Photosynthesis and Activation of Ribulose Bisphosphate Carboxylase in Wheat Seedlings : Regulation by CO(2) and O(2)

Photosynthetic carbon assimilation in plants is regulated by activity of the ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase. Although the carboxylase requires CO₂ to activate the enzyme, changes in CO₂ between 100 and 1,400 microliters per liter did not cause changes in activation of the leaf carboxylase in light. With these CO₂ levels and 21% O₂ or 1% or less O₂, the levels of ribulose bisphosphate were high and not limiting for CO₂ fixation. With high leaf ribulose bisphosphate, the K_act(CO₂) of the carboxylase must be lower than in dark, where RuBP is quite low in leaves. When leaves were illuminated in the absence of CO₂ and O₂, activation of the carboxylase dropped to zero while RuBP levels approached the binding site concentration of the carboxylase, probably by forming the inactive enzyme-RuBP complex.

The mechanism for changing activation of the RuBP carboxylase in the light involves not only Mg²⁺ and pH changes in the chloroplast stroma, but also the effects of binding RuBP to the enzyme. In light when RuBP is greater than the binding site concentration of the carboxylase, Mg²⁺ and pH most likely determine the ratio of inactive enzyme-RuBP to active enzyme-CO₂-Mg²⁺-RuBP forms. Higher irradiances favor more optimal Mg²⁺ and pH, with greater activation of the carboxylase and increased photosynthesis.

     

Modulation of proton efflux from chloroplasts in the light by external pH

The effects of external pH on the efflux of protons from illuminated spinach chloroplasts have been studied by monitoring the rates of proton-pumping electron transport under a variety of steady-state conditions. Phosphorylation-coupled proton efflux through the ATP synthase (CF₀-CF₁), determined from the rates of ATP formation and that portion of the total electron transport attributable to phosphorylation, is strongly dependent upon pH over the range 6–9, with little activity below pH 7 and half-maximal activity at pH ≈ 7.6. Noncoupled proton efflux through the ATP synthase, determined in the absence of ADP and phosphate, was also strongly pH sensitive, with little activity below pH 7.5 and half-maximal activity at pH ～- 7.9. When proton efflux via CF₀ was prevented by triphenyltin, the rate of passive proton leakage across the membrane was very low and practically insensitive to external pH indicating that the major pH-sensitive pathway(s) for proton efflux in the light involves CF₀ · CF₁. Modification of CF₁ sulfhydryls by Ag⁺ resulted in an apparent increase in proton efflux via the normally coupled CF₀ · CF₁ pathway (half-maximal activity = pH 7.6), whereas modification by Hg²⁺ resulted in an apparent increase in proton efflux via the noncoupled CF₀ · CF₁ pathway (half-maximal activity = pH 7.9).     

Inhibition of the transthylakoid gradient of electrochemical proton potential by the local anesthetic dibucaine

The effects of the local anesthetic dibucaine on coupling between electron transport and ATP synthesis-hydrolysis by the coupling-factor complex (CF₀CF₁ ATPase) were investigated in thylakoid membranes from Spinacia oleracea L. cv. Monatol. Evidence is presented that inhibition of ATP synthesis was produced by a specific uncoupling mechanism which was based on dibucaine-membrane surface interactions rather than on the interaction of dibucaine with the ATPase complex. Dibucaine reduced the osmotic space of thylakoid vesicles. At low pH of the medium it stimulated ATP hydrolysis beyond the rates obtained with optimum concentrations of ‘classical’ uncouplers. After addition of dibucaine, there was displacement of membrane-bound Mg²⁺ and strong thylakoid stacking in the presence of only low Mg²⁺ concentrations. Inhibition of ATP synthesis and transmembrane pH gradient increased with medium pH. Hydrolysis of ATP by isolated CF₁ and the CF₀CF₁ complex was only slightly affected by dibucaine. The data are discussed assuming the involvement of localized proton channels on the membrane surface in protonic coupling of electron transport and ATP synthesis. A hypothesis for the mechanisms of action of local anesthetics at the thylakoid membrane is presented.     

