Phospholipid base exchange enzyme activity in sarcolemmal membranes from the heart of cardiomyopathic hamsters

The activity of phospholipid base exchange enzymes has been evaluated in cardiac sarcolemmal membranes from Syrian Golden hamsters and from a hamster strain (UM-X7.1) characterized by a genetic form of hypertrophic cardiomyopathy. No choline base exchange activity and only a little serine base exchange activity were detected, whereas the ethanolamine base exchange enzyme was found highly active in membranes from both strains. For this reason, the present study is focussed on the ethanolamine base exchange enzyme. The apparent Km for ethanolamine of ethanolamine base exchange enzyme from Syrian Golden membranes and from UM-X7.1 strain membranes are 18 and 32 μM, respectively. The specific activity of the sarcolemmal ethanolamine base exchange enzyme is lower in the UM-X7.1 strain than in Syrian Golden hamsters. The calcium-dependence of the enzyme appears different when the membranes from the two strains are compared. Indeed, after removal of the membrane-bound divalent cations, comparable activities are found in both membrane preparations, whereas, upon addition of Ca²⁺ to the incubation mixtures, the activity of the enzyme is enhanced in the membranes from Syrian Golden strain more than in those from UM-X7.1 strain. The cholesterol content of sarcolemmal membranes is higher in the cardiomyopathic strain than in the Syrian Golden hamsters. A possible relation between changes of the membrane lipid composition and of the ethanolamine base exchange activity is discussed.     

Phospholipid metabolism of wheat grains: Enzymes of the CDP-diglyceride phospholipid biosynthetic pathway

Summary Enzymes of the CDP-diglyceride pathway of phospholipid synthesis, CDP-diacylglycerol synthetase, CDP-diacylglycerol: glycerol 3-phosphate phosphatidyl-transferase and enzymes of phosphatidylserine formation were initially of relatively high specific activities in aleurone cells of wheat and declined upon imbibition. Enzyme activity of phosphatidylinositol synthesis was not detected in dry grains but was present upon imbibition. CDP-diacylglycerol: glycerol 3-phosphate phosphatidyltransferase shifted during imbibition from 85% of the activity in the supernatant of aleurone layers from dry seeds to 98% associated with large particle fractions after 36 hours of imbibition. Phosphatidylserine formation shifted from a dominant location in the 1,500 x g fraction in the dry seed to a predominantly mitochondrial location after 36 hours of imbibition. The subcellular distribution of CDP-diacylglycerol synthetase did not change appreciably upon imbibition from that of the dry seed, 75 to 80% of the activity was found in the supernatant. Only CDP-diacylglycerol: glycerol 3-phosphate phosphatidyltransferase showed increased specific activity late in the imbibition period. GA₃ accelerated the decrease of already declining activities of the CDP-diglyceride enzymes and the changes in their patterns of distribution, augmented the activities of the phosphatidylinositol synthesizing enzyme, and both accelerated and augmented the increase in the activity of the enzyme of phosphatidylglycerol synthesis which occurred late in imbibition.Committee on Institutional Cooperation Travelling Scholar from the University of Chicago.     

Phospholipid methylation in MOPC-31C cell membranes with modified phospholipid composition

The presence of the methylation pathway from phosphatidylethanolamine to phosphatidylcholine was first shown in MOPC-31C cells. Intermediate phospholipids of this pathway, phosphatidyl-N-monomethylethanolamine and phosphatidyl-N,N′-dimethylethanolamine, were accumulated in the cell membranes by adding choline analogues such as N-monomethylethanolamine and N,N′-dimethylethanolamine to the culture medium. These modified membranes had a striking character of enhanced phospholipid methylation. This enhancement could be explained by increases in the second and the third step of the methylation pathway from phosphatidylethanolamine.     

Phospholipid composition and phospholipid asymmetry of ram spermatozoa plasma membranes

The phospholipid composition of ram spermatozoa plasma membranes has been investigated. An exclusively high participation of the choline- and ethanolamine-plasmalogens in the phosphatidylcholine and phosphatidylethanolamine fractions has been established. Phosphatidylcholine of ram spermatozoa plasma membranes contains a great amount of polyunsaturated fatty acids. The phospholipid distribution in spermatozoa plasma membrane was investigated. It was established that the choline containing phospholipids are situated mainly in the outer membrane lipid monolayer, whereas diphosphatidylglycerol and phosphatidylserine are localized predominantly in the inner monolayer. The rest of the phospholipids are evenly distributed among the two monolayers. Ram spermal plasma membranes exhibit high phospholipase A2 activity.     

