Control of plastidic glycolipid synthesis and its relation to chlorophyll formation

Mechanisms restricting the accumulation of chloroplast glycolipids in achlorophyllous etiolated or heat-treated 70S ribosome-deficient rye leaves (Secale cereale L. cv “Halo”) and thereby coupling glycolipid formation to the availability of chlorophyll, were investigated by comparing [¹⁴C]acetate incorporation by leaf segments of different age and subsequent chase experiments. In green leaves [¹⁴C]acetate incorporation into all major glycerolipids increased with age. In etiolated leaves glycerolipid synthesis developed much more slowly. In light-grown, heat-bleached leaves [¹⁴C]acetate incorporation into glycolipids was high at the youngest stage but declined with age. In green leaves [¹⁴C]acetate incorporation into unesterified fatty acids and all major glycerolipids was immediately and strongly diminished after application of an inhibitor of chlorophyll synthesis, 4,6-dioxoheptanoic acid. The turnover of glyco- or phospholipids did not differ markedly in green, etiolated, or heat-bleached leaves. The total capacity of isolated ribosome-deficient plastids for fatty acid synthesis was not much lower than that of isolated chloroplasts. However, the main products synthesized from [¹⁴C]acetate by chloroplasts were unesterified fatty acids, phosphatidic acid, and diacylglycerol, while those produced by ribosome-deficient plastids were unesterified fatty acids, phosphatidic acid, and phosphatidylglycerol. Isolated heat-bleached plastids exhibited a strikingly lower galactosyltransferase activity than chloroplasts, suggesting that this reaction was rate-limiting, and lacked phosphatidate phosphatase activity.     

Solubilization of a Proline Dehydrogenase from Maize (Zea mays L.) Mitochondria

L-Proline is oxidized to pyrroline-5-carboxylic acid in intact plant mitochondria by a proline dehydrogenase (EC 1.4.3) that is bound to the matrix side of the inner mitochondrial membrane (TE Elthon, CR Stewart [1981] Plant Physiol 67: 780-784). This investigation reports the first solubilization of the L-proline dehydrogenase (PDH) from plant mitochondria. The supernatant from NP-40-treated etiolated shoot mitochondria of maize, Zea mays L., reduced iodonitrotetrazolium violet in a proline dependent manner. The pH optimum for this activity was 8. The apparent K_m for proline was 6.6 millimolar. When supplied with proline, this solubilized PDH activity also synthesized pyrroline-5-carboxylic acid. The PDH activity was inhibited in vitro by 300 millimolar potassium chloride but not by 300 millimolar potassium acetate. The PDH activity had a molecular mass that was greater than 150 kilodaltons. Mitochondria were prepared from etiolated shoots grown in 100% water-saturated vermiculite (control) and 16% water-saturated vermiculite (stress). The specific activity of solubilized PDH from the stress treatment was 11% of the same activity from the control treatment. Oxygen uptake in the presence of proline and ADP (state 3 proline oxidation) by mitochondria from the stress treatment was 25% of the same rate by mitochondria from the control treatment. Mitochondria were also prepared 16 hours after rewatering the seedlings growing in the stress treatment. Both the solubilized PDH specific activity and state 3 proline oxidation returned to the control levels. The specific activities of the NAD⁺-dependent pyrroline-5-carboxylic acid dehydrogenase and cytochrome c oxidase in the solubilized preparations were unaffected by these stress and recovery treatments. Oxygen uptake rates by intact mitochondria in the presence of ADP and NADH, succinate or malate-pyruvate were also unaffected by these treatments.     

Effects of glyoxylate on photosynthesis by intact chloroplasts

Because glyoxylate inhibits CO₂ fixation by intact chloroplasts and purified ribulose bisphosphate carboxylase/oxygenase, glyoxylate might be expected to exert some regulatory effect on photosynthesis. However, ribulose bisphosphate carboxylase activity and activation in intact chloroplasts from Spinacia oleracea L. leaves were not substantially inhibited by 10 millimolar glyoxylate. In the light, the ribulose bisphosphate pool decreased to half when 10 millimolar glyoxylate was present, whereas this pool doubled in the control. When 10 millimolar glyoxylate or formate was present during photosynthesis, the fructose bisphosphate pool in the chloroplasts doubled. Thus, glyoxylate appeared to inhibit the regeneration of ribulose bisphosphate, but not its utilization.

