Uridine-diphospho-sulfoquinovose: diacylglycerol sulfoquinovosyltransferase activity is concentrated in the inner membrane of chloroplast envelopes

In experiments on the assembly of the sulfolipid sulfoquinovosyl diacylglycerol in envelope membranes of chloroplasts, UDP-sulfoquinovose (UDPS) was used with highest efficiency, and the corresponding enzyme, UDP-sulfoquinovose:diacylglycerol sulfoquinovosyltransferase, was partially characterized (E. Heinz et al., 1989, Eur J Biochem 184: 445–453). Here, we identified ³⁵S- and ³³P-labelled UDPS from various photosynthetically active organisms, suggesting that the sulfosugar nucleotide used for sulfolipid biosynthesis throughout the plant kingdom, including phototrophic bacteria, may indeed be UDPS. For attribution of the sulfolipid synthase to one of the two plastidial envelope membranes, these membranes were isolated from pea and spinach chloroplasts. The sulfoquinovosyltransferase was localized in the inner membrane of envelopes, which also contains the competing UDP-galactose:diacylglycerol galactosyltransferase. In contrast to the sulfoquinovosyltransferase, a substantial proportion of the galactosyltransferase was found in the outer membranes of envelopes from pea chloroplasts. Received: 6 October 1997 / Accepted: 31 January 1998     

Recombinant Arabidopsis SQD1 converts udp-glucose and sulfite to the sulfolipid head group precursor UDP-sulfoquinovose in vitro

The sulfolipid sulfoquinovosyldiacylglycerol is a component of plant photosynthetic membranes and represents one of the few naturally occurring sulfonic acids with detergent properties. Sulfolipid biosynthesis involves the transfer of sulfoquinovose, a 6-deoxy-6-sulfoglucose, from UDP-sulfoquinovose to diacylglycerol. The formation of the sulfonic acid precursor, UDP-sulfoquinovose, from UDP-glucose and a sulfur donor is proposed to be catalyzed by the bacterial SQDB proteins or the orthologous plant SQD1 proteins. To investigate the underlying enzymatic mechanism and to elucidate the de novo synthesis of sulfonic acids in biological systems, we developed an in vitro assay for the recombinant SQD1 protein from Arabidopsis thaliana. Among different possible sulfur donors tested, sulfite led to the formation of UDP-sulfoquinovose in the presence of UDP-glucose and SQD1. An SQD1 T145A mutant showed greatly reduced activity. The UDP-sulfoquinovose formed in this assay was identified by co-chromatography with standards and served as substrate for the sulfolipid synthase associated with spinach chloroplast membranes. Approximate K(m) values of 150 microm for UDP-glucose and 10 microm for sulfite were established for SQD1. Based on our results, we propose that SQD1 catalyzes the formation of UDP-sulfoquinovose from UDP-glucose and sulfite, derived from the sulfate reduction pathway in the chloroplast.     

Localization of sulfolipid labeling within cells and chloroplasts

Spinach chloroplasts were purified on gradients of Percoll which preserved envelope impermeability and CO₂-dependent oxygen evolution in the light. Application of ³⁵SO₄ to purified chloroplasts resulted in a light-dependent labeling of a lipid component which was indentified as sulfoquinovosyl diacylglycerol. Fractionation of chloroplasts showed that after 5 min of labeling most of the newly synthesized sulfolipid was present in thylakoids. Only a small percentage was recovered from the envelopes. Molecular species from envelopes and thylakoids were identical. The molecular species did not change during incubation times ranging from 5 min up to 4.5 h. Mesophyll protoplasts from ³⁵SO₄-labeled oat primary leaves were gently disrupted and separated into organelles by sucrose gradient centrifugation. Labeled sulfolipid was located almost exclusively in the chloroplasts. This, in combination with the experiments carried out with isolated chloroplasts, indicates that the final assembly steps in the biosynthesis of sulfolipid are confined to the chloroplasts.     

