Alterations in the molecular species of rat liver lecithin by corn-oil feeding to essential fatty acid-deficient rats as a function of time

The present paper describes, as a function of time, the qualitative and quantitative alterations in the molecular species pattern of rat liver lecithin which are observed when corn oil is fed to essential fatty acid-deficient rats. One of the most important changes observed was a very rapid replacement of (1-palmitoyl-2-eicosatrienoyl)- and (1-stearoyl-2-eicosatrienoyl)-lecithin by (1-palmitoyl-2-arachidonoyl)- and (1-stearoyl-2-arachidonoyl)-lecithin, respectively. Whereas this event occurred during a period of 9 days of corn oil feeding, the increase of (1-palmitoyl-2-linoleoyl)- and (1-stearoyl-2-linoleoyl)-lecithin had already reached the maximum level after 3 days. On the other hand a very rapid disappearance of the species (di-octadecenoyl)- and (1-hexadecenoyl-2-octadecenoyl)-lecithin was observed during this period. The decrease of the species (1-stearoyl-2-octadecenoyl)-lecithin was rather slow, whereas (1-palmitoyl-2-octadecenoyl)-lecithin started to diminish only 9 days after the change in diet. The results presented here suggest that different metabolic pathways may contribute, to a different extent, to the replacement of the individual molecular species of rat liver lecithin induced by a change of diet

Studies on phospholipase A and its zymogen from porcine pancreas III. Action of the enzyme on short-chain lecithins

1. 1. Short-chain lecithins (with C6, C7, and C8 fatty acid esters) have been used to study kinetically the enzymatic hydrolysis by pancreatic phospholipase A (EC 3.1.1.4) in aqueous systems, without the addition of emulsifiers. 2. 2. Although phospholipase A is able to attack these substrates in molecularly dispersed form, micellar solutions are hydrolyzed at a much higher rate. 3. 3. Of the three substrates examined, dioctanoyllecithin appeared to be the best substrate. Differences in maximal velocities might be interpreted in terms of interfacial area per molecule. 4. 4. Ca2+ is specifically required for activity of pancreatic phospholipase A. The kinetic results are consistent with a random mechanism in which the metal ion combines with the enzyme independently of the substrate. The substrate was found to combine with the enzyme independently of the metal ion concentration. 5. 5. Kinetic parameters were determined with diheptanoyllecithin as a substrate over a pH range from 5 to 9. Maximal binding of enzyme with substrate was observed at pH : 6. The affinity of the enzyme for Ca2+ decreased at pH values below 6.5. 6. 6. With diheptanoyllecithin as substrate, maximal velocities at infinite substrate and Ca2+ concentrations showed an optimum at pH 5.75. 7. 7. NaCl at high concentrations (up to 3.9 M) gave a 80-fold stimulation of the vmax (diheptanoyllecithin as substrate). The Ks value decreased slightly with increasing salt concentrations, while the KCa2+ increased very strongly. The activating effect of salt is presumed to be caused by a change of the properties of the lipid-water interface

Studies on phospholipase a and its zymogen from porcine pancreas IV. The influence of chemical modification of the lecithin structure on substrate properties

1. 1. A series of chemically modified lecithins were used to investigate by kinetic analyses their substrate c.q. inhibitor properties for porcine pancreatic phospholipase A. The substrate analogues used were systematically modified in: the stereochemical configuration, the susceptible ester bond, the phosphate moiety, the alkylchains, the glycerol backbone and in the position of the phosphorylcholine moiety. 2. 2. The desired relationship between chemical structure and inhibitory properties requires elimination of purely physical effects of the inhibitor on the organization of the substrate molecules at the lipid-water interface. 3. 3. Lecithins of the opposite stereochemical configuration and certain lecithin analogues with a modification of the susceptible ester bond were found to be purely competitive inhibitors. The 1-sn-phosphatidylcholines have Ki values identical to the K8 values of the corresponding 3-sn-phosphatidylcholines. The lecithin analogues with an acylamide linkage at the 2-position were found to be the most potent competitive inhibitors, while on the contrary substitution of the acylester bond by a sulfonyl ester linkage does not give rise to inhibitory properties. 4. 4. Lecithins with a modification in the glycerol-phosphate bond and in the position of the phosphorylcholine moiety are substrates, but exhibit much lower V values and their binding constants are similar to those of the corresponding normal substrates. 5. 5. Introduction of two methyl groups at the carbon atom adjacent to the carboxyl in the acyl chain of the potentially susceptible ester bond gives a lecithin which is not degraded by the enzyme. The presence of only one methyl branch in this position greatly diminishes the hydrolysis rate, probably due to steric hindrance. 6. 6. Increasing the distance between the susceptible ester bond and the phosphate moiety in a lecithin by introducing a methylene group completely abolishes enzymatic activity. These lecithin analogues were found to be competitive inhibitors. 7. 7. The minimal requirements for a phospholipid to be a substrate for phospholipase A, as established earlier, should be extended to include the fact that the phosphate moiety can be replaced by a phosphonate or sulfonate group

Studies on phospholipase a and its zymogen from porcine pancreas IV. The influence of chemical modification of the lecithin structure on substrate properties

1. 1. A series of chemically modified lecithins were used to investigate by kinetic analyses their substrate c.q. inhibitor properties for porcine pancreatic phospholipase A. The substrate analogues used were systematically modified in: the stereochemical configuration, the susceptible ester bond, the phosphate moiety, the alkylchains, the glycerol backbone and in the position of the phosphorylcholine moiety. 2. 2. The desired relationship between chemical structure and inhibitory properties requires elimination of purely physical effects of the inhibitor on the organization of the substrate molecules at the lipid-water interface. 3. 3. Lecithins of the opposite stereochemical configuration and certain lecithin analogues with a modification of the susceptible ester bond were found to be purely competitive inhibitors. The 1-sn-phosphatidylcholines have Ki values identical to the K8 values of the corresponding 3-sn-phosphatidylcholines. The lecithin analogues with an acylamide linkage at the 2-position were found to be the most potent competitive inhibitors, while on the contrary substitution of the acylester bond by a sulfonyl ester linkage does not give rise to inhibitory properties. 4. 4. Lecithins with a modification in the glycerol-phosphate bond and in the position of the phosphorylcholine moiety are substrates, but exhibit much lower V values and their binding constants are similar to those of the corresponding normal substrates. 5. 5. Introduction of two methyl groups at the carbon atom adjacent to the carboxyl in the acyl chain of the potentially susceptible ester bond gives a lecithin which is not degraded by the enzyme. The presence of only one methyl branch in this position greatly diminishes the hydrolysis rate, probably due to steric hindrance. 6. 6. Increasing the distance between the susceptible ester bond and the phosphate moiety in a lecithin by introducing a methylene group completely abolishes enzymatic activity. These lecithin analogues were found to be competitive inhibitors. 7. 7. The minimal requirements for a phospholipid to be a substrate for phospholipase A, as established earlier, should be extended to include the fact that the phosphate moiety can be replaced by a phosphonate or sulfonate group