Proton efflux through the chloroplast ATP synthase (CF0 · CF1) in the presence of sulfhydryl-modifying agents

The rate of photosynthetic electron transport measured in the absence of ADP and P_i is stimulated by low levels of Hg²⁺ or Ag⁺ (50% stimulation ≈ 3 Hg²⁺ or 6 Ag⁺/100 chlorophyll) to a plateau equal to the transport rate under normal phosphorylating conditions (i.e. +ADP, +P_i). Chloroplasts pretreated in the light under energizing conditions with N-ethylmaleimide show a similar stimulation of non-phosphorylating electron transport. The stimulations of non-phosphorylating electron transport by Hg²⁺, Ag⁺ and N-ethylmaleimide are reversed by the CF₁ inhibitor phlorizin, the CF₀ inhibitor triphenyltin chloride, and can be further stimulated by uncouplers such as methylamine. The Hg²⁺ and N-ethylmaleimide stimulations, but not the Ag⁺ stimulation, are completely reversed by low levels of ADP (2 μM), ATP (2 μM), and P_i (400 μM). Ag⁺, which is a potent inhibitor of ATP synthesis, has little or no effect upon phosphorylating electron transport (+ADP, +P_i). Concomitant with the stimulations of non-phosphorylating electron transport by Hg²⁺, Ag⁺ and ADP + P_i, there is a decrease in the level of membrane energization (as measured by atebrin fluorescence quenching) which is reversed when the CF₀ channel is blocked by triphenyltin. These results suggest that modification of critical CF₁ sulfhydryl residues by Hg²⁺, Ag⁺ or N-ethylmaleimide leads to the loss of intra-enzyme coupling between the transmembrane protontransferring and the ATP synthesis activities of the CF₀-CF₁ ATP synthase complex.     

Evidence for solvent-induced conformational changes of the soluble Dunaliella chloroplast coupling factor 1 (CF1)

The ATPase activity of the chloroplast coupling factor 1 (CF₁) isolated from the green alga Dunaliella is completely latent. A brief heat treatment irreversibly induces a Ca²⁺ -dependent activity. The Ca²⁺ dependent ATPase activity can be reversibly inhibited by ethanol, which changes the divalent cation dependency from Ca²⁺ to Mg²⁺. Both the Ca²⁺ -dependent and Mg²⁺ -dependent ATPase activities of heat-treated Dunaliella CF₁ are inhibited by monospecific antisera directed against Chlamydomonas reinhardi CF₁. However, when assayed under identical conditions, the Ca²⁺ -dependent ATPase activity is significantly more sensitive to inhibition by the antisera than is the Mg²⁺ -dependent activity. These data are interpreted as indicating that soluble Dunaliella CF₁ can exist in a variety of conformations, at least one of which catalyzes a Ca²⁺ -dependent ATPase and two or more of which catalyze an Mg²⁺ -dependent ATPase.     

Purification and Characterization of a Glycerol-Resistant CF(0)-CF(1) and CF(1)-ATPase from the Halotolerant Alga Dunaliella bardawil

The isolation of the chloroplast ATP synthase complex (CF₀-CF₁) and of CF₁ from Dunaliella bardawil is described. The subunit structure of the D. bardawil ATPase differs from that of the spinach in that the D. bardawil α subunit migrates ahead of the β subunit and ε-migrates ahead of subunit II of CF₀ when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The CF₁ isolated from D. bardawil resembles the CF₁ isolated from Chladmydomonas reinhardi in that a reversible, Mg²⁺-dependent ATPase is induced by selected organic solvents. Glycerol stimulates cyclic photophosphorylation catalyzed by D. bardawil thylakoid membranes but inhibits photophosphorylation catalyzed by spinach thylakoid membranes. Glycerol (20%) also stimulates the rate of ATP-P_i exchange catalyzed by D. bardawil CF₀-CF₁ proteoliposomes but inhibits the activity with the spinach enzyme. The ethanol-activated, Mg²⁺-ATPase of the D. bardawil CF₁ is more resistant to glycerol inhibition than the octylglucoside-activated, Mg²⁺-ATPase of spinach CF₁ or the ethanol-activated, Mg²⁺-dependent ATPase of the C. reinhardi CF₁. Both cyclic photophosphorylation and ATP-P_i exchange catalyzed by D. bardawil CF₀-CF₁ are more sensitive to high concentrations of NaCl than is the spinach complex.     

