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G Gros

Publications and source records attributed to G Gros.

At least 19 recordsLinked to original sources
PubMed2006

Red cell membrane CO2 permeability in normal human blood and in blood deficient in various blood groups, and effect of DIDS.

The red cell membrane has an exceptionally high permeability for CO2, PCO2 approximately 0.15 cm/s, which is two to three orders of magnitude greater than that of some epithelial membranes and similarly greater than the permeability of the red cell membrane for HCO3-. As shown previously, this high PCO2 can be drastically inhibited by 10 microM 4,4'-diisothiocyanato-2,2'-stilbenedisulfonate (DIDS), indicating that membrane proteins may be involved in this high gas permeability. Here, we have studied the possible contribution of several blood group proteins to CO2 permeation across the red cell membrane by comparing PCO2 of red cells deficient in specific blood group proteins with that of normal red cells. While PCO2 of normal red cells is approximately 0.15 cm/s and that of Fy(null) and Jk(null) red cells is similar, PCO2's of Colton null (deficient in aquaporin-1) and Rh(null) cells (deficient in Rh/RhAG) are both reduced to about 0.07 cm/s, i.e. to about one half. In addition, the inhibitory effect of DIDS is about half as great in Rh(null) and in Colton null red cells as it is in normal red cells. We conclude that aquaporin-1 and Rh/RhAG proteins contribute substantially to the high permeability of the human red cell membrane for CO2. Together these proteins are responsible for 50% or more of the CO2 permeability of red cell membranes. The CO2 pathways of both proteins can be partly inhibited by DIDS, which is why this compound very effectively reduces membrane CO2 permeability.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2006

Evidence that aquaporin 1 is a major pathway for CO2 transport across the human erythrocyte membrane.

We report here the application of a previously described method to directly determine the CO2 permeability (P(CO2)) of the cell membranes of normal human red blood cells (RBCs) vs. those deficient in aquaporin 1 (AQP1), as well as AQP1-expressing Xenopus laevis oocytes. This method measures the exchange of (18)O between CO2, HCO3(-), and H2O in cell suspensions. In addition, we measure the alkaline surface pH (pH(S)) transients caused by the dominant effect of entry of CO2 vs. HCO3(-) into oocytes exposed to step increases in [CO2]. We report that 1) AQP1 constitutes the major pathway for molecular CO2 in human RBCs; lack of AQP1 reduces P(CO2) from the normal value of 0.15 +/- 0.08 (SD; n=85) cm/s by 60% to 0.06 cm/s. Expression of AQP1 in oocytes increases P(CO2) 2-fold and doubles the alkaline pH(S) gradient. 2) pCMBS, an inhibitor of the AQP1 water channel, reduces P(CO2) of RBCs solely by action on AQP1 as it has no effect in AQP1-deficient RBCs. 3) P(CO2) determinations of RBCs and pH(S) measurements of oocytes indicate that DIDS inhibits the CO2 pathway of AQP1 by half. 4) RBCs have at least one other DIDS-sensitive pathway for CO2. We conclude that AQP1 is responsible for 60% of the high P(CO2) of red cells and that another, so far unidentified, CO2 pathway is present in this membrane that may account for at least 30% of total P(CO2).

Animals↗
PubMed2002

[Phylogeny of gas exchange systems].

