Small membrane-associated gtp-binding proteins of catecholamine-secreting cells

• 1.1. Four GTP-binding proteins (23–27 kDa) were identified in membranes from PC12 cells by [α³²P]GTP binding to nitrocellulose blots of SDS-polyacrylamide gels.
• 2.2. The GTP-binding proteins remained associated with membranes during stimulation of intact cells by K⁺-depolarization or even after addition of C²⁺to digitonin-permeabilized cells.
• 3.3. By two-dimensional gel electrophoresis, six GTP-binding proteins were resolved and based on their mobility, their phosphorylation state appeared independent of Ca²⁺.
• 4.4. Fractionation of PC12 membranes showed that these GTP-binding proteins were broadly distributed in post-nuclear membranes with the plasma membranes containing the highest specific GTP-binding activity.
• 5.5. Membrane fractions from bovine adrenal medulla contain similar GTP-binding proteins with GTP-binding intensity also being highest in the plasma membrane.
• 6.6. The GTP-binding proteins could be concentrated in the detergent-rich fraction upon Triton X-114 phase separation.

     

Protein tyrosine phosphorylation in normal rat tissues

• 1.1. Exogenous and endogenous tyrosine protein phosphorylation activities were examined in soluble and partieulate fractions from various normal tissues by using poly-[Glu-⁸⁰Na, Tyr²⁰] and a monoclonal antibody specific for phosphotyrosine.
• 2.2. Phosphorylation of the exogenous substrate by the partieulate forms of TPKs was 2- to 10-fold higher than by soluble forms. The activities of partieulate and soluble enzymes decreased in the following order: spleen > (thymus = kidney) > testes ⩾ (pancreas = liver = brain) > heart.
• 3.3. The level of endogenous phosphorylation in the tissues decreased respectively in the following order: thymus > brain ⩾ (pancreas = liver) > spleen > testes > kidney > heart for the partieulate fractions, and spleen > thymus > brain > pancreas ⩾ liver > testes > kidney > heart for the soluble fractions.
• 4.4. A large number of phosphotyrosine-containing proteins were detected. In addition, several phosphotyrosine-containing proteins of similar molecular weight were found in different tissues and fractions.

     

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.

     

Further studies on the mechanism of action of gossypol on mitochondrial membrane

• 1.1. In order to explore the mechanism of inhibition of hydroxylases involved in steroidogenesis, by gossypol, we studied the effect of this drug on adrenal cortex mitochondria, and compared it with those on kidney and heart.
• 2.2. The uncoupler effect of gossypol (collapse of Δψ and Ca²⁺ efflux) was found to be lower in adrenal cortex mitochondria than in kidney and heart mitochondria.
• 3.3. Gossypol produced more extensive changes on the membrane lipidic matrix (increase in the order parameter for 5-doxylstearic acid) in adrenal cortex mitochondria than in the other mitochondria studied.
• 4.4. The results described above indicate that the mechanism of inhibition of gossypol of steroidogenic adrenal enzymes could be attributed to an alteration of the lipidic matrix which, in turn, modifies protein function.

     

Rat liver endoplasmic reticulum protein kinases

• 1.1. Rat liver microsomal membranes were studied for the presence of protein kinases. Microsomal proteins solubilized with Triton X-100 were analyzed by means of ion exchange chromatography.
• 2.2. Protein kinase activity was detected in the column fractions using specific assays for cAMP-dependent protein kinase, cGMP-dependent protein kinase, protein kinase C, Ca²⁺/calmodulin-dependent protein kinase and casein kinases.
• 3.3. Fractions with protein kinase activity were further analyzed by SDS-polyacrylamide gel electrophoresis.
• 4.4. The results indicate that cAMP-dependent protein kinase type I and II, casein kinases I and II, protein kinase C proenzymes I and II and Ca²⁺ /calmodulin kinase II are associated with the membranes of endoplasmic reticulum (ER).

     

Studies on the nutrition of the supralittoral isopod Ligia pallasii using chemically defined artificial diets: Assessment of vitamin,carbohydrate, fatty acid,cholesterol and mineral requirements

• 1.1. An artificial diet, consisting of a dry aggregate of 59 chemical substances, was used to assess the requirements of the sea slater Ligia pallasii for vitamins, carbohydrates, fatty acids, cholesterol and minerals.
• 2.2. Good growth and survival of L. pallasii was obtained on the diet, comparable to that on seaweeds and to that shown by a field population.
• 3.3. No dietary requirements for vitamins, fatty acids or cholesterol were shown for periods of 40 weeks or more for L. pallasii.
• 4.4. Carbohydrates were shown to be required by L. pallasii in its diet, in the order: starch, lactose > maltose, glucose > sucrose, cellulose.
• 5.5. Dietary requirements for minerals were, in order: calcium, magnesium, phosphorus > copper, nickel, zinc > iron, manganese, sulphur > iodine, silicon.
• 6.6. The results are discussed in relation to the role of gut bacteria in supplying required nutrients to their isopod hosts and the enhancement of this process through coprophagic behaviour.

