A method to measure the diffusion coefficient of myoglobin in intact skeletal muscle cells.
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Animals↗
Biomedical subjects
G Gros
Publications and source records attributed to G Gros.
Sarcolemmal carbonic anhydrase in red and white rabbit skeletal muscle.
Sarcolemmal vesicles of white and red skeletal muscles of the rabbit were prepared by consecutive density gradient centrifugations in sucrose and dextran according to Seiler and Fleischer (1982, J. Biol. Chem. 257, 13,862-13,871). White and red muscle membrane fractions enriched in sarcolemma were characterized by high ouabain-sensitive Na+, K(+)-ATPase, by high Mg2(+)-ATPase activity, and by a high cholesterol content. Ca2(+)-ATPase activity, a marker enzyme for sarcoplasmic reticulum, was not detectable in the highly purified white and red muscle sarcolemmal fractions. White and red muscle sarcolemmal fractions exhibited no significant differences with regard to Na+, K(+)-ATPase, Mg2(+)-ATPase, and cholesterol. Specific activity of carbonic anhydrase in white muscle sarcolemmal fractions was 38 U.ml/mg and was 17.6 U.ml/mg in red muscle sarcolemma. Inhibition properties of sarcolemmal carbonic anhydrase were analyzed for acetazolamide, chlorzolamide, and cyanate. White muscle sarcolemmal carbonic anhydrase is characterized by inhibition constants, KI, toward acetazolamide of 4.6 X 10(-8) M, toward chlorzolamide of 0.75 X 10(-8) M, and toward cyanate of 1.3 X 10(-4) M. Red muscle sarcolemmal carbonic anhydrase is characterized by KI values toward acetazolamide of 8.1 X 10(-8) M, toward chlorzolamide of 6.3 X 10(-8) M, and toward cyanate of 0.81 X 10(-4) M. In contrast to the high specific carbonic anhydrase activities in sarcolemma, carbonic anhydrase activity in sarcoplasmic reticulum from white muscle varied between values of only 0.7 and 3.3 U.ml/mg. Carbonic anhydrase of red muscle sarcoplasmic reticulum ranged from 2.4 to 3.7 U.ml/mg.
Acetazolamide↗
Effects of carbonic anhydrase inhibitors on contraction, intracellular pH and energy-rich phosphates of rat skeletal muscle.
1. The effects of carbonic anhydrase inhibitors on contractile parameters, intracellular pH (pHi) and energy-rich phosphates were studied in isolated rat soleus and extensor digitorum longus (EDL) muscles. 2. The muscles were incubated either in Ringer solutions (95% O2/5% CO2 = control) or in solutions to which one of the inhibitors, 5 X 10(-4) M-chlorzolamide or 10(-2) M-NaCNO, had been added. Muscles were stimulated directly and contracted under isometric conditions. 3. Compared with control muscles, both inhibitor-treated muscles showed a significantly decreased tetanic force and an increased half-relaxation time of twitches and tetani. Chlorzolamide increased time-to-peak in both muscles. Cyanate decreased isometric twitch force in both muscles. 4. Both inhibitors decreased pHi in both muscles; chlorzolamide by 0.1 unit, cyanate by 0.4 unit in soleus and by 0.8 unit in EDL. 5. Chlorzolamide increased the concentrations of creatine and inorganic phosphate (Pi) in soleus (the effect of chlorzolamide was not studied in EDL). Cyanate caused these same changes in soleus as well as EDL and in addition decreased the concentrations of ATP and phosphocreatine in soleus and EDL. 6. In muscles acidified by either low external HCO3- (2 mM) or by elevated PCO2 (30% CO2 in the gas phase) in the bath, decreases in isometric force and increases in half-relaxation time of tetani were observed. In addition there were increases in muscle Pi. These effects were more pronounced with 30% CO2 than with 2 mM-HCO3-. 7. Neither acidifying solutions prolonged either half-relaxation time or time-to-peak of twitches. 8. We conclude that carbonic anhydrase inhibition exerts its effect (a) on isometric tension at least partly via an elevated Pi (perhaps in combination with lowered pHi); (b) on the half-relaxation time of tetani by means of lowered pHi and elevated concentration of Pi; (c) on relaxation and time-to-peak of twitches by some unknown mechanism, neither directly by a change in pHi nor in Pi.
