Carbonic anhydrase in the sarcoplasmic reticulum of rabbit skeletal muscle.

Abstract

SUMMARY1. Sarcoplasmic reticulum vesicles and mitochondria were prepared from red and white skeletal muscles of the rabbit. The preparations were characterized in terms of their specific activities of citrate synthase, basal (Mg2+-dependent) and Ca2+-dependent ATPase (the latter two in the presence of NaN3 and ouabain), and their specific carbonic anhydrase activities were determined.2. Skeletal muscle mitochondria had high specific activities of citrate synthase (700-1200 mu. mg protein-1) and low carbonic anhydrase activities (0 1-0 4 u. ml mg protein-1). The latter are likely to be due to a contamination of the preparations with sarcoplasmic reticulum (s.r.)3. Preparations of s.r. vesicles showed negligible activities of citrate synthase and the expected differing patterns of basal and Ca2+-dependent ATPase in red and white muscles. Specific carbonic anhydrase activities in s.r. from both muscle types were high (2-4 u. ml mg protein-'). The highest carbonic anhydrase activity, 11 u. ml mg protein-1, was found in s.r. from rabbit m. masseter. The inhibition constant of s.r. carbonic anhydrase towards acetazolamide was 4-6 x 10-8 M and similar but not identical to that of cytosolic carbonic anhydrase II.4. It appears possible that the carbonic anhydrase II-like enzyme previously found by us in muscle homogenates (Siffert & Gros, 1982) originates from the s.r.5. Histochemical studies using the dansylsuphonamide method described previously (Dermietzel, Leibstein, Siffert, Zamboglou & Gros, 1985) showed an intracellular pattern of carbonic anhydrase staining compatible with the presence of the enzyme in s.r.: spots homogeneously distributed across the fibre crosssections in transversely sectioned fibres and thin, longitudinally oriented, bands in longitudinally sectioned fibres.6. It is estimated that s.r. carbonic anhydrase accelerates C02 hydration within the s.r. approximately 1000-fold. Thus, CO2 and HCO3-react fast enough to provide a rapid source and sink for protons leaving and entering the s.r. in exchange for Ca2+.

Carbonic anhydrase in skeletal and cardiac muscle from rabbit and rat

“…In the case of skeletal muscles, there is evidence for both localizations. Sarcoplasmic reticulum of red as well as white muscles contains CA (Bruns et al, 1986), the K1 being in the same range as in our preparation.…”

“…This isoenzyme has about the same K1 towards ethoxzolamide as CA of rabbit erythrocytes (0.7 nM; Siffert & Gros, 1982), which, as mentioned above, under the experimental conditions employed exhibit CA II activity only. Similarly, the K. values towards acetazolamide for gastrocnemius and pooled white muscle supernatant, 28 nm and 20 nm, are nearly identical with those for 169 170 rabbit erythrocytes and for pure bovine CA 11 (17 nM;Bruns et at., 1986). Indeed, affinity chromatography studies of Siffert & Gros (1982) indicated that the sulphonamide-sensitive CA in homogenate supernatants of pooled white skeletal muscles of the rabbit is CA II, and that the other cytosolic sulphonamidesensitive isoenzyme, CA I, is absent from white muscle supernatants.…”

“…The K1 values with respect to acetazolamide obtained for this enzyme show, however, that it is different from cytosolic CA II. The K1 values, 73-94 nm, are three to six times higher than those for bovine CA II, rabbit erythrocyte CA II and cytosolic CA II of fast rabbit muscles (17-28 nM; Bruns et al, 1986;and above). On the other hand, they are two to three times lower than the K, value (200 nM) reported by Sanyal et al (1981) for cytosolic CA I.…”

Carbonic anhydrase in skeletal and cardiac muscle from rabbit and rat

“…In the case of skeletal muscles, there is evidence for both localizations. Sarcoplasmic reticulum of red as well as white muscles contains CA (Bruns et al, 1986), the K1 being in the same range as in our preparation.…”

“…This isoenzyme has about the same K1 towards ethoxzolamide as CA of rabbit erythrocytes (0.7 nM; Siffert & Gros, 1982), which, as mentioned above, under the experimental conditions employed exhibit CA II activity only. Similarly, the K. values towards acetazolamide for gastrocnemius and pooled white muscle supernatant, 28 nm and 20 nm, are nearly identical with those for 169 170 rabbit erythrocytes and for pure bovine CA 11 (17 nM;Bruns et at., 1986). Indeed, affinity chromatography studies of Siffert & Gros (1982) indicated that the sulphonamide-sensitive CA in homogenate supernatants of pooled white skeletal muscles of the rabbit is CA II, and that the other cytosolic sulphonamidesensitive isoenzyme, CA I, is absent from white muscle supernatants.…”

“…The K1 values with respect to acetazolamide obtained for this enzyme show, however, that it is different from cytosolic CA II. The K1 values, 73-94 nm, are three to six times higher than those for bovine CA II, rabbit erythrocyte CA II and cytosolic CA II of fast rabbit muscles (17-28 nM; Bruns et al, 1986;and above). On the other hand, they are two to three times lower than the K, value (200 nM) reported by Sanyal et al (1981) for cytosolic CA I.…”

Effects of carbonic anhydrase inhibitors on contraction, intracellular pH and energy‐rich phosphates of rat skeletal muscle.

