Hydrolysis Of Lysolecithin Research Articles

A binding study of phospholipase A2 with lecithin, lysolecithin and their tetrahedral intermediates using molecular modeling.

We used molecular modeling to examine the binding of 1,2-dioctanoyl-sn-glycero-3-phosphocholine (a lecithin), 1-octanoyl-sn-glycero-3-phosphocholine (a lysolecithin) and their tetrahedral intermediates in the catalytic site of phospholipase A2 (PLA2). We performed energy minimization on each complex, computed the binding energy, determined the relative binding energy among the complexes and calculated the difference in inter- and intramolecular energies of the components in the complexes. We found that the calculated orientation of the sn-1 ester bond of lysolecithin in the active site is similar to that of the sn-2 ester bond in lecithin, thus permitting PLA2 to hydrolyze lysolecithin using the same mechanism as it uses to hydrolyze lecithin. On the other hand, the binding of lecithin is energetically more favorable by 4.5 kcal/mol than the binding of lysolecithin to the enzyme, and the binding of the lecithin tetrahedral intermediate is also energetically more favorable by 19.7 kcal/mol than the binding of the lysolecithin tetrahedral intermediate to the enzyme, which explains why lecithin is a better substrate than lysolecithin in the catalytic site. These results indicate that the activation energy for the hydrolysis of lysolecithin is higher than that for lecithin, consistent with the observed slower rate for the hydrolysis of lysolecithin.

Is there a specific lysophospholipase in human pancreatic juice?

The existence of a specific lysophospholipase in human pancreatic juice was evaluated. The proteins were separated by a series of chromatographic steps including Sephacryl S-200, cholate-Sepharose 4B, Sephadex G-100 and CM-Sephadex G-50. The enzyme activities against 1-palmitoyl lysolecithin (LL) as well as tributyrin (TB) and p-nitrophenyl butyrate (PNPB) were determined in all the fractions of these purification procedures. Enzyme activity against LL was always eluted in parallel with activities against TB and PNPB, and no unique activity against LL could be found. The specific activity against LL was 40-times lower than that against PNPB and 200-times lower than that against TB. It is concluded that there is no unique lysophospholipase in human pancreatic juice and that the hydrolysis of lysolecithin is most likely performed by carboxyl ester lipase.

Enzymatic hydrolysis of 1-monoacyl-SN-glycerol-3-phosphoryl-choline (1-lysolecithin) by phospholipases from peanut seeds.

Hydrolysis of 1-lysolecithin (1-acyl glycerophosphorylcholine [1-acyl GPC]) by preparations of phospholipase D from peanut seeds was investigated. 1-Lysolecithin was hydrolyzed at a much slower rate than phosphatidylcholine (lecithin). Although Ca+2 ions are required for the cleavage of lecithin by the enzyme, their effect on the hydrolysis of lysolecithin depended upon the concentration of the substrate: at 0.2 mM 1-lysolecithin, Ca+2 ions increased the reaction rates, whereas at concentrations of the substrate lower than 0.1 mM, Ca+2 ions were inhibitory. A broad pH activity curve between 5 and 8 was obtained with higher rates in the alkaline range, both in the absence and presence of Ca+2 ions. The increased hydrolysis of lysolecithin due to Ca+2 was noticed over the entire pH range. Upon storage of the enzyme solutions at 4 C, decreased rates of hydrolysis of lecithin were observed, with t 1/2 values of ca. 50 and 100 days depending on the purity of the preparation. During the same period, no reduction occurred in the activity of these preparations on lysolecithin as substrate. The effects of Ca+2 ions and the analysis of the products of 1-acyl GPC cleavage by the enzyme preparations revealed the presence of more than one enzyme and the formation of the following compounds: lysophosphatidic acids (1 acyl glycerophosphoric acids), free fatty acids, glycerophosphorylcholine, and choline. The possible pathways leading to the degradation of lysolecithin and the formation of these products include reactions catalyzed by lysophospholipase A1 (lysophosphatidylcholine 1-acyl hydrolase, E.C. 3.1.1.5) and a phosphodiesterase (L-3-glycerylphosphorylcholine glycerophosphohydrolase, E.C.3.1.4.2), in addition to phospholipase D (phosphatidyl-choline phosphatidohydrolase, E.C. 3.1.4.4).