Enzymatic Characteristics of UDP-sulfoquinovose: Diacylglycerol Sulfoquinovosyltransferase from Chloroplast Envelopes*

Envelope membranes from chloroplasts contain UDP-sulfoquinovose: diacylglycerol sulfoquinovosyltransferase which catalyses the final step in sulfolipid assembly. In situ produced diacylglycerol served as radioactive acceptor to measure enzymatic activity. With this assay, several enzymatic parameters were investigated. The enzyme, which has maximal activity at pH 7.5, was stimulated by magnesium ions due to a decrease of the K_m for uridine 5′-diphospho-sulfoquinovose from 80 pM (no magnesium) to 10 μM (5 mM magnesium). This stimulation had a K_m of 0.7 mM magnesium and may be relevant in light/dark modulation of the enzymatic activity. The lower efficiency of guanosine 5′-diphospho-sulfoquinovose observed before can be ascribed to a higher K_m of this sugar nucleotide (400 μM). Under optimized and linearized conditions the sulfoquinovosyltransferase displayed about 10% of the activity of the UDP-galactose: diacylglycerol galactosyltransferase which competes in the same membrane system for diacylglycerols. Addition of acidic lipids, such as sulfolipid and phosphatidylglycerol, to envelope membranes resulted in an inhibition of the sulfoquinovosyltransferase, whereas the galactosyltransferase was not affected. In vivo this may contribute to an adjustment of the sulfolipid proportion in plastid membranes. In contrast to the galactosyltransferase the sulfoquinovosyltransferase did not discriminate against the dipalmitoyl molecular species of diacylglycerol when offered together with the oleoyl-palmitoyl species. Under conditions when oleoyl-palmitoyl-and dipalmitoyl-diacylglycerols were synthesized with concurrent conversion to monogalactosyl and sulfoquinovosyl diacylglycerol, the sulfolipid was highly enriched in the fully saturated species. This may explain the occurrence of dipalmitoyl species in sulfolipids, as found in many plants.     

Questions remaining in sulfolipid biosynthesis: a historical perspective

The plant sulfolipid sulfoquinovosyldiacylglycerol was discovered by A.A. Benson in the late 1950s. The increasing availability of radioisotope-containing biological substrates such as ³⁵S-sulfate provided the means to discover novel biological compounds and to sketch out their biosynthetic pathways. During this time the structure of sulfolipid with its 6-deoxy-6-sulfo-α-d-glucose (sulfoquinovose) headgroup was determined. Immediately, the origin of this unusual biological sulfonic acid mystified the scientific community and several proposals for its biosynthesis were developed and tested. Strong supportive evidence for the nucleotide pathway of sulfolipid biosynthesis became available with the discovery of the bacterial and plant genes encoding the enzymes of sulfolipid biosynthesis during the 1990s. This latter work was based on the foundations laid by A.A. Benson and confirmed one initial hypothesis on sulfolipid biosynthesis. An abbreviated summary of the turning points in defining the mechanism for sulfolipid biosynthesis and remaining issues in sulfolipid biochemistry are provided.     

Ferredoxin-dependent glutamate synthase moonlights in plant sulfolipid biosynthesis by forming a complex with SQD1

UDP-sulfoquinovose synthase, SQD1, catalyzes the transfer of sulfite to UDP-glucose giving rise to UDP-sulfoquinovose, which is the head group donor for the biosynthesis of the plant sulfolipid sulfoquinovosyldiacylglyerol. The native SQD1 enzyme of spinach exists as a 250 kDa heteroprotein complex with much higher affinity for the substrate sulfite than the recombinant SQD1 protein itself. The SQD1 protein co-purified with nine proteins. Likely binding partners included rubisco activase, HSP70, and ferredoxin-dependent glutamate synthase (FdGOGAT). While the first two proteins are known to interact with many other proteins, the identification of FdGOGAT was most intriguing because this 160kDa protein contains an FMN cofactor known to bind sulfite in vitro. Using different constructs expressing recombinant forms of the multidomain protein FdGOGAT, it was demonstrated that the FMN-binding domain of FdGOGAT is essential for specific binding of the protein to SQD1. A model suggests that FdGOGAT could channel sulfite to SQD1.     