Adenosine Triphosphate↗
Comparison of 18O exchange and pH stop-flow assays for carbonic anhydrase.
The hydration velocity of CO2 (0.002 M) catalyzed by bovine carbonic anhydrase (BCA) was measured at 25 degrees C and pH 7.4 by three different techniques: two initial-rate (steady-state) stop-flow methods, one using a glass pH electrode (in Hannover, method 1) and one using spectrophotometric measurements of a pH indicator (in Philadelphia, method 2), and an exchange method in which the disappearance of C18O16O from a bicarbonate solution was determined at equilibrium (in Philadelphia, method 3). The Michaelis-Menten constant (Km) and the inhibition constants for chloride (Ki,Cl) and ethoxzolamide (Ki,ez) were the same for methods 1, 2, and 3. The turnover numbers were 270,000, 400,000, and 555,000 s-1 by methods 1, 2, and 3, respectively. Values for CO2 hydration velocity measured by methods 2 and 3 on the same solution of BCA at the same time were the same. Km, maximal reaction velocity (Vmax), Ki,ez, and Ki,Cl obtained from normal human hemolysate at 37 degrees C and pH 7.2 by methods 2 and 3 were the same. Km and Vmax of the carbonic anhydrase isozyme CA III of homogenate from rabbit soleus were also identical by methods 1 and 3. According to Michaelis-Menten theory, the values of Km and Vmax obtained by method 3 should have been significantly smaller than those obtained by methods 1 and 2. We conclude that the catalytic step itself is apparently not rate limiting under physiological conditions and that method 3 can be used to obtain Michaelis-Menten characteristics of carbonic anhydrase.
Animals↗
Transport of SCFA across the large intestinal epithelium of guinea pig.
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Animals↗
Carbonic anhydrase inhibition affects contraction of directly stimulated rat soleus.
We have studied the contractile parameters of directly stimulated isolated rat soleus muscles incubated in media containing the carbonic anhydrase inhibitors chlorzolamide (5.10(-4)M) or cyanate (10(-2)M). Both inhibitors caused a decrease in isometric twitch and tetanic (5s) tensions and an increase in muscle relaxation time. It is speculated that among the three types of skeletal muscle carbonic anhydrase it may be the enzyme associated with the sarcoplasmatic reticulum whose inhibition caused the observed changes in contractile parameters.
Animals↗
Velocity of CO2 exchange in muscle and liver.
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Animals↗
Carbonic anhydrase in the sarcoplasmic reticulum of rabbit skeletal muscle.
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. 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.) 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-1). 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. 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. 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 cross-sections in transversely sectioned fibres and thin, longitudinally oriented, bands in longitudinally sectioned fibres. It is estimated that s.r. carbonic anhydrase accelerates CO2 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+.
Acetazolamide↗
Effects of dextran-bound inhibitors on carbonic anhydrase activity in isolated rat lungs.
Effects of macromolecular Prontosil-dextran inhibitors (PD) on carbonic anhydrase (CA) activity in isolated rat lungs were studied. Isolated lungs were perfused with Krebs-Ringer bicarbonate (KRB) solutions containing no inhibitor, PD 100,000 (mol wt 100,000), PD 5,000 (mol wt 5,000), or low-molecular-weight inhibitors (Prontosil or acetazolamide). The time course of effluent perfusate pH equilibration was measured in a stop-flow pH electrode apparatus. Pulmonary CO2 excretion (Vco2) was monitored by continuously recording expired CO2 concentration. The lungs were ventilated with room air and perfused at 37 degrees C with KRB prebubbled with 5% CO2- 20% O2- 75% N2. The results obtained show that both the low-molecular-weight inhibitors and PD's caused postcapillary pH disequilibria (delta pH) in effluent perfusate. However, only acetazolamide and Prontosil caused a reduction in Vco2. These results suggest that there is an intravascular CA, presumably associated with endothelial cell membranes, that is accessible to all inhibitors used and is responsible in part for equilibration of the CO2- HCO3- -H+ reactions in the perfusate but, under the conditions used, does not affect CO2 excretion; and there is an extravascular (possibly intracellular) CA that can be inhibited by low-molecular-weight inhibitors, is primarily responsible for enhanced CO2 transfer across the alveolar-capillary barrier (perhaps via facilitation of CO2 diffusion), and is in part responsible for pH equilibration.