Phospholipase B activity of a purified phospholipase A from Vipera palestinae venom

Phospholipase was isolated (in two fractions) from Vipera palestinae venom and it was shown to possess phospholipase A activity (hydrolyzing diacyl-sn-glycerophosphorylcholines, e.g., lecithin, in the 2-position) as well as lysophospholipase (phospholipase B) activity (hydrolyzing 1-monoacyl-sn-glycerophosphorylcholines, e.g., lysolecithin, yielding free fatty acid and glycerophosphorylcholine). Each of the two purified enzyme fractions was homogeneous as judged by electrophoresis on acrylamide gel and by immunodiffusion and immunoelectrophoresis, and both had essentially equal activities. The ratio of the specific activity, at various purification stages, to the specific activity of the whole venom was the same for A activity (substrate lecithin) as for B activity (substrate lysolecithin). The enzyme has a molecular weight of 16,000, six S-S bridges, and no free thiol groups. At pH 7, dimerization was observed in the ultracentrifuge. A dissociation constant of about 10(-5) m was estimated. The amino acid composition for both fractions (140 amino acid residues) was found to be essentially the same. The A activity had a pH optimum at 9; B activity was low at this pH but increased steadily beyond pH 10.5. For the hydrolysis of lysolecithin the Lineweaver-Burk plot was found to be linear, giving K(m) = 1.1 mm and k(cat) = 0.55 sec(-1) at 37 degrees C and pH 10. 2-Deoxylysolecithin was also hydrolyzed by the enzyme at pH 10, with k(cat) = 0.01 sec(-1) (zero-order kinetics in the range 0.5-2.5 mm). For lecithin these constants could not be determined, but at 0.25 mm substrate the hydrolysis rate (at pH 9) of lecithin was about 1000 times the hydrolysis rate of lysolecithin (at pH 10).

The Subcellular Distribution of Lysophospholipase in Bovine Adrenal Medulla

A study was made of lysophospholipase activity in bovine adrenal medulla. Maximum hydrolysis of lysolecithin was obtained at pH 6.5. The activity of the enzyme was 5.8 μmole × g tissue−1× hour−1. Differential centrifugation showed that about 90% of the enzyme was confined to particles. 55% of the activity present in the low speed supernatant was recovered in the “large granule fraction”. About 35% sedimented with the microsomes. Density gradient centrifugation (1.3–2.0 M sucrose) of the large granule fraction revealed that lysosomes and chromaffin granules contained no significant lysophospholipase activity. By the use of a second gradient (1.0–1.6 M), mitochondria could be partly separated from the microsomes which contaminate the large granule fraction. Lysophospholipase appeared to be concentrated in dense microsomal elements. It is concluded that most, if not all, of the lysophospholipase activity in bovine adrenal medulla is localised in microsomal elements.

Utilization of lysolecithin by landschütz ascites tumor in vivo and in vitro

1. 1. The metabolism of lysolecithin by ascites tumor cells and by the liver of tumor-bearing mice was studied. 2. 2. Following intraperitoneal injection of [1- 14C]palmitoyl-[ 32P]lysolecithin into tumor-bearing mice, up to 30% of the injected dose was taken up by the tumor cells and only about 3% by the liver. This relationship was reversed after intravenous injection. 3. 3. The lysolecithin taken up was converted to lecithin through the acylation pathway by both the liver and the tumor cells, as indicated by the conservation of the 14C/ 32P ratio in liver lecithin and only a slight rise of this ratio in tumor lecithin. 4. 4. The change in 14C/ 32P in tumor lecithin, especially in vitro, was shown to be due to reutilization of 14-labeled fatty acid following hydrolysis of lysolecithin. 5. 5. The lysolecithinase activity of intact tumor cells in vitro was inhibited partly by excess free fatty acid and completely by deoxycholate. 6. 6. It is proposed that the lysolecithinase activity of tumor cells is localized on the cell surface.

