Absence of Acid Phosphatase from Chloroplasts of Spinach and Iris.

The presence of phosphatases in leaves is well known. The acid phosphatase of leaves appears to lack specificity with respect to hydrolyzable substrates (1, 6, 7, 8). Glycerophosphate has always been found to be an effective substrate (1, 5, 6, 7, 8). However, there is some evidence for the plurality of the acid phosphatase of leaves in that specific effects of activators and inhibitors have been found (1). The objective of the present study was to determine whether or not chloroplasts contain acid phosphatase. This investigation was prompted by YIN'S (8) inability to discern whether most of the acid phosphatase of the leaves of Iris and Lamium was concentrated in the chloroplasts themselves or in the surrounding cytoplasm at the interface with the chloroplasts. Freehand sections of leaves of Iris and market spinach, and fractionated macerates of spinach leaves were tested for acid phosphatase by means of Gomori's microchemical method (3). All samples were washed with 0.2% versene and with several changes of distilled water, then incubated for periods of up to three hours at 370 C in the medium containing the substrate a-glycerophosphate at pH 5.1. Almost no phosphatase activity was found in the freehand sections of Iris leaves. Less than 1% of the chlorenchyma cells showed activity, and this activity did not appear to be associated with the chloroplasts. In sections of spinach leaves the phosphatase was active in irregular patches of chlorenchyma. The precipitated lead sulphide was seen to occur in the extra-chloroplastic cytoplasm and at nuclei. There was very high activity on the crystals (druses) which occurred regularly in enlarged cells in the mesophyll. As was observed by GLICK and FIsCHER (2), some cell walls, especially those of tracheids and epidermal cells, gave a positive reaction. However, this reaction was not the result of enzymatic activity since it also appeared in sections that had been boiled for three minutes. Spinach leaves were macerated with a Waring Blendor in 0.2%o versene. After filtration through muslin, the mixture was centrifuged at 6000 x g. for 15 minutes. The green sediment (fraction I), containing the chloroplasts and various unidentified particles, was resuspended in distilled water. The supernatant (fraction II) was light yellow in color. Both fractions were saturated with ammonium sulphate and the resulting precipitates were