Inhibiton of fatty acid biosynthesis in isolated chloroplasts by cycloxydim and other cyclohexane-1,3-diones

Kobek, K., Focke, M., Lichtenthaler, H.K., Retzlaff, G. and Würzer, B. 1988. Inhibiton of fatty acid biosynthesis in isolated chloroplasts by cycloxydim and other cyclohexane-1,3-diones. - Physiol. Plant. 72: 492–498.
The effect of the three cyclohexane-1,3-dione herbicides cycloxydim, sethoxydim and clethodim (proposed common name) on the de novo fatty acid biosynthesis of isolated chloroplasts as test system was investigated with intact chloroplasts isolated from sensitive grasses (Poaceae) and tolerant dicotyledonous plants. All three herbicides blocked the de novo fatty acid biosynthesis ([¹⁴C]-acetatc incorporation into total fatty acid fraction) in Avena sativa L. cv. Flämingnova chloroplasts in a dose-dependent manner. The I₅₀-values are lower for cycloxydim and clethodim than for sethoxydim. The rate of de novo fatty acid biosynthesis in isolated, intact and photosynthetically active Avena chloroplasts was higher in the light than in the dark, which appeared to be due to the light-dependent regeneration of the cofactors ATP and NADPH. The de novo fatty acid biosynthesis by isolated chloroplasts from the tolerant dicotyledonous species pea ( Pisum savivum L. cv. Kleine Rheinländerin), spinach ( Spinacea oleracea L. cv. Matador) and tobacco ( Nicotiana tabacum L. cv. su/su) was insensitive to the three herbicides. It is assumed that one of the enzymes of the fatty acid biosynthesis is modified in the dicotyledonous plants and not accessible to the cyclohexane-1,3-dione herbicides. In the case of Poa annua L., which as a whole plant is tolerant towards sethoxydim, the tolerance seems not to lie in the chloroplasts but in properties of the cytoplasm, since the isolated chloroplasts are sensitive to the herbicide.     

Isolation and genetic complementation of a sulfolipid-deficient mutant of Rhodobacter sphaeroides.

All photosynthetic organisms are thought to contain the sulfolipid 6-sulfo-alpha-D-quinovosyl diacylglycerol. However, the pathway of sulfolipid biosynthesis has not been elucidated, and the functional or structural significance of this lipid is not known. Mutants of Rhodobacter sphaeroides deficient in sulfolipid accumulation were isolated by directly screening for altered sulfolipid content. The mutants had no apparent phenotype except for the sulfolipid deficiency. A gene, designated sqdA, which complemented one of the mutations was isolated and characterized. The putative sqdA gene product is a protein with a molecular mass of 33.6 kDa that has no sequence similarity to any enzyme of known function.     

Disruption of a gene essential for sulfoquinovosyldiacylglycerol biosynthesis in Sinorhizobium meliloti has no detectable effect on root nodule symbiosis

The sulfolipid sulfoquinovosyldiacylglycerol is commonly found in the thylakoid membranes of photosynthetic bacteria and plants. While there is a good correlation between the occurrence of sulfolipid and photosynthesis, a number of exceptions are known. Most recently, sulfoquinovosyldiacylglycerol was discovered in the non-photosynthetic, root nodule-forming bacterium Sinorhizobium meliloti. This discovery raised the questions of the phylogenetic origin of genes essential for the biosynthesis of this lipid in S. meliloti and of a function of sulfolipid in root nodule symbiosis. To begin to answer these questions, we isolated and inactivated the sqdB gene of S. meliloti. This gene and two other genes located directly 3' of sqdB are highly similar to the sqdB, sqdC, and sqdD genes known to be essential for sulfolipid biosynthesis in the photosynthetic, purple bacterium Rhodobacter sphaeroides. This observation confirms the close phylogenetic kinship between these two species. Furthermore, the reduced similarity of sqdB to the plant ortholog SQD1 of Arabidopsis thaliana does not support a previous sqd gene transfer from the plant as a consequence of close symbiosis. A sulfolipid-deficient mutant of S. meliloti disrupted in sqdB is capable of inducing functional nodules and does not show an obvious disadvantage under different laboratory culture conditions. Thus far, no specific function can be assigned to bacterial sulfolipid, in either nodule-associated or free-living cells. S. meliloti contains a rich set of polar membrane lipids some of which, including sulfolipid, may become critical only under growth conditions that still need to be discovered.     