Several systems of gas transport have developed during evolution, all of which are able to sufficiently supply oxygen to the tissues and eliminate the CO2 produced by the metabolism, in spite of great distances between the environment and the individual cells of the tissues. Almost all these systems utilize a combination of convection and diffusion steps. Convection achieves an efficient transport of gas over large distances, but requires energy and cannot occur across tissue barriers. Diffusion, on the other hand, achieves gas transport across barriers, but requires optimization of diffusion paths and diffusion areas. When two convectional gas flows are linked via a diffusional barrier (gas/fluid in the case of the avian lung, fluid/fluid in the case of gills), the directions in which the respective convectional movements pass each other are important determinants of gas exchange efficiency (concurrent, countercurrent and cross-current systems). The tracheal respiration found in insects has the advantage of circumventing the convective gas transport step in the blood, thereby avoiding the high energy expenditure of circulatory systems. This is made possible by a system of tracheae, ending in tracheoles, that reaches from the body surface to every cell within the body. The last step of gas transfer in these animals occurs by diffusion from the tracheoles ("air capillaries") to the mitochondria of cells. The disadvantage is that the tracheal system occupies a substantial fraction of body volume and that, due to limited mechanical stability of tracheal walls, this system would not be able to operate under conditions of high hydrostatic pressures, i. e. in large animals. Respiration in an "open" system, i. e. direct exposure of the diffusional barrier to the environmental air, eliminates the problem of bringing the oxygen to the barrier by convection, as is necessary in the avian and mammalian lung, in the insects' tracheal system and in the gills. An open system is found in the respiration via the skin, which is of significance in some amphibians, but is limited by the thickness of the skin that constitutes a substantial diffusion path for O2 and CO2. The thick skin, on the other hand, provides mechanical protection as well as flexibility for the animals' body and helps avoid massive water loss via the body surface. The gills of fishes, in contrast, exhibit rather short diffusion distances, are located in a mechanically protected space, and the problem of water loss does not exist. The flows of blood and water occur in opposite direction (countercurrent flow) and this situation makes an arterial PO2 approaching the environmental PO2 possible. A major disadvantage is constituted by the environmental medium since water contains little O2 compared to air and, to compensate this, much energy is expended to maintain a high flow rate of water through the gills. In the mammalian lung ("pool system"), the presence of a dead space and the rhythmic ventilation that replaces only a small fraction of the gas volume of the lung per breath, are responsible for an arterial PO2 (2/3 of the atmospheric PO2) that cannot reach the expiratory PO2. However, an advantage of this feature is the constantly high alveolar and arterial PCO2, which provides a highly effective H(+) buffer system in the entire body. The apparent disadvantage of the mammalian lung is avoided by the avian lung, which uses an extended system of airways to establish continuous equilibration of a part of the capillary blood with fresh air (cross current system), during inspiration as well as during expiration. In this system, arterial PO2 can significantly exceed expiratory PO2. A disadvantage here is the enormous amount of space taken up by the avian lung, in animals of 1 kg body weight three times as much as taken up by the mammalian lung. All respiratory exchange systems considered here exhibit high degrees of optimization - yet follow highly diverse construction principles. There is no such thing as an ideal gas exchange system. The system that has evolved in each species depends to an impressive extent on environmental conditions, on body build and size, on the animal's patterns of movement and on its energy consumption.

Animals↗
PubMed2001

Calcineurin regulates slow myosin, but not fast myosin or metabolic enzymes, during fast-to-slow transformation in rabbit skeletal muscle cell culture.

The addition of cyclosporin A (500 ng ml(-1)) - an inhibitor of the Ca2+-calmodulin-regulated serine/threonine phosphatase calcineurin - to primary cultures of rabbit skeletal muscle cells had no influence on the expression of fast myosin heavy chain (MHC) isoforms MHCIIa and MHCIId at the level of protein and mRNA, but reduced the expression of slow MHCI mRNA. In addition, no influence of cyclosporin A on the expression of citrate synthase (CS) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was found. The level of enzyme activity of CS was also not affected. When the Ca2+ ionophore A23187 (4 x 10(-7) M) was added to the medium, a partial fast-to-slow transformation occurred. The level of MHCI mRNA increased, and the level of MHCIId mRNA decreased. Cotreatment with cyclosporin A was able to prevent the upregulation of MHCI at the level of mRNA as well as protein, but did not reverse the decrease in MHCIId expression. The expression of MHCIIa was also not influenced by cyclosporin A. Cyclosporin A was not able to prevent the upregulation of CS mRNA under Ca2+ ionophore treatment and failed to reduce the increased enzyme activity of CS. The expression of GAPDH mRNA was reduced under Ca2+ ionophore treatment and was not altered under cotreatment with cyclosporin A. When the myotubes in the primary muscle culture were electrostimulated at 1 Hz for 15 min periods followed by pauses of 30 min, a partial fast-to-slow transformation was induced. Again, cotreatment with cyclosporin A prevented the upregulation of MHCI at the level of mRNA and protein without affecting MHCIId expression. The nuclear translocation of the calcineurin-regulated transcription factor nuclear factor of activated thymocytes (NFATc1) during treatment with Ca2+ ionophore, and the prevention of the translocation in the presence of cyclosporin A, were demonstrated immunocytochemically in the myotubes of the primary culture. The effects of cyclosporin A demonstrate the involvement of calcineurin-dependent signalling pathways in controlling the expression of MHCI, but not of MHCIIa, MHCIId, CS and GAPDH, during Ca2+ ionophore- and electrostimulation-induced fast-to-slow transformations. The data indicate a differential regulation of MHCI, of MHCII and of metabolism. Calcineurin alone is not sufficient to mediate the complete transformation.

