A direct fluorometric assay of benzo[a]pyrene hydroxylase.

A technique to measure the activity of pyruvate carboxylase spectrophotometrically in crude liver homogenates is described. The assay is based on the transformation of oxaloacetate, which is formed during the carboxylation reaction, into citrate in the presence of excess acetyl CoA and citrate synthase. After removal of pyruvate with KBH₄ and of protein with HClO₄, citrate is cleaved with citrate lyase into oxaloacetate and acetate, and oxaloacetate then is measured spectrophotometrically. Optimal concentrations of pyruvate, Mg²⁺, ATP, and KHCO₃ for the carboxylation reaction and the V_max were in good correlation with the data found by others using [¹⁴C]pyruvate.     

The sensitivity of acetyl-coenzyme A carboxylase to citrate stimulation in a homogenate of rat liver containing subcellular particles

1. Although citrate is known to activate purified preparations of acetyl-CoA carboxylase, it had no stimulatory effect on the incorporation of [¹⁴C]acetate into long-chain fatty acids in a whole homogenate of rat liver (S_0.7) under conditions in which the activity of acetyl-CoA carboxylase was rate-limiting for fatty acid synthesis. 2. The rate of incorporation of acetyl carbon into fatty acids was estimated in S_0.7 preparations incubated with [¹⁴C]acetate, by measuring the specific radioactivity of the acetyl carbon of acetyl-CoA and the incorporation of ¹⁴C into fatty acids. These estimates were compared with estimates of acetyl-CoA carboxylase activity in the S_0.7 preparation obtained by direct assay in conditions in which the enzyme was in the fully activated state. 3. In the absence of citrate, incorporation of acetyl carbon into fatty acids was about 75% of the value expected if the acetyl-CoA carboxylase in the S_0.7 preparation were in the fully activated state. 4. Incorporation of acetyl carbon into fatty acids in the S_0.7 preparation was stimulated by citrate, but the effect was many times less than the stimulation of [¹⁴C]acetate incorporation by citrate in particle-free preparations. 5. When the mitochondria and microsomes were removed from the S_0.7 preparation, [¹⁴C]acetate incorporation into fatty acids fell to a negligible value and the preparation became highly sensitive to stimulation by citrate. 6. It is suggested that in the presence of mitochondria and microsomes, and in the intact liver cell, the degree of activation of acetyl-CoA carboxylase is such that citrate activation may not be of physiological significance.     

Acetyl-coenzyme A carboxylase in maize leaves

Purified chloroplasts from mesophyll and bundle sheath cells of maize leaves have been shown to be the location of acetyl-CoA carboxylase. In disrupted chloroplasts the enzyme was recovered in the stromal fraction, along with protein-bound biotin; acetyl-CoA carboxylase activity did not require a membrane component. Mg²⁺ and ATP are required for activity and sulfhydryl protecting agents enhance stability of the enzyme. Acetyl-CoA carboxylase activity was independent of leaf development in cell-free extracts of maize. Comparison of acetyl-CoA carboxylase activity with [¹⁴C]acetate incorporation into lipids, in isolated chloroplasts from developing leaves of maize, indicate that acetyl-CoA carboxylase is not limiting fatty acid synthesis.     

Relationship between fatty acid binding proteins,acetyl-CoA formation and fatty acid synthesis in developing human placenta

The relationship between fatty acid binding proteins, ATP citrate lyase activity and fatty acid synthesis in developing human placenta has been studied. Fatty acid binding proteins reverse the inhibitory efect of palmitoyl-CoA and oleate on ATP citrate lyase and fatty acid synthesis. In the absence of these inhibitors fatty acid binding proteins activate ATP citrate lyase and stimulate [ 1-¹⁴ C] acetate incorporation into placental fatty acids indicating binding of endogenous inhibitors by these proteins. Thus these proteins regulate the supply of acetyl-CoA as well as the synthesis of fatty acids from that substrates. As gestation proceeds and more lipids are required by the developing placenta fatty acid binding protein content, activity of ATP citrate lyase and rate of fatty acid synthesis increase indicating a cause and efect relationship between the demand of lipids and supply of precursor fatty acids during human placental development.     