Feedback inhibition of phosphatidate phosphatase from spinach chloroplast envelope membranes by diacylglycerol.

Because the envelope phosphatidate phosphatase plays a pivotal role in chloroplast glycerolipid metabolism, we have analyzed whether diacylglycerol could be a regulatory factor of the enzyme. Using isolated envelope membranes in which the level of diacylglycerol was modified by thermolysin treatment of intact chloroplasts to destroy the galactolipid:galactolipid galactosyltransferase, we have demonstrated that phosphatidate phosphatase activity was reduced when the membrane was enriched in diacylglycerol. All 1,2-diacylglycerol molecular species assayed were demonstrated to inhibit the enzyme to about the same extent. Kinetic studies with envelope from thermolysin-treated chloroplasts were performed in the absence and presence of diacylglycerol, and diacylglycerol was shown to be a powerful competitive inhibitor of the reaction. Finally, using isolated intact spinach chloroplasts, we have demonstrated that in situ phosphatidate phosphatase activity can be modulated by the level of diacylglycerol present in the membrane. The relevance of phosphatidate phosphatase inhibition by diacylglycerol in the regulation of chloroplast glycerolipid biosynthesis is discussed.     

Mechanism responsible for the hypoglycemic action of 2-alkoxy-2-propenylidene methanaminiums

2-Alkoxy-2-propenylidene methanaminiums inhibited gluconeogenesis and stimulated glycolysis by hepatocytes isolated from 48-h-fasted rats and fasted-refed rats, respectively. The order of effectiveness of these compounds was the same as the hypoglycemic response of intact rats found in other studies, i.e., butoxy greater than propoxy greater than ethoxy derivative. Lactate/pyruvate and beta-hydroxybutyrate/acetoacetate ratios were elevated whereas cellular ATP concentration was decreased by these compounds. The butoxy derivative inhibited the oxidation of [U-14C]glucose to 14CO2 but increased glucose utilization and lactate accumulation by isolated rat diaphragms. The butoxy derivative also inhibited site I reversed electron transfer and the oxidation of NAD+-linked substrates but not succinate by isolated rat liver mitochondria. Methanaminium-induced hypoglycemia in intact rats was accompanied by an increase in blood lactate concentration as well as blood beta-hydroxybutyrate to acetoacetate ratio. The hypoglycemia caused by these compounds is proposed to be due to inhibition of glucose synthesis in the liver along with increased glucose utilization in peripheral tissues, both for want of ATP as a consequence of inhibition of site I electron transfer.     

Fatty Acid Synthesis in Hydrilla Chloroplasts

The rates of carboxylation, photophosphorylation and acetate incorporation have been compared in the intact and broken chloroplasts of Hydrilla verticillata Royle leaves in the presence and absence of certain inhibitors and metabolites. The intact chloroplasts showed low rates of photophosphorylation, high rates of carboxylation, and exhibited normal capacity for fatty acid biosynthesis. In broken chloroplasts a drastic decrease was observed in the rates of carboxylation and acetate incorporation. However, the rate of photophosphorylation was considerably increased. In the presence of light, inhibitors such as iodoacetamide, arsenite and sodium azide decreased the photophosphorylation rate. F-1,6-di-P and PGA stimulated CO₂ fixation rate. In the absence of artificial light, inhibitors such as sodium arsenite, gluconate-6-phosphate, sodium azide and iodoacetamide decreased the rate of CO₂ fixation. CoA, ATP, G-6-P, F-1,6-di-P Stimulated the synthesis of fatty acids. Exogenous supply of ADP. NADH, NADP and NADPH did not stimulate fatty acid biosynthesis probably because these compounds could not gain entry into the chloroplasts. Light was necessary for the in vitro fatty acid biosynthesis.     

