Phospholipid substrate-specificity of the L-serine base-exchange enzyme in rat liver microsomal fraction

The specificity of the L-serine base-exchange enzyme towards the fatty acid composition of the phospholipid substrate was investigated with a rat liver microsomal fraction. The relative rates of L-serine incorporation into saturated-hexaenoic, saturated-pentaenoic, saturated-tetraenoic, saturated-trienoic, dienoic-dienoic, monoenoic-dienoic, saturated-dienoic and saturated-monoenoic + saturated-saturated phosphatidylserine molecular species were 42, 5, 23, 4, 5, 4, 5 and 11% respectively. This is similar to, but not identical with, the relative mass abundance of these molecular species in total liver cell phosphatidylserines. The results indicate that the substrate-specificity of the L-serine base-exchange enzyme can at least in part explain the observed fatty acid composition of rat liver phosphatidylserines.     

Sidedness of ceramide-phosphoethanolamine synthesis on rat liver and brain microsomal membranes

Phosphatidylethanolamine:ceramide-ethanolamine-phosphotransferase catalyzes the synthesis of ceramide-phosphoethanolamine, a sphingomyelin analogue. Its localization was studied in rat liver and brain microsomes. After testing the integrity and the sidedness of microsomal vesicles, trypsin treatment of intact or deoxycholate-disrupted microsomes made it possible to conclude that both the transferase and the ceramide-phosphoethanolamine are located in the cisternal leaflet of the membrane bilayer. Using trinitrobenzenesulfonic acid as a probe, no trace of newly synthesized ceramide-phosphoethanolamine was detectable on the cytoplasmic side of the microsomes.     

Structural state of rat liver mitochondrial and microsomal membranes in aging and after treatment with thyroxine

Rotation mobility of stearic acid spin labelling in mitochondria and microsomes of 24 months old rats is shown to decrease in comparison with that of 1 month rats. This decrease correlates with an increase in the free cholesterol content in membranes. The phospholipid composition is practically unchanged. With age the osmoresistance of organelles decreases and the cryoresistance increases, supporting indirectly the existence of structural changes in membranes with ageing. The most pronounced activation of NADPH-cytochrome C reductase in the microsomal membrane after the treatment with triton X-100 is observed in case of old animals. The thyroxine treatment of old rats causes a "rejuvenative" effect on the structural state of mitochondrial and microsomal membranes, evidencing for the important role of hormonal regulation in mechanisms of their age rearrangement.     

Enzymic synthesis of ether types of choline and ethanolamine phosphoglycerides by microsomal fractions from rat brain and liver.

The formation of product by ethanolamine phosphotransferases (EC 2.7.8.1) and cholinephosphotransferases (EC 2.7.8.2) in microsomal fractions from brains and livers of mature rats is increased several fold by 1,2-diacyl-sn-glycerols. With the addition of 1-alkyl-2-acyl-sn-glycerols, we have found an 11-fold increase with brain microsomes and a 20-fold increase with lvier microsomes in the synthesis of choline ether lipids (1-alkyl-2-acyl- and 1-alk-1'-enyl-2-acyl-sn-glycero-3-phosphorylcholines). For the synthesis of ethanolamine ether lipids (1-alkyl-2-acyl and 1-alk-1'-enyl-2-acyl-sn-glycero-3-phosphorylethanolamines), the stimulation of alkylacylglycerols was 7-fold for brain microsomes and 18-fold for liver microsomes. The alkylacyl glycerols (8 mM) also inhibited the synthesis of diacyl phosphoglycerides by 44 to 65%, indicating that the same ethanolaminephosphotransferases and cholinephosphotransferases are utilized for the synthesis of alkylacyl phosphoglycerides and diacyl phosphoglycerides. A desaturation of the alkyl groups may take place in the same reaction mixture. The rate of incorporation of phosphorylcholine into alkenylacyl glycerophosphorylcholines (choline plasmalogens) with alkylacylglycerols, cytidine diphosphate choline, and liver microsomes was 15 nmoles per mg protein per hour. The in vitro synthesis of choline plasmalogens with alkylacylglycerols had not been observed previously. The corresponding rate of incorporation of phosphorylethanolamine into ethanolamine plasmalogens was 10 nmoles per mg protein per hour, a value greater than any of the previously reported values for ethanolamine plasmalogen formation from alkylacyl glycerophosphorylethanolamines.     

gamma-Aminobutyric acid receptors in cerebellar membranes of rat brain after a treatment with Triton X-100.

