Influence of Carbicron (O-[(2-butenoic acid)-N,N-dimethylamide-3-YL] O,O-dimethylphosphate) on some biochemical and biophysical parameters of rat liver membranes

• 1.1. Treatment of rats with carbicron induced a reduction of the phospholipids in both microsomal and plasma membranes.
• 2.2. A decrease of the structural order parameter (S_DPH) and an increase of the pyrene excimer-to-monomer fluorescence ratio (I_E/I_M) was also observed, indicating membrane fluidization.
• 3.3. The specific activity of membrane-bound phospholipase A₂ and phospholipase C were decreased in both types of membranes, whereas acyl-CoA:lysophosphatidylcholine acyltransferase activity was augmented due to carbicron treatment.

     

Palmitic acid metabolism in hepatopancreas of the freshwater shrimp Macrobrachium borellii

• 1.1. The palmitic acid fate as substrate for the synthesis of either glycerides or other fatty acids was studied in vivo and in the microsomal fraction from hepatopancreas of Macrobrachium borellii.
• 2.2. Most of the palmitic acid administered in vivo circulated to the hepatopancreas, being incorporated mainly in the triacylglycerol (TG) fraction.
• 3.3. Palmitic acid transformations into palmitoleic, stearic and oleic acids were observed in the hepatopancreas.
• 4.4. The in vitro biosynthesis of TG in hepatopancreas was more active than in other tissues. In the microsomal fraction, palmitic acid was also incorporated mainly in TG, and followed the α-glycerophosphate pathway.

     

Transport of cholesterol in the hemolymph of the mollusc Diplodon delodontus

• 1.1. The dietary and inter-organ cholesterol transport in the hemolymph of the bivalve mollusc Diplodon delodontus, was studied. Plasma and hemocytes were obtained after feeding labeled cholesterol to animals or injecting it into the posterior adductor muscle.
• 2.2. In both cases, cholesterol was incorporated either into plasma or hematic cells.
• 3.3. Two plasmatic fractions differing in their hydrated densities were recognized as cholesterol carriers and were isolated. They have characteristics of high density (HDL) and very high density (VHDL) lipoproteins, respectively.
• 4.4. The major lipids in the different classes of lipoproteins were free sterols in HDL and phospholipids in VHDL.
• 5.5. Neither low nor very low density lipoprotein transporting cholesterol was detected.

     

PLA2 activity in rat liver nuclei

• 1.1. Subcellular fractions of rat liver were assayed for PLA₂ activity.
• 2.2. The PLA₂ assay measures the release of [³ H]oleic acid from phospholipids, using labeled E. coli as substrate.
• 3.3. Nuclear fractions contained PLA₂ activity, which was Ca²⁺ dependent and could not be explained from mitochondrial, microsomal or plasma membrane contamination.
• 4.4. The V_max value of nuclear PLA₂ is 0.30 ± 0.04 pmol oleic acid/min/mg protein; its K_m value is 0.86±0.12μM, similar to that of mitochondrial PLA₂.
• 5.5. We conclude that rat liver nuclei contain PLA₂ activity.

     

Seasonal changes in condition and lipids composition of the bivalve Macoma balthica L. from the gulf of Gdańsk (Southern Baltic)

• 1.1. The total lipids content and composition of lipid classes, their per cent in dry wt of soft tissues and level in standard animal, as well as composition of fatty acids and sterols were studied in Macoma balthica collected from three sites in the Gulf of Gdańsk, in the years, 1983–1984.
• 2.2. The increase in the content of total lipids, triacylglycerols, oleopalmitic, 16:1 and eicosapentaenoic acids, 20:5, C27 sterols (mainly cholest-5en-3β-ol), in spring and early summer and their decrease in autumn and winter were observed.
• 3.3. Content of phospholipids, sterols and hydrocarbons in the tissue dry wt of Macoma balthica remained nearly constant.