Lipid synthesis and metabolism in the plastid envelope

Plastid envelope membranes play a major role in the biosynthesis of glycerolipids. In addition, plastids are characterized by the occurrence of plastid-specific membrane glycolipids (galactolipids, a sulfolipid). Plant lipid metabolism therefore has unique features, when compared to that of other eukaryotic organisms, such as animals and yeast. However, the glycerolipid biosynthetic pathway in chloroplasts is almost identical to that found in cyanobacteria, and reflects the prokaryotic origin of the chloroplast. Fatty acids generated in the plastid stroma are substrates for a whole set of enzymes involved in the synthesis of polar lipids of plastid membranes such as galactolipids, the sulfolipid, the phosphatidylglycerol. In addition, fatty acids are exported outside the plastid where they are used for extraplastidial polar lipid synthesis (phosphatidylcholine, phosphatidylethanolamine, etc.). Various desaturation steps leading to the formation of polyunsaturated fatty acids occur in various cell compartments, especially in chloroplasts, using fatty acids esterified to polar lipids as substrates. Furthermore, plant glycerolipids can be metabolized by a series of very active envelope enzymes, such as the galactolipid:galactolipid galactosyltransferase and the acyl-galactolipid forming enzyme. The physiological significance of these enzymes is however largely unknown. One of the most active pathways involved in lipid metabolism and present in envelope membranes is the oxylipin pathway: polyunsaturated fatty acids that are released from polar lipids under various conditions (injury, pathogen attack) are converted to oxylipin. Thus, the plastid envelope membranes are also involved in the formation of signalling molecules.     

Biosynthesis of sulfoquinovosyldiacylglycerol in higher plants: the incorporation of 35SO4 by intact chloroplasts

Isolated spinach chloroplasts have been found to incorporate 35SO4 into the plant sulfolipid, sulfoquinovosyldiacylglycerol, at rates of up to 700 pmol mg chlorophyll-1 h-1. The reaction is light-dependent, requires that the chloroplasts be intact, and is slightly stimulated by ATP and UTP. UDP-galactose inhibits the formation of sulfoquinovosyldiacylglycerol, presumably by competing for the diacylglycerol pool. The rates of synthesis observed are sufficient to conclude that the chloroplast is autonomous with respect to the synthesis of sulfoquinovose, the headgroup moiety of sulfoquinovosyldiacylglycerol. No evidence could be obtained to support the concept that the proposed sulfoglycolytic pathway is the biosynthetic route for sulfoquinovose formation.     

The effect of some platinum compounds on the biosynthesis of RNA and its precursors.

The effect of cis-DDP (cis-diamminedichloroplatinum(II)), trans-DDP (trans-diamminedichloroplatinum(II)), SPC (spermine-platinum(II) complex), and K2PtCl4 on the ribomononucleotide and RNA metabolism was studied. When Ehrlich ascites tumor cells were preincubated with the aforementioned compounds and then labeled with [C14]uridine a clear-cut suppression of the radioactive labeling of RNA was observed. As radioactivity incorporated into the pool of the free uridine nucleotides in the cells treated with platinum compounds was even higher in comparison with that of the non-treated cells a conclusion may be drawn with certainty that the platinum compounds studied inhibit RNA biosynthesis. It was also found that under the effect of these compounds in the in vivo-assessed rate of the conversion of uridine nucleotides into cytidine nucleotides was considerably diminished. Using NaH14CO3 as a radioactive precursor it was shown that platinum compounds also inhibited purine biosynthesis de novo, in particular the conversion of IMP into GMP and AMP. The pronounced inhibitory effect of the platinum compounds on essential steps of the pyrimidine and purine biosynthesis de novo may be at least partly responsible for the firmly established inhibition in the present study of RNA biosynthesis by platinum compounds. The inhibition of the synthesis of the mononucleotides and RNA by the platinum compounds may be closely related to their cytostatic and cytotoxic activities.     

