1. Chloroplasts isolated from spinach leaves by using the low-ionic-strength buffers of Nakatani & Barber [(1977) Biochim. Biophys. Acta.461, 510-512] had higher rates of HCO(3) (-)-dependent oxygen evolution (up to 369mumol/h per mg of chlorophyll) and higher rates of [1-(14)C]acetate incorporation into long-chain fatty acids (up to 1500nmol/h per mg of chlorophyll) than chloroplasts isolated by using alternative procedures. 2. Acetate appeared to be the preferred substrate for fatty acid synthesis by isolated chloroplasts, although high rates of synthesis were also measured from H(14)CO(3) (-) in assays permitting high rats of photosynthesis. Incorporation of H(14)CO(3) (-) into fatty acids was decreased by relatively low concentrations of unlabelled acetate. Acetyl-CoA synthetase activity was present 3-4 times in excess of that required to account for rates of [1-(14)C]acetate incorporation into fatty acids, but pyruvate dehydrogenase was either absent or present in very low activity in spinach chloroplasts. 3. Rates of long-chain-fatty acid synthesis from [1-(14)C]acetate in the highly active chloroplast preparations, compared with those used previously, were less dependent on added cofactors, but showed a greater response to light. The effects of added CoA plus ATP, Triton X-100 and sn-glycerol 3-phosphate on the products of [1-(14)C]acetate incorporation were similar to those reported for less active chloroplast preparations. 4. Endogenous [(14)C]acetyl-CoA plus [(14)C]malonyl-CoA was maintained at a constant low level even when fatty acid synthesis was limited by low HCO(3) (-) concentrations. Endogenous [(14)C]acyl-(acyl-carrier protein) concentrations increased with increasing HCO(3) (-) concentration and higher rates of fatty acid synthesis, but were slightly lower in the presence of Triton X-100. It is proposed that rates of long-chain-fatty acid synthesis in isolated chloroplasts at saturating [1-(14)C]acetate concentrations and optimal HCO(3) (-) concentrations may be primarily controlled by rates of removal of the products of the fatty acid synthetase.
A potent cellulase solution was prepared from culture filtrates of an artificiallyproduced mutant of Trichoderma species. The filtrates were diluted to provide a standardised, simulated rumen liquor which was then used to study the correlation between cellulase digestibility and in-vivo digestibility of a range of plant materials. Cell walls of whole, dried plant material were either not attacked by the cellulase or were attacked only very slowly, but cell walls isolated by neutral-detergent extraction were readily hydrolysed. Cellulase digestibility, defined as the percentage of whole, dry plant material solubilised by neutral-detergent extraction followed by exhaustive hydrolysis with standardised cellulase, was highlycorrelatedwith in-vivo dry matter digestibility (DMD) (r = 0.98) and predicted that parameter with reasonable accuracy (r.s.d,, residual standard deviation = 2.83). The form of the regression equation was in-vivo DMD=0.98 x cellulase solubility-10.12, suggesting that the same factors limited cellulase and in-vivo digestibility. The method was simple and reliable and results were known within 48 h.
1. Isolated spinach (Spinacia oleracea) chloroplasts were incapable of accumulating polar lipids when incubated with [1-14C]acetate in a cofactor-free medium. When CoA, ATP and glycerol 3-phosphate were added to incubation media, the accumulated products were non-esterified fatty acids, acyl-CoA and 1,2-diacylglycerol, all intermediates of lipid metabolism. 2. Chloroplast acyl-CoA was used to synthesize phosphatidylcholine only when a microsomal fraction was added back to the incubation medium. 3. The 1,2-diacylglycerol synthesized by isolated chloroplasts was converted almost quantitatively into diacylgalactosylglycerol when exogenous UDP-galactose was available. 4. Stereospecific analyses of the isolated lipids suggested that the diacylglycerol synthesized by isolated chloroplasts may be an important precursor for the synthesis in vivo of diacylgalactosylglycerol and phosphatidylglycerol but was unlikely to be a precursor of phosphatidylcholine. 5. A scheme for plant-lipid biosynthesis is presented that integrates the functions of chloroplasts, the cytoplasm and the endoplasmic reticulum.