The fixation of CO₂ by intact chloroplasts was inhibited by salts of several weak acids, and the inhibition was more severe at pH 6.0 than at pH 8.0. At pH 6.0, glyoxylate inhibited CO₂ fixation by 50% at 50 micromolar, and glycolate caused 50% inhibition at 150 micromolar. This inhibition of CO₂ fixation seems to be a general effect of salts of weak acids.

Radioactive glyoxylate was reduced to glycolate by chloroplasts more rapidly in the light than in the dark. Glyoxylate reductase (NADP⁺) from intact chloroplast preparations had an apparent K_m (glyoxylate) of 140 micromolar and a V_max of 3 micromoles per minute per milligram chlorophyll.

     

Photosynthesis by isolated protoplasts, protoplast extracts, and chloroplasts of wheat: influence of orthophosphate, pyrophosphate, and adenylates

Protoplasts, protoplast extracts (intact chloroplasts plus extrachloroplastic material), and chloroplasts isolated from protoplasts of wheat (Triticum aestivum) have rates of photosynthesis as measured by light-dependent O₂ evolution of about 100 to 150 micromoles of O₂ per milligram of chlorophyll per hour at 20 C and saturating bicarbonate. The assay conditions sufficient for this activity were 0.4 molar sorbitol, 50 millimolar N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid KOH (pH 7.6), and 10 millimolar NaHCO₃ with protoplast, plus a requirement of 1 to 10 millimolar ethylenediaminetetraacetate (EDTA) and 0.2 to 0.5 millimolar inorganic orthophosphate (Pi) with protoplast extracts and chloroplasts. Protoplast extracts evolved approximately 6 micromoles of O₂ per milligram of chlorophyll before photosynthesis became largely dependent on exogenous Pi while photosynthesis by chloroplasts had a much stronger dependence on exogenous Pi from the outset.

Photosynthesis by chloroplasts from 6-day-old wheat plants under optimum levels of Pi was similar to that with the addition of 5 millimolar inorganic pyrophosphate (PPi) plus 0.2 millimolar adenosine-5′-diphosphate (ADP). Either PPi or ADP added separately inhibited photosynthesis. When chloroplasts were incubated in the dark for 2 to 6 minutes, photosynthesis was strongly inhibited by 5 millimolar PPi and this inhibiting was relieved by including adenosine-5′-triphosphate (ATP) or ADP (0.2 to 0.6 millimolar). Chloroplasts from 9-day-old wheat leaves were slightly less sensitive to inhibition by PPi and showed little or no inhibition by ADP.

Chloroplasts isolated from protoplasts and assayed with 0.3 millimolar Pi added before illumination have an induction time from less than 1 minute up to 16 minutes depending on the time of the assay after isolation and the components of the medium. In order to obtain maximum rates of photosynthesis and minimum induction time, NaHCO₃ and chelating agents, EDTA or PPi (+ATP), are required in the chloroplast isolation, resuspension and assay medium. With these inclusions in the isolation and resuspension medium the induction time decreased rapidly during the first 20 to 30 minutes storage of chloroplasts on ice. Requirements for isolating intact and photosynthetically functional chloroplasts from wheat protoplasts are discussed.

     

Steady state proline levels in salt-shocked barley leaves

Excised barley (Hordeum vulgare var Larker) leaves were treated with salt solutions or wilted. After the treatment period, the leaves were allowed to recover in a 50 millimolar sucrose and 1 millimolar glutamate solution, and proline, Na⁺, and K⁺ were measured at intervals. Na⁺ and K⁺ concentrations stayed at a constant high level after the salt treatments, and proline increased to a steady state concentration in response. The relationship between the maximum rate of proline accumulation and the Na⁺ concentration reached in each experiment was linear. The final steady state proline concentration reached was also directly proportional to the Na⁺ concentration. For a given Na⁺ concentration in the leaves, the steady state proline level was greater when 410 millimolar NaCl was added to the leaves than when 205 millimolar NaCl was added. These results are consistent with proline acting as a compatible cytoplasmic solute, balancing an accumulation of salts outside of the cytoplasm.

