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1. As part of their characterization, the effect of histamine (H) on both soluble and membrane-associated carbonic anhydrase (CA) activity of pig and bovine gastric light microsomal membranes was investigated. 2. H did not affect the activity of soluble CA purified from pig oxyntic mucosa, whereas 10(-7) and 10(-4) M H produced a significant enhancement of pig gastric firmly-membrane-associated CA activity at 20 and 30 degrees C (about 2-3-fold), but it failed at 0 degree C. The increase of activity produced by H occurred within 1 min and it was maintained for at least 15 min. 3. H also stimulated bovine gastric firmly-membrane-associated CA activity, the stimulation also being dependent on temperature.

1. The occurrence and characteristics of carbonic anhydrase (CA) activity were studied in light microsomal membranes (LMM) purified from bovine gastric mucosa. 2. Bovine gastric LMM contained a high activity of CA ranging from 170 to 400 mumol.H+/min/mg protein when assayed at 0 degree C by pH-stat technique. 3. The addition of 2mM EDTA to the assay mixture increased the enzyme activity. Lower concentrations (0.5-1 mM) had no effect. 4. The enzyme activity was dose-dependently inhibited by acetazolamide and furosemide (I50: 5 x 10(-10) M and 4.8 x 10(-7) M, respectively) and by chloride ion (Ki 85 mM) and appeared to be quite stable to treatment with alkaline Triton X-100. 5. Most of the CA activity is loosely associated with the LMM since it was removed by different washing treatments. Nevertheless, after extensive washes, gastric LMM still contained CA activity suggesting the existence of a firmly membrane-associated form of CA. 6. Values of CA activity higher than those reported previously were found in pig gastric LMM. Furthermore, the washing treatments described in this work were more effective in washing CA activity off pig gastric LMM than procedures described previously.

1. The effect of pH on the association of carbonic anhydrase (CA) with bovine gastric light microsomal membranes (LMMs) was investigated (a) by washing LMMs containing CA activity with solutions of different pHs; (b) by studying the adsorption at various pHs of soluble bovine erythrocyte CA to washed gastric LMMs. In both cases, the association of CA with gastric LMMs was dependent on pH, being lower at neutral or alkaline pH. 2. The amount of soluble CA associated with gastric LMMs at pHs 8.0 and 9.0 was reduced when 140 mM K+/10 mM Na+ was added to the incubation medium. 3. Two sources of CA activity in bovine gastric LMMs were assumed: a loosely- and a firmly-membrane-associated activity. Both CA activities were dose-dependently inhibited by acetazolamide (I50: 3.6 x 10(-9) and 8.4 x 10(-9) M, respectively) and by chloride, acetate, iodide, bromide and nitrate at 100 mM. Firmly-membrane-associated activity appeared to be less sensitive to inhibition by acetazolamide, chloride and iodide. 4. Both activities exhibited different behavior and stability following treatment with alkaline Triton X-100. 5. The possible importance of a membrane-associated CA activity in gastric LMMs related to gastric acid secretion is discussed.

Mucosal cells were isolated from pig stomachs and then fractionated on linear density gradients of Percoll. Different types of cells were identified by their typical staining and morphology. In disrupted cell fractions, hydration of CO2 by carbonic anhydrase was measured by means of pH-stat technique. Localization of carbonic anhydrase to certain cell fractions was also studied by histochemical staining. Both parietal cells and carbonic anhydrase were confined to the low and intermediate density fractions of the gradients. Purified membranes from pig gastric mucosa, which contained the acid pump of the stomach, the H,K-ATPase, also contained a firmly bound carbonic anhydrase of high activity. The enzyme activity in the membranes was inhibited by acetazolamide, furosemide and KSCN. The molecular mass of the carbonic anhydrase was 33 kDA as estimated by its binding of [14C]furosemide followed by polyacrylamide gel electrophoresis. Previous suggestions of a role of carbonic anhydrase as a supplier of H+ in the secretion of acid are supported by its high activity and its localization to the same membrane as the acid pump of the stomach.