Total Carbonic Anhydrase Activity Research Articles

Effect of inhibitors of carbonic anhydrase activity on photosynthesis in the red alga Soliera filiformis (Gigartinales: Rhodophyta)

The effect of light intensity, pH and carbonic anhydrase (CA) inhibitors on photosynthesis of the red marine macroalgae Solieria filiformis (Kutzing) Gabrielson, collected from Taliarte (Gran Canaria, Canary Islands) in 1991, has been investigated. Plants taken from the sea (“wild phenotype”) developed spherical morphology (“ball phenotype”) after 2 mo culture in aerated tanks. The photosynthetic oxygen evolution in the wild phenotype was saturated at 100 μmol photons m-2s-1, while the “ball” phenotype displayed saturation at 200 μmol photons m-2s-1. The inhibitors of total CA activity (6-ethoxizolamide) and extracellular CA activity (dextran-bound sulfonamide) inhibited photosynthesis at pH 8.2, to 90 and 50% respectively, in both phenotypes. No inhibition of the photosynthetic oxygen evolution was detected at pH 6.5. CA activity was associated with both supernatant and pellet fractions of crude extracts of S. filiformis. The rate of alkalization of the medium by the algae was dependent on light intensity. We suggest that carbon dioxide is the general form of inorganic carbon transported across the plasmamembrane in S. filiformis. HCO3 transport into the cell takes place simultaneously by an “indirect” mechanism (dehydration to CO2 catalyzed by CAext) and by direct uptake. Extracellular (CAext) and intracellular (CAint) CAs are involved in the mechanisms of inorganic carbon assimilation by S. filiformis.

Differential regulation of hepatic carbonic anhydrase isozymes in the streptozotocin-diabetic rat

Most work with the male rat liver carbonic anhydrase isozymes in the past decade has centered on the cytosolic CA III and the mitochondrial CA V. This paper reports that the relative activity of both isozymes is altered in streptozotocin-diabetes. Carbonic anhydrase activity of perfused liver homogenates and disrupted, isolated mitochondria was measured by the mass spectrometric 18O decay technique at 37 °C. The contributions of the different isozymes were determined based on intracellular location and sensitivity to acetazolamide inhibition. Diabetes resulted in a twofold increase in the activity of CA V but a halving in the activity of CA III. This is the first time that liver CA V has been shown to be altered by physiological stress. The total carbonic anhydrase activity in the diabetic rat liver was unaltered compared with control rats; however, CA III never accounted for more than 50% of this activity. Since CA isozymes I, II, and IV together account for 30% of the CA activity in control rats and 70% in diabetic rats it is concluded that one or more of these isozymes is subject to regulation in the diabetic male rat. The increase in CA V during diabetes is in accord with this isozyme having an important function in provision of substrate for hepatic gluconeogenesis and ureagenesis.

Localization of carbonic anhydrase in the gills of Carcinus maenas

1. 1. Gill hemogenates of Carcinus maenas were centrifuged at 100,000 g. Pellets and supernatants were analyzed for carbonic anhydrase (CA) activity. Approximately 70% of total CA activity occurred in a bound form, the rest being cytosolic. 2. 2. Repeated hemogenization and sonication prior to recentrifugation did not result in additional liberation of CA from the bound state. 3. 3. Centrifugation of a 7500 g pellet in a sucrose density gradiant provided evidence that the bound form of CA is present in plasma membranes.

Distribution of carbonic anhydrase in British marine macroalgae.