     

The dynamics of the permeation of an analogue of insect juvenile hormone into nematodes

• 1.1. ¹⁴C-dichlorofarnesoate permeated rapidly into Haemonchus contortus (infective juveniles) and Panagrellus redivivus (mixed cultures) and was strongly bound by hydrophobic association (K_s > 10⁻⁴M).
• 2.2. Uptake rose linearly with increases in temperature (5–38°C) and external concentration (C₀; 0.07–2.15 × 10⁻⁴ M). Within 1 hr the internal concentration, C₁ was >C C₀.
• 3.3. The pH of the medium (6–8) did not affect uptake.
• 4.4. Efflux of dichlorofarnesoate was low: the half-time of release was > 18 hr.
• 5.5. The uptake curve approximated to the expression C₁/C₀ = a(1 − e^−bt) with a and b as constants and t in hr.
• 6.6. These results clarify previous work on the inhibitory action of juvenile hormone on the development of nematodes.

     

Myofibril and sarcoplasmic reticulum changes during muscle development: Activity vs inactivity

• 1.1. The purpose of this study was to determine whether biochemical changes of skeletal muscle that occur as a result of exercise in young rats persist into adulthood.
• 2.2. Littermates (10 days old) were assigned to a 3, 6 and 12 week control or training group. In addition, a rest-exercise group (R-E) and exercise-rest (E-R) group were included.
• 3.3. The rest-exercise and exercise-rest rats were maintained for the 12 weeks with the first 6 weeks being either rest or exercise and the condition reversed during the last 6 weeks of the experiment.
• 4.4. Myofibril ATPase activity of rat plantaris increased from the 10d to 12 week animals (P < 0.05). As anticipated, training resulted in a lowered activity at 6 and 12 weeks compared to controls.
• 5.5. The Ca²⁺ uptake and Ca²⁺-ATPase activity of the sarcoplasmic reticulum followed a similar pattern.
• 6.6. With regard to the exercise-rest rats, the myofibril and SR ATPase activities at 12 weeks were comparable to the 12 weeks control rats.
• 7.7. The rest-exercise group approximated the 12 week training group with regard to myofibril and SR ATPase activities (P > 0.05).
• 8.8. The results suggest that the training adaptations that occur during development of skeletal muscle return to normal, when training ceases in the adult rat.
• 9.9. Furthermore, animals that started to train prior to puberty do not have a greater capacity to adapt than animals which initiated training during adulthood.

     

The influence of calcium and magnesium on sea bass (Dicentrarchus labrax) intestinal brush border membrane purification and activity

• 1.1. The activity of brush border enzymes (alkaline phosphatase, maltase, sucrase, trehalase, leucine amino peptidase) was higher in purified membranes prepared with calcium. The contamination of these membranes with basolateral membranes was also lower (1.27 for Na-K-ATPase activity ratio).
• 2.2. The extraction of brush border lipids was carried out according to Folch adapted method. Two dimensional thin layer chromatography was used to separate the phospholipidic fractions. Fatty acids of phospholipids were analysed using gas chromatography after acid transmethylation (column SP 2330).
• 3.3. Phospholipids are composed of phosphatidylcholine (PC: 33%), phosphatidylethanolamine (PE: 30%), sphingomyeline (SM: 21%), phosphatidylserine (PS: 14%) and phosphatidylinositol (PI: 2%). 4. PC, PE and PS are characterized by high levels of unsaturated fatty acids (monounsaturated MUFA: 21.5% and polyunsaturated PUFA: 34.9%). The most abundant PUFA belong to the (n-3) family [18:3 (n-3), 20:5 (n-3) and 22:6 (n-3)].
• 4.5. Fatty acids from sphingomyelin of purified membranes have low proportions of PUFA (13.5%) but higher proportions of MUFA (39.5%).
• 5.6. No specific differences were found between calcium and magnesium prepared membranes.
• 6.7. The low content in LPC and the absence of LPE confirmed the absence of major structural lipids transformation during the membrane purification with calcium or magnesium.
• 7.8. Glycine transport was measured during 10 sec at different temperatures using the rapid filtration technique. Glycine transport was higher with Na⁺ than with K⁺. In the presence of Na⁺, this transport increases with temperature.
• 8.9. Arrhenius curves were mono phasic without obvious breakpoint and indicated no phase transition in the lipid bilayer.
• 9.10. A significant Na⁺ dependent glycine transport has been characterized at low temperatures (0°C) which suggests a possible role of membrane polyunsaturated fatty acids in the control of glycine transport.