In contrast to the proline levels in salt-shocked leaves, the concentrations in wilted leaves decreased to near control levels within 24 hours of relief of stress.

     

Properties of the Isolated Intact Chloroplast at Cytoplasmic K Concentrations : I. Light-Induced Cation Uptake into Intact Chloroplasts is Driven by an Electrical Potential Difference

Photosynthesis, stroma-pH, and internal K⁺ and Cl⁻ concentrations of isolated intact chloroplasts from Spinacia oleracea, as well as ion (K⁺, H⁺, Cl⁻) movements across the envelope, were measured over a wide range of external KCl concentrations (1-100 millimolar).

Isolated intact chloroplasts are a Donnan system which accumulates cations (K⁺ or added Tetraphenylphosphonium⁺) and excludes anions (Cl⁻) at low ionic strength of the medium. The internally negative dark potential becomes still more negative in the light as estimated by Tetraphenylphosphonium⁺ distribution. At 100 millimolar external KCl, potentials both in the light and in the dark and also the light-induced uptake of K⁺ or Na⁺ and the release of protons all become very small. Light-induced K⁺ uptake is not abolished by valinomycin suggesting that the K⁺ uptake is not primarily active. Intact chloroplasts contain higher K⁺ concentrations (112-157 millimolar) than chloroplasts isolated in standard media. Photosynthetic activity of intact chloroplasts is higher at 100 millimolar external KCl than at 5 to 25 millimolar. The pH optimum of CO₂ fixation at high K⁺ concentrations is broadened towards low pH values. This can be correlated with the observation that high external KCl concentrations at a constant pH of the suspending medium produce an increase of stroma-pH both in the light and in the dark. These results demonstrate a requirement of high external concentrations of monovalent cations for CO₂ fixation in intact chloroplasts.

     

Protection of Pyruvate,Pi Dikinase from Maize against Cold Lability by Compatible Solutes

Most C₄ species are chilling sensitive and certain enzymes like pyruvate,Pi dikinase of the C₄ pathway are also cold labile. The ability of cations and compatible solutes to protect maize (Zea mays) dikinase against cold lability was examined. The enzyme in desalted extracts at pH 8 from preilluminated leaves could be protected against cold lability (at 0°C) by the divalent cations Mn²⁺, Mg²⁺, and Ca²⁺. There was substantial protection by sulfate based salts but little protection by chloride based salts of potassium or ammonium (concentration 250 millimolar). The degree of protection against cold lability under limiting MgCl₂ (5 millimolar) was pH sensitive (maximum protection at pH 8), but independent of ionic strength (up to 250 millimolar by addition of KCl). In catalysis Mg²⁺ is required and Mn²⁺ could not substitute as a cofactor. Several compatible solutes reduced or prevented the cold inactivation of dikinase (in desalted extracts and the partially purified enzyme), including glycerol, proline, glycinebetaine and trimethylamine-N-oxide (TMAO). TMAO and Mg²⁺ had an additive effect in protecting dikinase against cold inactivation. TMAO could largely substitute for the divalent cation and addition of TMAO during cold treatment prevented further inactivation. Cold inactivation was partially reversed by incubation at room temperature; with addition of TMAO reversal was complete. The temperature dependence of inactivation at pH 8 and 3 millimolar MgCl₂ was evaluated by incubation at 2 to 17°C for 45 minutes, followed by assay at room temperature. At preincubation temperatures below 11°C there was a progressive inactivation which could be prevented by TMAO (450 millimolar). The results are discussed relative to possible effects of the solutes on the quaternary structure of this enzyme, which is known to dissociate at low temperatures.     

