pharmacology*; D002256:Carbonic Anhydrases / Q000187:drug effects* / Q000378:metabolism*; D005260:Female; D007425:Intracellular Membranes / Q000737:chemistry / Q000201:enzymology*; D018656:Muscle Fibers, Fast-Twitch / Q000737:chemistry / Q000201:enzymology; D018482:Muscle, Skeletal / Q000201:enzymology*; D011817:Rabbits; D012508:Sarcolemma / Q000737:chemistry / Q000201:enzymology; D012519:Sarcoplasmic Reticulum / Q000737:chemistry / Q000201:enzymology; D013347:Subcellular Fractions / Q000737:chemistry / Q000201:enzymology
D002257:Carbonic Anhydrase Inhibitors; D002256:Carbonic Anhydrases