     

Purification and some properties of an atypical alkaline p-nitrophenylphosphate phosphatase activity from Halobacterium halobium

• 1.1. An alkaline p-nitrophenylphosphate phosphatase has been purified 440-fold from extracts of Hatobacterium halobium.
• 2.2. The enzyme has an apparent molecular weight of 24,000.
• 3.3. A K_m value for p-nitrophenylphosphate of 1.12mM has been found under optimal conditions.
• 4.4. The enzyme is selectively activated and stabilized by Mn²⁺.
• 5.5. It requires high salt concentrations for stability and maximum activity.
• 6.6. It displays an unusual restricted substrate specificity of 25 phosphate esters tested, only phosphotyrosine and casein were hydrolysed besides p-nitrophenylphosphate.

     

Ascorbate (vitamin c) and dehydroascorbate stimulation of copper induced hemolysis protective action of ceruloplasmin,albumin and apotransferrin

• 1.1. Copper ion induced lysis of rat erythrocytes was markedly stimulated by low concentrations of ascorbate and dehydroascorbate.
• 2.2. Ascorbate oxidase, superoxide dismutase, catalase or scavengers of hydroxyl radicals protected erythrocytes against copper-ascorbate stimulated lysis.
• 3.3. It is proposed that superoxide radicals and hydrogen peroxide cooperate in producing hydroxyl radicals, which are directly involved in hemolysis.
• 4.4. The serum proteins, ceruloplasmin. albumin and apotransferrin, also reduced the hemolytic action of copper-ascorbate, the order of effectiveness being; ceruloplasmin > albumin > apotransferrin.

     

The isolation of skeletal muscle plasma membranes from rainbow trout (Salmo gairdneri)

• 1.1. Plasma membranes were isolated from caudal flank skeletal musculature of rainbow trout by discontinuous sucrose gradient centrifugation.
• 2.2. Na⁺−K⁺-ATPase was enriched 8-fold and 5′-nucleotidase activities 4-fold in a fraction isolated at the 8–25% sucrose interface.
• 3.3. A cholesterol: phospholipid ratio of 0.37 in the plasma membrane fraction was 85% greater than that observed in adjacent subcellular fractions.
• 4.4. Electron microscopy provided morphological confirmation of enrichment and integrity of skeletal muscle plasma membranes at the 8–25% sucrose interface.

     

Serum lipoproteins of sea bass (Dicentrarchus Labrax L.). Purification and partial characterization by density gradient ultracentrifugation and agarose column chromatography

• 1.1. Five classes of sea bass serum lipoproteins were purified by single vertical spin ultracentrifugation and agarose column chromatography
• 2.2. VLDL, beta migrating, are the larger and less dense lipoproteins.
• 3.3. LDL are the more heterogeneous in size, ranging from 11 × 10⁶ to 1 × 10⁶.
• 4.4. HDL represent the predominant class which, on the basis of density and electrophoresis migration, is differentiated in three subclasses.
• 5.5. VHDL float at a density > 1.22 mg/ml, which corresponds to the density of the other serum lipoproteins. This subclass, with an apparent molecular weight of 1.5 × 10⁵, resembles the albumin-like fatty acids binding proteins, shown in mammals and teleosts and absent in elasmobranchs.

     

The digestive enzymes of the pacific brown shrimp Penaeus californiensis.: I—Properties of amylase activity in the digestive tract

• 1.1. Crude extract of the whole digestive tract from the brown shrimp (P. californiensis) was investigated for digestive amylase activity.
• 2.2. Considerable amylase activity was found at pH 6.5–8.0, with optimum pH at around 7.5.
• 3.3. Optimum temperature was found between 30–40°C, similar to amylases from other crustaceans.
• 4.4. Amylase activity was highly halotolerant, having 50% maximum activity at 3 M NaCl.
• 5.5. Maximum amylase activity was found at 0.01 M NaCl.
• 6.6. Amylase activity was partially inhibited by the divalent ions Hg²⁺, Zn²⁺, Cu²⁺ and Cr²⁺.
• 7.7. Mg²⁺ and Ca²⁺ ions seemed to enhance amylase activity.

     

Uncovering Thousands of New Peptides with Sequence-Mask-Search Hybrid De Novo Peptide Sequencing Framework

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Highlights

• •Deep learning-based hybrid de novo sequencing with database search strategy.
• •Accurate identifications via ability to revise confidence scores and amino acids.
• •Discovery of >10,000 potential new HLA antigens and human phosphopeptides.
• •A dataset of >26 million annotated HCD spectra from Q Exactive instruments.