Activation of ribulose bisphosphate carboxylase in intact chloroplasts by CO2 and light

The activity of ribulose 1,5-bisphosphate (RuBP) car?ylase in intact spinach chloroplasts is shown to depend on light and CO₂. This activity was measured upon lysis of chloroplasts and assay of the initial activity using nonlimiting substrate concentrations. Incubation of chloroplasts at 25 °C in the absence of CO₂ results in a gradual inactivation of the RuBP car?ylase. In the presence of CO₂ the initial activity is preserved or increased. CO₂ is also able to reactivate the chloroplast car?ylase previously inactivated in the absence of CO₂. Upon illumination of the chloroplasts, additional activation was observed. This light activation results from an increased affinity for CO₂ of the chloroplast car?ylase. At pH 7.8, the enzyme in dark-adapted chloroplasts required 112 μ m CO₂ for half activation, while in the light it required 24 μ m CO₂. The light activation was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, carbonylcyanide 3-chlorophenylhydrazone, or dl-glyceraldehyde. Part of the light activation is most likely due to increased Mg²⁺ in the stroma. dl-Glyceraldehyde inhibition also suggests that some intermediate of the photosynthetic carbon cycle is involved. These results suggest that photosynthetic CO₂ assimilation in the chloroplast depends upon the amount of activation of the RuBP car?ylase. This activation is regulated by CO₂ and light-induced changes in the chloroplast stroma such as pH, Mg²⁺, and intermediates of the photosynthetic carbon cycle.     

Light/dark labeling differences in chloroplast membrane polypeptides associated with chloroplast coupling factor 0

The fluorogenic reagent fluorescamine has been used to determine the labeling patterns of Type C spinach chloroplast membrane polypeptides. Membrane polypeptides labeled with fluorescamine were detected by scanning high resolution sodium dodecyl sulfate polyacrylamide gradient slab gels for fluorescence emission.Three membrane polypeptides show a decrease in the extent of labeling when chloroplast membranes are labeled in the light compared to when they are labeled in the dark. These polypeptides have apparent molecular weights of 32 000, 23 000 and 15 000.The decrease in labeling observed in the light is abolished or reduced by treatments which inactivate the light-generated transmembrane pH gradient. CF₁-depleted chloroplasts show neither a light-activated pH gradient nor a light/dark difference in labeling of these three polypeptides. Both a light-activated pH gradient and light/dark differences in labeling are observed in CF₁-depleted chloroplasts which have been treated with N,N′-dicyclohexylcarbodiimide.The same ammonium sulfate fractions of a 2% sodium cholate extract, which are believed to be enriched in the membrane-bound sector of the chloroplast ATPase (CF_o) are also found to be enriched in the 32 000, 23 000 and 15 000 molecular weight polypeptides. The three polypeptides are believed to be components of CF_o, and the light/dark labeling differences may indicate conformational changes within CF_o. Such conformational changes may reflect a mechanism which couples light-generated proton gradients to ATP synthesis.     

Activation of kidney guanylate cyclase by cobalt.

Guanylate cyclase activity and cyclic nucleotide content were studied in individual slices from guinea pig kidneys. Basal guanylate cyclase activity, assayed in homogenates or in particulate fractions (100,000g × 1 h), and the tissue content of cGMP and cAMP were greater in the inner than in the outer (entirely cortical) slices. The fraction of guanylate cyclase activity recovered in the supernatant was greater in the cortex. Taurodeoxycholate increased activity of the particulate cyclase but decreased that of the supernatant enzyme. Activity of the particulate was increased ca. 200% and that of the supernatant >500% by 1 mm NaN₃. Supernatant activity was markedly increased by 0.1 mm Co²⁺, which had no effect on the particulate enzyme. (Incubation of kidney slices with 2 mm Co²⁺ did not alter their cGMP content, but caused a small increase in the cAMP content of slices containing medullary tissue.) Basal guanylate cyclase activity in fresh supernatants increased linearly with pH from 5.9 to 9, whereas in the presence of Co²⁺ there was a clear maximum at pH 7.3 to 7.5. Incubation of fresh supernatant fractions at 37 °C for 3 h increased guanylate cyclase activity and abolished Co²⁺ activation. The relationship between Co²⁺ activation and that resulting from incubation remains to be defined. It seems probable, however, that these phenomena reflect regulatory properties of the supernatant guanylate cyclases of kidney and other tissues.     