Acetazolamide↗
Extracellular carbonic anhydrase of skeletal muscle associated with the sarcolemma.
We report here 1) the synthesis and properties of a new macromolecular carbonic anhydrase inhibitor, Prontosil-dextran, 2) its application to determine the localization of a previously described extracellular carbonic anhydrase in skeletal muscle, and 3) the application of a recently published histochemical technique using dansylsulfonamide to the same problem. Stable macromolecular inhibitors of molecular weights of 5,000, 100,000 and 1,000,000 were produced by covalently coupling the sulfonamide Prontosil to dextrans. Their inhibition constants towards bovine carbonic anhydrase II are 1-2 X 10(-7) M. The Prontosil-dextrans, PD 5,000, PD 100,000, and PD 1,000,000, were used in studies of the washout of H14CO3-) from the perfused rabbit hindlimb. This washout is slow due to the presence of an extracellular carbonic anhydrase and can be markedly accelerated by PD 5,000 but not by PD 100,000 and PD 1,000,000. Since PD 5,000 is accessible to the entire extracellular space and PD 100,000 and PD 1,000,000 are confined to the intravascular space, we conclude that the extracellular carbonic anhydrase of skeletal muscle is located in the interstitium. The histochemical studies show a strong staining of the sarcolemma of the muscle fibers with high oxidative capacity. It appears likely, therefore, that the extracellular carbonic anhydrase of skeletal muscle is associated with muscle plasma membranes with its active site directed toward the interstitial space.
Animals↗
A fast screening method for histochemical localization of carbonic anhydrase. Application to kidney, skeletal muscle, and thrombocytes.
A simple method for histochemical localization of carbonic anhydrase using 5-dimethyl-amino-naphthalene-1-sulfonamide (DNSA) is described. Cryosections of tissues, or cell smears, are incubated in 3 to 10 X 10(-5) M DNSA and viewed in a fluorescence microscope. Upon excitation with ultraviolet light, sites of carbonic anhydrase localization can be identified by an intense blue fluorescence, which is due to the emission of blue light (lambda max = 470 nm) by carbonic anhydrase-DNSA complexes. This fluorescence can be largely suppressed by simultaneous incubation with 1 X 10(-4) to 2 X 10(-3) M concentrations of nonfluorescent carbonic anhydrase inhibitors, displacing DNSA from its binding site on the enzyme. Application of the method to kidney, skeletal muscle, and thrombocytes yields patterns of carbonic anhydrase localization that are in good agreement with results that have been obtained with a variety of other techniques.
Animals↗
The effect of carbonic anhydrase inhibition on the velocity of thrombin-stimulated platelet aggregation under physiological conditions.
We have studied the effect of ethoxzolamide , a specific carbonic anhydrase inhibitor, on the velocity of thrombin-stimulated platelet aggregation. After preincubation of platelet rich plasma with 10(-6) M ethoxzolamide the velocity of platelet aggregation was reduced by about 40%. Between 10(-11) M and 10(-10)M ethoxzolamide was necessary to achieve a half-maximal diminution of the aggregation velocity. An identical maximal reduction of the velocity of aggregation as with ethoxzolamide could be achieved by a nearly complete removal of CO2 from the platelet rich plasma. These results suggest that the intracellular CO2 hydration-dehydration reaction is involved in the activation of human platelets by thrombin. It is possible that the cytosolic carbonic anhydrase of platelets provides a rapid source of the protons that are transferred across the plasma membrane during the activation process.
Blood Platelets↗
Carbonic anhydrase in human platelets.
The carbonic anhydrase activity of human platelets was investigated by measuring the kinetics of CO2 hydration in supernatants of platelet lysates by using a pH stopped-flow apparatus. An average carbonic anhydrase concentration of 2.1 microM was determined for pellets of human platelets. Analysis of the kinetic properties of this carbonic anhydrase yielded a Km value of 1.0 mM, a catalytic-centre activity kcat. of 130000 s-1 and an inhibition constant Ki towards ethoxzolamide of 0.3 nM. From these values, CO2 hydration inside platelets is estimated to be accelerated by a factor of 2500. When platelet lysates were subjected to affinity chromatography, only the high-activity carbonic anhydrase II could be eluted from the affinity column, whereas the carbonic anhydrase isoenzyme I, which is known to occur in high concentrations in human erythrocytes, appeared to be absent.