Envelope membranes from mature spinach chloroplasts contain a NADPH:protochlorophyllide reductase on the cytosolic side of the outer membrane

Using fluorescence spectroscopy, we have demonstrated that isolated envelope membranes from mature spinach chloroplasts catalyze the phototransformation of endogenous protochlorophyllide into chlorophyllide in presence of NADPH, but not in presence of NADH. Protochlorophyllide reductase was characterized further using monospecific antibodies (anti-protochlorophyllide reductase) raised against the purified enzyme from oat. In mature spinach chloroplasts, protochlorophyllide reductase is present only in envelope membranes. We have demonstrated that the envelope protochlorophyllide reductase, a 37,000-dalton polypeptide, is only a minor envelope component and is present on the outer surface of the outer envelope membrane. This conclusion is supported by several lines of evidence: (a) the envelope polypeptide that was immunodecorated with anti-protochlorophyllide reductase can be distinguished from the major 37,000-dalton envelope polypeptide E37 (which was identified by monospecific antibodies) only after two-dimensional polyacrylamide gel electrophoresis; (b) the envelope protochlorophyllide reductase was hydrolyzed when isolated intact chloroplasts were incubated in presence of thermolysin; and (c) isolated intact chloroplasts strongly agglutinate when incubated in presence of antibodies raised against protochlorophyllide reductase. These results demonstrate that major differences exist between chloroplasts and etioplasts with respect to protochlorophyllide reductase levels and localization. The presence on the chloroplast envelope membrane of both the substrate (protochlorophyllide) and the enzyme (protochlorophyllide reductase) necessary for chlorophyllide synthesis could have major implications for the understanding of chlorophyll biosynthesis in mature chloroplasts.     

Events surrounding the early development of Euglena chloroplasts. 15. Origin of plastid thylakoid polypeptides in wild-type and mutant cells.

Techniques are described for the isolation of plastid thylakoid membranes from light-grown and dark-grown cells of Euglena gracilis var. bacillaris, and from mutants affecting plastid development. These membranes, which have minimal contamination with other cell fractions, are localized in sucrose gradients by using the thylakoid membrane sulfolipid as a specific marker. The plastid thylakoid membrane polypeptides isolated from these membranes were separated on SDS polyacrylamide gels and yielded patterns containing 30-40 polypeptides. Light-grown strain Z gave patterns identical with bacillaris. Since the plastid thylakoid polypeptide patterns obtained from dark-grown wild-type cells and from a bleached mutant W3BUL in which plastid DNA is undetectable are identical, it appears that the proplastid thylakoid polypeptides of wild-type cannot be coded in plastid DNA and are probably coded in nuclear DNA. The plastid thylakoid polypeptide patterns obtained from various dark-grown mutants, making large but abnormal chloroplasts, show a correlation between the amount of chlorophyll formed and the amount of a plastid thylakoid polypeptide thought to be associated wtth one of the pigment-protein light-harvesting complexes. Treatment with SAN 9789 (4-chloro-5-(methylamino)-2(alpha, alpha, alpha,-trifluoro-m-tolyl)-3-(2H(pyridazinone) known to block carotenoid synthesis at the level of phytoene, causes a progressive loss of all plastid thylakoid polypeptides during growth in darkness and results in the establishment of a new, lowere steady-state level of sulfolipid. At least ten of the plastid thylakoid polypeptides become labeled when isolated chloroplasts are supplied with radioactive amono acids; of these six are undectable in W3BUL and are, therefore, candidates for coding by plastid DNA.     

DEGRADATION AND FORMATION OF SULFOLIPID OCCURRING CONCURRENTLY WITH DE- AND RE-GENERATION OF CHLOROPLASTS IN THE CELLS OF CHLORELLA PROTOTHECOIDES

