Fatty Acyl Derivatives Research Articles

Purification and physiological properties of two lipolytic enzymes of Solanum tuberosum

Two lipolytic enzymes have been separated and partially purified from potato tubers. One enzyme of higher isoelectric value, possessed acyl hydrolase activity toward a number of p-nitrophenyl fatty acyl derivatives, the relative activity depending on the fatty acyl chain length. There was also some activity towards phosphatidyl choline. The other enzyme possessed phospholipase and galactolipase activity, but showed a low acyl hydrolase activity towards p-nitrophenyl fatty acyl derivatives. When applied to plant tissues, the enzyme with the greater acyl hydrolase activity caused rapid ion efflux from discs of potato tuber and beetroot, foflowed by reabsorption of ions by the tissues. The purified phospholipase did not produce this effect but induced acid phosphatase leakage from lysosome-enriched fractions of potato sprout tissue. No maceration of tissue or protoplast disruption was observed when either of the two enzymes were incubated with a variety of plant preparations.

Fatty acyl-acyl carrier protein and fatty acyl-CoA as acyl donors in the biosynthesis of phosphatidic acid in [formula omitted

In the synthesis of phosphatidic acid catalyzed by a particulate fraction from Clostridium butyricum in the presence of fatty acyl derivatives of an acyl carrier protein (ACP) and sn -glycero-3-phosphate, the first acylation of glycero-3-P is more rapid with an unsaturated acyl-ACP than with a saturated acyl-ACP. In the phosphatidic acid formed, carbon-1 is preferentially acylated with an unsaturated fatty acid and carbon-2 with a saturated fatty acid, as they are in the cellular phospholipids. Acylation by the saturated fatty acid at C 2 is not very specific for the acyl carrier: palmityl-CoA competes effectively with palmityl-ACP.

The oxidation of fatty-acyl derivatives by mitochondria from bovine fetal and calf hearts.

Functional activity of the "external" carnitine palmitoyltransferase in intact mitochondria, prepared from hearts from various sources, was estimated by measuring respiration by mitochondria in the presence of palmitoyl-CoA plus or minus l-carnitine. When palmitoyl-CoA alone was substrate, respiration was not increased above the basal rate under all conditions examined. Addition of l-carnitine increased respiration, provided the ionic strength of the incubation medium was sufficiently high. In the presence of palmitoyl-CoA plus l-carnitine, the rate of respiration increased as the ionic strength was increased to physiological levels. In contrast, the increase in rate of oxygen consumption by heart mitochondria which followed the addition of palmitoyl-l-carnitine was relatively independent of the ionic strength of the medium.Mitochondrial fractions prepared from fetal bovine hearts were shown to possess "external" carnitine palmitoyltransferase activity, as judged by the ability of l-carnitine to stimulate respiration by mitochondria incubated with palmitoyl-CoA under various conditions. These data were discussed in relation to information available concerning the functions of different carnitine acyltransferases in mitochondria.

Mutations enabling Escherichia coli to grow on medium chain fatty acids

New spontaneous mutants of Escherichia coli able to use butyrate or valerate as carbon source have been studied for their growth, adaptation lag and the level of enzymes which may participate in the utilisation of these compounds.Each mutant acquires the simultaneous constitutivity for uptake of these fatty acids, CoA transferase and high thiolase activities. This mutation designated as butR does not affect the regulation of the β‐oxidation nor that of the glyoxylate cycle.Induction or mutation for constitutivity (oleR) of β‐oxidation act multiplicatively on the level of thiolase derepressed by the butR mutation.If the presence of CoA transferase, thiolase and C4–C5 fatty acids entry system is a prerequisite of the growth ability on these fatty acids, the genetic constitutivities (and not the preinduction) of the β‐oxidation (oleR) and particularly of the glyoxylic cycle (aceD) greatly increase the growth capacity. This implies that not only the C, and C, fatty acyl derivatives are very poor inducers of β‐oxidation, as already reported by others, but that some products of their catabolism repress the glyoxylic cycle.The acquisition of the new growth capacity is thus accomplished by spontaneous mutations affecting regulatory processes; one of these discloses activities which are cryptic in the normal parental strains; the physiological significance of these enzymatic potentialities in wild strains and the mechanism of the interaction between two different regulatory units brought to light here are some of the open issues.

Antimicrobial activity of hydroxamic acids.

o-, m- and p-alkyloxybenzohydroxamic acids were synthesized and subjected to the examination for antibacterial and antifungal activity. All the hydroxamic acids tested were almost ineffective for pathogens examined of Enterobacteriaceae, but some of alkyloxybenzo- and fatty acyl-hydroxamic acid were found to be as highly effective for pathogenic fungi as butyl p-hydroxybenzoate being used as a comparison. The increase of carbon number of p-alkyloxybenzo- and fatty acyl-hydroxamic acid led to the increase in their antifungal activity, reached to the maximum at C6 in alkyloxy moiety and C10 in fatty acyl derivatives, and then the gradual decrease in both series. Considering the inhibitory power of hydroxamic acid on plant and bacterial urease, we discussed the possible correlation between antimicrobial activity and inhibitory powers on urease activity of the compounds.