Activation of Barnea candida (mollusca,pelecypoda) oocytes by sperm or KCl,but not by NH4Cl,requires a calcium influx

Artificial activation of Barnea candida oocytes by NH₄Cl and the addition of excess KCl are, respectively, independent and dependent upon external Ca²⁺ concentration. The activating efficiency of NH₄Cl increases when external pH is raised which suggests that the proportion of unionized penetrating NH₃ is the key factor for activation under these conditions. The external Ca²⁺-dependent period for KCl or sperm-induced activation does not exceed 3–4 min. The transmembrane Ca²⁺ flux inhibitor D-600 reversibly inhibits KCl- or sperm-induced activation and the D-600-sensitive period closely corresponds to the Ca²⁺-dependent period. Ca²⁺ ions alone can also trigger activation provided external Mg²⁺ concentration is lowered. Finally, a direct demonstration is presented for the existence of a D-600-sensitive Ca²⁺ uptake following KCl- or sperm-induced activation, by using the radioactive tracer ⁴⁵Ca. The significance of that Ca²⁺ influx is discussed with respect to the possible involvement of an intracellular pH shift as a main factor in the activation of lamellibranch mollusc oocytes.     

Activation of the CF0-CF1, ATP synthase from spinach chloroplasts by chloroplast lipids

The interactions of CF₀-CF₁ with different lipids were studied by following the stimulation of Mg-ATPase and of P_i-ATP exchange activities of reconstituted CF₀-CF₁ proteoliposomes. The following results were obtained: (1) Both P_i-ATP exchange and Mg-ATPase activities are stimulated by lipids. Furthermore, the inhibition of Mg-ATPase by N,N′-dicyclohexylcarbodiimide is dependent on the interactions of CF₀-CF₁ with lipids. (2) A polar lipid extract of thylakoid membranes stimulates Mg-ATPase activity of CF₀-CF₁ more efficiently than phospholipids. The relative effectiveness of Mg-ATPase stimulation is: chloroplast lipids > soybean phospholipids > phosphatidylcholine/phosphatidylserine (4: 1) > phosphatidylcholine. The rate of P_i-ATP exchange in chloroplast lipids CF₀-CF₁ proteoliposomes is, however, lower than in soybean lipids CF₀-CF₁ proteoliposomes, due to their higher permeability to protons. Addition of 10% phosphatidylserine to chloroplast lipids reduces their permeability to protons and stimulates P_i-ATP exchange. (3) The kinetic mechanism of ATPase stimulation by chloroplast lipids is by decreasing the K_m (ATP) and by increasing V_max in comparison to soybean lipid proteoliposomes. This may explain the low affinity for ATP and the slow turnover rate of the purified enzyme in artificial lipids in comparison to the native enzyme in chloroplast thylakoids. (4) Chloroplast lipids lacking monogalactosyldiacylglycerols only poorly activate CF₀-CF₁. A large stimulation of P_i-ATP exchange is obtained by a mixture of 60% monogalactosyldiacylglycerol and 40% of the rest of the chloroplast lipids, but not by mixtures of monogalactosyldiacylglycerol with phospholipids. Hydrogenation of the unsaturated fatty acids of monogalactosyldiacylglycerol inhibits the activation of CF₀-CF₁. (5) The results suggest that: (a) interactions of specific chloroplast lipids with CF₀-CF₁ activates the enzyme by increasing its turnover and its affinity for ATP; (b) specific requirements for CF₀-CF₁ activation are the presence of monogalactosyldiacylglycerols together with another chloroplast lipid component and of highly unsaturated fatty acids.     