     

A study of glycerolphosphate acyltransferase in rat liver mitochondria-submitochondrial localization and some properties of the solubilized enzyme

• 1.1. Glycerolphosphate acyltransferase (GPAT) was solubilized from the rat liver mitochondrial membranes using sodium cholate. Dithiothreitol was necessary to stabilize the solubilized enzyme on storage.
• 2.2. Unlike the enzyme in situ in mitochondrial membranes, the solubilized mitochondrial GPAT was susceptible to inhibition by N-ethylmaleimide; a property more characteristic of the distinct microsomal form of GPAT.
• 3.3. Solubilized mitochondrial GPAT retained its very high preference for saturated acyl-CoA substrate (palmitoyl-CoA) and had no activity whatever with any tested concentration of the unsaturated substrate oleoyl-CoA.
• 4.4. Solubilization increased the affinity of mitochondrial GPAT for palmitoyl-CoA whilst decreasing the K_m for glycerol phosphate.
• 5.5. After separation of liver mitochondrial outer and inner membranes and estimation of cross-contamination by appropriate markers it was concluded that the mitochondrial inner membrane contains significant GPAT activity. This was established with preparations from fed, 48 hr-starved and streptozotocin-diabetic rats.

     

Steroid metabolites of the far eastern Holothurian Stichopus japonicus Selenka

• 1.1. Sulphated and etherified sterols were isolated from the far eastern holothurian Stichopus japonicus S. The sterol composition of both fractions was determined using gas-liquid chromatography and mass-spectroscopic methods. The structures of individual sterols were proved on the basis of mass-spectrometry and ¹H-NMR-spectroscopy data.
• 2.2. The structures of 29 sterols were established.
• 3.3. Sterols (22E, 24R)-23,24-dimethyl-5α-cholest-22-en-3β-ol, 23,24-dimethyl-cholesta-5,22-dien-3β-ol, 24-methyl-cholesta-5,24(28)-dien-3β-ol, (24Z)-24-ethyl-cholesta-5,24(28)-dien-3β-ol, 24-nor-cholesta-5,22-dien-3β-ol, 24-ethyl-cholesta-5,25-dien-3β-ol were described for holothurians for the first time.
• 4.4. Δ⁵-sterols were shown to be the main components of the sulphated alcohol fractions (67.61%), while the saturated and Δ⁷-sterols were there in less quantities (14.72 and 9.52%, respectively).
• 5.5. The etherified sterols were represented, mainly, by saturated and Δ⁷-sterols (37.82% and 33.95%, respectively). Δ⁵-sterols were 19%.
• 6.6. The sensitivity of liposomal membranes, containing steroid metabolites of the holothurian St. japonicus (Δ⁷-, sulphated and glycosilated sterols) to the action of endotoxin-stichoposide A, was studied.

     

Plasma membrane: Can its structure and function be modulated by dietary fat?

• 1.1. Compositional analysis of plasma membranes from rats fed nutritionally adequate diets different in fatty acid composition establishes that fundamentally different dietary fat intake results in alteration in structural lipid composition of plasma membranes in brain, liver and the intestinal mucosa.
• 2.2. Dietary differences in fatty acid intake altered the fatty acyl tail composition of plasma membrane phospholipids in brain, liver and intestinal mucosa.
• 3.3. Diet altered the phospholipid profile observed in brain synaptosomal and liver plasma membrane.
• 4.4. Feeding high vs low polyunsaturated to saturated fat diets for 7 days altered the fatty acid composition of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin and mono-glucosylceramide isolated from plasma membrane of the intestinal mucosa

     

Effects of Echinostoma trivolvis (Trematoda) infection on neutral lipids,sterols, and carotenoids in Helisoma trivolvis (Gastropoda)

• 1.1. Histochemical, thin layer and gas-liquid chromatographic studies were done on neutral lipids, sterols and carotenes in the digestive gland-gonad (DGG) complex of Helisoma trivolvis infected with Echinostoma trivolvis vs uninfected DGG.
• 2.2. Hitochemical Oil Red O staining showed the presence of neutral lipids in the redial body wall and in the digestive cells of the DGG.
• 3.3. TLC showed that free sterols and triacylglycerols were major neutral lipid fractions along with lesser amounts of steryl esters and free fatty acids in the DGG of both populations. The percentage composition of all neutral lipid fractions was greater in infected than uninfected DGG.
• 4.4. Infected DGG contained more carotenoid fractions than uninfected DGG, but only beta-carotene was identified from both.
• 5.5. GLC studies showed that the major sterol present in snail DGG was cholesterol (about 70%) along with lesser amounts of stigmasterol, campesterol, beta-sitosterol and desmosterol. No clear cut distinction was seen in sterols from infected vs uninfected DGG.