Studies on anti-plasminic agents.

It was shown that dilauroyl- and dimyristoyl-L-lysine were more powerful inhibitors than the other fattyacyl derivatives of L-lysine in the proteolytic system of plasmin. In the case of carbon atom number of fattyacyl radical, upon increasing of carbon atom number the inhibitory activity was enhanced to maximum at the C-12 acyl radical, however, higher homologs decreased the inhibitory activity gradually. The inhibitory effect of dilauroyl-L-lysine given to rabbits intravenously was more remarkable than that of e-aminocaproic acid when the effect was examined by the streptokinase activation test of blood samples taken at various intervals. Reversal by dilauroyl-L-lysine of the accelerated fibrinolysis in circulatroy rabbit blood produced by streptokinase was demonstrated. Dicarbobenzoxy-L-lysine and dilauroyl-L-lysine were shown to be potent antiinflammatory compounds. These compounds were intravenously active as the inhibitor of extravasation of trypan blue in a localized skin area of the rabbit, where the lung extract was injected intracutaneously.

Studies on the Control of Fatty Acid Synthesis: I. STIMULATION BY (+)-PALMITYLCARNITINE OF FATTY ACID SYNTHESIS IN LIVER PREPARATIONS FROM FED AND FASTED RATS

Abstract Hepatic long chain fatty acid synthesis from labeled acetate but not labeled malonyl coenzyme A was greatly increased by dl-palmitylcarnitine or (+)-palmitylcarnitine. Only long chain fatty acyl carnitine derivatives were effective in stimulating lipogenesis. The increase induced by 50 µm (+)-palmitylcarnitine was approximately 5-fold in liver supernatant fractions prepared from fed rats and 10-fold in preparations from fasted animals. (-)-Carnitine and (-)-palmitylcarnitine also increased fatty acid synthesis from acetate, but to a considerably lesser extent. Deoxycarnitine decreased acetate incorporation into fatty acids. Rates of acetate incorporation into fatty acids by fasted liver preparations supplemented with (+)-palmitylcarnitine were higher than those obtained in nonsupplemented livers from fed rats, but were still less than values achieved in (+)-palmitylcarnitine-treated livers from fed animals. Stimulation of fatty acid synthesis by (+)-palmitylcarnitine was shown to be at the level of malonyl-CoA formation. Partially purified preparations of acetyl-CoA carboxylase were activated equally well by either (+)- or (-)-palmitylcarnitine at all citrate concentrations tested, provided that enzyme preparations were free of carnitine palmityltransferase activity. Palmityl-CoA inhibition of acetyl-CoA carboxylase was partially relieved by (+)-palmitylcarnitine in the presence or absence of citrate or malonate. It was concluded that inhibition of hepatic fatty acid synthesis observed in fasted rats could not be accounted for by a decrease in enzyme capacity. Enzymes required for lipogenesis were present but masked in livers from fasted animals. It appeared that acetyl-CoA carboxylase in the complex system was inhibited by a factor (factors) such as palmityl-CoA which could be reversed by (+)-palmitylcarnitine addition, but not by citrate or malonate. (+)-Palmitylcarnitine stimulation of fatty acid synthesis could not be accounted for by invoking inhibition of carnitine palmityltransferase. The possible role of (-)-palmitylcarnitine formation from palmityl-CoA and relationships between effects of the two fatty acyl derivatives on the control of fatty acid synthesis are discussed.

The Identification of Glycerophosphorylglycerol Phosphate as the Deacylation Product of a New Brain Lipid

Abstract A compound has been isolated from mild alkaline methanolysates of brain lipids and identified as 1-(l-3-glycerophosphoryl)-l-glycerol 3-phosphate. It is suggested that it is derived from a corresponding fatty acyl derivative, probably 1-(l-phosphatidyl)-l-glycerol 3-phosphate.

On the Mechanism of Dehydrogenation of Fatty Acyl Derivatives of Coenzyme A: VII. THE NATURE OF THE GREEN COLOR OF BUTYRYL DEHYDROGENASE

On the Mechanism of Dehydrogenation of Fatty Acyl Derivatives of Coenzyme A: VII. THE NATURE OF THE GREEN COLOR OF BUTYRYL DEHYDROGENASE

On the Mechanism of Dehydrogenation of Fatty Acyl Derivatives of Coenzyme A: VI. ISOLATION AND PROPERTIES OF STABLE ENZYME-SUBSTRATE COMPLEXES

On the Mechanism of Dehydrogenation of Fatty Acyl Derivatives of Coenzyme A: VI. ISOLATION AND PROPERTIES OF STABLE ENZYME-SUBSTRATE COMPLEXES