Animals↗
PubMed2001

Radial and longitudinal diffusion of myoglobin in single living heart and skeletal muscle cells.

We have used a fluorescence recovery after photobleaching (FRAP) technique to measure radial diffusion of myoglobin and other proteins in single skeletal and cardiac muscle cells. We compare the radial diffusivities, D(r) (i.e., diffusion perpendicular to the long fiber axis), with longitudinal ones, D(l) (i.e., parallel to the long fiber axis), both measured by the same technique, for myoglobin (17 kDa), lactalbumin (14 kDa), and ovalbumin (45 kDa). At 22 degrees C, D(l) for myoglobin is 1.2 x 10(-7) cm(2)/s in soleus fibers and 1.1 x 10(-7) cm(2)/s in cardiomyocytes. D(l) for lactalbumin is similar in both cell types. D(r) for myoglobin is 1.2 x 10(-7) cm(2)/s in soleus fibers and 1.1 x 10(-7) cm(2)/s in cardiomyocytes and, again, similar for lactalbumin. D(l) and D(r) for ovalbumin are 0.5 x 10(-7) cm(2)/s. In the case of myoglobin, both D(l) and D(r) at 37 degrees C are about 80% higher than at 22 degrees C. We conclude that intracellular diffusivity of myoglobin and other proteins (i) is very low in striated muscle cells, approximately 1/10 of the value in dilute protein solution, (ii) is not markedly different in longitudinal and radial direction, and (iii) is identical in heart and skeletal muscle. A Krogh cylinder model calculation holding for steady-state tissue oxygenation predicts that, based on these myoglobin diffusivities, myoglobin-facilitated oxygen diffusion contributes 4% to the overall intracellular oxygen transport of maximally exercising skeletal muscle and less than 2% to that of heart under conditions of high work load.

Animals↗
PubMed2001

Extracellular carbonic anhydrase activity facilitates lactic acid transport in rat skeletal muscle fibres.

1. In skeletal muscle an extracellular sarcolemmal carbonic anhydrase (CA) has been demonstrated. We speculate that this CA accelerates the interstitial CO2/HCO3- buffer system so that H+ ions can be rapidly delivered or buffered in the interstitial fluid. Because > 80 % of the lactate which crosses the sarcolemmal membrane is transported by the H+-lactate cotransporter, we examined the contributions of extracellular and intracellular CA to lactic acid transport, using ion-selective microelectrodes for measurements of intracellular pH (pHi) and fibre surface pH (pHs) in rat extensor digitorum longus (EDL) and soleus fibres. 2. Muscle fibres were exposed to 20 mM sodium lactate in the absence and presence of the CA inhibitors benzolamide (BZ), acetazolamide (AZ), chlorzolamide (CZ) and ethoxzolamide (EZ). The initial slopes (dpHs/dt, dpHi/dt) and the amplitudes (DeltapHs, DeltapHi) of pH changes were quantified. From dpHi/dt, DeltapHi and the total buffer factor (BFtot) the lactate fluxes (mM min-1) and intracellular lactate concentrations ([lactate]i) were estimated. 3. BFtot was obtained as the sum of the non-HCO3- buffer factor (BFnon-HCO3) and the HCO3- buffer factor (BFHCO3). BFnon-HCO3 was 35 +/- 4 mM pH-1 for the EDL (n = 14) and 86 /- 16 mM pH-1 for the soleus (n = 14). 4. In soleus, 10 mM cinnamate inhibited lactate influx by 44 % and efflux by 30 %; in EDL, it inhibited lactate influx by 37 % and efflux by 20 %. Cinnamate decreased [lactate]i, in soleus by 36 % and in EDL by 45 %. In soleus, 1 mM DIDS reduced lactate influx by 18 % and efflux by 16 %. In EDL, DIDS lowered the influx by 27 % but had almost no effect on efflux. DIDS reduced [lactate]i by 20 % in soleus and by 26 % in EDL. 5. BZ (0.01 mM) and AZ (0.1 mM), which inhibit only the extracellular sarcolemmal CA, led to a significant increase in dpHs/dt and pHs by about 40 %-150 % in soleus and EDL. BZ and AZ inhibited the influx and efflux of lactate by 25 %-50 % and reduced [lactate]i by about 40 %. The membrane-permeable CA inhibitors CZ (0.5 mM) and EZ (0.1 mM), which inhibit the extracellular as well as the intracellular CAs, exerted no greater effects than the poorly permeable inhibitors BZ and AZ did. 6. In soleus, 10 mM cinnamate inhibited the lactate influx by 47 %. Addition of 0.01 mM BZ led to a further inhibition by only 10 %. BZ alone reduced the influx by 37 %. 7. BZ (0.01 mM) had no influence on the Km value of the lactate transport, but led to a decrease in maximal transport rate (Vmax). In EDL, BZ reduced Vmax by 50 % and in soleus by about 25 %. 8. We conclude that the extracellular sarcolemmal CA plays an important role in lactic acid transport, while internal CA has no effect, a difference most likely attributable to the high internal vs. low extracellular BF(non-HCO3). The fact that the effects of cinnamate and BZ are not additive indicates that the two inhibitors act at distinct sites on the same transport pathway for lactic acid.

