The pH in the cytoplasmic and thylakoid spaces of the blue-green alga, Anacystis nidulans, has been determined in the light and in the dark by uptake of 5,5-dimethyloxazolidine-2,4-dione and methylamine into the sucrose-impermeable 3H-H20 space, as measured by silicon layer filtering centrifugation.Illumination causes an linization in the cytoplasm which is accompanied by an The principle of the method of pH measurement, utilizing the distribution of DMO and methylamine inside and outside the cell, has been described elsewhere (3). The intracellular space was defined as the sucrose-impermeable 3H-H20 space (2). The thylakoid space was assumed to be 7% of the intracellular space; this value is based on a planimetric estimation by Allen (1). There are two factors that complicate the pH measurement with Anacystis. First, it is possible that methylamine could undergo metabolic changes. For this reason, the substance was not incubated for longer than 2 min. During this period of time, any errors due to metabolic conversion were within the standard deviation of the method. Any change of the pH or redistribution of methylamine between inside and outside the cell caused by light-dark alterations occurs within 40 sec. Secondly, the accuracy of the absolute pH values may be affected to a certain extent by the influence of the several layers of the cell wall on the sucrose space.A linear relationship between the 5,5-dimethyloxazolidine-2,4-dione anion concentration in the medium and in the large sucrose-impermeable 3H-H20 space was noted over a large concentration range (0.1-6.0 mm 5,5-dimethyloxazolidine-2,4-dione); that is, the results of the pH measurement in the cytoplasm are independent of the DMO concentration. With methylamine, such a linearity exists only when the methylamine concentration is below 70 AM, almost certainly as a result of the uncoupling effect of methylamine at higher concentrations. The concentration of methylamine employed for pH measurement in the thylakoid space was, therefore, 70 A.M. RESULTS AND DISCUSSIONValues for the pH in the cytoplasmic and in the thylakoid space in the light and in the dark are given in Table I

Investigations of the energy-dependent accumulation of orthophosphate by the blue-green alga Anacystis nidulans have established: 1. The transport through the cell membrane is the rate-limiting step in the incorporation of phosphate.-2. This transport is facilitated by a "carrier" that can be activated by Ca(2+) and Mg(2+) and inhibited by EDTA.-3. The activation of the carrier in the light is associated with changes of the cytoplasmic Mg(2+) content.-4. Intracellular phosphate is shown to be present in bound form.-5. The energy-dependent accumulation of orthophosphate within the cell depends strictly on the cytoplasmic pH and not on the energy conversion at the thylakoid membrane which is responsible for the energy supply. The cytoplasmic pH is different in the light, in the dark, and in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). Orthophosphate accumulation can most readily be explained in terms of a pH dependent precipitation into a complex with bivalent cations rather than by an active transport against a concentration gradient.

ZusammenfassungDas von der Blaualge Anacystis nidulans aufgenommene Phosphat wurde in den anorganischen und in den metabolisierten Anteil durch Extraktion mit Isobutanol‐Benzol in Gegenwart von Ammoniummolybdat aufgetrennt. Bezog man in Anwesenheit von Calciumionen die Menge an aufgenommenem intrazellulären Phosphat auf das Cytoplasmavolumen, so errechnete sich in den meisten Fällen eine oft wesentlich höhere Konzentration als im Medium. In Abwesenheit von Calciumionen jedoch hört der Einstrom von Phosphat auf, wenn die Konzentration im Cytoplasma die Konzentration im Medium erreicht hat. Daran schließt sich ein lichtabhängiger Einbau in organische Phosphorverbindungen an, der mit dem pH‐Gradienten über die Thylakoidmembran korreliert werden kann. Die vorliegenden Daten zeigen, daß die Akkumulation von anorganischem Phosphat, die in Gegenwart von Calciumionen auftritt, keine notwendige Voraussetzung für die nachfolgende Photophosphorylierung bildet und vermutlich durch Präzipitation von Calciumphosphat in der Zelle bewirkt wird.

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