Thirty-four species of marine macroalgae from around St. Andrews, Scotland, have been assayed for their external activity and thirty-three species for their total activity of carbonic anhydrase. Activity was detected in all the Rhodophyta tested apart from Chondrus crispus, but was absent in Codium fragile, Enteromorpha sp. and Monostroma fuscum (Chlorophyta), and Alaria esculenta, Laminaria digitata, L. saccharina and L. hyperborea (Phaeophyta). Total activity of carbonic anhydrase per unit fresh weight tended to be higher in the Rhodophyta than in the Chlorophyta or Phaeophyta. External activity was present in two of the six Chlorophyta, four of the twelve Phaeophyta and four of the sixteen Rhodophyta tested. On average, when present, external carbonic anhydrase activity represented 2.7% of the total activity. A relationship was found between total carbonic anhydrase activity and habitat. Species from the high intertidal and the low-light subtidal habitats had significantly higher activity than species from the mid and low intertidal, rockpools, or high-light region of the subtidal. External carbonic anhydrase activity did not vary significantly with habitat. There appeared to be no strong relationship between carbonic anhydrase activity and the ability of a species to use HCO -3 in photosynthesis under water.

Carbonic anhydrase in the normal rat stomach and duodenum and after treatment with omeprazole and ranitidine

A low pH in the lumen of the stomach and duodenum stimulates gastroduodenal mucosal secretion of bicarbonate, particularly in the duodenum. Long-term deprivation of this acid stimulus might affect the ability of the mucosa to secrete bicarbonate, with a consequent decrease in mucosal protection against the acid. This could occur by 'down-regulation' of carbonic anhydrase (CA) activity in the bicarbonate-transporting cells. Levels of CA activity and amounts of CA isoenzymes in rat gastric and duodenal mucosa were determined by biochemical assay and histochemical and immunohistochemical staining. Control animals and animals pre-treated for 4-6 weeks with the histamine H2-receptor antagonist ranitidine (600 mg kg-1 daily) or the H+,K+-ATPase inhibitor omeprazole (28 mg kg-1 daily) were examined. Both drugs are potent inhibitors of gastric secretion of acid. Both gastric and duodenal mucosal total CA activity and the distribution of isoenzymes were very similar in control animals and animals treated with these drugs. In the stomach, CA II was found in the surface epithelial and parietal cells. In the duodenum both CA I and CA II were observed. The staining for CA I was restricted to a small number of villus cells which looked like ordinary duodenal enterocytes. CA II in the duodenum was found in all villus cells, except the goblet cells. The staining decreased gradually from the top to the bottom of the villi and was absent in the crypts. Duodenal bicarbonate secretion is dependent on mucosal CA activity, and the distribution of CA II thus suggests that this alkaline secretion is of villous rather than cryptal origin.

Acetazolamide-sensitive and resistant carbonic anhydrase activity in rat and rabbit skeletal muscles of different fiber type composition

1. 1. Acetazolamide (ACET)-resistant and -sensitive carbonic anhydrase (CA) activity was measured in post-mitochondrial supernatants from the soleus (SOL), deep vastus lateralis (DVL) and superficial vastus lateralis (SVL) muscles of rats and rabbits. 2. 2. The relative total CA activity in the three muscles of both species can be summarized as SOL > DVL > SVL. 3. 3. ACET-resistant CA activity was found in the SOL and DVL muscles of both species whereas a low level of ACET-sensitive CA activity was detected in the SVL muscle. 4. 4. ACET-sensitive CA activity was also found in sarcoplasmic reticulum preparations from rat and rabbit SOL muscles.

Total CA activity in isolated perfused guinea pig lung by 18O-exchange method.

The rate of exchange of 18O between alveolar CO2 and lung water was measured in isolated perfused guinea pig lungs to quantify carbonic anhydrase (CA) activity. The average lung CA activity, with a reaction velocity constant of 5.32 +/- 2.2 s-1, is sufficient to accelerate CO2 reactions in lung water by two orders of magnitude over the uncatalyzed rate at 22 degrees C and a PCO2 of 40 Torr. Three sulfonamide inhibitors of CA with different human erythrocyte membrane permeabilities were used to determine the availability of the enzyme to the perfusate. Ethoxzolamide, the most permeable at 0.1 microM (100 times its inhibition constant, of Ki) inhibited 85% of enzyme activity after exposure of the lung for 3 min and 94% of enzyme activity after 30 min, whereas 1.25 microM (320 times its Ki) acetazolamide (1/165 as permeable) only inhibited CA 28% at 3 min and 75% at 30 min. Benzolamide (less than 1/1,000 as permeable) at 4 microM (1,000 times its Ki) inhibited only approximately 17% of control CA activity by 5 min and 48% by 30 min after the start of perfusion. These data indicate the CA available to pulmonary capillary plasma is approximately 10% of the total lung CA activity, in agreement with published measurements on the homogenized lung.