Evidence for light-dependent reversible conformational change in spinach chloroplast CF1

We have investigated an inhibition of photophosphorylation which occurs during preillumination of isolated spinach chloroplasts. Preillumination for 4–6 min in the absence of a complete set of components required for ATP synthesis inhibits photophosphorylation to a maximum of 25–40%; no inhibition occurs if all components for phosphorylation are present from the time illumination begins. The inhibition is about 40% recoverable by imposing a dark (“rebound”) period after the preillumination. Photoinhibition is accompanied by an increased leakiness of the thylakoid membrane to protons and is prevented by the presence of FCCP during the preillumination. Several lines of evidence implicate changes in conformation of chloroplast coupling factor (CF₁) as the cause of both photoinhibition and dark rebound. Conditions which result in photoinhibition also result in a loss of Mg²⁺-dependent ATPase activity which can be elicited from chloroplasts. Both photoinhibition and dark rebound are accompanied by changes in the K_m of CF₁ for both ADP and P_i. Photoinhibition precludes further inhibition of phosphorylation by light plus N-ethylamleimide (NEM) while phosphorylating activity regained by dark rebound is sensitive to subsequent inhibition by light plus NEM. The results are consistent with the conformational coupling hypothesis in indicating that CF₁ may be able to store energy in a conformational state which can be released by the reversal of that state. The photoinhibition we observe may represent conformational changes in CF₁ which are related to conformational coupling but which lead to photoinhibition under our conditions of preillumination.     

Characterization of the Activation of Membrane-Bound and Soluble CF(1) by Thioredoxin

The activation of spinach (Spinacia oleracea) chloroplast coupling factor 1 (CF₁) by thioredoxin (ThR) was characterized using membrane-bound and soluble CF₁. Light generates an electrochemical proton gradient across the thylakoid membrane, which increases the accessibility of the disulfide bond on the γ-subunit of CF₁ to reduced ThR. The proton gradient substantially accelerates the activation of CF₁ compared with thylakoids incubated in the dark with similar concentrations of dithiothreitol and ThR. The interaction of soluble CF₁ with ThR was studied using fluorescent probes. CF₁ in solution, with and without its associated ε-subunit, was labeled at Cys-322 of the γ-subunit with fluoresceinyl maleimide. ThR from Escherichia coli was labeled with eosin isothiocyanate. Labeled ThR and CF₁ showed normal activities. Fluorescence energy transfer between donor fluoresceinyl maleimide and acceptor eosin isothiocyanate, manifested by a quenching of the donor fluorescence, was detected, suggesting that ThR and CF₁ form an intermolecular complex. When the ε-subunit was absent, quenching of donor fluorescence was approximately doubled, indicating that labeled ThR could approach more closely to the γ-subunit of CF₁. The distance between the fluorescent probes on CF₁ and ThR was calculated to be approximately 65 Å when ε-subunit was present and 52 Å when ε was absent. These values are consistent with other distance measurements and energy transfer values reported previously for fluorescent probes on CF₁. Whereas the extent of quenching increased by removal of the ε-subunit, the apparent dissociation constant was unchanged. The quenching effect was reversed when the ε-subunit was added back to the titration mixture. Similarly, the addition of unlabeled ThR decreased donor quenching.     

Comparison of Properties of the Proteolytic Degradation of Unassembled Nuclear-encoded Subunits of Ribulose-1,5-bisphosphate Carboxylase and of the Coupling Factor of Photophosphorylation CF1*