Phospholipid base exchange activity in rat liver plasma membranes. Evidence for regulation by G-protein and P2y-purinergic receptor.

Phospholipid base exchange activity using choline as substrate was detected in plasma membranes (PM) and other subcellular fractions of rat liver, with microsomes (MS) showing the highest specific activity. In contrast, phospholipase D activity was only detected in PM. In PM, choline exchanged for phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS), whereas ethanolamine exchanged for PE and PS, and serine exchanged for PS. Ca2+ (10 microM or higher) stimulated choline incorporation into PC in MS and PM, whereas Mg2+ (10 microM or higher) stimulated it only in PM. Ethanolamine and serine incorporation into PM phospholipids was also stimulated by Ca2+, and inositol incorporation by Mn2+. Phospholipase D activity was substantial in the presence of EGTA and was slightly stimulated by Ca2+ concentrations less than 500 microM. It was undetectable without Mg2+. Low concentrations of oleate (1 mM or less) stimulated phospholipase D activity. These concentrations inhibited choline base exchange activity, whereas higher concentrations (3-8 mM) were stimulatory. Comparison of the subcellular distribution and Ca2+, Mg2+, and oleate effects on choline base exchange and phospholipase D activities supports the view that they are catalyzed by different enzymes. The incorporation of choline, but not ethanolamine or serine, into the phospholipids of PM, but not MS, was stimulated by micromolar concentrations of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) and other slowly hydrolyzable analogues of GTP. GDP, GMP, and other nucleoside triphosphates and their analogues were ineffective. GTP gamma S stimulation of base exchange activity was dependent upon Mg2+ and was inhibited by high concentrations of guanosine 5'-O-2-(thio)diphosphate. In the presence of low concentrations of GTP gamma S, ATP and its slowly hydrolyzable analogues stimulated base exchange activity. Dose-response curves for these nucleotides revealed a potency order consistent with mediation by purinergic receptors of the P2Y type. Base exchange activity stimulated by ATP plus GTP gamma S or GTP gamma S alone was not altered by treatment with pertussis or cholera toxins. These results suggest that the choline base exchange activity of liver PM is regulated by a pertussis toxin-insensitive G-protein linked to P2Y purinergic receptors.     

Phospholipid diversity: correlation with membrane-membrane fusion events

The transport of various metabolically important substances along the endocytic and secretory pathways involves budding as well as fusion of vesicles with various intracellular compartments and plasma membrane. The membrane-membrane fusion events between various sub-compartments of the cell are believed to be mainly mediated by so-called "fusion proteins". This study shows that beside the proteins, lipid components of membrane may play an equally important role in fusion and budding processes. Inside out (ISO) as well as right side out (RSO) erythrocyte vesicles were evaluated for their fusogenic potential using conventional membrane fusion assay methods. Both fluorescence dequenching as well as content mixing assays revealed fusogenic potential of the erythrocyte vesicles. Among two types of vesicles, ISO were found to be more fusogenic as compared to the RSO vesicles. Interestingly, ISO retained nearly half of their fusogenic properties after removal of the proteins, suggesting the remarkable role of lipids in the fusion process. In another set of experiments, fusogenic properties of the liposomes (subtilosome), prepared from phospholipids isolated from Bacillus subtilis (a lower microbe) were compared with those of erythrocyte vesicles. We have also demonstrated that various types of vesicles upon interaction with macrophages deliver encapsulated materials to the cytosol of the cells. Membrane-membrane fusion was also followed by the study, in which a protein synthesis inhibitor ricin A (that does not cross plasma membrane), when encapsulated in the erythrocyte vesicles or subtilosomes was demonstrated to gain access to the cytosol.     

Crosstalk of DNA glycosylases with pathways other than base excision repair

While a role for DNA glycosylase activity in base excision repair (BER) is well understood, there is mounting evidence to implicate cooperation of DNA glycosylases with components of repair pathways other than BER. The mechanisms by which DNA glycosylases interact with non-BER pathways are, in many cases, poorly understood. However, accumulating evidence indicates that crosstalk is common and may be as important in signaling repair as the canonical pathways themselves.     

