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The physiological relevance of a novel thiol methyltransferase from cabbage, and its possible role in sulphur metabolism have been investigated. The enzyme was absent from the chloroplast, the site of sulphate reduction, and was localized in the cytosol. Potential substrates were initially screened on the basis of their ability to inhibit the methylation of iodide, a previously known substrate for the enzyme. Thiocyanate, 4,4′-thiobisbenzenethiol, thiophenol, and thiosalicylic acid were identified as possible substrates. Methylation of these thiols by the purified enzyme using [Methyl-3H]S-adenosyl- L-methionine confirmed their nature as substrates. The purified enzyme strongly preferred thiocyanate as a methyl acceptor. The enzyme had Km values of 11, 51, 250 and 746 mmol m−3 for thiocyanate, 4,4′-thiobisbenzenethiol, thiophenol and thiosalicylic acid, respectively. The identity of methylthiocyanate as the product of thiocyanate methylation by the purified enzyme was confirmed by mass spectrometry. The enzyme was strictly associated with glucosinolate-containing plants. Thiol substrates of the enzyme are known products of glucosinolate hydrolysis. Our observations indicate that this enzyme could be involved in the detoxification of reactive thiols produced upon glucosinolate degradation in these plants.

The pyruvate dehydrogenase complex (PDC) and acetyl-CoA carboxylase (ACC, EC 6.4.1.2) have been characterized in pea root plastids. PDC activity was optimum in the presence of 1.0 mM pyruvate, 1.5 mM NAD + , 0.1 mM CoA, 0.1 mM TPP, 5 mM MgCI2, 3.0 mM cysteine-HCI, and 0.1 M Tricine (pH 8.0) and represents approximately 47% of the total cellular activity. ACC activity was greatest in the presence of 1.0 mM acetyl-CoA, 4 mM NaHC03, 3 mM ATP, 10 mM MgCI2, 2.5 mM dithiothreito l, and 100 mM Tricine (pH 8.0). Both enzymes were stimulated by reduced sutphydryl reagents and inhibited by sulphydryl inhibitors. ACC was also inhibited by malonyl-CoA while PDC was inhibited by both malonyl-CoA and NADH. Both enzymes were stimulated by DHAP and UDP-galactose while ACC was also stimulated by PEP and F1,6P. Palmitic acid and oleic acid both inhibited ACC, but had essentially no effect on PDC. Palmitoyl-CoA inhibited both enzymes while PA and Lyso-PA inhibited PDC, but stimulated ACC. The results presented support the hypothesis that PDC and ACC function in a co-ordinated fashion to promote glycolytic carbon flow to fatty acid biosynthesis in pea root plastids.

Fatty acid and glycerolipid biosynthesis from [ 14 C]acetate by isolated pea root plastids is completely dependent on exogenously supplied ATP. CTP, GTP, and UTP are ineffective in supporting fatty acid biosynthesis, all resulting in

The glycerolipid composition of pea (Pisum sativum L.) root plastids and their capacity to synthesize glycerolipids from [UL-14C]glycerol-3-phosphate were determined. Pea root plastids primarily consist of monogalactosyldiacylglycerol, triacylglycerol, phosphatidylcholine, digalactosyldiacylglycerol, and diacylglycerol. Maximum rates of total glycerolipid biosynthesis were obtained in the presence of 2.4 mM glycerol-3-phosphate, 15 mM KHCO3, 0.2 mM sodium-acetate, 0.5 mM each of NADH and NADPH, 0.05 mM coenzyme A, 2 mM MgCl2, 1 mM ATP, 0.1 M Bis-Tris propane (pH 7.5), and 0.31 M sorbitol. Glycerolipid biosynthesis was completely dependent on exogenously supplied ATP, coenzyme A, and a divalent cation, whereas the remaining cofactors improved their activity from 1.3- to 2.4-fold. Radioactivity from glycerol-3-phosphate was recovered predominantly in phosphatidic acid, phosphatidylglycerol, diacylglycerol, and triacylglycerol with lesser amounts in phosphatidylcholine and monoacylglycerol. The proportions of the various radiolabeled lipids that accumulated were dependent on the pH and the concentration of ATP and glycerol-3-phosphate. The data presented indicate that pea root plastids can synthesize almost all of their component glycerolipids and that glycerolipid biosynthesis is tightly coupled to de novo fatty acid biosynthesis. pH and the availability of ATP may have important roles in the regulation of lipid biosynthesis at the levels of phosphatidic acid phosphatase and in the reactions that are involved in phosphatidylglycerol and triacylglycerol biosynthesis.