Expression of glutamine synthetase and carbonic anhydrase in isolated periportal and perivenous hepatocytes

lmmunohistochemical studies first demonstrated that, both in human and rat liver, glutamine synthetase is expressed only in a narrow zone surrounding the terminal hepatic venule (Gebhardt & Mecke, 1983). This peculiar heterogeneous distribution of the enzyme protein has recently been confirmed by direct analysis of glutamine synthetase activity in periportal and perivenous cell lysates (Quistorff & Grunnet, 1987) or hepatocytes (Ugele et al., 1987; K. Lindros unpublished work) isolated by digitonin-collagenase perfusion (Lindros & Penttila, 1985). The so-called glutamine cycle encompassing periportal ammonia fixation and glutamine utilization for urea synthesis and perivenous glutamine synthesis has been established (Haussinger, 1983) and has been considered to have an important role in hepatic and systemic pH regulation. The factors governing the local expression of glutamine synthetase are, however, not known. Recently, a heterogeneous hepatic expression of another enzyme, carbonic anhydrase (CA), resembling that of glutamine synthetase, was described (Carter et al., 1987). The highand low-activity cytoplasmic forms CAI1 and CAIII, identified by specific radioimmunoassay, were shown to exhibit growth hormone-mediated sexual dimorphism in rat liver. While there was 3-4 times more CAI1 in female than in male liver, CAI11 was up to 30 times more abundant in male liver. Immunohistochemistry revealed that although weak staining was observed all over the lobule, both CAI1 and CAI11 stained intensively around the central veins. However, while livers from males exhibited much higher CAI11 activities than females in the perivenous zones, CAI1 activities were much higher in livers from females. The mitochondria1 CA isoenzyme, CAIV serves the production of urea (Dodgson et al., 1983). This process is more active in the periportal zone (Haussinger, 1983; Poso et al., 1986). Since glutamine synthetase and carbonic anhydrase are both intimately involved in regulation of pH and metabolism, and since both are differentiation markers in embryonic neural retina (Vardimon et al., 1986), their possible coexpression in rat liver was investigated. Glutamine synthetase was measured radioisotopically, by separating [ 14C]glutamate and [ 14C]glutamine by ionexchange (Pishak & Phillips, 1979). Total CA activity was measured by a pH indicator method and CAI1 and CAIII isoenzymes by specific radioimmunoassay. Periportal and perivenous hepatocytes were isolated by the digitonincollagenase perfusion technique (Lindros & Penttila, 1985; Quistorff, 1985). Hepatocytes were inoculated on culture dishes coated with rat tail collagen at a density of 6.3 x lo4/ cm2. A 22 mwbicarbonate/l5 mM-Hepes buffered 1 : 1 mixture of Waymouth MB 752/1 and Ham F-12 was ordinarily supplemented with a 5% (v/v) fetal calf serum, 5% (v/v) newborn calf serum (Gibco), insulin (10 units/l), dexamethasone (1 p ~ ) , gentamycin (10 mg/l) and nystatin ( 1 mg/l). Cultures were always supplemented with serum for the first 4 h after plating. Analysis of freshly isolated cells revealed a striking and consistent difference both in total COz hydrase activity and in CAI1 and CAI11 concentrations between periportal and

Effect of vitamin D status on the activity of carbonic anhydrase in chicken epiphysis and kidney.

