Histochemical Technique

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Abstract
Biochemists who study single cells were constrained by the lack of a general methodology of high time resolution and high measurement sensitivity for quantitatively assaying enzyme activities using natural substrates in solution. The methods described will remove this limitation. Nanogram tissue samples are dissected from frozen-dried tissue. The samples are extracted in microdroplets of assay cocktail. The enzyme activity, indicated fluorometrically by the oxidation/reduction of NAD(P), is followed in real time on a computer display. In the development of this method, several parameters required for optimization were evaluated, the most important of these evaluations, including numerous empirically derived relationships, are reported here and in supplemental material provided with reprints. With these methods, assays of pyruvate orthophosphate dikinase on samples enriched in bundlesheath cells and mesophyll cells of Flaveria brownii yielded the predictable results. Assays of this enzyme in guard cells dissected from Vicia faba leaflets gave results like those recently reported by another laboratory for protoplasts derived from these cells. The results of assays by this method and by enzymic cycling for NAD(P)triose-P dehydrogenase were comparable. Phosphoenolpyruvate carboxylase, the most extensively studied enzyme activity, was present at high levels in guard cells, which was demonstrated previously in other reports based on diverse assay approaches.