     

Comparative study on the fatty acid and lipid composition of four marine fish larvae

• 1.1. Fatty acid and lipid class composition were determined in larvae of four marine species: Atlantic halibut (Hippoglossus hippoglossus L.), plaice (Pleuronectes platessa), cod (Gadus morhua) and turbot (Scophthalmus maximus) at hatching and prior to first feeding.
• 2.2. Total fatty acid content decreased in the four species with up to 50% reduction in one of the halibut groups. Docosahexanaoic acid (22:6 n-3) was especially utilized.
• 3.3. Low lipid utilization was found in turbot in relation to the other three species.
• 4.4. Water environmental temperature may explain some of the differences in the fatty acid utilization and the source of metabolic energy between cold water species (halibut, cod, and plaice) and temperate species (turbot), in the period from hatching to prior to first feeding.
• 5.5. Relative amounts of neutral lipids and phospholipids were similar in plaice, cod and halibut, approximately 25% and 75% of total lipids, respectively, and were approximately constant during the yolk-sac stage. Neutral lipids were dominant for turbot at hatching, accounting for 53–55% of the total lipids, while phospholipids predominated prior to first feeding, being 56–59%.
• 6.6. Phosphatidylcholine was catabolized in halibut, plaice and cod but not in turbot, while phosphatidylethanolamine tended to be synthesized in all four species.

     

Locust vitellogenin receptor: an acidic glycoprotein with N- and O-linked oligosaccharides

• 1.1. The locust vitellogenin (VTG) receptor which is embedded in oocyte plasma membranes is a glycoprotein.
• 2.2. With various lectins oligosaccharide units have been identified, among them neuraminic acid linked to Gal or GalNAc, mannose chains, Gal linked to GalNAc or GlcNAc and fucose linked to GlcNAc.
• 3.3. With specific enzymes it could be shown that mannose and most other oligosaccharides are O-linked while others like fucose are N-linked.
• 4.4. Enzymatic removal of all O-linked carbohydrates resulted in a drop of the molecular mass of the receptor protein from 200,000 to 110,000.
• 5.5. A total of N- and O-linked oligosaccharides of 54% was calculated.
• 6.6. The isoelectric point of the receptor was found to be at pH 3.4 increasing slightly after removal of neuraminic acid.
• 7.7. Removal of neuraminic acids destroyed the binding ability for VTG.

     

IDentification of a microsomal retinoic acid synthase as a microsomal cytochrome P-450-linked monooxygenase system

• 1.1. To characterize an enzyme which metabolizes retinal in liver microsomes, several properties of the enzymatic reaction from retinal to retinoic acid were investigated using rabbit liver microsomes.
• 2.2. The maximum pH of the reaction in the liver microsomes was 7.6.
• 3.3. The K_m and V_max values for all-trans, 9-cis and 13-cis-retinals were determined.
• 4.4. The reaction proceeded in the presence of NADPH and molecular oxygen.
• 5.5. The incorporation of one atom of molecular oxygen into retinal was confirmed by using oxygen-18, showing that the reaction comprised monooxygenation, not dehydrogenation.
• 6.6. The monooxygenase activity was inhibited by carbon monoxide, phenylisocyanide and antiNADPH-cytochrome P-450 reductase IgG, but not by anti-cytochrome b₅ IgG.
• 7.7. The enzymatic activity inhibited by carbon monoxide was photoreversibly restored by light of a wavelength of around 450 nm.
• 8.8. The retinal-induced spectra of liver microsomes with three isomeric retinals were type I spectra.
• 9.9. The microsomal monooxygenase activity induced by phenobarbital or ethanol were more effective than that by 3-methylcholanthrene, clotrimazole or β-naphthoflavone.
• 10.10. These results showed that the monooxygenase reaction from retinal to retinoic acid in liver microsomes is catalyzed by a cytochrome P-450-linked monooxygenase system.

