A method for the determination of ApH in chloroplasts which is based on the distribution of [Wlmethylamine between the osmotic water and the medium was developed. It was found that [14C]sorbitol penetrates only the nonosmotic space and can be used to determine the osmotic space.[14CJMethylamine was found to be somewhat concentrated in the chloroplasts in the dark but was extensively concentrated in the light, indicating a ApH of more than 2 units. Electrontransport cofactors increased this gradicnt while uncouplers decreased it.The maximal ApH was found when the medium p H was around 8.5, thus agreeing with the optimal p H for phosphorylation and electron transport. Comparison of the pH-dependence and the light-intensity-dependence of the extent of proton uptake and ApH indicates that the extent of proton uptake is essentially a measure of the chloroplasts buffering power but not of the d p H . The membrane potential across the chloroplast membrane was determined from the distribution of 36Cl and E6Rb and was found t o be negligibly small in the light.The demonstration of light-induced p H rise in chloroplast suspensions [ 1,2], is normally interpreted as indicating a n acidification of the compartment that is enclosed by the thylakoid membrane. This interpretation forms thc basis of the hypothesis that a proton gradient controls the energy-conversion process of photophosphorylation [2,3 etc.]. However, the actual determination of the internal p H has not been successful so far. It is not possible t o estimate the internal p H from the extent of proton disappearance, since the internal buffer capacity and the extent of possible surface binding are unknown. The application of the 5,5-dimethyl-2,4-oxazolidinadione distribution method for the determination of internal p H in whole cells [4] and mitochondria [ 5 ] was also tried for chloroplasts [Sl. It could not be expected to give reliable results since in the light the p H inside should be much lower and most of the dimethyloxazolidinadione would therefore be outside the thylakoid membranes. I n this case it would be technically impossible to determine the exact internal concentration of dirnethyloxazolidinadione.We have developed a method which is similar to the dimethyloxazolidinadione technique except for the use of amines instead of a weak acid. The distribution of amines between the osmotic compartment and the medium is determined and the d p H calculated assuming that the non-charged amine concentration is equal on both sides due to permeability of this form. This assumption is based on the results of numerous studies on the mechanism of amine transport in chloroplasts [7--91. It follows from the above assumption that when the nonprotonated amine is in equilibrium the following relation holds :Since the internal p H is expected to be lower than that of the medium the amine would be more concentrated in the pellet and this would allow the measurement of the concentration ratio. The p K of methylamine (10.6) is much higher than physiological p H values and th...
A new method for the determination of ApH in chloroplasts and related particles was developed. It is based on measurement, with a cationic electrode of the light-induced uptake of NH,+ in low external concentrations. This, together with a separate determination of the osmotic space by the use of [14C]sorbitol and 3H,0 allow the calculation of the internal NH,+ concentration, and the ApH. I n sub-chloroplast particles the light-induced A pH determined by this method was only somewhat lower than in chloroplasts. Furthermore, inhibition of phosphorylation by methylamine in these particles was parallel to that of the ApH.Two methods for the determination of ApH between the suspension medium and the thylakoid space in chloroplasts were described in detail [1-31. Both were based on determining the distribution of amines across the thylakoid space. I n this study another method, which is based on the same principle, is described. A cationic electrode or a specific ammonium electrode is utilized to determine the extent of the light-induced NH,+ uptake, a t low external NH,+ concentration, well below uncoupling concentrations. The calculation of the internal concentration is done as previously, assuming that the NH,+ is concentrated in the non-sorbitol space [ 1,3]. The underlying assumption is that the principle permeating form is NH, and thus This new method enables one to continuously monitor the ApH in the suspension medium without separation of the particles. It is however, limited by the selectivity of the available electrodes which a t present do not allow the use of potassium ion as a component of the incubation medium. METHODSChloroplasts were prepared as previously described [2] but washed once or twice with a cholinechloride (I00 mM), Tris tricinate (10 mM, pH 8.0) medium. The osmotic space was determined as described [2]. Sub-chloroplast particles were prepared Unusual Abbreviation. Tricine, N-tris(hydroxymethy1)-methylglycine.from digitonin-treated chloroplasts by differential centrifugation according t o the method of Nelson et al. [4]. The ApH determination by the use of fluorescence quenching of 9-aminoacridine was performed as described [3]. NH,+ uptake was measured with the aid of cationic electrodes. E.I.L. as well as a Beckman cationic electrode were used. Some experiments were performed with a new experimental ammonium-selective membrane electrode, kindly provided by Hydronautics Israel Ltd (Rehovot, Israel). A calomel electrode served as reference electrode and was connected to the medium by a choline chloride (100 mM), Tris-tricinate (10 mM, pH 8.0) liquid salt bridge. The signal was recorded through a Radiometer pH meter, model TTT1. Light was supplied by a 500 W projector lamp and filtered through either water or a 680nm Baird Atomic Interference filter (30 nm half-band width). All experiments were performed a t room temperature. Fig.1 shows the tracing of a typic1 experiment demonstrating light-induced NH,+ uptake. Aliquots of a standard solution of NH,C1 were added consecutively to the chloropl...
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