The proteolytic degradation of unassembled small subunit polypeptides of ribulose-1,5-bisphosphate carboxylase and of the δ-subunit of the coupling factor of photophosphorylation CF₁ were analyzed and compared in vitro in the presence of stroma or membrane preparations from ribosome-deficient plastids isolated from 32°C-grown rye leaves (Secale cereale L.). Extracts obtained from 70S ribosome-deficient rye leaves after radioactive labeling were used as substrate source for the unassembled polypeptides. Soluble stroma as well as membrane preparations from isolated plastids contained proteolytic activities catalyzing the degradation of both the small subunits of ribulose-1,5-bisphosphate carboxylase and CF₁-δ in vitro. Maximal in vitro degradation was observed at pH 2–3 for the unassembled small subunits, but at pH 6–7 for the purified holoprotein of ribulose-1,5-bisphosphate carboxylase, and at pH 6.0 for unassembled CF₁-δ. Degradation of unassembled small subunits of ribulose-1,5-bisphosphate carboxylase at pH 3.0 was stimulated by Cu²⁺ but not by Ca²⁺, Mg²⁺ or ATP. At pH 3.0 the degradation of unassembled small subunits of ribulose-1,5-bisphosphate carboxylase was not inhibited by various protease inhibitors but was even stimulated. At pH 7.0 its degradation was inhibited by HgCl₂ and diazoacetyl nor-leucine methyl ester + Cu-acetate. The degradation of CF₁-δ was markedly inhibited by phenylmethylsulphonyl fluoride (PMSF) and to a lesser extent by 1,10-phenanthroline. According to present results different proteolytic systems appear to be involved in the degradation of unassembled small subunits of ribulose-1,5-bisphosphate carboxylase and of unassembled CF₁-δ.     

Activation of ribulose-1, 5-bisphosphate oxygenase, The role of Mg2+, CO2, and pH.

Ribulose-1,5-bisphosphate oxygenase was activated by incubation with CO₂ and Mg²⁺ and inactivated upon removal of CO₂ and Mg²⁺ by gel filtration. The activity of the enzyme was dependent upon the preincubation concentrations of CO₂ and Mg²⁺ and upon the preincubation pH. This indicated that activation involved the reversible formation of an equilibrium complex of enzyme-CO₂-Mg. The kinetics of the activation process were the same as those described by G. H. Lorimer et al. ((1976) Biochemistry15, 529–536), for ribulose bisphosphate carboxylase and are consistent with the ordered reversible reaction sequence:

The activity of the enzyme, after preincubation at constant concentrations of CO₂ and Mg²⁺, increased as the pH was raised, suggesting that CO₂ reacted with an enzyme group having an alkaline pK. Since CO₂ and O₂ interact competitively at the catalytic site, the activation of ribulose bisphosphate oxygenase by CO₂ and Mg²⁺ indicates that the CO₂ molecule which takes part in the activation process is not the same as that which becomes fixed during the carboxylase reaction. These results also indicate that the oxygenase and carboxylase functions of the catalytic site are tightly coupled rather than independent of one another.     

Synergistic activation of an Mg-specific ATPase activity in chloroplast coupling factor by octylglucoside and tentoxin

(1) Octylglucoside stimulates an Mg²⁺-specific ATPase activity with CF₁ preparations from different higher plants and the alga Chlamydomonas reinhardii. (2) Tentoxin at high concentrations (10^?4–10^?3 M) in the presence of octylglucoside further stimulates the Mg²⁺-ATPase activity of CF₁ from tentoxin-sensitive species and inhibits the activity of CF₁ from tentoxin-resistant species. The extent of tentoxin stimulation and inhibition varies among species. A maximal stimulation of over 2-fold was obtained with spinach CF₁ and a maximal inhibition of 50% was obtained with C. reinhardii CF₁. In Nicotiana spp., tentoxin had only a marginal effect on the Mg²⁺-ATPase activity induced by octylglucoside.     

A Novel Method for the Activation and Solubilization of Coupling Factor 1 from Chloroplasts of Euglena gracilis

A method was found for the in situ activation of the latent Ca²⁺-ATPase of the coupling factor from chloroplasts of Euglena gracilis and its resultant solubilization. The activation causes the concomitant solubilization of the enzyme and facilitates easy and nearly complete extraction. The activation consists of lowering the pH of isolated chloroplasts for 2 minutes to pH 4.5 to 4.7 with acetic acid, followed by neutralization. Increases in activity as high as 18-fold can be obtained. The method does not appear to work with chloroplasts from other sources.     