Chickens were raised for 6 weeks from the date of hatch under red light on a vitamin D-free diet; controls were given an oral vitamin D supplement. Vitamin D-deficient animals showed decreased total serum calcium concentration and decreased DNA content in epiphysis and kidney homogenates. In calcifying epiphysis, total carbonic anhydrase (CA) activity was decreased, but activity per microgram DNA was slightly increased and specific activity was double that of the controls. Polyacrylamide gel isoelectric focusing after preparation of the enzyme showed a picture similar to that seen after parathyroid hormone (PTH) administration in chicks; therefore, this could be considered a secondary hyperparathyroidism. The CA activation was not seen in the kidney which can be explained by induction of an endogenous inhibitor protein of the cyclic AMP-dependent protein kinase exclusively in the kidney in vitamin D deficiency. In an additional experiment, chickens were raised for 3 weeks from the date of hatch under red light on a vitamin D-free diet. Daily oral substitution by different vitamin D metabolites (1,25(OH)2D3, 25OHD3, 24,25(OH)2D3) over 7 days led to CA activation compared with controls probably by restoring protein kinase activity in the kidney. Our results show that CA activity is inversely correlated with serum calcium concentrations which is in agreement with a regulatory mechanism recently proposed by us.

Quaternary ammonium sulfanilamide: a membrane-impermeant carbonic anhydrase inhibitor.

A novel carbonic anhydrase (CA) inhibitor, quaternary ammonium sulfanilamide (QAS), was tested for potency as a CA inhibitor and for its ability to be excluded from permeating biological membranes. Inhibitor titration plots of QAS vs. pure bovine CA II and CA from the gills of the blue crab, Callinectes sapidus, yielded Ki values of approximately 15 microM; thus QAS is a relatively weak but effective CA inhibitor. Permeability of the QAS was directly tested by two independent methods. The inhibitor was excluded from human erythrocytes incubated in 5 mM QAS for 24 h as determined using an 18O-labeled mass spectrometer CA assay for intact cells. Also QAS injected into the hemolymph of C. sapidus (1 or 10 mM) did not cross the basal membrane of the gill. The compound was cleared from the hemolymph by 96 h after injection, and at no time during that period could the QAS be detected in homogenates of gill tissue. Total branchial CA activity was only slightly reduced following the QAS injection. These data indicate that QAS is a CA inhibitor to which biological membranes are impermeable and that can be used in vivo or in vitro in the study of membrane-associated CA.

Inhibition of CA V decreases glucose synthesis from pyruvate

The carbonic anhydrase inhibitor acetazolamide reduces citrulline synthesis by intact guinea pig liver mitochondria and also inhibits mitochondrial carbonic anhydrase (CA V) and the more lipophilic carbonic anhydrase inhibitor ethoxzolamide reduces urea synthesis by intact guinea pig hepatocytes in parallel with its inhibition of total hepatocytic carbonic anhydrase activity. Intact hepatocytes from 48-h starved male guinea pig livers were incubated at 37 °C in Krebs-Henseleit with 95% O 2/5% CO 2 at pH 7.1 with 5 m m pyruvate, 5 m m lactate, 3 m m ornithine, 10 m m NH 4Cl, 1 m m oleate; with these inclusions both urea and glucose synthesis start with HCO 3 −-requiring enzymes, carbamyl phosphate synthetase I and pyruvate carboxylase, respectively. Urea and glucose synthesis were inhibited in parallel by increasing concentrations of ethoxzolamide, estimated K i for each approximately 0.1 m m. In other experiments hepatocytes were incubated at 37 °C in Krebs-Henseleit with 95% O 2/5% CO 2 at pH 7.1 with 10 m m glutamine, 1 m m oleate; with these inclusions glucose synthesis no longer starts with a HCO 3 −-requiring enzyme. Urea synthesis was inhibited by ethoxzolamide with an estimated K i of 0.1 m m, but glucose synthesis was unaffected. Intact mitochondria were prepared from 48-h starved male guinea pig livers. Pyruvate carboxylase activity of intact mitochondria was determined in isotonic KCl-Hepes buffer, pH 7.4, 25 °C, with 7.5 m m pyruvate, 3 m m ATP, and 10 m m NaHCO 3. Inclusion of ethoxzolamide resulted in reduction in the rate of pyruvate carboxylation in intact mitochondria, but not in disrupted mitochondria. It is concluded that carbonic anhydrase is functionally important for gluconeogenesis in the male guinea pig liver when there is a requirement for bicarbonate as substrate.

