Diana H. Petkova

Changes in the Phospholipid Composition and Phospholipid Asymmetry of Ram Sperm Plasma Membranes after Cryopreservation

The changes in the phospholipid composition of spermatozoa plasma membranes after freezing were determined by thin-layer chromatography. The results showed an augmentation of the diphosphatidylglycerol and a diminution of phosphatidylglycerol, phosphatidylserine, and phosphatidylethanolamine in sperm plasma membranes after freezing. In intact sperm cells we observed an elevation of the sphingomyelin and phosphatidylinositol levels and a diminution of the phosphatidylethanolamine and diphosphatidylglycerol levels. The effect of freezing on the phospholipid distribution between the inner and outer monolayers of the plasma membrane was also studied using exogenous phospholipases and trinitrobenzene sulfonate. The most important change we observed after freezing, was the translocation of diphosphatidylglycerol from the inner to the outer monolayer of the plasma membrane.

Phospholipid composition modifications influence phospholipase A2 activity in rat liver plasma membranes

The influence of the phospholipid composition and the physico-chemical properties of rat liver plasma membranes on the activity of membrane-bound phospholipase A2 has been investigated. The plasma membrane composition was modified by the aid of exogenous phospholipases A2, C and D, and by butanol treatment. The partially delipidated membranes thus obtained were enriched with different phospholipids. The steady-state fluorescent anisotropy of 1,6-diphenyl-1,3,5-hexatriene and the lipid order parameter-SDPH in the modified membranes were calculated. It was established that the activity of the membrane-bound phospholipase A2 was higher in rigid membranes and was decreased when the membrane lipid bilayer was fluidized.

Age-related changes in rat liver plasma membrane sphingomyelinase activity

The age-induced changes of some phospholipid fractions, membrane fluidity and neutral membrane-bound sphingomyelinase (EC 3.1.4.12) activity in rat liver plasma membranes have been investigated. Alterations in the percentage participation of phosphatidylcholine and sphingomyelin with aging have been established. Regression analysis indicated a positive linear correlation (r = 0.927) between the membrane-bound neutral sphingomyelinase activity and the phosphatidylcholine percent in the total plasma membrane phospholipids, as well as a negative linear correlation (r = -0.937) between the enzyme activity and the sphingomyelin/phosphatidylcholine ratio.

Age-related changes in rat liver plasma membrane phospholipase A2 activity

The alterations of the lipid composition and fluidity of liver plasma membranes as well as the activity of membrane-bound phospholipase A2 (EC 3.1.1.4) in male and female rats of different ages are investigated. An age-induced increase of the protein/phospholipid and cholesterol/phospholipid ratios is observed, as well as a decrease in the unsaturation index of plasma membrane phosphatidylcholine. These changes induce an augmentation of the steady-state fluorescent anisotropy-rs and structural order parameter for 1,6-diphenyl-1,3,5-hexatriene-SDPH in plasma membranes. The membrane-bound phospholipase A2 activity is enhanced with aging, correlating well with the reduced membrane fluidity. The sex-determined alterations in all these parameters is also investigated.

Phospholipase A2 activity in ram spermatozoa plasma membranes.

A highly active phospholipase A2 in ram spermatozoa plasma membranes has been established. Phospholipase A2 was optimally active at pH 7.4 and was Ca2+ dependent. Low concentrations of K+ (25 mM) were found to inhibit phospholipase A2 activity, whereas higher concentrations induced enzyme reactivation. An inhibitory effect of Zn2+ has also been observed.

Effect of liver plasma membrane fluidity on endogenous phospholipase A2 activity

Investigations have been carried out on the influence of the phospholipid composition and the physicochemical properties of rat liver plasma membranes on the endogenous activity of membrane-bound phospholipase A2. The membrane phospholipid composition was modified by the incorporation of different phospholipids in the lipid bilayer by the aid of lipid transfer proteins. The results indicate that the endogenous activity of phospholipase A2 in liver plasma membranes depends upon membrane fluidity and not upon the presence of a specific phospholipid in the enzymes microenvironment.