The role of lysolecithin in phospholipid metabolism of human umbilical and dog carotid arteries

1. 1. The metabolism of lysolecithin by human umbilical and dog carotid arteries was studied. 2. 2. The umbilical artery extracted lysolecithin preferentially from biosynthetically 32P-labelled serum phospholipids, a finding which might have a bearing on the origin of arterial phospholipids under various conditions. 3. 3. The fate of labelled lysolecithin complexed to albumin introduced into the lumen of umbilical arteries was determined with [1- 14C]palmitoyl-[ 32P]lysolecithin; it was shown that a major part of the lysolecithin had been hydrolysed and a part taken up by the artery and converted to lecithin. Evidence was obtained indicating that hydrolysis of lysolecithin occurred mainly on the cell membrane. The labelled lysolecithin was converted to lecithin directly through acylation and was shown also to serve as fatty acid donor for complex lipid synthesis after hydrolysis. 4. 4. Uptake, acylation and hydrolysis of intraluminally introduced labelled lysolecithin was demonstrated in dog carotid artery. The hydrolytic activity was less pronounced than in the umbilical artery. 5. 5. Serum [ 131I] albumin was taken up by human umbilical arteries, and to a much lesser extent by dog carotid arteries. In the latter the uptake of lysolecithin exceeded that of albumin, indicating that they proceed independently. No definite information regarding the interdependence of lysolecithin and albumin uptake by human umbilical arteries was obtained.

Chemical structure and biochemical significance of lysolecithins from rat liver

1. 1. Synthetic lecithins containing in 2-position a [ 14C]fatty acid constituent were found to be hydrolysed by rat-liver homogenates so as to form both 1-acyl-glycero-3-phosphorylcholine and 2-acyl-glycero-3-phosphorylcholine. 2. 2. A comparison of the fatty acid pattern of lysolecithin obtained from rat liver by thin-layer chromatography with the fatty acid composition of 1-acyl-glycero-3-phosphorylcholine prepared by breakdown of liver lecithin with snake venom phospholipase A (EC 3.1.1.4) suggested that two structurally isomeric lysolecithins occur. 3. 3. Phospholipase A degradation of lysolecithin from rat liver previously labelled with 32P demonstrated that, apart from 1-acyl-glycero-3-phosphorylcholine, 2-acyl-glycero-3-phosphorylcholine was also present. Hydrolysis of lysolecithin with phospholipase C (EC 3.1.4.3) and separation of the monoglycerides formed confirmed that rat-liver lysolecithin consists of two structural isomers. 4. 4. A combination of these findings with the results obtained by Lands and co-workers on the selective enzymic transacylation of isomeric lysophosphoglycerides allows a monoacyl-diacyl phospholipid cycle to be formulated. This mechanism is postulated to play a part in maintaining the liquid-crystalline state of membraneous lipids.

The enzymatic acylation and hydrolysis of lysolecithin

The present experiments show that lysolecithin is converted to lecithin by ratliver preparations which are free from cell particles. In this process lysolecithin is esterfied directly rather than being degraded to simpler compounds which are subsequently incorporated into lecithin. Two separate reactions are postulated whereby lysolecithin is converted to lecithin. These reactions are as follows: 2 lysolecithin → lecithin + glycerophosphorylcholine (1) lysolecithin + acyl-CoA → lecithin (2) Reaction 1 is from a quantitative standpoint the more important reaction although Reaction 2 has a faster rate. The Michaelis-Menten plot of lecithin synthesis vs. lysolecithin concentration gave support for Reactions 1 and 2 since the rate curve was anomalous, being composed of two separate parts which gave K m values of 0.08 mM (Reaction 2) and 3.3. mM (Reaction 1). The value of K m obtained for lysolecithin hydrolysis was 1.03 mM. When [ 32P]lysolecithin was used as substrate, the synthesis of lecithin as measured by either 32P-incorporation or by chemical phosphorus was the same and either method could be used to quantitate lysolecithin esterification or lysolecithin hydrolysis. The studies given here show that the rate of metabolism of lysolecithin was essentially constant at pH values between 6.2 and 6.9 but at values greater than 6.9 there was suppression of both lecithin synthesis and lysolecithin hydrolysis. Iodoacetic acid failed to inhibit either lecithin synthesis or lysolecithin hydrolysis whereas HgCl 2 inhibited both. CN- inhibited lecithin synthesis but not lysolecithin hydrolysis. Stearylglycollecithin was found to be inactive as a substrate for lysolecithinase in the rat-liver supernatant fluid but inhibited lysolecithin hydrolysis and lecithin synthesis 73 and 80% respectively. Transesterification has been shown to occur by the use of doubly-labeled lysolecithin.