Biochemical and immunocytochemical evidence for a deficiency of normal interfascicular oligodendroglia in the CNS of the dysmyelinating mutant (md) rat.

Carbonic anhydrase was assayed and carbonic anhydrase and 5'-nucleotidase were localized in the CNS of myelin-deficient mutant rats and normal littermates. The carbonic anhydrase specific activities were reduced by 61% and 29% in the mutants' forebrains and cerebella, respectively, and the total carbonic anhydrase activity in the spinal cords was reduced by 35%. Immunostained cells were found in gray matter from both normal and mutant rats, but, in the mutants, there was a marked deficiency of interfascicular oligodendrocytes in the regions that are normally occupied by white matter. It is suggested that a developmental study could indicate the step(s) at which normal differentiation of interfascicular oligodendroglia is blocked in this mutant.

Inhibition of carbonic anhydrase in dog plasma.

Using a pH stat method, we measured the activity of carbonic anhydrase (CA) from dog erythrocytes in the presence of various amounts of dog plasma. A plasma factor appeared to be able to inhibit about 86% of the total CA activity, corresponding to the relative activity of CA II. Naiodoacetate was shown to inhibit the total CA activity up to about 13%, corresponding to the relative activity of CA I. C1- inhibited the total CA activity up to about 20%, presumably mainly through its strong influence on Ca I. It is concluded that with a degree of haemolysis of up to 3%, no appreciable plasma CA activity will occur.

Carbonic anhydrase isozymes of osteoclasts and erythrocytes of osteopetrotic microphthalmic mice

The content of carbonic anhydrase isozymes I (CA I) and II (CA II) in red blood cells and bone of osteopetrotic microphthalmic ( mi mi ) mice was analyzed. Monospecific rabbit polyclonal antibodies against purified rat carbonic anhydrase I or II detected both isozymes in hemolysates of both normal and mi mi mice. Total carbonic anhydrase activity measurements of hemolysates from normal or mi mi mice were identical. A procedure based on bromopyruvate inactivation was devised to measure the relative contributions of CA I and II isozymes to the carbon dioxide hydration activity of hemolyzates. CA II dominates the observed activity of hemolysates of normal and mi mi mice. Immunohistochemical studies showed that CA II was present in osteoclasts of tibial and calvarial bones of both normal and mi mi mice. Thus, in contrast to the several cases of inherited osteopetrosis in humans, there is no lack of active CA II in erythrocytes of mi mi mice. The absence of CA II, therefore, does not appear to play a role in the etiology of osteopetrosis in the mi mi mouse.

Effect of short physical exercise on the levels of zinc and carbonic anhydrase isoenzyme activities in human erythrocytes