Glucose 6-phosphate dehydrogenase isozymes of maize leaves : some comparative properties

Two different forms of glucose 6-phosphate dehydrogenase (EC 1.1.1.49) have been purified from etiolated and green leaves, respectively, of 6-day maize (Zea mays L. cv Fronica) seedlings. The procedure includes an ammonium sulfate step, an ion exchange chromatography, and a second gel filtration in Sephadex G-200 in the presence of NADP⁺ to take advantage of the corresponding molecular weight increase of the enzyme. The isozyme from etiolated leaves is more stable and has been purified up to 200-fold. Subunit molecular weight, measured by sodium dodecyl sulfate-gel electrophoresis, is 54,000. The active protein, under most conditions, has a molecular weight 114,000, which doubles to molecular weight 209,000 in the presence of NADP⁺. The association behavior of enzyme from green leaves is similar, and the molecular weight of the catalytically active protein is also similar to the form of etiolated leaves.

Glucose 6-phosphate dehydrogenase of dark-grown maize leaves isoelectric point (pI) 4.3 is replaced by a form with pI 4.9 during greening. The isozymes show some differences in their kinetic properties, K_m of NADP⁺ being 2.5-fold higher for pI 4.3 form. Free ATP (K_m = 0.64 millimolar) and ADP (K_m = 1.13 millimolar) act as competitive inhibitors with respect to NADP⁺ in pI 4.3 isozyme, and both behave as less effective inhibitors with pI 4.9 isozyme. Magnesium ions abolish the inhibition.

     

Ion Homeostasis in Chloroplasts under Salinity and Mineral Deficiency: II. Solute Distribution between Chloroplasts and Extrachloroplastic Space under Excess or Deficiency of Sulfate, Phosphate, or Magnesium

Spinach (Spinacia oleracea var “Yates”) plants grown hydroponically were exposed to an excess or deficiency of various mineral ions. Solutes were measured in leaf extracts and in isolated intact chloroplasts. Under phosphate (120 millimoles per liter NaH₂ PO₄), sulfate (200 millimolar per liter (Na₂ SO₄), or magnesium excess (150 millimolar per liter MgCl₂), concentrations of these ions in leaf extracts increased, but in chloroplasts, concentrations of all ions remained constant. Concentrations of quarternary ammonium compounds in chloroplasts increased. Under mild phosphate or magnesium deficiency, concentrations of these ions decreased in chloroplasts less than in whole leaf extracts. Under severe sulfate deficiency causing chlorosis in younger leaves, sulfate concentrations in chloroplasts remained even unchanged, despite a drastic decrease of sulfate concentrations both in green and in chlorotic leaves. Together with results from a companion study (G Schröppel-Meier, WM Kaiser 1988 Plant Physiol 87: 822-827) our data demonstrate that leaf cells are able to keep the concentrations of several mineral ions rather constant in metabolically active compartments even at extremely large variations of ion concentrations in the culture solution and in the leaves.     

Properties of Ornithine Carbamoyltransferase from Pisum sativum L

Some properties of ornithine carbamoyltransferase from chloroplasts isolated from leaves of Pisum sativum L. (cv Marzia) were compared with those of the enzyme partially purified (316-fold) from shoots of seedlings after 3 weeks of cultivation.

Both preparations showed a pH optimum at pH 8.3 and had the same affinity to ornithine (K_m = 1.2 millimolar) as well as to carbamoyl phosphate (K_m = 0.2 millimolar). The approximate molecular weight determined by gel sieving was 77,600.

A desalted ammonium sulfate precipitate from 14-day seedlings (inclusive roots and senescing cotyledons) was applied on a column of anion exchanger. The elution pattern showed one peak of ornithine carbamoyl-transferase activity. This elution pattern was the same as observed for the enzyme from chloroplasts.

The results suggest the presence of one form of ornithine carbamoyl-transferase in pea seedlings.

     

Effects of the Proline Analog l-Thiazolidine-4-carboxylic Acid on Proline Metabolism

The effect of various proline analogs on proline oxidation in mitochondria isolated from etiolated barley (Hordeum vulgare) shoots was investigated. Of the analogs tested, only l-thiazolidine-4-carboxylic acid (T4C) was an effective inhibitor. T4C (1 millimolar) inhibited proline (10 millimolar) -dependent 0₂ uptake an average of 67%. T4C was also oxidized to some degree (12.9 nanoatoms oxygen per minute per milligram protein for 10 millimolar). The effect of T4C on the oxidation of other mitochondrial substrates was also tested. T4C inhibited ¹-pyrrolidine-5-carboxylic acid-dependent oxygen uptake slightly (13%), the oxidation of malate plus pyruvate even less (6%), and stimulated the oxidation of succinate (+11%), exogenous NADH (+19%), and citrate (+20%). Thus, inhibition by T4C in mitochondria is relatively specific to proline oxidation. T4C was found to inhibit proline dehydrogenase and not the transport of proline into the matrix.     