Permeability of rabbit polymorphonuclear leukocyte membranes to urea and other nonelectrolytes

The diffusional permeability coefficients of rabbit polymorphonuclear leukocyte membranes to urea, methylurea and thiourea have been measured. It was found that the permeability coefficient of these membranes to urea is very low and that thiourea was more permeable than methylurea which was, in turn, more permeable than urea. These results suggest that there is no need to postulate a carrier-mediated mechanism for urea transport across biological membranes and that the concept of “aqueous pores” is not a general property of biological membranes but restricted only to certain cases.     

Phospholipid metabolism in V79-R membranes composed of phospholipid molecular species containing trans-monoenoic fatty acids

The interrelationship between the inhibition of cell growth and changes in phospholipid molecular species was studied in the presence of elaidic, trans-11-eicosenoic, or brassidic acids in Chinese hamster V79-R cells. The addition of trans-monoenoic fatty acids to the medium inhibited cell growth and caused an increase in the total cellular content of phospholipids. However, there was no difference in the polar head group composition of these phospholipids among all the cells supplemented with trans-monoenoic fatty acids. Exogenous trans-monoenoic fatty acids were incorporated into cellular phospholipids to form novel phospholipid molecular species. Phospholipid synthesizing enzyme activities bound to the membranes composed of phospholipid molecular species of trans-monoenoic fatty acids were determined. Cholinephosphotransferase [EC 2.7.8.2] and ethanolaminephosphotransferase [EC 2.7.8.1] activities were decreased by trans-11-eicosenoic acid, but not changed by elaidic acid. Glycerophosphate acyltransferase [EC 2.3.1.15] activity was increased by elaidic acid and decreased by trans-11-eicosenoic acid. Cholinephosphate cytidylyltransferase [EC 2.7.7.15] activity was not changed by trans-monoenoic fatty acids.     

Molecular interactions of peptides with phospholipid vesicle membranes as studied by fluorescence correlation spectroscopy

Interactions of the peptides melittin and magainin with phospholipid vesicle membranes have been studied using fluorescence correlation spectroscopy. Molecular interactions of melittin and magainin with phospholipid membranes are performed in rhodamine-entrapped vesicles (REV) and in rhodamine-labelled phospholipid vesicles (RLV), which did not entrap free rhodamine inside. The results demonstrate that melittin makes channels into vesicle membranes since exposure of melittin to vesicles causes rhodamine release only from REV but not from RLV. It is obvious that rhodamine can not be released from RLV because the inside of RLV is free of dye molecules. In contrast, magainin breaks vesicles since addition of magainin to vesicles results in rhodamine release from both REV and RLV. As the inside of RLV is free of rhodamine, the appearance of rhodamine in solution confirms that these vesicles are broken into rhodamine-labelled phospholipid fragments after addition of magainin. This study is of pharmaceutical significance since it will provide insights that fluorescence correlation spectroscopy can be used as a rapid protocol to test incorporation and release of drugs by vesicles.     

The interaction of phospholipid membranes with poly(ethylene glycol). Vesicle aggregation and lipid exchange

(1) The water soluble polymer, poly(ethylene glycol), causes aggregation of sonicated vesicles of dimyristoylphosphatidylcholine in a manner consistent with a steric exclusion mechanism. (2) Poly(ethylene glycol) promotes the exchange of lipids between multilamellar vesicles of dimyristoylphosphatidylcholine and dipalmitoylphosphatidylcholine when the lipids are in the liquid-crystalline state. (3) ³¹P-NMR studies demonstrate that the bilayer configuration of smectic mesophases of dipalmitoylphosphatidylcholine is substantially maintained in the presence of poly(ethylene glycol).     

Permeability of rabbit polymorphonuclear leukocyte membranes to urea and other nonelectrolytes.

The diffusional permeability coefficients of rabbit polymorphonuclear leukocyte membranes to urea, methylurea and thiourea have been measured. It was found that the permeability coefficient of these membranes to urea is very low and that thiourea was more permeable than methylurea which was, in turn, more permeable than urea. These results suggest that there is no need to postulate a carrier-mediated mechanism for urea transport across biological membranes and that the concept of "aqueous pores" is not a general property of biological membranes but restricted only to certain cases.     