Animals↗
PubMed2000

Reversible Ca2+-induced fast-to-slow transition in primary skeletal muscle culture cells at the mRNA level.

1. The adult fast character and a Ca2+-inducible reversible transition from a fast to a slow type of rabbit myotube in a primary culture were demonstrated at the mRNA level by Northern blot analysis with probes specific for different myosin heavy chain (MyHC) isoforms and enzymes of energy metabolism. 2. No non-adult MyHC isoform mRNA was detected after 22 days of culture. After 4 weeks of culture the fast MyHCIId mRNA was strongly expressed while MyHCI mRNA was virtually absent, indicating the fast adult character of the myotubes in the primary skeletal muscle culture. 3. The data show that a fast-to-slow transition occurred in the myotubes at the level of MyHC isoform gene expression after treatment with the Ca2+ ionophore A23187. The effects of ionophore treatment were decreased levels of fast MyHCII mRNA and an augmented expression of the slow MyHCI gene. Changes in gene expression started very rapidly 1 day after the onset of ionophore treatment. 4. Levels of citrate synthase mRNA increased and levels of glyceraldehyde 3-phosphate dehydrogenase mRNA decreased during ionophore treatment. This points to a shift from anaerobic to oxidative energy metabolism in the primary skeletal muscle culture cells at the level of gene expression. 5. Withdrawal of the Ca2+ ionophore led to a return to increased levels of MyHCII mRNA and decreased levels of MyHCI mRNA, indicating a slow-to-fast transition in the myotubes and the reversibility of the effect of ionophore on MyHC isoform gene expression.

Animals↗
PubMed2000

Protein diffusion in living skeletal muscle fibers: dependence on protein size, fiber type, and contraction.

Sarcoplasmic protein diffusion was studied under different conditions, using microinjection in combination with microspectrophotometry. Six globular proteins with molecular masses between 12 and 3700 kDa, with diameters from 3 to 30 nm, were used for the experiments. Proteins were injected into single, intact skeletal muscle fibers taken from either soleus or extensor digitorum longus (edl) muscle of adult rats. No correlation was found between sarcomere spacing and the sarcoplasmic diffusion coefficient (D) for all proteins studied. D of the smaller proteins cytochrome c (diameter 3.1 nm), myoglobin (diameter 3.5 nm), and hemoglobin (diameter 5.5 nm) amounted to only approximately 1/10 of their value in water and was not increased by auxotonic fiber contractions. D for cytochrome c and myoglobin was significantly higher in fibers from edl (mainly type II fibers) compared to fibers from soleus (mainly type I fibers). Measurements of D for myoglobin at 37 degrees C in addition to 22 degrees C led to a Q(10) of 1.46 for this temperature range. For the larger proteins catalase (diameter 10.5 nm) and ferritin (diameter 12.2 nm), a decrease in D to approximately 1/20 and approximately 1/50 of that in water was observed, whereas no diffusive flux at all of earthworm hemoglobin (diameter 30 nm) along the fiber axis could be detected. We conclude that 1) sarcoplasmic protein diffusion is strongly impaired by the presence of the myofilamental lattice, which also gives rise to differences in diffusivity between different fiber types; 2) contractions do not cause significant convection in sarcoplasm and do not lead to increased diffusional transport; and 3) in addition to the steric hindrance that slows down the diffusion of smaller proteins, diffusion of large proteins is further hindered when their dimensions approach the interfilament distances. This molecular sieve property progressively reduces intracellular diffusion of proteins when the molecular diameter increases to more than approximately 10 nm.