     

Human placental atp-diphosphohydrolase: Biochemical characterization,regulation and function

• 1.1. Kinetic and physico-chemical studies on human placental microsomal fraction confirmed that the ATPase and ADPase activities detected in this fraction correspond to the enzyme ATP-diphosphohydrolase or apyrase (EC 3.6.1.5). These include substrate specificity, and coincident M_r and pI values of both ATPase-ADPase activities.
• 2.2. This enzyme hydrolyses both the free unprotonated and cation-nucleotide complex, the catalytic efficiency for the latter being considerably higher.
• 3.3. Microsomal apyrase is insensitive to ouabain and Ap5A. The highly purified enzyme was only inhibited by o-vanadate, DBS and slightly by DCCD.
• 4.4. Apyrase seems to be a glycoprotein from its interaction with Concanavalin-A.
• 5.5. Preliminary studies on the essential amino acid residues suggest the participation of Arg, Lys and His residues, and discard the requirement of −SH, COO⁻, −OH, and probably also Tyr and Trp.
• 6.6. Two kinetic modulatory proteins of apyrase were detected in placental tissue. An activating protein was found in the soluble fraction and an inhibitory protein was loosely bound to the membranes.
• 7.7. The proposed in vivo function for apyrase is related to the inhibition of platelet aggregation due to its ADPase activity, which is supported by the direct effect on washed platelets and by its plasma membrane localization.

     

Isolation and characterization of phospholipase A2, from Agkistrodon bilineatus (common cantil) venom

• 1.1. Phospholipase A₂ was isolated from Agkistrodon bilineatus venom by Sephadex G-75 and CM-Cellulose column chromatographies.
• 2.2. The purified phospholipase A₂-I gave a single band on disc polyacrylamide gel electrophoresis, isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis.
• 3.3. The enzyme preparation had a molecular weight of 14,000, isoelectric point of pH 8.77 and possessed 123 amino acid residues.
• 4.4. The purified phospholipase A₂ possessed lethal, indirect hemolytic and anticoagulant activities.
• 5.5. The enzyme hydrolyzed the phospholipids phosphatidyl choline (PC), phosphatidyl ethanolamine (PE), phosphatidyl inositol (PI) and phosphatidyl serine (PS).
• 6.6. The concentration of mouse diaphragm was inhibited and the contraction of guinea pig left atrium was increased by phospholipase A₂-I.
• 7.7. Phospholipase A₂ activity of this preparation was inhibited by ethylenediamine tetraacetic acid, p-bromo phenacyl bromide, n-bromo succinimide or dithiothreitol, but not by diisopropyl fluorophosphate or benzamidine.

     

Subcellular and developmental studies of the tyrosyl protein sulfotransferase in rat brain

• 1.1. Tyrosyl protein sulfotransferase (TPS) activity in the newborn and mature rat brain was studied using the cholecystokinin derivative terbutyloxycarbonyl-Asp-Tyr-Met-Gly-Trp-Met-Asp-PheNH₂, BocCCK-8(ns), as the peptide substrate.
• 2.2. TPS activity was enriched 4 times in the microsomal and synaptic vesicular enriched fractions of rat cerebral cortex.
• 3.3. CCK-8 content, in the subcellular fractions and the peptide sulfation activity distribution was in accord with the hypothesis that tyrosyl protein sulfotransferase plays a key role in the maturation process of bioactive CCK.
• 4.4. TPS activity measured in membranes from newborn brain was 2.5 times higher than the activity observed in the mature brain membranes with a V_max = 0.83 ± 0.05 and 0.31 ± 0.02 respectively. The apparent K_M for the sulfate donor, 3'-phosphoadenosine 5'-phosphosulfate (PAPS), was similar, 94 ± 4 nM and 90 ± 6 nM and the k_M for the peptide substrate, BocCCK-8(ns), was 234 ± 16 μM and 160 ± 12 μM in the newborn and adult brain membranes respectively.
• 5.5. TPS activity reached normal mature values within 20 days of age.
• 6.6. These data support the idea that tyrosyl protein sulfation is an important process in the secretion mechanism and in the CCK maturation.

     

On the mechanism of decreasing the lithogenicity of bile in Camelus dromedarius

• 1.1. Analysis of camel bile revealed that it is highly concentrated.
• 2.2. The molar fraction of phospholipids in camel bile was low and cholesterol was very high relative to rat bile.
• 3.3. Cholate is the main primary bile acid secreted. Moderate quantities of the secondary bile acid deoxycholate were found but no lithocholate was detectable possibly secondary to rapid recycling of the bile acid pool in the enterohepatic circulation.
• 4.4. Glycoconjugated bile acids predominated over tauroconjugates.
• 5.5. The hepatic bile from the camel may be concentrated as part of the general mechanism of water conservation exhibited by that species. The increased concentration of bile acids helps maintain cholesterol in solution, thus reducing lithogenicity.

