Alcohol-resistant phosphatase activity in chloroplasts

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Abstract

When isolated spinach chloroplasts were incubated with 32PO43− and subsequently treated with methanol, the formation of methyl phosphate was observed chromatographically. The ester is formed even without incubation. Other simple alcohols give the corresponding alkyl phosphates, the yields decreasing with increasing molecular weights. This formation of alkyl phosphates is due to a phosphatase in the chloroplasts which survives treatment with up to 80% alcohol even at room temperature for periods up to several days and stays active under these conditions. It catalyses the reaction: phosphate + alcohol ⇋ alkyl phosphate + water, to give, under appropriate conditions, an equilibrium which can be reached from both sides as well. The equilibrium is strictly dependent on the alcohol concentration and meets the condition ΔF = 0 in methanol concentrations below 50% by volume. In mixtures containing over 50% methanol, the turn-over is incomplete because of denaturation of the enzyme before it can reach the equilibrium. There is no high-energy phosphate donor involved in the reaction. Phosphatases of similarly high stability, giving the same reaction, are shown to exist in several other biological materials, e.g., blood. Thus, simple addition of cold alcohol, to make its concentration 80% of the final volume, cannot be considered as sufficient to kill every enzyme contained in samples of biochemical experiments.

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