Characteristics of light-dependent inorganic carbon uptake by isolated spinach chloroplasts

The light-dependent accumulation of radioactively labeled inorganic carbon in isolated spinach (Spinacia oleracea L.) chloroplasts was determined by silicone oil filtering centrifugation. Intact chloroplasts, dark-incubated 60 seconds at pH 7.6 and 23°C with 0.5 millimolar sodium bicarbonate, contained 0.5 to 1.0 millimolar internal inorganic carbon. The stromal pool of inorganic carbon increased 5- to 7-fold after 2 to 3 minutes of light. The saturated internal bicarbonate concentration of illuminated spinach chloroplasts was 10- to 20-fold greater than that of the external medium. This ratio decreased at lower temperatures and with increasing external bicarbonate. Over one-half the inorganic carbon found in intact spinach chloroplasts after 2 minutes of light was retained during a subsequent 3-minute dark incubation at 5°C. Calculations of light-induced stromal alkalization based on the uptake of radioactively labeled bicarbonate were 0.4 to 0.5 pH units less than measurements performed with [¹⁴C]dimethyloxazolidine-dione. About one-third of the binding sites on the enzyme ribulose 1,5-bisphosphate carboxylase were radiolabeled when the enzyme was activated in situ and ¹⁴CO₂ bound to the activator site was trapped in the presence of carboxypentitol bisphosphates. Deleting orthophosphate from the incubation medium eliminated inorganic carbon accumulation in the stroma. Thus, bicarbonate ion distribution across the chloroplast envelope was not strictly pH dependent as predicted by the Henderson-Hasselbach formula. This finding is potentially explained by the presence of bound CO₂ in the chloroplast.     

Partial Purification and Characterization of Uracil-DNA Glycosylase Activity from Chloroplasts of Zea mays Seedlings

A uracil-DNA glycosylase activity has been purified about 750-fold from the chloroplasts of light-grown Zea mays seedlings. This report represents the first direct demonstration of a DNA-glycosylase repair activity in chloroplasts. The activity, in part, was associated with a chloroplast Triton X-100 sensitive membrane. Its apparent K_m was 1.0 micromolar for a poly(dA-dT/U) substrate, and its molecular weight, as determined by gel filtration, was 18,000. The enzyme exhibited optimal activity at pH 7.0 with an atypically narrow pH tolerance. Activity was inhibited greater than 60% by 10 millimolar NaCl, 5 millimolar MgCl₂, or 5 millimolar EDTA. Enzyme activity was inhibited 80% by 10 millimolar N-ethylmaleimide, a sulfhydryl group-blocking agent. The activity removed uracil more rapidly from single-stranded DNA than from double-stranded DNA. With this report, uracil-DNA glycosylase activity has now been attributed to all three DNA-containing organelles of eucaryotic cells.     

Elevated Accumulation of Proline in NaCl-Adapted Tobacco Cells Is Not Due to Altered Delta-Pyrroline-5-Carboxylate Reductase

Tobacco (Nicotiana tabacum L. var Wisconsin 38) cells that are adapted to 428 millimolar NaCl accumulate proline mainly due to increased synthesis from glutamate. These cells were used to evaluate the possible role of Δ¹-pyrroline-5-carboxylate reductase in the regulation of proline biosynthesis. No increase in the specific activity of Δ¹-pyrroline-5-carboxylate reductase in crude extracts throughout the growth cycle was observed in NaCl-adapted cells compared to unadapted cells. The enzyme from both cell types was purified extensively. On the basis of affinity for the substrates NADPH, NADH, and Δ¹-pyrroline-5-carboxylate, pH profiles, chromatographic behavior during purification, and electrophoretic mobility of the native enzyme, the activities of the enzyme from the two sources were similar. These data suggest that the NaCl-dependent regulation of proline synthesis in tobacco cells does not involve induction of pyrroline-5-carboxylate isozymes or changes in its kinetic properties.     