Blood Platelets↗
Inhibition properties and inhibition kinetics of an extracellular carbonic anhydrase in perfused skeletal muscle.
We have investigated the properties of the carbonic anhydrase which is functionally available to CO2 and HCO3- in the capillary bed of skeletal muscle. We used essentially the indicator-dilution technique of Effros and Weissman (J. Appl. Physiol. 47, 1090-1098, 1979). Into hindlimbs of rabbits perfused with dextran solution we injected boli containing H14CO3- or 36Cl-, and 3H-dextran (MW 80 000) as an intravasal indicator, and observed the washout of these indicators by fractionated collection and analysis of the venous effluent. In agreement with previous studies we found that addition of 10(-4) M of the carbonic anhydrase inhibitor acetazolamide to the perfusate considerably speeds up the washout of 14C, reducing the extraction of 14C from 0.72 to 0.45. A half-maximal effect on 14C extraction was achieved with 1 x 10(-6) M acetazolamide (IC50). The carbonic anhydrase inhibitors methazolamide and benzolamide both yielded IC50 values of 10(-5) M. This pattern of inhibitory potency of the three sulfonamides is incompatible with their inhibitory effects on the two known cytosolic isoenzymes of skeletal muscle, CAII and CAIII. While cells take up acetazolamide and benzolamide extremely slowly, with half-times of several minutes to hours, the effect of both sulfonamides on 14C washout occurred very rapidly: less than 1 min, probably not more than a few seconds, were necessary to achieve inhibitory effects. We conclude that (1) a tissue carbonic anhydrase converts the injected H14CO3- quickly into 14CO2 which then diffuses into the intracellular space thus causing a washout of 14C that is much slower than that of the intravasal indicator or that of 36Cl-, (2) this carbonic anhydrase is not intra- but extracellular and presumably membrane-bound, and (3) its properties suggest that it is distinct from the well-known cytosolic carbonic anhydrases and represents a different isoenzyme.
Acetazolamide↗
Evidence for rotational contribution to protein-facilitated proton transport.
Two modes of molecular motion of carrier molecules can, in principle, lead to a facilitated transport of a substrate: translational and rotational diffusion. In the present study, which deals with the mechanism of the facilitated diffusion of H+ and O2 in solutions of earthworm hemoglobin, examples for both types of facilitation are presented. Only translational, not rotational, diffusion of earthworm hemoglobin appears to lead to a facilitated O2 flux. In contrast, substantial facilitated H+ fluxes of comparable size arise from rotational diffusion as well as from translational diffusion of this large protein. This is derived from measurements of facilitated H+ and O2 fluxes in earthworm hemoglobin solutions and determinations of the rotational and translational diffusion coefficients of earthworm hemoglobin with the help of a theoretical treatment of facilitated diffusion by rotational carrier diffusion. H+ transport by rotational protein diffusion appears to be a case where the often-postulated mechanism of facilitated transport by rotation of a carrier lends itself to experimental verification.
Biological Transport↗
Carbonic anhydrase in human platelets: effects of carbonic anhydrase inhibition on platelet aggregation.
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Blood Platelets↗
Extracellular, extravascular carbonic anhydrase in skeletal muscle.
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Animals↗
The use of prontosil for the visualization of carbonic anhydrase bands in polyacrylamide gels. Application to the carbonic anhydrase isozymes of erythrocytes and white skeletal muscle of the rabbit.
Prontosil, a carbonic anhydrase inhibitor of orange-red colour, is used to visualize carbonic anhydrase bands during isoelectric focusing in polyacrylamide gels. 5-60 ng of the sulfonamide Prontosil are added to the 100-200 microliters samples before application to the gels. Bound Prontosil moves into the gel together with carbonic anhydrase and stains the enzyme bands formed there, while unbound Prontosil remains on top of the gels. The method is specific, no proteins other than carbonic anhydrase were observed to be stained, and it requires no special equipment. It was applied to chloroform/ethanol extracts of erythrolysates and white muscle homogenates from rabbits. Densitometric evaluation of the Prontosil-stained bands obtained with these extracts showed that rabbit red cells contain roughly equal amounts of carbonic anhydrase isoenzymes B and C while in rabbit white skeletal muscle isoenzyme C is predominant and little B enzyme occurs. These results confirm previous findings obtained by affinity chromatography of erythrolysates and muscle homogenates.
Animals↗