Molecular and catalytic properties of the acetoacetyl-coenzyme A thiolase of Escherichia coli.

The acetoacetyl-CoA-thiolase, a product of the acetoacetate degradation operon (ato) was purified to homogeneity as judged by polyacrylamide-gel electrophoresis at pH 4.5, 7.0, and 8.3. The enzyme has a molecular weight of 166,000 and is composed of four identical subunits. The subunit molecular weight is 41,500. Histidine was the sole N-terminal amino acid detected by dansylation. The thiolase contains eight free sulhydryl residues and four intrachain disulfide bonds per mole. The ato thiolase catalyzes the CoA- dependent cleavage of acetoacetyl-CoA and the acetylation of acetyl-CoA to form acetoacetyl-CoA. The maximal velocity in the direction of acetoacetyl-CoA cleavage was 840 nmol min^? (enzyme unit)^?1 and the maximal velocity in the direction of acetoacetyl CoA formation was 38 nmol min^?1 (enzyme unit)^?1. Like other thiolases, the ato thiolase was inactivated by sulfhydryl reagents. The enzyme was protected from inactivation by sulfhydryl reagents in the presence of the acyl-CoA substrates, acetyl-CoA and acetoacetyl-CoA; however, no protection was obtained when the enzyme was incubated with the acetyl-CoA analog, acetylaminodesthio-CoA. Consistent with these results was the demonstration of an acetyl-enzyme compound when the thiolase was incubated with [1-¹⁴C]acetyl-CoA. The sensitivity of the acetyl-enzyme bond to borohydride reduction and the protection afforded by acyl-CoA substrates against enzyme inactivation by sulfhydryl reagents indicated that acetyl groups are bound to the enzyme by a thiolester bond.     

Elongation of acyl-CoAs by microsomes from etiolated leek seedlings

Long chain fatty acid synthesis was studied using etiolated leek seedling microsomes. In the presence of ATP, [2-¹⁴C]malonyl-CoA was incorporated into fatty acids of C₁₆C₂₆. The omission of ATP, even in the presence of acetyl-CoA, led to a complete loss of activity, which was restored by addition of exogeneous acyl-CoAs. Comparison of acyl-CoA (C₁₂C₂₄) elongation showed that stearoyl-CoA, in the presence of [2-¹⁴C]malonyl-CoA, was the more efficient precursor leading to the formation of fatty acids having a chain length of C₂₀C₂₆. [1-¹⁴C]C₁₆CoA and [1-¹⁴C]C₁₈CoA were elongated in the presence of malonyl-CoA, without degradation of the acyl chain. The time-course and the malonyl-CoA concentration curves showed that [1-¹⁴C]C₁₈CoA was a better primer than [1-¹⁴C]C₁₆CoA. Acyl-CoA elongation was also studied over the concentration range 4.5–45 μM [1-¹⁴C]C₁₈CoA. Comparison of the radioactivity incorporated into the fatty acids formed using [2-¹⁴C]malonyl-CoA in the presence of C₁₈CoA, on the one hand, and [1-¹⁴C]C₁₈CoA in the presence of malonyl-CoA, on the other, demonstrated clearly that the acyl chain of the acyl-CoA was elongated by malonyl-CoA.     