“…The CA associated with the sarcoplasmic reticulum (Bruns et al 1986) could explain some of the results reported here. It has been shown (Somlyo, GonzalezSerratos, Shuman, McClellan & Somlyo, 1981) that only about one-half of the calcium released from sarcoplasmic reticulum during contraction is electrically compensated for movements of Mg2+ and K+ in the opposite direction.…”

Carbon Dioxide Transport and Carbonic Anhydrase in Blood and Muscle

“…A membrane-bound CA was detected in preparations of SR vesicles from red as well as white skeletal muscle of the rabbit (17). This finding was confirmed by histochemical results employing the fluorescent CA inhibitor dansylsulfonamide for CA staining (17,26).…”

Carbonic anhydrase in skeletal and cardiac muscle from rabbit and rat

“…In the case of skeletal muscles, there is evidence for both localizations. Sarcoplasmic reticulum of red as well as white muscles contains CA (Bruns et al, 1986), the K1 being in the same range as in our preparation.…”

“…This isoenzyme has about the same K1 towards ethoxzolamide as CA of rabbit erythrocytes (0.7 nM; Siffert & Gros, 1982), which, as mentioned above, under the experimental conditions employed exhibit CA II activity only. Similarly, the K. values towards acetazolamide for gastrocnemius and pooled white muscle supernatant, 28 nm and 20 nm, are nearly identical with those for 169 170 rabbit erythrocytes and for pure bovine CA 11 (17 nM;Bruns et at., 1986). Indeed, affinity chromatography studies of Siffert & Gros (1982) indicated that the sulphonamide-sensitive CA in homogenate supernatants of pooled white skeletal muscles of the rabbit is CA II, and that the other cytosolic sulphonamidesensitive isoenzyme, CA I, is absent from white muscle supernatants.…”

“…The K1 values with respect to acetazolamide obtained for this enzyme show, however, that it is different from cytosolic CA II. The K1 values, 73-94 nm, are three to six times higher than those for bovine CA II, rabbit erythrocyte CA II and cytosolic CA II of fast rabbit muscles (17-28 nM; Bruns et al, 1986;and above). On the other hand, they are two to three times lower than the K, value (200 nM) reported by Sanyal et al (1981) for cytosolic CA I.…”

Effects of carbonic anhydrase inhibitors on contraction, intracellular pH and energy‐rich phosphates of rat skeletal muscle.

“…The CA associated with the sarcoplasmic reticulum (Bruns et al 1986) could explain some of the results reported here. It has been shown (Somlyo, GonzalezSerratos, Shuman, McClellan & Somlyo, 1981) that only about one-half of the calcium released from sarcoplasmic reticulum during contraction is electrically compensated for movements of Mg2+ and K+ in the opposite direction.…”

Carbon Dioxide Transport and Carbonic Anhydrase in Blood and Muscle

“…A membrane-bound CA was detected in preparations of SR vesicles from red as well as white skeletal muscle of the rabbit (17). This finding was confirmed by histochemical results employing the fluorescent CA inhibitor dansylsulfonamide for CA staining (17,26).…”

Carbonic anhydrase in skeletal and cardiac muscle from rabbit and rat

“…In the case of skeletal muscles, there is evidence for both localizations. Sarcoplasmic reticulum of red as well as white muscles contains CA (Bruns et al, 1986), the K1 being in the same range as in our preparation.…”

“…This isoenzyme has about the same K1 towards ethoxzolamide as CA of rabbit erythrocytes (0.7 nM; Siffert & Gros, 1982), which, as mentioned above, under the experimental conditions employed exhibit CA II activity only. Similarly, the K. values towards acetazolamide for gastrocnemius and pooled white muscle supernatant, 28 nm and 20 nm, are nearly identical with those for 169 170 rabbit erythrocytes and for pure bovine CA 11 (17 nM;Bruns et at., 1986). Indeed, affinity chromatography studies of Siffert & Gros (1982) indicated that the sulphonamide-sensitive CA in homogenate supernatants of pooled white skeletal muscles of the rabbit is CA II, and that the other cytosolic sulphonamidesensitive isoenzyme, CA I, is absent from white muscle supernatants.…”

“…The K1 values with respect to acetazolamide obtained for this enzyme show, however, that it is different from cytosolic CA II. The K1 values, 73-94 nm, are three to six times higher than those for bovine CA II, rabbit erythrocyte CA II and cytosolic CA II of fast rabbit muscles (17-28 nM; Bruns et al, 1986;and above). On the other hand, they are two to three times lower than the K, value (200 nM) reported by Sanyal et al (1981) for cytosolic CA I.…”

Effects of carbonic anhydrase inhibitors on contraction, intracellular pH and energy‐rich phosphates of rat skeletal muscle.

“…The CA associated with the sarcoplasmic reticulum (Bruns et al 1986) could explain some of the results reported here. It has been shown (Somlyo, GonzalezSerratos, Shuman, McClellan & Somlyo, 1981) that only about one-half of the calcium released from sarcoplasmic reticulum during contraction is electrically compensated for movements of Mg2+ and K+ in the opposite direction.…”

Carbon Dioxide Transport and Carbonic Anhydrase in Blood and Muscle

“…A membrane-bound CA was detected in preparations of SR vesicles from red as well as white skeletal muscle of the rabbit (17). This finding was confirmed by histochemical results employing the fluorescent CA inhibitor dansylsulfonamide for CA staining (17,26).…”