Studies of the influences of physical exercise of short duration (bicycle ergometer, 200 W for 30 min) on the activities of carbonic anhydrase isoenzymes (CA-B and CA-C types) and zinc concentration in erythrocytes were made on 5 untrained healthy male volunteers. The subjects were rested for 30 min after the exercise. There were significant decreases in the levels of zinc, CA-B, total carbonic anhydrase activity and CA-B-dependent activity immediately after exercise, but after 30 min of rest they all returned to their pre-exercise levels. No significant change in CA-C level or CA-C-dependent activity was found after exercise. Immediately after exercise, total carbonic anhydrase activity and CA-B-dependent activity following the addition of Zn2+ showed significant increases, compared with their respective activities without Zn2+ addition. However, no such effects were observed just before exercise or after rest; the addition of Zn2+ had no effect on CA-C-dependent activity at any time. A significant correlation was found between the changes in concentration of zinc and CA-B-dependent activity after exercise (r=0.711). The findings of the present study suggest that active CA-B enzymes are converted in part to inactive enzymes during acute physical exercise, possibly by decreased zinc binding. Moreover, the change in CA-B-dependent activity correlated well with the changes in pH and HCO3 concentrations in venous blood (r=0.853 and r=0.718, respectively). One may speculate that an adaptive decrease in CA-B-dependent activity in erythrocytes occurs with increased acidification in blood during heavy physical exercise of short duration.

Carbonic anhydrase isozymes of rabbit renal microvillus membranes: inhibition by sulfonamides

The microvillus fraction isolated from rabbit renal cortex contained about 5% of the total carbonic anhydrase activity in the homogenate, which could not be explained by contamination with the cytoplasm or erythrocyte soluble fractions. The carbonic anhydrase of the microvillus fraction showed two K1 values for each of the three sulfonamide inhibitors tested (sulfanilamide, acetazolamide, and ethoxzolamide). The values were essentially the same as those shown by the cytoplasm and erythrocyte soluble fractions, though the activity ratio of low to high K1 isozymes of the microvillus fraction seemed to be different from those of the other two fractions.

The importance of carbonic anhydrase B and C for the unloading of CO2 by the human erythrocyte.

Carbonic anhydrase (carbonate dehydratase, EC 4.2.1.1) isoenzymes HCA B and HCA C from human erythrocytes were purified by affinity chromatography and characterized kinetically at 37 degrees C in 25 mM N-methylimidazole buffer, I = 0.15, pH 7.1, using a pH-indicator stopped-flow technique. The rate constants for the uncatalyzed hydration of CO2 and dehydration of H2CO3 were 0.12 . s-1 and 58 . s-1, respectively, Km and turnover numbers in the hydration reaction were 14.0 mM and 19.1 . 10(5) . s-1 for HCAC and 3.3 mM and 0.56 . 10(5) . s-1 for HCA B. Km and turnover numbers in the dehydration reaction were 70 mM and 5 . 10(5) . s-1 for HCA C and 16.8 mM and 0.27 . 10(5) . s-1 for HCA B. Ki for chloride was 14 mM for HCA B and greater than 200 mM for HCA C. Ki for acetazolamide was 0.9 microM against HCA B and 16 nM against HCA C. The rates of hydration of CO2 in hemolysates with known concentrations of HCA B and HCA C, and in mixtures of purified HCA B and HCA C with concentrations near those in the erythrocyte, were similar to the rates calculated from the kinetic parameters of each isoenzyme. HCA B is 86% inhibited by chloride in vivo. This strong inhibition, together with the low specific activity, explains why HCA B only accounts for 10% of the total carbonic anhydrase activity of the erythrocyte, en spite the cellular concentration HCA B is 8 times higher than that of HCA C. HCA B and HCA C can together accelerate the intracellular CO2-reaction 23 000-fold, which gives a margin of 25-fold over physiological needs in hard work and 50-fold at rest. Perceptible physiological effects on respiration should therefore be seen when total carbonic anhydrase activity of erythrocytes is 96 to 98% inhibited. These degrees of inhibition are achieved when plasma concentrations of acetazolamide reach 2 and 5 microM, respectively.