Affinity labelling of rat liver acetyl-CoA car☐ylase by a 2′,3′-dialdehyde derivative of ATP

The interaction of rat liver acetyl-CoA car☐ylase with a 2′,3′-dialdehyde derivative of ATP (oATP) has been studied. The degree of the enzyme inactivation has been found to depend on the oATP concentration and the incubation time. ATP was proved to be the only substrate which protected the inactivation. Acetyl-CoA did not effect inactivation, while HCO₃⁻ accelerated the process. K_i values for oATP in the absence and presence of HCO₃⁻ were 0.35 ± 0.04 and 0.5 ± 0.06 mM , and those of the modification constant (k_mod) were 0.11 and 0.26 min⁻¹ respectively. oATP completely inhibited the [¹⁴C]ADP ⇌ ATP exchange and did not effect the [¹⁴C]acetyl-CoA ⇌ malonyl-CoA exchange. Incorporation of ∼1 equivalent of [³H]oATP per acetyl-CoA car☐ylase subunit has been shown. No recovery of the modified enzyme activity has been observed in Tris or β-mercaptoethanol containing buffers, and treatment with NaB³H₄ has not led to³H incorporation. The modification elimination of the ATP triphosphate chain. The results indicated the affinity modification of acetyl-CoA car☐ylase by oATP. It was shown that the reagent apparently interacted selectively with the ɛ-amino group of lysine in the ATP-binding site to form a morpholine-like structure.     

Genetic enhancement of fatty acid synthesis by targeting rat liver ATP:citrate lyase into plastids of tobacco

ATP:citrate lyase (ACL) catalyzes the conversion of citrate to acetyl-coenzyme A (CoA) and oxaloacetate and is a key enzyme for lipid accumulation in mammals and oleaginous yeasts and fungi. To investigate whether heterologous ACL genes can be targeted and imported into the plastids of plants, a gene encoding a fusion protein of the rat liver ACL with the transit peptide for the small subunit of ribulose bisphosphate carboxylase was constructed and introduced into the genome of tobacco. This was sufficient to provide import of the heterologous protein into the plastids. In vitro assays of ACL in isolated plastids showed that the enzyme was active and synthesized acetyl-CoA. Overexpression of the rat ACL gene led to up to a 4-fold increase in the total ACL activity; this increased the amount of fatty acids by 16% but did not cause any major change in the fatty acid profile. Therefore, increasing the availability of acetyl-CoA as a substrate for acetyl-CoA carboxylase and subsequent reactions of fatty acid synthetase has a slightly beneficial effect on the overall rate of lipid synthesis in plants.     

Isolation and properties of naphthoate synthetase from Mycobacterium phlei

Cell-free extracts obtained by sonication of Mycobacterium phlei cells contain an important enzyme of the menaquinone (= vitamin K₂) biosynthetic pathway. This enzyme, naphthoate synthetase (1,4-dihydroxy-2-naphthoate synthetase), was partially purified by chromatography on Sepharose 6BCL. Conversion of o-succinylbenzoate to 1,4-dihydroxy-2-naphthoate was followed by a radioactivity assay using o-[2,3-¹⁴C₂]succinylbenzoate, or by a spectrophotofluorometric assay. o-[1-¹³C]Succinylbenzoate was converted intact by the extracts to dihydroxynaphthoate containing ¹³C only in the carboxyl carbon atom. For maximum activity, the enzyme requires ATP, Mg²⁺, and coenzyme A. The pH optimum is 6.9 and the molecular weight approximately 44,000. In the presence of farnesyl pyrophosphate, the extracts convert o-[2,3-¹⁴C₂]succinylbenzoate to ¹⁴C-containing menaquinone.     