Abstract— The treatment of cerebellar membranes of rat brain with a low concentration of Triton X-100 followed by sufficient washing results in an increase of the Na⁺-independent binding of [³H]GABA and a total loss of the Na ⁺-dependent binding of [³H]GABA. The Na⁺-independent binding of [³H]GABA was more abundant in membranes of cerebellum than in membranes of other rat brain regions and mainly localized in the synaptic membrane fraction of a cerebellar homogenate. In the Triton-treated membranes, the Na⁺-independent binding of [³H]GABA was a saturable process, which could be resolved into two components, a high and a low affinity component with dissociation constants of 4.5 and 30 nm , respectively. The neurophysiological agonists, muscimol, GABA, and imidazole acetic acid, and the antagonist, bicuculline, inhibited the high affinity Na⁺-independent binding of [³H]GABA by 50% at 0.003, 0.012, 0.3 and 10 μm respectively. These data suggest that the Na⁺-independent binding of [³H]GABA in the Triton-treated cerebellar membranes represents the synaptic receptors of GABA. It is emphasized that extensive washing of the membranes after a Triton treatment is necessary in order to detect the high affinity Na⁺-independent binding of [³H]GABA.     

Studies on base-exchange reactions of phospholipids in rat brain particles and a solubilized system.

A filtration-method on Millipore-membranes for the assay of the base-exchange reaction was described. Its advantage over the usual procedure based upon the extraction and the washing of lipids was discussed with the viewpoint of processing many samples, which would be indispensable for purifying the enzyme.The reaction showed an absolute dependency for calcium ion with different optimal concentrations for each of the three bases, a sensitivity to inhibition by high ionic strength, and a pH optimum around 9.0. Exogenously added phospholipid, asolectin, gave a slight stimulation for ethanolamine and l-serine incorporation at a low concentration while choline incorporation was essentially inhibited at all concentrations examined. In heat-denaturation experiments with the particulate and soluble the incorporation of choline into lipid was more sensitive than that of ethanolamine and l-serine. A developmental study showed that brain particles sedimenting between 10,000 and 35,000g prepared from rats aged 22–27 days readily incorporated ethanolamine, l-serine, and choline into their corresponding phosphatidyl compound.Several procedures for solubilization of the “base-exchange” enzyme were examined. The most effectively solubilized preparation was obtained by the use of an ionically balanced detergent, Miranol H2M. This preparation showed a marked dependency on exogenously added phospholipids for its maximal enzymic activity, had a pH optimum at around 7.2, and had an absolute requirement for Ca²⁺. This particular detergent at a concentration of 1% ($\text{w}\text{v}$) solubilized approximately 50% of the protein, and about 30% of the phospholipids, 40% of the cerebrosides, and only 11% of the cholesterol originally present in the particles. The relative proportions of different phospholipids solubilized by the detergent were, however, similar to those present in the original particles.The base-exchange reaction catalyzed by the solubilized enzyme was found to be highly sensitive to ionic strength, and the inhibitory effect of a specific monovalent cation paralleled its ionic size. Substantial differences in the K_m value for each of the substrates with only slight differences in V were observed.The choice of solubilizing agents in relation to these properties and to the maintenance of the activity of the base-exchange reaction was discussed.     

A spin label study of rat brain membranes. Effects of temperature and divalent cations.