On the Mechanism of Dehydrogenation of Fatty Acyl Derivatives of Coenzyme A. IV. Kinetic Studies

On the Mechanism of Dehydrogenation of Fatty Acyl Derivatives of Coenzyme A. IV. Kinetic Studies

FATTY ACID OXIDATION IN SOLUBLE SYSTEMS OF ANIMAL TISSUES

Summary1. The half‐century of investigations directed towards an understanding of the mechanism of β‐oxidation of fatty acids may be divided into three periods: (a) from 1904 to 1939 when the oxidation could be studied at the level of the intact animal or isolated organ or tissue slice; (b) from 1939 to 1952 when it could be studied at the mitochondrial level; and (c) from 1952 onwards when it could be reconstructed in non‐mitochondrial and soluble enzyme systems.2. The Knoop‐Dakin theory of β‐oxidation could not be directly confirmed owing to the non‐accumulation of any intermediates. The theory was based on deductions from the nature of the end‐products of the metabolism of phenyl fatty acids.3. The study of fatty acid oxidation at the mitochondrial level led to the recognition that the fatty acids are not oxidized as such but only in the form of some derivative whose formation is tied up with oxidative phosphorylation and the production of adenosine triphosphate (ATP).4. The transition from the mitochondrial system to soluble enzymes was facilitated first by the discovery that coenzyme A (CoA) was concerned in acyl transfer reactions and later by the recognition that the active fatty acids are indeed the fatty acyl derivatives of CoA.5. There are four known enzymatic processes by which fatty acyl CoA's are formed: (a) oxidation of pyruvate to acetyl CoA; (b) conversion of fatty acids to fatty acyl CoA's by ATP; (c) replacement of the succinyl group of succinyl CoA by short‐chain fatty acids; and (d) cleavage of β‐ketoacyl CoA's by CoA with formation of a fatty acyl CoA and acetyl CoA.6. Two separate enzymes are known to catalyse the oxidation of fatty acyl CoA's to their corresponding transα, β‐unsaturated derivatives. The first is a green dehydrogenase containing copper and flavin as prosthetic groups which is active upon acyl CoA's from C3 to C8. The metal is essential for the interaction of this dehydrogenase with cytochrome c. The second is a yellow flavoprotein which is active upon acyl CoA's from C4 to C18.7. Unsaturated fatty acyl CoA hydrase catalyses the hydration of transα, β‐ or β, γ‐unsaturated CoA's to their corresponding l(+)‐β‐hydroxyacyl CoA derivatives. The enzyme acts upon all unsaturated derivatives from C4 to at least C12. At equilibrium (pH 9, 250) the ratio β‐hydroxyacyl CoA:total unsaturated acyl CoA is 1. 4:1.8. The β‐hydroxyacyl CoA dehydrogenase catalyses the oxidation of l(+)‐β‐hydroxyacyl CoA by DPN+. The product of oxidation is the corresponding β‐ketoacyl CoA. The enzyme is active over the entire range of fatty acid chain length. The E0of the reaction couple at pH 7.0 and 220 is – 0.224 V. The equilibrium point of the oxidation is strongly pH dependent.9. The β‐ketoacyl CoA cleavage enzyme catalyses the reversible cleavage of β‐ketoacyl CoA's by another molecule of CoA to form acetyl CoA and a new acyl CoA with two carbon atoms less than the parent β‐ketoacyl CoA.10. The new fatty acyl CoA generated in the cleavage reaction undergoes a repeat cycle of β‐oxidation while the C2 unit (acetyl CoA) undergoes condensation with oxalacetate to form citrate.11. Each of the component reactions in the β‐oxidation cycle has been shown to be reversible.12. The asymmetric labelling of acetoacetate formed during oxidation of labelled fatty acids by liver homogenates or mitochondrial suspensions is a phenomenon which can readily be explained in terms of the mechanism of the β‐ketoacyl CoA cleavage enzyme.13. The factors which militate against the accumulation of intermediates during fatty acid oxidation are discussed.14. The accumulation of acetoacetate in any tissue requires a combination of two essential conditions: (a) presence of acyl CoA deacylase; and (b) absence of a β‐ketoacid activation enzyme.15. Assuming that in the diabetic the operation of the citric acid cycle is subnormal by virtue of reduced conversion of glucose to pyruvate, it is possible to explain the accumulation of ketone bodies and its abolition by insulin in terms of the known enzyme reactions of the β‐oxidation cycle.16. The predominance of C16 and C18 fatty acids in lipids may be due to the fact that only the acyl CoA's of these particular acids dissociate to a sufficient degree from combination with the enzymes of the fatty acid oxidizing system as to become available for ester synthesis.It is a great pleasure to acknowledge my indebtedness to Dr Helmut Beinert for his advice and assistance in the preparation of the manuscript and to Drs H. R. Mahler, D. R. Sanadi and S. J. Wakil for their suggestions.