Animals↗
PubMed2000

Immunochemical evidence for a unique GPI-anchored carbonic anhydrase isozyme in human cardiomyocytes.

To clarify the controversial question of cell-specific distribution of carbonic anhydrase (CA) in the heart, endothelial cells and cardiomyocytes were isolated from porcine and human hearts and were characterized with cell-specific markers. CA activity was found in the microsomal fraction of both cell types. It was shown by Triton X-114 phase separation that both cell types possess a membrane-bound form of CA. These CAs share the same mechanism of membrane-anchoring via glycosylphosphatidylinositol (GPI), which excludes identity with transmembrane isoforms CA IX or CA XII. Western blotting analysis of human microsomes with anti-human CA IV antibodies revealed a marked difference in immunoreactivity. Endothelial CA activity resulted in 11-fold stronger CA IV bands compared with identical amounts of myocytic CA activity, indicating that cardiac endothelium and cardiomyocytes possess immunologically distinct forms of CA. We conclude that in human hearts CA IV is associated with the endothelium, whereas most of the CA in myocytes is not identical with one of the known CA isozymes. This suggests that cardiomyocytic CA is a novel isozyme.

Animals↗
PubMed2000

Carbon dioxide transport and carbonic anhydrase in blood and muscle.

CO(2) produced within skeletal muscle has to leave the body finally via ventilation by the lung. To get there, CO(2) diffuses from the intracellular space into the convective transport medium blood with the two compartments, plasma and erythrocytes. Within the body, CO(2) is transported in three different forms: physically dissolved, as HCO(3)(-), or as carbamate. The relative contribution of these three forms to overall transport is changing along this elimination pathway. Thus the kinetics of the interchange have to be considered. Carbonic anhydrase accelerates the hydration/dehydration reaction between CO(2), HCO(3)(-), and H(+). In skeletal muscle, various isozymes of carbonic anhydrase are localized within erythrocytes but are also bound to the capillary wall, thus accessible to plasma; bound to the sarcolemma, thus producing catalytic activity within the interstitial space; and associated with the sarcoplasmic reticulum. In some fiber types, carbonic anhydrase is also present in the sarcoplasm. In exercising skeletal muscle, lactic acid contributes huge amounts of H(+) and by these affects the relative contribution of the three forms of CO(2). With a theoretical model, the complex interdependence of reactions and transport processes involved in CO(2) exchange was analyzed.

Animals↗
PubMed2000

Hagfish (Myxine glutinosa) red cell membrane exhibits no bicarbonate permeability as detected by (18)O exchange.

The bicarbonate permeability of the plasma membrane of intact hagfish (Myxine glutinosa) red blood cells and the intracellular carbonic anhydrase activity of these cells were determined by applying the (18)O exchange reaction using a special mass spectrometric technique. When the macromolecular carbonic anhydrase inhibitor Prontosil-Dextran was used to suppress any extracellular carbonic anhydrase activity, the mean intracellular acceleration of the CO(2) hydration/HCO(3)(-) dehydration reaction over the uncatalyzed reaction (referred to as intracellular carbonic anhydrase activity A(i)) was 21 320+/-3000 at 10 degrees C (mean +/- s.d., N=9). The mean bicarbonate permeability of the red blood cell membrane (P(HCO3)-) was indistinguishable from zero. It can be concluded that CO(2) transport within hagfish blood does not follow the classical scheme of CO(2) transport in vertebrate blood. It is suggested that the combination of considerable intraerythrocytic carbonic anhydrase activity and low P(HCO3)- may serve to enhance O(2) delivery to the tissue in the exceptionally hypoxia-tolerant hagfish.

Animals↗
PubMed2000

Properties of a carbonic anhydrase inhibitor protein in flounder serum.

The blood serum of the European flounder Platichthys flesus strongly inhibits soluble erythrocytic carbonic anhydrase from the same species. The inhibition is of the uncompetitive type. Hence, the mechanism of the carbonic anhydrase inhibition is different from that of all other known carbonic anhydrase inhibitors. The serum showed no inhibitory effect on carbonic anhydrase from human and bovine red blood cells. By applying the (18)O exchange reaction, it could be demonstrated that the presence of the carbonic anhydrase inhibitor in the extracellular fluid has no effect on carbonic anhydrase in intact red blood cells. Thus, this carbonic anhydrase inhibitor seems to act only within the plasma space of the circulatory system. However, the carbonic anhydrase inhibitor does appear to reduce the bicarbonate permeability of flounder red cells to approximately one-quarter of normal levels as measured by the (18)O exchange reaction. The 28 kDa carbonic anhydrase inhibitor was isolated from the serum by gel filtration. The isolated inhibitor was detected in acrylamide gels as a single band representing a 7 kDa protein. The denaturing conditions used in electrophoresis presumably led to a dissociation of the native protein into subunits.

