Inorganic pyrophosphatase and photosynthesis by isolated chloroplasts. I. Characterisation of chloroplast pyrophosphatase and its relation to the response to exogenous pyrophosphate
Department of Botany, The University, Sheffield, S10 2TN, Great Britain
Abstract:
1. 1. A soluble, alkaline, Mg2+-dependent inorganic pyrophosphatase (EC 3.6.1.1) has been isolated from the stroma of intact spinach and pea chloroplasts and purified some 100-fold. The enzyme has a high specificity for inorganic pyrophosphate and Mg2+, and exhibits maximal activity at pH 8.2–8.6. The enzyme shows allosteric characteristics with Mg2+ as activator and optimal rates are obtained with a ratio of Mg2+ to PPi of approximately 4 to 1. The enzyme is inhibited by anionic PPi and by its own reaction product, orthophosphate.
2. 2. If Mg2+ is excluded from the medium in which isolated chloroplasts are assayed, active photosynthetic oxygen evolution can still be observed. The addition of Pi, but not PPi, will then offset a phosphate deficiency. If external Mg2+ is present PPi will also offset a phosphate deficiency and in these circumstances the rapidity and nature of the response is related to the external pyrophosphatase activity.
3. 3. Evidence is presented that the chloroplast envelope is relatively impermeable to PPi and that the response to added PPi is due to external hydrolysis followed by entry of Pi to the chloroplast. These results have significance concerning proposed mechanisms for control of photosynthesis.