Monoterpene and sesquiterpene biosynthesis in glandular trichomes of peppermint (Mentha x piperita) rely exclusively on plastid-derived isopentenyl diphosphate

The subcellular compartmentation of isopentenyl diphosphate (IPP) synthesis was examined in secretory cells isolated from glandular trichomes of peppermint (Mentha x piperita L. cv. Black Mitcham). As a consequence of their anatomy and the conditions of their isolation, the isolated secretory cells are non-specifically permeable to low-molecular-weight water-soluble metabolites. Thus, the cytoplasm is readily accessible to the exogenous buffer whereas the selective permeability of subcellular organelles is maintained. With the appropriate choice of exogenous substrates, this feature allows the assessment of cytoplasmic and organellar (e.g. plastidic) metabolism in situ. Glycolytic substrates such as [¹⁴C]glucose-6-phosphate and [¹⁴C]pyruvic acid are incorporated into both monoterpenes and sesquiterpenes with a monoterpene:sesquiterpene ratio that closely mimics that observed in vivo, indicating that the correct subcellular partitioning of these substrates is maintained in this model system. Additionally, exogenous [¹⁴C]mevalonic acid and [¹⁴C]IPP, which are both intitially metabolized in the cytoplasm, produce an abnormally high proportion of sesquiterpenes. In contrast, incubation with either [¹⁴C]citrate or [¹⁴C]acetyl-CoA results in the accumulation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) with no detectable isoprenoids formed. Taken together, these results indicate that the cytoplasmic mevalonic acid pathway is blocked at HMG-CoA reductase and that the IPP utilized for both monoterpene and sesquiterpene biosynthesis is synthesized exclusively in the plastids.     

N-acetylation of dopamine and tyramine by mosquito pupae (Aedes togoi)

The $\text{N-}\text{acetyltransferase}$ (NAT) activity of mosquito pupae was measured by a radioenzymatic assay, using [¹⁴C]-, [³H]dopamine, [¹⁴C]tyramine or [¹⁴C]acetyl-CoA. The pupal extract could also generate acetyl-CoA from ATP, acetate and CoA for this acetylation reaction. Both the dopamine- and tyramine-NAT reactions proceeded linearly up to 20 min at an optimum pH of 8.4. It is possible that the same enzyme is involved in the acetylation of both biogenic amines as shown by the competitive inhibition kinetics obtained, and the similarities of the NAT reaction with both amines, in the presence of metal chelators, metal ions, SH reagents and MAO inhibitors. Mn²⁺ stimulated and Zn²⁺ inhibited the reaction. The specific activity of NAT in individual pupae measured soon after pupation showed no significant difference between the male and female pupae: the values obtained were, respectively, $\text{893 ± 57}$ and $\text{861 ± 30}$ pmol [¹⁴C]NAcT formed/min per mg protein and $\text{21.9 ± 1.2}$ and $\text{22.0 ± 1.4}$ pmol [³H]NADA formed/min per mg protein.     

Effect of carnitine on mitochondrial oxidation of palmitoylcarnitine

The effects of carnitine on the metabolism of palmitoylcarnitine were studied by using isolated rat liver mitochondria. Particular attention was given to carnitine acyltransferase-mediated interactions between carnitine and the mitochondrial CoA pool. Carnitine concentrations less than 1.25mm resulted in an increased production of acetylcarnitine during palmitoylcarnitine oxidation. Despite this shunting of C₂ units to acetylcarnitine formation, no change was observed in the rate of oxygen consumption or major product formation (citrate or acetoacetate). Further, no changes were observed in the mitochondrial content of acetyl-CoA, total acid-soluble CoA or acid-insoluble acyl-CoA. These observations support the concept, based on studies in vivo, that the carnitine/acylcarnitine pool is metabolically sluggish and the acyl-group flux low as compared with the CoA/acyl-CoA pool. Acid-insoluble acyl-CoA content was decreased and CoA content increased at carnitine concentrations greater than 1.25mm. When [¹⁴C]carnitine was used in the incubations, it was demonstrated that this resulted from acid-insoluble acylcarnitine formation from intramitochondrial acid-insoluble acyl-CoA mediated by carnitine palmitoyltransferase B. Again, the higher carnitine concentrations resulted in no changes in the rates of oxygen consumption or major product formation. The above effects of carnitine were observed whether citrate or acetoacetate was the major product of oxidation. In contrast, an increase in acetyl-CoA concentration was observed at high carnitine concentrations only when acetoacetate was the product. Since the rate of acetoacetate production was not changed, these higher acetyl-CoA concentrations suggest that a new steady state had been established to maintain acetoacetate-production rates. Since there was no change in acetyl-CoA concentration when citrate was the major product, a change in the activity of the pathway utilizing acetyl-CoA for ketone-body synthesis and the potential regulation of this pathway must be considered.     

