NaH'4CO3 (105 Cpm) to 1.3 ml of the enzyme preparation. At intervals, 0.4-ml samples were removed and acidified with 0.1 ml of 6 N acetic acid. Controls consisted of a zero time sample that was acidified immediately upon mixing the reaction components, and a reaction mixture lacking ribulose-1, 5-diP which was acidified at the end of the experimental period. The same enzyme preparation procedure was used for phosphopyruvate carboxylase except that dithioerythritol was sometimes substituted for reduced glutathione to provide an alternative test for sulfhydryl protection. The assay system for this enzyme, after Slack and Hatch (25), contained 1.55 ,umoles of phosphopyruvate and 15 ,umoles of sodium glutamate in substitution for the ribulose-1,5-diP used above. Controls were the same. For the assay of glycolate oxidase activity, leaves were homogenized in 0.067 M phosphate buffer (pH 7.8) and filtered through four layers of 60 mesh cheesecloth. This homogenate was used directly in a polarographic assay (10) with the Clark electrode. The reaction was initiated by adding 10 .moles of sodium glycolate in 0.2 ml of the phosphate buffer to 2.8 ml of the enzyme preparation. Controls were samples to which no glycolate was added. Addition of the enzyme cofactor, flavin mononucleotide, was not found to be stimulatory, and this was omitted from the assay system. Reagents were from Sigma.For "4CO0 assimilation experiments, leaves were preilluminated in 2 X 106 ergs cm-2 sec-l of light with their base in distilled water or a glycolate oxidase inhibitor, 0.01 M a-hydroxypyridinemethanesulfonic acid (Aldrich), for 30 min prior to release of "4CO2 by the acidification of a bicarbonate solution. Carbon dioxide concentration was 370 ,ul liter-' after addition of bicarbonate, to eliminate artifacts arising from high CO2 concentrations. Following 10 min of photosynthesis with water or 20 min with the inhibitor, the leaves were killed with boiling 80%-ethanol. In the controls, 1.8 AC of "CO, were used. In the experiments with inhibitor, 2.02 ,C of "CO2 were used. The different time periods and specific radioactivities of "CO2 were used to assure that approximately the same amount of radioactivity would be recovered from chromatograms of both treatments without overloading with plant extracts. Total cpm recovered from chromatograms per g of leaf extracted were 43,200 + 21,600 (0.95 confidence interval) for the water controls and 53,600 + 8,300 for experiments in inhibitor. For
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