SUMMARY1. The total carbonic anhydrase activity in some guinea-pig tissues has been measured using a pH-stat procedure. Stomach, gall bladder, proximal colon and caecum all possess more carbonic anhydrase activity per unit amount of protein than does whole blood.2. The carbonic anhydrase activity of the small intestine is low. Reasons are given for supposing that activity found there is not entirely due to contamination by whole blood, and it is suggested that in this tissue the enzyme may be localized in some cell type other than the columnar absorbing cells.3. Evidence is presented which indicates that heavy metals interfere with the activity of the enzyme as measured in tissue homogenates.4. The distribution and concentration of the two major isoenzymes of carbonic anhydrase have been measured in different tissues. Blood and proximal colon contain both isoenzymes in comparable concentrations, the ratio of the concentration of the 'low activity' isoenzyme to that of the 'high activity' being about 2. The gastric mucosa contains much 'high activity' carbonic anhydrase, but only a negligible amount of the 'low activity' isoenzyme. In the caecal mucosa, the 'low activity' isoenzyme is predominant, the ratio of its concentration to that of the 'high activity' isoenzyme being about 9. It is also found that more than 15 % of the protein in the caecal mucosa is accounted for as carbonic anhydrase enzymes.5. It is found that some 45 % of the total carbonic anhydrase activity of sucrose homogenates of the guinea-pig colon is bound to particles. The activity is located mainly in the nuclear and microvillous fraction and in the 'high-speed supernatant' fraction. The form of enzyme bound is M. J. CARTER AND D. S. PARSONS largely of the 'high activity' variety. When the tissue is homogenized in potassium chloride solutions less than 4 % of the total activity is recovered in particulate fractions. The amount of activity which is bound to particulate fractions increases as the ionic strength or pH of the homogenate is lowered.6. The findings are discussed in relation to the possible physiological roles of the isoenzymes in tissues other than blood. Possible relationships between the presence of the enzymes and the metabolism and transport of ammonium and fatty acids are considered.
1. Details are given of an electrometric method for measuring the activity of isoenzymes of carbonic anhydrase (EC 4.2.1.1) in catalysing the hydration of carbon dioxide under different conditions at 0° C. In the method, a measured volume of water saturated with carbon dioxide at a known partial pressure and appropriate temperature is introduced into a buffered solution. Using a sensitive electrometer and recording instrument, the subsequent change in hydrogen ion concentration is recorded as a function of time. Under the conditions of assay, the pH change induced in the presence of substrate is very small (ΔpH < 0·05 units) and the period of observation need not exceed 10 sec. 2. For enzymes isolated from guinea‐pig tissues, it is found that the specific activity of the ‘high activity’ isoenzyme (carbonic anhydrase C, carbonic anhydrase II, HACA) is about eighteen times that of the ‘low activity’ counterpart (carbonic anhydrase B, carbonic anhydrase I, LACA) when measured at 0° C, pH 7·2, and ionic strength 0·19. Under the same conditions, the Km was found to be 10 m M for the ‘high activity’ isoenzyme and 23 m M for the ‘low activity’ isoenzyme. No differences were found between the equivalent kinetic parameters of the corresponding isoenzymes isolated from different tissues. 3. The isoenzymes isolated from guinea‐pig tissues are found to be inhibited by acetazolamide in a non‐competitive manner. It is also found that the ‘high activity’ isoenzyme is many times more sensitive to this inhibitor than is the ‘low activity’ isoenzyme. Evidence is presented which indicates that one acetazolamide binding site is present on each molecule of either isoenzyme. 4. While chloride ions specifically inhibit the ‘low activity’ component of guinea‐pig carbonic anhydrase (I0·5 = 40 m M), acetate, butyrate and pyruvate inhibit both isoenzymes. Under the conditions employed, acetate and pyruvate are more strongly inhibitory to the ‘low activity’ isoenzyme than to the ‘high activity’ isoenzyme, while butyrate is more strongly inhibitory to the ‘high activity’ isoenzyme. 5. The findings are discussed with particular reference to the physiological significance of the presence of the isoenzymes in the gastro‐intestinal tract. Also considered are possible relationships between the distribution of the ‘low activity’ isoenzyme in these tissues and the transport and metabolism of products of fermentation occurring in the intestinal lumen.
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