Application of enzymatically synthesized short-chain-length hydroxy fatty acid coenzyme A thioesters for assay of polyhydroxyalkanoic acid synthases

Various hydroxyacyl coenzyme A (CoA) thioesters were synthesized from the corresponding hydroxyalkanoic acid (such as e.g. [3-¹⁴C]d-(–)-hydroxybutyric acid, [1-¹⁴C]d-lactic acid, [1-¹⁴C]l-lactic acid, etc.) and from acetyl-CoA employing the propionate CoA transferase of Clostridium propionicum. Preparative isolation of the thioesters on hydrophobic matrices and analysis by HPLC are reported. These thioesters were subjected to a radiometric or a spectrometric assay of polyhydroxyalkanoic acid (PHA) synthase activity. The latter was based on the release of CoA from, for example, d-(–)-3-hydroxybutyryl-CoA, which was detected spectroscopically at 412 nm by reduction of 5,5-dithiobis(2-nitrobenzoic acid) and provided a convenient assay of poly(3-hydroxybutyrate) synthase. When [1-¹⁴C]lactyl-CoA was used as substrate in a PHA synthase assay employing crude extracts obtained from various wild-type strains, [1-¹⁴C]lactyl-CoA was used as a substrate at a rate that was only less than 10^–4 of the rate than with [3-¹⁴C]d-(–)-3-hydroxybutyryl-CoA or was negligible. One exception was a recombinant strain of Escherichia coli, which overexpressed the PHA synthase complex of Chromatium vinosum and which used [1-¹⁴C]d-lactyl-CoA as substrate at a relatively high rate. Correspondence to: A. Steinbüchel     

Escherichia coli coenzyme A-transferase: Kinetics,catalytic pathway and structure

The inducible acetyl-CoA:acetoacetate CoA-transferase of Escherichia coli catalyzes the transfer of CoA from acetyl-CoA to acetoacetate by a mechanism involving a covalent enzyme-CoA compound as a reaction intermediate. Acetyl-CoA + enzyme ? enzyme-CoA + Acetate Enzyme-CoA + acetoacetate ? acetoacetyl-CoA + enzyme These conclusions are based on the following data: 1) In the absence of acetoacetate, the maximal velocity of exchange of [¹⁴C]acetate into acetyl-CoA was comparable with maximal velocity of the complete reaction. 2) Incubation of the enzyme with NaBH₄ after preincubation with an acyl-CoA substrate inactivated the enzyme by reduction of a glutamate residue in the β subunit of the CoA-transferase to α-amino-δ-hydroxyvaleric acid. Given the susceptibility of thioesters to borohydride reduction, the enzyme-CoA bond is a γ-glutamyl thiolester 3) Following incubation of the enzyme with a fluorescent derivative of acetyl-CoA, 1,N⁶-ethenoacetyl-CoA, etheno-CoA was bound to the CoA-transferase. Free etheno-CoA did not bind to the enzyme.     