     

Differential post-receptor responses of adenylate cyclase in white and brown adipose tissue membranes of rats fed high-energy diets

• 1.1. Adenylate cyclase activity was determined in membranes of white and brown adipose tissue (WAT and BAT, respectively) from rats fed a high-energy diet (EXP group) vs those fed a nutritionally balanced one (CON group).
• 2.2. The isoproterenol- and guanine nucleotide-induced adenylate cyclase activity in WAT membranes of EXP rats was lower than that in CON rats.
• 3.3. Relative adenylate cyclase activity in like treated BAT membranes was higher in EXP than in CON rats.
• 4.4. It is concluded that feeding high-energy diets to rats induces similar post-receptor modifications of adenylate cyclase as found in genetic obese rodents.

     

Phosphatidylethanolamine: Ceramide-ethanolaminephosphotransferase activity in synaptic plasma membrane vesicles. Influence of some cations and phospholipid environment on transferase activity. Further proof of its location

• 1.1. Synaptic plasma membrane vesicles (SPMV) from rat brain synthesized ceramide-phosphoethanolamine (SpE), an analogue of sphingomyelin (SpC) from phosphatidylethanolamine (PE) and ceramide.
• 2.2. This reaction was catalyzed by PE: ceramide-phosphotransferase.
• 3.3. The presence of PC did not modify the SpE synthesis and PI and PS at twice PE concentration seemed to be activators; only PG was an inhibitor at all concentrations.
• 4.4. Some cations (Mg²⁺, Mn²⁺) were without effect, while Ca²⁺ increased transferase activity, so was interesting to study.
• 5.5. Transferase was compared with sialidase (external enzyme).
• 6.6. Kinetics other than those already performed by us were undertaken in order to confirm its location.

     

Characterization of lactoferrin binding to the MAC-T bovine mammary epithelial cell line using a biotin-avidin technique

• 1.1. A non-radioisotopic method utilizing a biotin-avidin approach was used to characterize lactoferrin binding to the clonal MAC-T bovine mammary epithelial cell line.
• 2.2. Binding of lactoferrin to MAC-T cells and isolated membranes was specific and saturable.
• 3.3. Unlabeled lactoferrin competed for and displaced biotin-labeled lactoferrin from binding sites on mammary epithelial cells. In contrast, unlabeled transferrin did not compete.
• 4.4. Scatchard analysis of lactoferrin binding to MAC-T cell crude membranes was nonlinear, revealing two classes of binding sites with association constants (K_a) of 2.36 × 10⁷ and 3.36 × 10⁶M⁻¹.
• 5.5. Binding of lactoferrin to MAC-T cells may be associated with the initial events which result in decreased MAC-T cell proliferation.

     

Time-dependent cadmium-neurotoxicity in rat brain synaptosomal plasma membranes

• 1.1. The modulation of lipid dynamics and lipid protein interactions were studied in rat brain synaptosomal plasma membranes (SPM) up to 24 hr after exposure to cadmium (Cd).
• 2.2. The activity of acetylcholinesterase and adenylate cyclase showed a considerable decrease after 6 hr of Cd exposure, followed by a progressive increase up to 24 hr.
• 3.3. SPM chemiluminescence showed a maximum decrease at 12 hr, demonstrating a considerable increase in lipid peroxidation.
• 4.4. SPM of Cd-exposed animals showed a statistical significant increase in fluorescence anisotropy parameter [(r₀/r) — 1]⁻¹ at 18 and 24 hr compared to SPM of the control, indicating a decrease of membrane fluidity.

     

Demonstration of highly specific and sensitive antibodies to a naturally occurring tetrahydroisoquinoline alkaloid,salsolidine

• 1.1. We report for the first time on the production and characterization of antibodies against a naturally occurring tetrahydroisoquinoline, namely salsolidine (6,7-dimethoxy-1-methyl-1,2,3,4-tetrahydroisoquinoline).
• 2.2. Immunogen synthesis was carried out by coupling the hapten salsolidine to bovine serum albumin (BSA) as carrier protein on the basis of reductive amination.
• 3.3. By immunization of rabbits with salsolidine-BSA conjugate antisalsolidine antibodies were produced.
• 4.4. At a final dilution of 1:1700 the highest-litre antiserum bound 35% of 0.21 pmol [³H] salsolidine. This antiserum was used to develop a radioimmunoassay for salsolidine.
• 5.5. Cross-reactivity studies revealed a high specificity of the antiserum to the hapten.
• 6.6. The antibodies had a high affinity to salsolidine (K_a = 1.5 × 10⁹ M⁻¹).
• 7.7. Standard curves covered a measuring range of 0.5–70 pmol/tube and the detection limit was found to be 0.27 pmol/tube.