Radiolabeled pyruvate, glucose, glucose-6-phosphate, acetate, and malate are all variously utilized for fatty acid and glycerolipid biosynthesis by isolated pea (Pisum sativum L.) root plastids. At the highest concentrations tested (3-5mM), the rates of incorporation of these precursors into fatty acids were 183, 154, 125, 99 and 57 nmol h-1 mg-1 protein, respectively. In all cases, cold pyruvate consistently caused the greatest reduction, whereas cold acetate consistently caused the least reduction, in the amounts of each of the other radioactive precursors utilized for fatty acid biosynthesis. Acetate incorporation into fatty acids was approximately 55% dependent on exogenously supplied reduced nucleotides (NADH and NADPH), whereas the utilization of the remaining precursors was only approximately 10 and 20% dependent on added NAD(P)H. In contrast, the utilization of all precursors was greatly dependent (85-95%) on exogenously supplied ATP. Palmitate, stearate, and oleate were the only fatty acids synthesized from radioactive precursors. Higher concentrations of each precursor caused increased proportions of oleate and decreased proportions of palmitate synthesized. Radioactive fatty acids from all precursors were incorporated into glycerolipids. The data presented indicate that the entire pathway from glucose, including glycolysis, to fatty acids and glycerolipids is operating in pea root plastids. This pathway can supply both carbon and reduced nucleotides required for fatty acid biosynthesis but only a small portion of the ATP required

Fatty acid biosynthesis from Na[1-(14)C]acetate was characterized in plastids isolated from primary roots of 7-day-old germinating pea (Pisum sativum L.) seeds. Fatty acid synthesis was maximum at 82 nanomoles per hour per milligram protein in the presence of 200 micromolar acetate, 0.5 millimolar each of NADH, NADPH, and coenzyme A, 6 millimolar each of ATP and MgCl(2), 1 millimolar each of MnCl(2) and glycerol-3-phosphate, 15 millimolar KHCO(3), 0.31 molar sucrose, and 0.1 molar Bis-Tris-propane, pH 8.0, incubated at 35 degrees C. At the standard incubation temperature of 25 degrees C, fatty acid synthesis was essentially linear for up to 6 hours with 80 to 120 micrograms per milliliter plastid protein. ATP and coenzyme A were absolute requirements, whereas divalent cations, potassium bicarbonate, and reduced nucleotides all variously improved activity two- to 10-fold. Mg(2+) and NADH were the preferred cation and nucleotide, respectively. Glycerol-3-phosphate had little effect, whereas dithiothreitol and detergents generally inhibited the incorporation of [(14)C]acetate into fatty acids. On the average, the principal radioactive products of fatty acid biosynthesis were approximately 39% palmitic, 9% stearic, and 52% oleic acid. The proportions of these fatty acids synthesized depended on the experimental conditions.

Intact mitochondria from the endosperm of castor bean were isolated on linear sucrose gradients. These mitochondria were ruptured and the membranes separated on discontinuous sucrose gradients into outer membrane, intact inner membrane, and ruptured inner membrane fractions. Each membrane fraction was examined for its capacity to synthesize phosphatidylglycerol, CDP-diglyceride, phosphatidylcholine via methylation, and phosphatidic acid. The syntheses of phosphatidylglycerol, CDP-diglyceride, and phosphatidylcholine were localized exclusively in the inner mitochondrial membrane fractions while phosphatidic acid synthesis occurred in both the inner and outer mitochondrial membranes.