Subcellular Location of NADP-Isocitrate Dehydrogenase in Pisum sativum Leaves

The subcellular location of NADP⁺-isocitrate dehydrogenase was investigated by preparing protoplasts from leaves of pea seedlings. Washed protoplasts were gently lysed and the whole lysate separated on sucrose gradients by a rate-zonal centrifugation. Organelles were located by marker enzymes and chlorophyll analysis. Most of the NADP⁺-isocitrate dehydrogenase was in the soluble fraction. About 10% of the NADP⁺-isocitrate dehydrogenase was present in the chloroplasts as a partially latent enzyme. Less than 1% of the activity was found associated with the peroxisome fraction. NADP⁺-isocitrate dehydrogenase was partially characterized from highly purified chloroplasts isolated from shoot homogenates. The enzyme exhibited apparent K_m values of 11 micromolar (NADP⁺), 35 micromolar (isocitrate), 78 micromolar (Mn²⁺), 0.3 millimolar (Mg²⁺) and showed optimum activity at pH 8 to 8.5 with Mn²⁺ and 8.8 to 9.2 with Mg²⁺. The NADP⁺-isocitrate dehydrogenase activity previously claimed in the peroxisomes by other workers is probably due to isolation procedures and/or nonspecific association. The NADP⁺-isocitrate dehydrogenase activity in the chloroplasts might help supply α-ketoglutarate for glutamate synthase action.     

Ion Relations of Symplastic and Apoplastic Space in Leaves from Spinacia oleracea L. and Pisum sativum L. under Salinity

Salt tolerant spinach (Spinacia oleracea) and salt sensitive pea (Pisum sativum) plants were exposed to mild salinity under identical growth conditions. In order to compare the ability of the two species for extra- and intracellular solute compartmentation in leaves, various solutes were determined in intercellular washing fluids and in aqueously isolated intact chloroplasts. In pea plants exposed to 100 millimolar NaCl for 14 days, apoplastic salt concentrations in leaflets increased continuously with time up to 204 (Cl⁻) and 87 millimolar (Na⁺), whereas the two ions reached a steady concentration of only 13 and 7 millimolar, respectively, in spinach leaves. In isolated intact chloroplasts from both species, sodium concentrations were not much different, but chloride concentrations were significantly higher in pea than in spinach. Together with data from whole leaf extracts, these measurements permitted an estimation of apoplastic, cytoplasmic, and vacuolar solute concentrations. Sodium and chloride concentration gradients across the tonoplast were rather similar in both species, but spinach was able to maintain much steeper sodium gradients across the plasmamembrane compared with peas. Between day 12 and day 17, concentrations of other inorganic ions in the pea leaf apoplast increased abruptly, indicating the onset of cell disintegration. It is concluded that the differential salt sensitivity of pea and spinach cannot be traced back to a single plant performance. Major differences appear to be the inability of pea to control salt accumulation in the shoot, to maintain steep ion gradients across the leaf cell plasmalemma, and to synthesize compatible solutes. Perhaps less important is a lower selectivity of pea for K⁺/Na⁺ and NO₃⁻/Cl⁻ uptake by roots.     

Localization of ferredoxin isoproteins in mesophyll and bundle sheath cells in maize leaf

Four ferredoxin isoproteins were identified in the C₄ plant Zea mays L. by analysis of extracts from leaves, mesocotyls, and roots of the young seedlings. The relative amounts of the isoproteins isolated from the photosynthetic and nonphotosynthetic organs were different. All the isoproteins were present in the leaves of green and etiolated plants, whereas two out of the four isoproteins were not detected in the roots or in the mesocotyls. During the greening of etiolated seedlings, the level of the two isoproteins unique to the leaf increased markedly. Analysis of the cellular and subcellular distribution of the two major leaf isoproteins showed that one isoprotein was present in the chloroplasts of both mesophyll and bundle sheath cells, whereas the other was only found in the chloroplasts of bundle sheath cells. This is the first report of the cell-specific expression of ferredoxin isoproteins in the leaves of a C₄ plant.     