If isolated rat hepatocytes are preincubated for 90 min before addition of hormone, glucagon causes a significant (50 %) decrease in fatty acid synthesis without concomitant large decreases in the cellular content of the allosteric activator, citrate. We present evidence that this inhibition can be entirely accounted for by the phosphorylation of the rate-limiting enzyme, acetyl-CoA carboxylase, by cyclic-AMP-dependent protein kinase. In particular : (1) the effect is associated with a 50 % decrease in acetyl-CoA carboxylase activity (measured at physiological citrate concentration) which survives purification of the enzyme; (2) the effect is associated with a selective increase in the phosphorylation of a chymotryptic peptide (peptide 1) which is identical to the peptide containing the major site phosphorylated on purified acetyl-CoA carboxylase by cyclic-AMP-dependent protein kinase ; (3) the effects of glucagon on the kinetic parameters of the enzyme are very similar to the effect of phosphorylation of the purified enzyme, i.e. a decrease in V and an increase in K, for citrate; and (4) all of these effects occur at physiological concentrations of glucagon identical to those producing inhibition of fatty acid synthesis.The main function of fatty acid synthesis in mammals is the conversion of excess carbohydrate into fat, which provides a more concentrated store of energy for long-term utilization. During starvation, carbohydrate must be spared for more critical needs, such as the supply of glucose to the brain. It has been known for some years that glucagon, which is released on starvation, inhibits fatty acid synthesis in the liver [1,2] and that this correlates with a decreased activity of hepatic acetylCoA carboxylase [3,4]. However, the mechanism of this inhibition has not been clear. Acetyl-CoA carboxylase is subject to allosteric regulation, being completely dependent on the fatty acid precursor, citrate, for activity, and being potently inhibited by the end product, long-chain acyl-CoA [5]. Watkins et al. [6] reported that glucagon dramatically inhibited fatty acid synthesis in isolated chicken hepatocytes, and this correlated with a 90% decrease in cytosolic citrate levels. More recently, it has been proposed that phosphorylation of acetyl-CoA carboxylase is an important mechanism of regulation of fatty acid synthesis. This was first suggested by experiments with partially purified rat liver acetyl-CoA carboxylase [7] and our laboratories have now shown that the homogeneous rat mammary [8] and rat liver [9] enzymes can be phosphorylated and inactivated by cyclic-AMP-dependent protein kinase, both effects being reversed by addition of purified protein phosphatase. We have also characterized the protein phosphatases in rat liver which catalyze the dephosphorylation and reactivation [lo].Acetyl-CoA carboxylase becomes labelled when isolated rat [4] and chicken [Ill hepatocytes are incubated with [32P]phosphate. However, while glucagon clearly stimulates this phosphorylation in rat hepatocytes [4], i...
The gene encoding the major intracellular tributyrin esterase of Lactococcus lactis was cloned using degenerate DNA probes based on 19 known N-terminal amino acid residues of the purified enzyme. The gene, named estA, was sequenced and found to encode a protein of 258 amino acid residues. The transcription start site was mapped 233 nucleotides upstream of the start codon, and a canonical promoter sequence was identified. The deduced amino acid sequence of the estA product contained the typical GXSXG motif found in most lipases and esterases. The protein was overproduced up to 170-fold in L. lactis by use of the nisin-controlled expression system recently developed for lactic acid bacteria. The estA gene was inactivated by chromosomal integration of a temperature-sensitive integration vector. This resulted in the complete loss of esterase activity, which could then be recovered after complementation of the constructed esterase-deficient strain with the wild-type estA gene. This confirms that EstA is the main enzyme responsible for esterase activity in L. lactis. Purified recombinant enzyme showed a preference for short-chain acyl esters, surprisingly also including phospholipids. Medium-and long-acyl-chain lipids were also hydrolyzed, albeit less efficiently. Intermediate characteristics between esterases and lipases make intracellular lactococcal EstA difficult to classify in either of these two groups of esterolytic enzymes. We suggest that, in vivo, EstA could be involved in (phospho)lipid metabolism or cellular detoxification or both, as its sequence showed significant similarity to S-formylglutathione hydrolase (FGH) of Paracoccus denitrificans and human EstD (or FGH), which are part of a universal formaldehyde detoxification pathway.
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