Rat liver microsomal phospholipase A2 and membrane fluidity

The influence of the physical state and the lipid composition on microsomal membrane-bound phospholipase A2 activity has been investigated. It was established that the decrease of membrane fluidity expressed by the alterations of the steady-state fluorescent anisotropy (rs) and the structural order parameter for DPH (SDPH) leads to augmentation of phospholipase A2 specific activity. Phosphatidylinositol is the only phospholipid having a specific activating effect on microsomal membrane-bound phospholipase A2.

Resveratrol alters the lipid composition, metabolism and peroxide level in senescent rat hepatocytes

Investigations were performed on the influence of resveratrol on the lipid composition, metabolism, fatty acid and peroxide level in plasma membranes of hepatocytes, isolated from aged rats. Hepatocytes were chosen due to the central role of the liver in lipid metabolism and homeostasis. The obtained results showed that the level of sphingomyelin (SM) and phosphatidylserine (PS) was augmented in plasma membranes of resveratrol-treated senescent hepatocytes. The saturated/unsaturated fatty acids ratio of the two most abundant membrane phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), was decreased as a result of resveratrol treatment. The neutral sphingomyelinase was found to be responsible for the increase of SM and the decrease of ceramide in plasma membranes of resveratrol-treated senescent hepatocytes. Using labeled acetate as a precursor of lipid synthesis we demonstrated, that resveratrol treatment resulted in inhibition mainly of phospholipid synthesis, followed by fatty acids synthesis. Resveratrol induced reduction of specific membrane-associated markers of apoptosis such as localization of PS in the external plasma membrane monolayer and ceramide level. Finally, the content of lipid peroxides was investigated, because the unsaturated fatty acids, which were augmented as a result of resveratrol treatment, are an excellent target of oxidative attack. The results showed that the lipid peroxide level was significantly lower, ROS were slightly reduced and GSH was almost unchanged in resveratrol-treated hepatocytes. We suggest, that one possible biochemical mechanism, underlying the reported resveratrol-induced changes, is the partial inactivation of neutral sphingomyelinase, leading to increase of SM, the latter acting as a native membrane antioxidant. In conclusion, our studies indicate that resveratrol treatment induces beneficial alterations in the phospholipid and fatty acid composition, as well as in the ceramide and peroxide content in plasma membranes of senescent hepatocytes. Thus, the presented results imply that resveratrol could improve the functional activity of the membrane lipids in the aged liver by influencing specific membrane parameters, associated with the aging process.

Phospholipid dependence of the neutral sphingomyelinase in rat liver plasma membranes

Investigations have been carried out on the influence of the phospholipid composition of rat liver plasma membranes and of their physico-chemical properties on the activity of membrane-bound neutral sphingomyelinase. The membrane phospholipid composition was modified by the incorporation of different phospholipids into the membrane bilayer by means of lipid transfer proteins, n-butanol delipidation or exogenous sphingomyelinase (Staphylococcus aureus) treatment. The results indicate that the activity of neutral sphingomyelinase in liver plasma membranes depends upon phosphatidyl choline presence in the membrane bilayer and not upon membrane fluidity.

Effects of age-dependent or liposome-induced alterations in the phospholipid composition on sphingomyelin biosynthesis in rat liver microsomal and plasma membranes

1. The effects of age-dependent or liposome-induced alterations in the phospholipid composition of rat liver plasma and microsomal membranes on the phosphatidylethanolamine:ceramide-phosphoethanolamine (PE:Cer-PEt) and phosphatidylcholine:ceramide-phosphocholine (PC:Cer-PCh) transferase activities were studied. 2. In all cases under study the PE:Cer-PEt transferase activity was found to be several times higher than that of PC:Cer-PCh transferase in both plasma and microsomal rat liver membranes. 3. The presence of phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) in plasma membranes was observed to enhance the PE:Cer-PEt transferase activity, while phosphatidylserine (PS) inhibited it.