ATP synthesis in a succinate decarboxylase system from Fasciola hepatica mitochondria

Köhler P. B.,Ryant C. and Behm Carolyn A. 1978. ATP synthesis in a succinate decarboxylase system from Fasciola hepatica mitochondria. International Journal for Parasitology8: 399–404. Succinate decarboxylation was measured by the formation of ¹⁴CO₂ from 1,4-¹⁴C-succinate in a particle free, dialysed mitochondrial extract from liver fluke. It has an absolute requirement for Mg²⁺ and CoA. ATP, ADP and inorganic phosphate are essential for optimal activity. Ap₅A, an inhibitor of adenylate kinase, and glutathione are also necessary. GTP supports decarboxylation as well as ATP, provided ADP is also present. The formation of CO₂ and propionate greatly exceeds the amount of ATP and CoA initially present in the reaction mixture. A net, substrate-level phosphorylation of ADP occurs, the amount of ATP formed being equivalent to the production of CO₂ or propionate. This system is inhibited in flukes incubated in vitro with mebendazole.It is concluded that ATP is required to spark the fermentation system when succinate is the initial substrate and intermediate substrates are absent; that the terminal step in propionate formation is catalysed by a transferase which transfers CoA from propionyl CoA to succinate; and that ATP formation is coupled to the decarboxylation of methylmalonyl-CoA. A reaction scheme is presented.     

ACETYL-CoA SYNTHESIZING ENZYME ACTIVITIES IN SINGLE NERVE CELL BODIES OF RABBIT

Activities of five enzymes (pyruvate dehydrogenase complex; citrate synthase, EC 4.1.3.7; carnitine acetyltransferase, EC 2.3.1.7; acetyl-CoA synthetase, EC 6.2.1.1; and ATP citrate lyase, EC 4.1.3.8) were determined in cell bodies of anterior horn cells and dorsal root ganglion cells from the rabbit. For comparison, molecular layer, granular layer and white matter from rabbit and mouse cerebella and cerebral cortex and striatum from the mouse were analyzed. Samples (3–85 ng dry weight) were assayed in 180 to 370 ml of assay reagents containing CoASH and other substrates in excess. By using ‘CoA cycling’, the assay systems were devised to amplify and measure small amounts of acetyl-CoA formed during the enzyme reactions. Carnitine acetyltransferase was the most active enzyme in single nerve cell bodies and all layer samples, except for rabbit and mouse cerebellar white matter. Citrate synthetase was the lowest in single cell bodies. The activities of carnitine acetyltransferase and acetyl-CoA synthetase (656 and 89.8 mmoles of acetyl-CoA formed/kg of dry weight/h at 38°C) from dorsal root ganglion cells were about 2-fold higher than those from anterior horn cells. The activity of ATP citrate lyase (134mmol of acetyl-CoA formed/kg of dry weight/h at 38°C) from anterior horn cells was approximately twice that from dorsal root ganglion cells. The activity of this enzyme was distributed in a wider range in anterior horn cells than dorsal root ganglion cells. The second highest activity (80.0 mmol of acetyl-CoA formed/kg of dry weight/h at 38°C) of ATP citrate lyase was found in striatum where cholinergic interneurones are abundant. Relatively higher activities of this enzyme were found in cerebellar granular layer and white matter which are known to contain the cholinergic mossy fibers. These results suggested that cholinergic neurones contain higher activity of ATP citrate lyase which is thought to supply acetyl-CoA to choline acetyltransferase (EC 2.3.1.6) as a substrate to form acetylcholine.     

Formation of coA esters of cinnamic acid derivatives by extracts of Brassica napo-brassica root tissue

An enzyme fraction from aged swede root disks catalyses the formation of CoA thioesters of cinnamic acids in the presence of CoA, ATP and Mg²⁺. The enzyme shows activity only to those cinnamic acid derivatives bearing a phenolic OH group, p-coumaric and ferulic acids being the most active substrates. The requirement for Mg²⁺ can be replaced by Mn²⁺, Co²⁺ or Ni²⁺. The requirement for ATP could not be replaced by GTP, CTP, UTP, ADP or AMP. ADP and AMP, but not pyrophosphate, inhibited the ATP dependent activation of p-coumarate. The activity was inhibited by N-ethylmaleimide and p-chloro-mercuribenzoate which suggests a requirement for -SH groups for activation. The activity of the enzyme is low in freshly prepared disks but rises during ageing, particularly if the ageing is carried out in the presence of low concentrations of ethylene.     