Inorganic Carbon Accumulation and Photosynthesis in a Blue-green Alga as a Function of External pH

The blue-green alga Coccochloris peniocystis photosynthesizes optimally over the pH range of 7.0 to 10.0, but the O₂-evolution rate is inhibited below pH 7.0 and ceases below pH 5.25. Measurement of the inorganic carbon pool in this alga in the light, using the silicone-fluid filtration technique demonstrated that the rate of accumulation of dissolved inorganic carbon remained relatively constant over a wide pH range. At external dissolved inorganic carbon concentrations of 0.56 to 0.89 millimolar the internal concentration after 30 seconds illumination was greater than 3.5 millimolar over the entire pH range. Intracellular pH measured in the light using [¹⁴C]5,5-dimethyloxazolidine-2,4-dione and [¹⁴C]methylamine dropped from pH 7.6 at an external pH of 7.0 to pH 6.6 at an external pH of 5.25. Above an external pH of 7.0 the intracellular pH rose gradually to pH 7.9 at an external pH 10.0. Ribulose-1,5-bisphosphate carboxylase activity of cell-free algal extracts exhibited optimal activity at pH 7.5 to 7.8 but was inactive below pH 6.5. It is suggested that the inability of Coccochloris to maintain its intracellular pH when in an acidic environment restricts its photosynthetic capacity by a direct pH effect on the principal CO₂ fixing enzyme.     

Studies on H-Translocating ATPases in Plants of Varying Resistance to Salinity : I. Salinity during Growth Modulates the Proton Pump in the Halophyte Atriplex nummularia

Membrane vesicles were isolated from the roots of the halophyte Atriplex nummularia Lindl. H⁺-translocating Mg²⁺-ATPase activity was manifested by the establishment of a positive membrane potential (measured as SCN⁻ accumulation); and also by the establishment of a transmembrane pH gradient (measured by quinacrine fluorescence quenching). H⁺-translocation was highly specific to ATP and was stable to oligomycin. Growing the plants in the presence of 400 millimolar NaCl doubled the proton-translocating activity per milligram of membrane protein and otherwise modulated it in the following ways. First, the flat pH profile observed in non-salt-grown plants was transformed to one showing a peak at about pH 6.2. Second, the lag effect observed at low ATP concentration in curves relating SCN⁻ accumulation to ATP concentration was abolished; the concave curvature shown in the double reciprocal plot was diminished. Third, sensitivity to K-2 (N-morpholino)ethanesulfonic acid stimulation was shown in salt-grown plants (about 40% stimulation) but was absent in non-salt-grown plants. Fourth, the KCl concentration bringing about 50% dissipation of ATP-dependent SCN⁻ accumulation was 20 millimolar for salt-grown plants and 50 millimolar for non-salt-grown plants. Vanadate sensitivity was shown in both cases. No clear NO₃⁻ inhibition was observed.     

Intracellular localization of serine acetyltransferase in spinach leaves

Intact chloroplasts isolated from spinach leaves by a combination of differential and Percoll density gradient centrifugation and free of mitochondrial and peroxisomal contamination contained about 35% of the total leaf serine acetyltransferase (EC 2.3.1.30) activity. No appreciable activity of the enzyme could be detected in the gradient fractions containing broken chloroplasts, mitochondria, and peroxisomes. L-cysteine added to the incubation mixture at 1 mM almost completely inhibited serine acetyltransferase activity, both of leaf and chloroplast extracts. D-cysteine was much less inhibitory. L-cystine up to 5 mM and O-acetyl-L-serine up to 10 mM had no effect on the enzyme activity. When measured at pH 8.4, the enzyme extracted from the leaves had a K _m for L-serine of 2.4, the enzyme from the chloroplasts a K _m of 2.8 mM.Abbreviations NAS N-acetyl-L-serine - NADP-GPD NADP-dependent glyceraldehyde-3-phosphate dehydrogenase - OAS O-acetyl-L-serine - OASSase O-acetyl-L-serine sulfhydrylase - 3-PGA D-3-phosphoglycerate - SATase serine acetyltransferase