Senita cactus: a plant with interrupted sterol biosynthetic pathways

Locereol (4α-methylcholesta-8,14-dien-3β-ol) and 5α-cholesta-8,14-dien-3β-ol, not previously isolated from plants, 24-methylenelophenol, lathosterol, 5α-campest -7-en-3β-ol and spinasterol are present in senita cactusin addition to the lophenol and schottenol described previously.     

Membrane integrity and phospholipid movement influence the base exchange reaction in rat liver microsomesý

Properties of Ca2+-stimulated incorporation of amincalcohols, serine and ethanolamine, into phospholipids, and factors regulating the reaction were studied in endoplasmic reticulum membranes isolated from rat liver. In contrast to apparent Km values for either aminoalcohol, maximal velocities of the reaction were significantly affected by Ca2+ concentration. No competition between these two soluble substrates used at equimolar concentrations close to their Km values was observed, suggesting the existence of two distinct phospholipid base exchange activities. The enzyme utilizing the electrically neutral serine was not sensitive to changes of membrane potential evoked by valinomycin in the presence of KCI. On the other hand, when positively charged ethanolamine served as a substrate, the enzyme activity was inhibited by 140 mM KCI and this effect was reversed by valinomycin. The rates of inhibition of phospholipid base exchange reactions by various thiol group modifying reagents were al so found to differ. Cd2+ and lipophylic p-chloromercuribenzoic acid at micromolar concentrations were most effective. It can be suggested that -SH groups located within the hydrophobic core of the enzymes molecules are essential for the recognition of membrane substrates. However, the influence of the -SH group modifying reagents on the protein-facilitated phospholipid motion across endoplasmic reticulum membranes can not be excluded, since an integral protein-mediated transverse movement of phospholipids within the membrane bilayer and Ca2+-mediated changes in configuration of the phospholipid polar head groups seem to be a regulatory step of the reaction. Indeed, when the membrane integrity was disordered by detergents or an organic solvent, the reaction was inhibited, although not due to the transport of its water-soluble substrates is affected, but due to modulation of physical state of the membrane bilayer and, in consequence, the accessibility of phospholipid molecules.     

Abnormalities in mitochondria-associated membranes and phospholipid biosynthetic enzymes in the mnd/mnd mouse model of neuronal ceroid lipofuscinosis

Endoplasmic reticulum-like membranes (MAM) that are associated with mitochondria have been implicated as intermediates in the import of lipids, particularly phosphatidylserine, from the endoplasmic reticulum to mitochondria (Vance, J.E. (1990) J. Biol. Chem. 265, 7248–7256; Shiao, Y.-J. et al. (1995) J. Biol. Chem. 270, 11190–11198). We have now examined further the role of MAM in lipid metabolism using the mnd/mnd mouse, a model for the human degenerative disease neuronal ceroid lipofuscinosis. The biochemical phenotype of the mnd/mnd mutant mouse (in which lipids and proteins accumulate abnormally in storage bodies in cells of affected tissues) suggested that the mutation might lead to impaired mitochondrial import of lipids and proteins as a result of a defective linkage between MAM and mitochondria. We, therefore, investigated the status of MAM and phospholipid metabolism in mnd/mnd mice livers. Separation of MAM from livers of older, but not younger, mnd/mnd mice was aberrant. In addition, the amount of the MAM-specific protein, phosphatidylethanolamine N-methyltransferase-2 (PEMT2), was greatly reduced in homogenates and MAM from livers of mnd/mnd mice of all ages, although PEMT2 mRNA abundance was normal. Moreover, PEMT activity in MAM from mnd/mnd mice was 60% less than in control mice. Activities of two additional phospholipid biosynthetic enzymes — CTP:phosphocholine cytidylyltransferase and phosphatidylserine synthase — were also reduced by >50% in mnd/mnd microsomes. Radiolabeling experiments in hepatocytes indicated that neither the mitochondrial import nor the subsequent metabolism of phosphatidylserine was grossly affected in mnd/mnd mice. However, 3 proteins (cytochrome b₅, NADH:cytochrome b₅ reductase and mitochondrial F₁F₀-ATP synthase c subunit) which are normally present in mitochondria were partially redistributed to microsomes in mnd/mnd mouse liver. These studies indicate that MAM are defective in the mnd/mnd mutant mouse in which the biochemical phenotype includes an abnormal accumulation of lipids and proteins in storage bodies.