1. It has been demonstrated that when the cells of Chlorella protothecoidesare grown mixotrophically under illumination in a medium richin nitrogen source (urea) and poor in glucose, the normal greencells are obtained, while in a medium rich in glucose and poorin the nitrogen source, entirely chlorophyll-less cells withprofoundly degenerated plastids ("glucose-bleached" cells) areproduced, irrespective of whether in the light or in darkness.The "glucose-bleached" cells turn green with regeneration offully organized chloroplasts when incubated in a nitrogen-enrichedmedium in the light ("light-greening"), while in the dark theybecome pale green with formation of only partially organizedchloroplasts ("dark-greening"). When, on the other hand, thegreen cells are transferred into a medium enriched with glucose,they are bleached fairly rapidly with degeneration of chloro-plastsin the light as well as in darkness ("bleaching"). Using ³⁵Sas a tracer, investigations were made on the changes of contentsof the algal cells in sulfolipid and other sulfur compoundsduring the processes of the greening and bleaching.
2. By determiningthe radioactivities of chromatographically separatedsulfur-containingcompounds of the uniformly ³⁵S-labeled green("G") and "glucose-bleached"("W") cells, it was found thatthe concentration of a speciesof sulfolipid (discovered byBENSON et al.) as well as thoseof glutathione, sulfotriosesand most of the other sulfur-containingcompounds were at least5 times higher in the "G" cells thanin the "W" cells, whilesulfoquinovosyl glycerol was presentin approximately equalamounts in the two types of cells.
3. Phospholipidcontents and compositions in the two types of algalcells werefound to be practically identical.
4. The sulfolipid contentof algal cells increased and decreasedalmost in parallel withthe processes of greening and bleaching,respectively.
5. Studyingthe mode of incorporation of radiosulfate into varioussulfurcompounds of algal cells during the processes of "light-anddark-greening" and "bleaching" (lasting about 70 hr), itwasfound that active ³⁵S-incorporation into sulfolipid occurredthroughout the process of "light-greening," while in the "dark-greening"and "bleaching" the active incorporation abruptly ceased afterthe initial 24 hr period of experiments. It was suggested thatthe biosynthesis of the sulfolipid is closely related to theformation of photosynthetic apparatus in chloroplast.
6. Whenthe ³⁵S-labeled green cells were bleached in a medium containingno radiosulfate, the ³⁵S-sulfolipid and most of other ³⁵S-sulfurcompounds decreased markedly but the ³⁵S-sulfoquinovosyl glycerolincreased considerably. It was inferred that the deacylationof the sulfolipid, a surfactant lipid, with formation of watersoluble sulfoquinovosyl glycerol may be a cardinal event ofbleaching process, causing a disintegration of the intact architechtureof photosynthetic apparatus.
7. Based on these observations itwas concluded that the sulfolipidis an integral component ofphotosynthetic structure.

¹This work was partly reported at the Symposium on Biochemistryof Lipids, sponsored by the Agricultural Chemical Society ofJapan, Sapporo, July, 1964.     

Stimulation of carbon dioxide fixation in isolated pea chloroplasts by catalytic amounts of adenine nucleotides

Carbon dioxide-dependent O(2) evolution by isolated pea (Pisum sativum var. Massey Gem) chloroplasts was increased two to 12 times by the addition of ATP. O(2) evolution was also stimulated by ADP and to a lesser extent by AMP. The ATP effects were not due to broken chloroplasts present in the preparations nor was ATP acting as a phosphate source. We concluded that the adenine nucleotides were acting catalytically. The concentration of ATP required for half-maximum rate of O(2) evolution was 16 to 25 mum. The degree to which ATP stimulated O(2) evolution depended on the age of pea plants from which the chloroplasts were isolated. Spinach (Spinacia oleracea var. True Hybrid 102) chloroplasts did not show a consistent stimulation of O(2) evolution by adenine nucleotides.The adenine nucleotide content of pea chloroplasts was not lower than that of spinach chloroplasts, but pea chloroplasts which showed a large stimulation of O(2) evolution by ATP contained an ATP-hydrolyzing reaction with rates of 10 to 50 mumol ATP hydrolyzed mg chlorophyll(-1) hour(-1). The rate of the ATP-consuming reaction was much lower in spinach chloroplasts and in chloroplasts from older pea plants which did not show large stimulation of O(2) evolution by ATP. We propose that the ATP-consuming reaction, with a high affinity for ATP, decreased the effective size of the ATP pool available for CO(2) fixation. Added adenine nucleotides could be transported into the chloroplasts increasing the concentration of internal nucleotides. Calculations showed that the adenine nucleotide transporter on the outer chloroplast membranes could operate at a sufficient rate to produce such an effect.