On the light dependence of Fatty Acid synthesis in spinach chloroplasts

The capacity of intact chloroplasts to synthesize long chain fatty acids from acetate depends on the stroma pH in Spinacia oleracea, U. S. hybrid 424. The pH optimum is close to 8.5. Lowering of the stroma pH leads to a reduction of acetate incorporation but does not suffice to eliminate fatty acid synthesis completely. Chain elongation from palmitic to oleic acid shows the same pH dependence. Fatty acid synthesis is activated in the dark upon the simultaneous addition of dihydroxyacetone phosphate and orthophosphate supplying ATP and oxaloacetate for reoxidation of NADPH in the stroma. Under these conditions both dark fatty acid synthesis and synthesis of oleate from palmitate show the same pH dependence as in the light. Dark fatty acid synthesis is further stimulated by increasing the stromal Mg²⁺ concentration with the ionophore A 23187. In contrast to CO₂ fixation, dark fatty acid synthesis is considerably reduced by dithiothreitol (DTT). This observation may be due to an acetyl-CoA deficiency, caused by a nonenzymic acylation of DTT, and a competition for ATP between DTT-activated CO₂ fixation and fatty acid synthesis. Because d,l-glyceraldehyde as inhibitor of CO₂ fixation compensates the DTT effect on dark fatty acid synthesis, reducing equivalents may be involved in the light dependence of acetate activation.     

Properties of the solvent-stimulated ATPase activity of chloroplast coupling factor 1 from Chlamydomonas reinhardii

The effects of solvents on the ATPase activity of chloroplast coupling factor 1 (CF₁) isolated from wild-type Chlamydomonas reinhardii have been studied. Of the solvents examined, the following order summarizes their maximal ability to stimulate the ATPase activity of CF₁: ethanol > methanol>allyl alcohol >n-propanol > acetone≈dioxane > ethylene glycol. Glycerol inhibits the CF₁ activity at all concentrations. In the absence of organic solvents, 50% of the activity of the enzyme is irreversibly lost after a 10 min incubation at 65–70°C. Ethanol (23%) causes a 30°C drop in the temperature required for 50% inactivation. ATP partially stabilizes the CF₁ in the presence, but not in the absence, of ethanol. In the absence of organic solvents, both free Mg²⁺ and ADP inhibit the CF₁-ATPase. Mg²⁺ is a noncompetitive inhibitor with respect to MgATP, and the kinetic constants are: V, 6.3 μmol ATP hydrolyzed/mg protein per min; K_m(MgATP), 0.23 mM; K_ii(Mg²⁺), 27 μM; and K_is(Mg²⁺), 50 μM. In the presence of ethanol, double-reciprocal plots are no longer linear and have a Hill coefficient of about 1.8±0.1. V increases about 10–12-fold. The pattern of inhibition by Mg²⁺ appears to change from noncompetitive to competitive with respect to MgATP. In addition, ADP no longer inhibits the MgATPase activity of CF₁.     

JNK and Ceramide Kinase Govern the Biogenesis of Lipid Droplets through Activation of Group IVA Phospholipase A2

The biogenesis of lipid droplets (LD) induced by serum depends on group IVA phospholipase A₂ (cPLA₂α). This work dissects the pathway leading to cPLA₂α activation and LD biogenesis. Both processes were Ca²⁺-independent, as they took place after pharmacological blockade of Ca²⁺ transients elicited by serum or chelation with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester). The single mutation D43N in cPLA₂α, which abrogates its Ca²⁺ binding capacity and translocation to membranes, did not affect enzyme activation and formation of LD. In contrast, the mutation S505A did not affect membrane relocation of the enzyme in response to Ca²⁺ but prevented its phosphorylation, activation, and the appearance of LD. Expression of specific activators of different mitogen-activated protein kinases showed that phosphorylation of cPLA₂α at Ser-505 is due to JNK. This was confirmed by pharmacological inhibition and expression of a dominant-negative form of the upstream activator MEKK1. LD biogenesis was accompanied by increased synthesis of ceramide 1-phosphate. Overexpression of its synthesizing enzyme ceramide kinase increased phosphorylation of cPLA₂α at Ser-505 and formation of LD, and its down-regulation blocked the phosphorylation of cPLA₂α and LD biogenesis. These results demonstrate that LD biogenesis induced by serum is regulated by JNK and ceramide kinase.