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For the first time, the 31P nuclear magnetic resonance technique has been used to study the properties of isolated vacuoles of plant cells, namely the vacuolar pH and the inorganic phosphate content. Catharanthus roseus cells incubated for 15 hours on a culture medium enriched with 10 millimolar inorganic phosphate accumulated large amounts of inorganic phosphate in their vacuoles. Vacuolar phosphate ions were largely retained in the vacuoles when protoplasts were prepared from the cells and vacuoles isolated from the protoplasts. Vacuolar inorganic phosphate concentrations up to 150 millimolar were routinely obtained. Suspensions prepared with 2 to 3 x 106 vacuoles per milliliter from the enriched C. roseus cells have an intemal pH value of 5.50 ± 0.06 and a mean trans-tonoplast ApH of 1.56 ± 0.07. Reliable determinations of vacuolar and extemal pH could be made by using accumulation times as low as 2 minutes. These conditions are suitable to follow the kinetics of H exchanges at the tonoplast. The 31P nuclear magnetic resonance technique also offered the possibility of monitoring simultaneously the stability of the trans-tonoplast pH and phosphate gradients. Both appeared to be reasonably stable over several hours. The buffering capacity of the vacuolar sap around pH 5.5 has been estimated by several procedures to be 36 ± 2 microequivalents per milliliter per pH unit. The increase of the buffering capacity due to the accumulation of phosphate in the vacuoles is, in large part, compensated by a decrease of the intravacuolar malate content.

The vacuolar pH and the trans-tonoplast ApH modifications induced by the activity of the two proton pumps H+-ATPase and H -PPase and by the proton exchanges catalyzed by the Na+/H+ and Ca2+/H+ antiports at the tonoplast of isolated intact vacuoles prepared from Catharanthus roseus cells enriched in inorganic phosphate (Y Mathieu et al 1988 Plant Physiol [in press]) were measured using the 31P NMR technique. The H+-ATPase induced an intravacuolar acidification as large as 0.8 pH unit, building a trans-tonoplast ApH up to 2.2 pH units. The hydrolysis of the phosphorylated substrate and the vacuolar acidification were monitored simultaneously to estimate kinetically the apparent stoichiometry between the vectorial proton pumping and the hydrolytic activity of the H+-ATPase. A ratio of H+ translocated/ ATP hydrolyzed of 1.97 ± 0.06 (mean ± standard error) was calculated. Pyrophosphate-treated vacuoles were also acidified to a significant extent. The H+-PPase at 2 millimolar PPi displayed hydrolytic and vectorial activities comparable to those of the HATPase, building a steady state ApH of 2.1 pH units. Vacuoles incubated in the presence of 10 millimolar Na+ were alkalinized by 0.4 to 0.8 pH unit. It has been shown by using 23Na NMR that sodium uptake was coupled to the H+ efflux and occurred against rather large concentration gradients. For the first time, the activity of the Ca2+/H+ antiport has been measured on isolated intact vacuoles. Ca2+ uptake was strongly inhibited by NH4CI or gramicidin. mulation and anion retrieval (6). However, aside the relatively well studied tonoplast proton pumping ATPase (26, 28 and references therein), our knowledge of the other ionic exchanges involving fluxes of protons or proton-equivalents is more limited. A second proton pump, the H+-PPase, present on tonoplast-enriched microsomal vesicles, has been described several times as distinct from the H+-ATPase (12,25,31). Its hydrolytic activity has also been measured on isolated intact vacuoles of beet roots (32) and tulipa petals (31) but, in these few cases, the possible effect of PPi hydrolysis on the vacuolar pH has not been determined.The study of H+-coupled secondary transport of cations through the functioning of Na+/H+ or Ca2+/H' antiports has been restricted to tonoplast vesicles isolated from beet (8, 9) carrot (1 1), or oat (27) Figure 1 illustrates the evolution with time of 31P NMR spectra of a vacuolar suspension incubated with Mg-ATP. The extravacuolar and intravacuolar inorganic phosphate peaks were clearly distinct from the peaks corresponding to the three phosphate groups of ATP. As ATP was hydrolyzed, the two ADP peaks appeared along with a weak AMP signal. The main interest of using 31P NMR was that it allowed simultaneously (a) the measurement of ATP hydrolysis, (b) the measurement ofvacuolar pH modifications linked to ATP hydrolysis, and (c) the estimation of the apparent stoichiometry between vectorial H+-pumping and the hydrolytic activities of the H+-ATPase.The rate of ATP hydrolysis was measured from...

Vitamin B E starvation inIn severe anaemia that is due to B12 avitaminosis, the block of mitosis and the maturation of the erythrocyte line is followed by a diminution of red blood cells, while large cells with abnormal nuclei ~fmegaloblasts") appear in the blood and the bone marrow. These giant cells have a high RNA/DNA ratio (7,26). Defective DNA synthesis in the megaloblastic state has been reviewed (30,40). In vitamin B12-deprived Euglena cells, divisions slow down and then stop, while RNA and protein content, number of chloroplasts, and mitochondria continue to increase (14). For the study of biological and biochemical disorders induced by vitamin B12 deficiency, Euglena cells, whose alterations resemble those observed in the megaloblasts, are a very suitable model for observing where "DNA replication cycle" is decoupled from the "growth cycle" (36), as they are easy to cultivate and even to synchronize. In the recovering cells, after addition of vitamin B12, the DNA replication is completed, followed by cell divisions (9). Two relatively synchronous divisions occur immediately without a new G1 phase (15,32,41).In the model of the metabolic cell cycle block, biochemical analyses have given a mean DNA content of 1.8 or twice that of G1 cells (5,161, with no information on cell heterogeneity. In contrast, microcytofluorometry or flow cytometric analyses can provide data on DNA content of individual cells. These two types of analysis complement each other and can lead to a better understanding of the cell cycle arrest, in light of independent results concerning the dispersed structure of chromatin in starved giant cells (4,6,12), the modification of histones (101, and the nucleosomal organization in control and vitamin B12-starved chromatin (241, all of which could affect DNA stainability.The fluorochrome Hoechst 33258 for DNA labeling was chosen in present studies in order to avoid, as far as possible, nonstoichiometric DNA stainability owing to differences in the conformation of the chromatin (23).Initial studies that used microfluorometry contributed to a better knowledge of the material because individual cells could be observed, and so, for example, mitotic cells could be distinguished from cells in GB. Later, flow cytofluorometric measurements allowed analysis of large samples, and thus statistically more reliable results could be obtained. The data indicated that starved Euglena cells were distributed in the S and G2 phases. No cells were found either in the G1 phase or in the M phase. The unbalanced growth of vitamin B12-deficient cells was characterized by a block in the DNA replication and another block at the transition from G2 to M.The significance of cell arrest is discussed in relation to DNA replication. MATERIALS AM) METHODSControl Cells Euglena gracilis strain Z (Cambridge culture collection No. 1224-5D) were cultivated, either on a mineral

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