Effect of Physical Exercise on Erythrocyte Carbonic Anhydrase Isozymes and 2,3-Diphosphoglycerate in Men

Untrained healthy male volunteers were studied to observe the effects of muscular exercise (bicycle ergometer, 150 W for 15 min and 30 min) upon the levels of carbonic anhydrase B(CA-B) and C(CA-C) isozymes and 2,3-diphosphoglycerate (2,3-DPG) in erythrocyte cells. Significant decreases in the levels of both CA-B and total carbonic anhydrase activity were observed after 15 min as well as after 30 min of exercise whereas the level of CA-C did not vary substantially. The level of 2,3-DPG increased after 30 min of exercise. A negative correlation was found between the levels of CA-B and 2,3-DPG after 30 min of exercise (r = -0.635) while no significant correlation was found after 15 min of exercise. The blood lactate-pyruvate ratio increased above the pre-exercise values after 15 min of ex-ecercise but not after 30 min of exercise. After 30 min of exercise, the increase in lactate concentration and the change of 2,3-DPG correlated well (r = 0.668). These observations suggest that the steady-state level of CA-B is affected by physical exercise more readily than that of CA-C and that the changes in the levels of these isozymes and 2,3-DPG in erythrocyte cells are requisite for adaptation to prolonged exercise.

Changes in pH with age in Drosophila and the influence of buffers on longevity

The pH of whole homogenates of Drosophila melanogaster declines from 7.44 during the first instar larval stage to 6.77 during the late pupal stage. In adult flies the pH declines from 6.92 to 6.70 with increasing age. Total carbonic anhydrase activity declines during the first 10 days of the adult stage. The carbonic anhydrase inhibitor acetazolamide failed to shorten life span. Low molecular weight buffers prolong the median life span when fed to flies in the pH range 7.0–8.3. The greatest prolongation occurs when the flies are given the buffers beginning at 2 weeks of age. 0.1 M HEPES (pH 7.5) increased life span by 22.9% and 0.01 M KHCO 3 (pH 7.0) by 17.8%.

ATPase and carbonic anhydrase activities of bulk-isolated neuron, glia and synaptosome fractions from rat brain

The (Na + + K +) ATPase, carbonic anhydrase and HCO 3 −-stimulated ATPase activities of bulk-isolated neuronal perikarya, glial and synaptosome fractions from 20–26-day-old rats were studied. The effects of varying K +, Na + and ATP concentrations were investigated to determine limits on how these enzymes might respond to changes in the levels of these substances in vivo. The (Na + + K +) ATPase activity of all three fractions had a similar high affinity for K +, with K m values in the range of 0.7–1.7mM. The K m for Na + was around 10-fold higher, in the range of 10–15mM. K m values for ATP were also not markedly different between the different fractions, being 1.2mM for the neurons and 1.8mM for the glia and synaptosome fractions, respectively. The V max at infinite [ATP] in the presence of 10mM K + was 2.9-fold higher for the glia as compared to the neuron fraction and 1.5-fold higher than the synaptosome fraction. At infinite K + in the presence of 3.3mM ATP the corresponding figures were 3.0 and 2.1. Arrhenius plots of (Na + + K +) ATPase activity were different for the neuron and glia fractions as compared to the synaptosome fraction,, suggesting subtle differences in themembrane environment and/or the enzyme molecule itself. The HCO 3 −-stimulated ATPase activity was only 14% higher in the glial fraction compared to the neurons and was variably stimulated by added K + at concentrations < 10mM. The glial-enriched cell fraction had a 2-fold higher specific carbonic anhydrase activity than the neuron fraction, but the combined activity of these fractions only represented < 2% of the total brain activity. Total brain carbonic anhydrase activity was not stimulated by added K + in the range of 5–50mM K +. These data indicate that none of the enzymes studied are likely to directly respond to extracellular K + levels in excess of 10mM, and therefore would only be secondarily involved in cerebrocortical swelling caused by increasing K + concentrations 20mM. The relation of these findings to previous work on increased (Na + + K +) ATPase and carbonic anhydrase activity in glial cells, the role of glial cells in passive or mediated transport processes related to increased levels of extracellular K + and the suitability of bulk-isolated glia and neuron-enriched fractions as experimental models are discussed.