Molecular biology of cytosolic acetyl-CoA generation

ATP citrate lyase (ACL) catalyses the ATP-dependent reaction between citrate and CoA to form oxaloacetate and acetyl-CoA. Our molecular characterizations of the cDNAs and genes coding for the Arabidopsis ACL indicate that the plant enzyme is heteromeric, consisting of two dissimilar subunits. The A subunit is homologous to the N-terminal third of the animal ACL, and the B subunit is homologous to C-terminal two-thirds of the animal ACL. Using both ACL-A- and ACL-B-specific antibodies and activity assays we have shown that ACL is located in the cytosol, and is not detectable in the plastids, mitochondria or peroxisomes. During seed development, ACL-A and ACL-B mRNA accumulation is co-ordinated with the accumulation of the cytosolic homomeric acetyl-CoA carboxylase mRNA. Antisense Arabidopsis plants reduced in ATP citrate lyase activity show a complex phenotype, with miniaturized organs, small cell size, aberrant plastid morphology and reduced cuticular wax. Our results indicate that ACL generates the cytosolic pool of acetyl-CoA, which is the substrate required for the biosynthesis of a variety of phytochemicals, including cuticular waxes and flavonoids.     

Increased Fatty Acid beta-Oxidation after Glucose Starvation in Maize Root Tips

The effects of glucose starvation on the oxidation of fatty acids were studied in excised maize (Zea mays L.) root tips. After 24 hours of glucose starvation, the rate of oxidation of palmitic acid to CO₂ by the root tips was increased 2.5-fold. Different enzyme activities were tested in a crude particulate fraction from nonstarved root tips and those starved for 24 hours. The activities of the β-oxidation enzymes crotonase, hydroxyacyl-coenzyme A (CoA) dehydrogenase, and thiolase and those of catalase, malate synthase, and peroxisomal citrate synthase were higher after starvation. However, no isocitrate lyase activity was detected, thus suggesting that the glyoxylate cycle does not operate. The overall β-oxidation activity was assayed as the formation of [¹⁴C]acetyl-CoA from [¹⁴C]palmitic acid after high-performance liquid chromatography analysis of the CoA derivatives. An activity was detected in sugar-fed root tips, and it was increased by two-to fivefold in starved roots. Because the recovery of enzyme activities is only marginally better in starved roots compared with nonstarved roots, these results indicate that the β-oxidation activity in the tissues is increased during sugar starvation. This increase is probably an essential part of the response to a situation in which lipids and proteins replace carbohydrates as the major respiratory substrates. These results are discussed in relation to the metabolic changes observed in senescing plant tissues.     

Synthesis of Long-Chain Acyl-CoA in Chloroplast Envelope Membranes

The chloroplast envelope is the site of a very active long-chain acylcoenzyme A (CoA) synthetase. Furthermore, we have recently shown that an acyl CoA thioesterase is also associated with envelope membrane (Joyard J, PK Stumpf 1980 Plant Physiol 65: 1039-1043). To clarify the interacting roles of both the acyl-CoA thioesterase and the acyl-CoA synthetase, the formation of acyl-CoA in envelope membranes was examined with different techniques which permitted the measurement of the actual rates of acyl-CoA formation. Using [¹⁴C]ATP or [¹⁴C]oleic acid as labeled substrates, it can be shown that the envelope acyl-CoA synthetase required both Mg²⁺ and dithiothreitol. Triton X-100 slightly stimulated the activity. The specificity of the acyl-CoA synthetase was determined either with [¹⁴C]ATP or with [³H]CoA as substrates. The results obtained in both cases were similar, that is, as substrates, the unsaturated fatty acids were more effective than saturated fatty acids, the velocity of the reaction increased from lauric acid to palmitic acid, and the maximum velocity was obtained with unsaturated C₁₈ fatty acids.