Animals↗
PubMed1999

Carbonic anhydrase in the gills of seawater- and freshwater-acclimated flounders Platichthys flesus: purification, characterization, and immunohistochemical localization.

Flounders Platichthys flesus were investigated with respect to isolation, purification, and cellular localization of carbonic anhydrase (CA) in the respiratory system. CA was purified from gills and erythrocytes and was shown to exclusively represent a soluble enzyme with an apparent molecular weight of 30 kD. Inhibition constants (KI) towards acetazolamide (ACTZ) were 8.4.10(-9) M for erythrocyte CA and 7.6.10(-9) M for gill CA, indicating a high sensitivity to sulfonamides, as exhibited by human CA II. Specific CA activity did not differ significantly in seawater- and freshwater-acclimated fish. Antibodies were raised against purified gill and erythrocyte CA. Both antisera crossreacted and were used to localize CA in the gills of seawater and freshwater flounders at the light microscopic level. Independent of the salinity, a positive reaction of variable intensity was found in the following cell types: pavement cells (PVCs), forming the gill epithelial surface layer; mucous cells (MCs); pillar cells (PCs), bordering the vascular channels of the secondary lamellae; and chloride cells (CCs), mitochondria-rich cells located in the primary epithelium, the interlamellar regions, and at the bases of the secondary lamellae.(J Histochem Cytochem 47:43-50, 1999)

Animals↗
PubMed1998

The effect of 4,4'-diisothiocyanato-stilbene-2,2'-disulfonate on CO2 permeability of the red blood cell membrane.

It has long been assumed that the red cell membrane is highly permeable to gases because the molecules of gases are small, uncharged, and soluble in lipids, such as those of a bilayer. The disappearance of 12C18O16O from a red cell suspension as the 18O exchanges between labeled CO2 + HCO3- and unlabeled HOH provides a measure of the carbonic anhydrase (CA) activity (acceleration, or A) inside the cell and of the membrane self-exchange permeability to HCO3- (Pm,HCO-3). To test this technique, we added sufficient 4, 4'-diisothiocyanato-stilbene-2,2'-disulfonate (DIDS) to inhibit all the HCO3-/Cl- transport protein (Band III or capnophorin) in a red cell suspension. We found that DIDS reduced Pm,HCO-3 as expected, but also appeared to reduce intracellular A, although separate experiments showed it has no effect on CA activity in homogenous solution. A decrease in Pm,CO2 would explain this finding. With a more advanced computational model, which solves for CA activity and membrane permeabilities to both CO2 and HCO3-, we found that DIDS inhibited both Pm,HCO-3 and Pm,CO2, whereas intracellular CA activity remained unchanged. The mechanism by which DIDS reduces CO2 permeability may not be through an action on the lipid bilayer itself, but rather on a membrane transport protein, implying that this is a normal route for at least part of red cell CO2 exchange.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1998

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.

Animals↗
PubMed1998

18O exchange in suspensions of red blood cells: determination of parameters of mass spectrometer inlet system.

A membrane inlet system to a mass spectrometer (MIMS) allows us to monitor the abundance of 18O in CO2 during the exchange of 18O between HCO3-, dissolved CO2 and H2O that occurs after dissolving 18O-labelled NaHCO3 in aqueous solution. Using a mathematical model we quantitate intracellular carbonic anhydrase activity and membrane permeabilities for HCO3-, CO2 and H2O of isolated cells or intact epithelia added to the solution. For these calculations it was necessary to determine (a) the relation between MS signal and [C18O16O], (b) MIMS response kinetics and (c) CO2 leakage during the experiment. The three parameters were determined by stepwise addition of HCl to HCO3- solution: (a) was found to be linear, (b) response times were 7.5 +/- 2s (10 degrees C), 4.5 +/- 1s (20 degrees C), 3.5 +/- 0.6s (37 degrees C), and (c) CO2 leakage was < 1/1000 s-1.

Carbon Dioxide↗