Phosphatidylethanolamine synthesis by CDP-ethanolamine:1,2-diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1) from the endoplasmic reticulum of castor bean (Ricinus communis L. var. Hale) endosperm was characterized. The Michaelis-Menten constant of the enzyme for CDP-ethanolamine was approximately 8.0 micromolar. The pH optimum was 6.5 and a divalent cation was an absolute requirement for activity, with Mg(2+) giving the greatest stimulation at 3 millimolar. Sulfhydryl reagents variously affected enzyme activity. No discernible differences were detected between the responses of the ethanolaminephosphotransferase and CDP-choline:1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) to a variety of treatments. CDP-choline and CDP-ethanolamine were competitive inhibitors of the ethanolaminephosphotransferase and cholinephosphotransferase reactions, respectively.

Subcellular fractions from pea (Pisum sativum L.) roots have been prepared by differential centrifugation techniques. Greater than 50% of the recovered plastids can be isolated by centrifugation at 500g for 5 minutes. Plastids of this fraction are largely free from mitochondrial and microsomal contamination as judged by marker enzyme analysis. De novo fatty acid biosynthesis in pea roots occurs in the plastids. Isolated pea root plastids are capable of fatty acid synthesis from acetate at rates up to 4.3 nanomoles per hour per milligram protein. ATP, bicarbonate, and either Mg(2+) or Mn(2+) are all absolutely required for activity. Coenzyme A at 0.5 millimolar improved activity by 60%. Reduced nucleotides were not essential but activity was greatest in the presence of 0.5 millimolar of both NADH and NADPH. The addition of 0.5 millimolar glycerol-3-phosphate increased activity by 25%. The in vitro and in vivo products of fatty acid synthesis from acetate were primarily palmitate, stearate, and oleate, the proportions of which were dependent on experimental treatments. Fatty acids synthesized by pea root plastids were recovered in primarily phosphatidic acid and diacylglycerol or as water soluble derivatives and the free acids. Lesser amounts were found in phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and monogalactosyldiacylglycerol.

[(14)C]Acetate, [(14)C]formate, and methyl[(14)C]methionine all serve as precursors of pyrenocines A, B, and C when added to cultures of Pyrenochaeta terrestris (Hansen) Gorenz, Walker, and Larson, the pathogen responsible for disease known as pink root of onion (Allium cepa L.). This information supports the hypothesis that these metabolites are methyl-substituted polyketides in origin. Pyrenocine A arises from acetate via uncharacterized intermediates and is subsequently converted to pyrenocine B. The biosynthetic role of pyrenocine C remains uncertain.

CDPcholine:1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) and CDPethanolamine:1,2-diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1) were detected in mitochondrial fractions from castor bean (Ricinus communis) endosperm. These activities were not due to contamination of the fractions with endoplasmic reticulum. The enzymes were localized on both the inner and outer mitochondrial membranes.Only minor kinetic differences between the phosphatidylcholine-synthesizing activities of intact mitochondria and of the endoplasmic reticulum were found. The K(m) of the mitochondrial enzyme for CDP-choline was about 2 units less than that for the enzyme of the endoplasmic reticulum (8.0 and 10.0 micromolar, respectively). The mitochondrial enzyme activity was maximal above 10 millimolar Mg(2+), whereas maximum endoplasmic reticulum activity was achieved by 4 millimolar. The endoplasmic reticulum enzyme was more stable at 37 C than was that of the mitochondria. The mitochondrial cholinephosphotransferase represented about 1 to 2% of the total activity isolated from castor bean endosperm.

Intact chloroplasts from spinach (Spinacia oleracea L., hybrid 424) readily incorporate [(14)C]glycerol-3-phosphate and [(14)C]acetate into diacylglycerol, monoacylglycerol, diacylglycrol, free fatty acids (only when acetate is the precursor), phosphatidic acid, phosphatidylcholine, and most notably phosphatidylglycerol. The fraction of phosphatidylglycerol synthesized is greatly increased by the presence of manganese chloride in the reaction mixture. Glycerol-3-phosphate-labeled phosphatidylglycerol is equally labeled in the two glycerol moieties of the molecule. Acetate-labeled phosphatidylglycerol is equally labeled in both acyl groups. Position one contains primarily oleate, linoleate and small amounts of palmitate. Position two contains primarily palmitate. No radioactive trans-Delta(3)-hexadecenoate was detected. The labeling patterns indicate that the radioactive phosphatidylglycerol is the product of de novo chloroplast lipid biosynthesis and furthermore, phosphatidylglycerol may be a substrate for fatty acid desaturation.