Rat brain myelin, synaptosomal plasma membranes and synaptic vesicles were spin labelled with stearic acid nitroxide derivatives. Their electron spin resonance spectra were studied as a function of temperature and devalent ions (Ca2+ and Mg2+) concentrations. (1) Synaptosomal plasma membranes and synaptic vesicles show identical temperature variations of their order parameter (S = 0.58 at 35 degrees C and S = 0.72 AT 22 DEGREES C). Myelin appears more rigid (S = 0.66 at 35 degrees C and S = 0.76 at 22 degrees C). A discontinuity of the order parameter variation as a function of temperature, is observed between 14.5 degrees C and l9.5 degrees C with the three types of membranes. (2) The hydrophobic core of these membranes is very fluid. No transition temperature is observed. The measured values of the spin label rotation correlation times and rotational activation energies are 2.1 and 2.8 ns at 35 degrees C and 3.1 and 3.6 kcal/mol respectively for synaptosomal plasma membranes and myelin. (3) Ca2+ enhances the membrane rigidity (12+/-0.7% increase of the order parameter at 35 degrees C in the presence of 10(-3) M Ca2+) and increases the transition temperature. At a lower extend, similar effects are observed with Mg2+.     

Effects of sulfhydryl reagents on Na+-Ca2+ exchange in rat brain microsomal membranes

Effects of six thiol reagents with different physico-chemical properties were tested on the Na+-dependent 45Ca2+ transport into the rat brain microsomal membrane vesicles. The mercurials p-chlormercuribenzoate and Mersalyl effectively inhibited 45Ca2+ uptake with IC50 values in the order of 10(-4) mol X l-1 in the medium. N-ethylmaleimide and its more lipophilic analog N-(4-(2-benzoxazolyl)phenyl)maleimide were much less effective at the same concentrations. 2,2'-dithiodipyridine markedly reduced 45Ca2+ uptake already at concentrations below 10(-4) mol X l-1, whereas 5,5'-dithiobis-2-nitrobenzoate in a concentration range 10(-6)-10(-3) mol X l-1 was a weak inhibitor. Inhibitory effects of the most potent inhibitors p-chlormercuribenzoate and 2,2'-dithiodipyridine were readily reversed by 1 mmol X l-1 dithiothreitol. The results suggest that free SH groups of membrane polypeptides are involved in the functioning of the Na+-Ca2+ exchanger in the nerve tissue cell membranes.     

A study on the topological distribution of phospholipids in microsomal membranes of chick brain using phospholipase C and trinitrobenzenesulfonic acid

The transbilayer distribution of phospholipids in chicken brain microsomal membranes has been investigated using trinitrobenzenesulfonic acid and phospholipase C from Clostridium weichii. The exposure of intact microsomes to trinitrobenzenesulfonic acid showed that the labelling of aminophospholipids followed biphasic kinetics, indicating that these membranes contain a fast- and a slow-reacting pool of aminophospholipids. Use of microsomes radioiodinated on their surface led to the conclusion that the fast-reacting pool may be located on the outer leaflet of the microsomal vesicles. It contains about 35% of the phosphatidylethanolamine, 29% of the ethanolamine plasmalogens and 18% of the phosphatidylserine. The treatment of intact microsomes with the phospholipase C Cl. welchii produced the hydrolysis of 50% of the phospholipids without any loss of their permeability properties, indicating that they are not permeable to the hydrolase. Phospholipids extracted from the microsomes were hydrolyzed rapidly by the phospholipase C with the exception of phosphatidylserine and phosphatidylinositol. In intact microsomes about 90% of phosphatidylcholine, 32% of ethanolamine phospholipids and 60% of sphingomyelin were accessible to the phospholipase. These results suggest that the phospholipids have an asymmetric distribution in chicken brain microsomes, the external leaflet containing about 75% of the choline phospholipids and 25% of the aminophospholipids, whereas an opposite distribution is observed in the inner leaflet.     

Structural and functional state of microsomal membranes in the rat brain cortex during long-term ethanol consumption

The range of the Na+, K(+)-ATPase functional stability in microsomal fraction of rat brain cortex under long-term chronic ethanol (15%, v/v) consumption was ascertained. The enzyme activity decreased only after 15 months of alcoholisation on the background of the stable structural membrane characteristics (on the basis of the intrinsic and 1-anilinonaphthalene-8-sulfonate fluorescence parameters) and SH-content in postmitochondrial supernatant. The cellular homeostatic mechanisms under ethanol effect are discussed.