     

Characterization of NaK ATPase from the gills of the freshwater prawn Macrobrachium rosenbergii (de Man)

• 1.1. The specific activity of Na-K ATPase was determined from the microsomal preparation of gills dissected from adult Macrobrachium rosenbergii.
• 2.2. Maximal ATPase activity was achieved at a substrate concentration of 0.5 mM ATP.
• 3.3. Optimal enzyme activity was obtained at pH of 7.5.
• 4.4. The Arrhenius plot of Na-K ATPase activity revealed a marked discontinuity at 30°C. “Mg” ATPase activity did not exhibit a marked discontinuity.
• 5.5. The E_a for Na-K ATPase and “Mg” ATPase was 14.6 kCal/mole and 9.31 kCal/mole respectively. Q₁₀ values for Na-K ATPase was 2.34 and for “Mg” ATPase 1.65.
• 6.6. ATPase activity and gill homogenate protein concentration exhibited a linear relationship up to 130 μg protein/ml.
• 7.7. Na-K ATPase activity was inhibited by 10⁻³ M ouabain. It was equally inhibited by the removal of K⁺ from the reaction medium.

     

The binding and degradation of 125I-labelled insulin by rat kidney brush-border membranes

• 1.1. The interaction of insulin with purified brush-border membranes from rat kidney was studied with the use of [¹²⁵I]insulin.
• 2.2. The specific binding of insulin by brush-borders could be demonstrated, and was time- and temperature-dependent.
• 3.3. [¹²⁵I]insulin was displaced by unlabelled insulin. A₁-B₂₉ dodecoyl insulin and insulin A- and B-chains in proportion to their relative bioactivity.
• 4.4. Brush-border membranes showed high insulin-degrading activity with an apparent K_m of 2.2 μM.
• 5.5. A number of proteinase inhibitors were effective in inhibiting insulin degradation but the greatest degree of inhibition was achieved by the use of thiol-blocking reagents.
• 6.6. No evidence was obtained for the involvement of the enzyme glutathione-insulin transhydrogenase.

     

The sterol constituents and Δ5,7-sterol content of some bivalve molluscs

• 1.1. The bivalve molluscs Cerastoderma edula, Chlamys opercularis, Ensis soliqua, Modiolus modiolus, Mya arenaria, Mytilus edulis and Pecten maximus contained mixtures of C₂₆-, C₂₇-, C₂₈- and C₂₉-sterols. Cholesterol, 24-methylcholesta-5,22-dien-3β-ol and 24-methylene-cholesterol were the major sterols.
• 2.2. The sterols of Cerastoderma edula, Mya arenaria and Mytilus edulis contained 6–16% of cholesta-5,24-dien-3β-ol.
• 3.3. All the molluscs contained Δ^5,7-sterols in amounts ranging from 2 to 21% of the total sterols.
• 4.4. Cholesta-5,7-dien-3β-ol and 24-methylcholesta-5,7,22-trien-3β-ol were identified in Mytilus edulis. 25-Norcholesta-5,7,22-trien-3β-ol was detected in Modiolus modiolus.

     

Purification and characterization of carbonyl reductases from bovine liver cytosol and microsome. the cytosolic enzyme has a novel 3α/17β-hydroxysteroid dehydrogenase activity

• 1.1. Carbonyl reductase, which is distributed in both cytosolic and microsomal fractions in bovine liver, were purified to homogeneity on 12.5% sodium dodecylsulfate-polyacrylamide gel electrophoresis and shown to have molecular weights of 32 kDa and 68 kDa, respectively.
• 2.2. Both carbonyl reductases can catalyze the reduction of many carbonyl compounds including ketone, quinones and aldehyde with relatively low K_m values.
• 3.3. From the absorption spectrum result, microsomal carbonyl reductase closely resembles cytochrome P-450 reductase.
• 4.4. Cytosolic carbonyl reductase is a novel enzyme which can act on both testosterone and androsterone at low concentration.