The identification of an active enzyme site by rapid freeze substitution enzyme histochemistry on rat retina.

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Since cell function is supported by many constituent enzymes in organized array, it is important that the site of enzyme activities in combination with fine ultrastructures be studied.<br>In the retina, the cGMP metabolizing enzymes included in phosphodiesterase are estimated to play a key role in action potential formation. Therefore, the site and arrangement of these enzymes in the rod outer segment are of histochemical interest and have yet to be clarified. The new tool, freeze substitution enzyme histochemistry developed in our laboratory enables us to visualize the exact activity and also the sites of enzymes as they are in the living state. With this method, the active sites of guanylate cyclase and phosphodiesterase have been demonstrated, but before having been able to specify the precise active sites at the ultrastructured level, the contrast obtained by rapid freeze substitution enzyme histochemistry on the biological membranes had to be increased. This contrast enhancement has been successfully achieved applying higher concentrations of fixatives in combination with tannic acid. Other attempts were the extension of fixation time as well as the application of higher temperatures. Potassium ferrocyanide osmium post-fixation also appeared effective. With the contrast enhancement achieved by various combinations in pretreatment, the reaction products of guanylate cyclase and phosphodiesterase were demonstrated at the cytoplasmic side of the disc membranes, which result is in good agreement with the biochemical estimations. Rapid freeze substitution enzyme histochemistry may be useful in detecting the precise locations of substances in living conditions.

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