Reconstitution of pig lymphocyte plasma membranes from solubilized components, with particular reference to membrane-associated immunoglobulins.
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ABSTRACT:
SUBMITTER: Chavin SI
PROVIDER: S-EPMC1172468 | biostudies-other | 1975 Nov
REPOSITORIES: biostudies-other
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Project description:The degree of solubilization of pig lymphocyte plasma membrane by sodium deoxycholate was determined at a variety of temperatures and detergent concentrations. Approx. 95% of the membrane protein was soluble in 2% deoxycholate at 23 degrees C. Some of the biological activities of the membrane survived this treatment. The leucine beta-naphthylamidase activity was more readily soluble than the 5'-nucleotidase and these enzymes could be separated by extraction with 0.5% deoxycholate at 0 degrees C. Membrane solubilized in 2% deoxycholate at 23 degrees C was fractionated by sucrose-density-gradient centrifugation in 1% deoxycholate. The phospholipid was separated from the protein, which formed a fairly symmetrical peak that sedimented slightly slower than ovalbumin; the leucine naphthylamidase and 5'-nucleotidase activities were resolved from each other and from the main protein peak. Similar separations were achieved by elution from Sephadex G-200 and Sepharose 6B in 1% deoxycholate. The main proteins, however, appeared to possess much higher molecular weights than those indicated by sucrose-density-gradient centrifugation. This disparity suggests that many of the membrane proteins have a rod-like shape, especially since the results of experiments with [(14)C]deoxycholate revealed that the proteins did not bind significant amounts of deoxycholate. In contrast, 5'-nucleotidase and leucine naphthylamidase appeared to be globular. Polyacrylamide-gel electrophoresis of membrane solubilized in sodium dodecyl sulphate gave a similar distribution of protein to that achieved by sucrose-density-gradient centrifugation. Trace amounts only of polypeptides of molecular weight less than 10000 were detected.
| S-EPMC1177097 | biostudies-other
Project description:Plasma-membrane preparations purified from pig lymphocytes contained a major polypeptide component of mol.wt. about 68 000. This component was identified as pig albumin by the following comparisons with authentic pig serum albumin: (a) co-migration when analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under reducing and non-reducing conditions; (b) identical isoelectric points; (c) similar "fingerprints" of arginine-containing tryptic peptides; (d) reactivity with anti-(pig albumin) serum. The albumin was bound tightly to the plasma membrane. Biosynthetic labelling of pig lymphocytes under a variety of conditions failed to provide evidence that albumin was synthesized by lymphocytes, suggesting that the plasma-membrane-associated albumin was of extraneous origin. Radiolabelled pig serum albumin, however, failed to bind to the plasma-membrane fraction when added before cell disruption. Although lymphocyte plasma membrane preparations from other species possessed a polypeptide of about 68 000 mol.wt., this was judged not to be albumin on the basis of electrophoretic mobility under non-reducing conditions; also, no polypeptide was precipitated by anti-albumin sera. It is concluded that pig lymphocyte plasma-membrane preparations possess albumin which, although firmly attached, was probably of extraneous origin. This association appeared not to be common to lymphocytes from other species.
| S-EPMC1162306 | biostudies-other
Project description:Pig lymphocyte plasma-membrane vesicles that are impermeable to large molecules were prepared by means of centrifugation on a continuous dextran gradient. They were isolated in a yield of 60--80% of the total plasma membrane recovered, and were characterized as being impermeable to macromolecules by their exclusion of inulin (mol.wt. 5200) and Dextran T10 (mol.wt. 10 000).
| S-EPMC1162062 | biostudies-other
Project description:Plasma membranes from pig mesenteric lymph-node lymphocytes contain a large number of polypeptide chains ranging in molecular weight from 20,000 to greater than 470,000, with a major component of 46,000. There are approx. 12-15 glycoproteins. The membranes contain immunoglobulin G, which comprises 0.6% of the total protein. Immunoglobulin M is also detected, but has not been accurately quantified for technical reasons. Possible origins of the membrane-associated immunoglobulin are briefly discussed. It is concluded that the immunoglobulin is probably associated with the plasma membrane in the intact cell.
| S-EPMC1165558 | biostudies-other
Project description:1. The lipid fraction of the plasma membrane of pig mesenteric lymph-node lymphocytes contained primarily (94%) neutral lipids and phospholipids in about equal weights. The remianing lipid comprised glycosphingolipids (1.8%), gangliosides (o.27%)and probably ceramides (1.3%). The major phospholipid was phosphatidylcholine (46% of the total), and mono- and tri-hexosylceramides accounted for 72% of the glycosphingolipids. Haematoside was distributed between the glycosphingolipid and ganglioside fractions. The major ganglioside was monosialoganglioside. About 90% of the sialic acid was N-glycollylated. 2. A comparision of the lipid composition of the plasma-membrane fraction with that of the initial lymph-node homogenate showed that the purified membrane contained increased proportions of phospholipids, especially sphingomyelin, glycosphingolipids and gangliosides. 3. Fatty acid analyses showed that the membrane phosphatidylcholine was rich in palmitic acid, that the sphingomeyelin and phosphatidylethanolamine were high in myristic acid and that the glycosphingolipids were rich in oleic acid.
| S-EPMC1163721 | biostudies-other
Project description:Pig lymphocyte plasma membrane isolated from mesenteric lymph node contained 69 mug of carbohydrate/mg dry wt., which was made up of neutral sugar, amino sugar and sialic acid in the molar proportions 5:1.7:1. The neutral sugar comprised fucose, ribose, mannose, glucose, galactose and inositol (molar proportions 2:9:11:15:26:1), and the amino sugar glucosamine and galactosamine (molar ratio 2:1). The ribose was most probably derived from RNA. All of the fucose and mannose and almost all of the glucosamine were associated with the membrane protein whereas the membrane lipid contained all of the inositol. The remaining sugars were distributed in various ratios between the protein and lipid fractions.
| S-EPMC1172542 | biostudies-other
Project description:There is an increasing demand for non-antibiotics solutions to control infectious disease in intensive pig production. Here, one such alternative, namely pig antibodies purified from slaughterhouse blood was investigated in order to elucidate its potential usability to control post-weaning diarrhoea (PWD), which is one of the top indications for antibiotics usage in the pig production. A very cost-efficient and rapid one-step expanded bed adsorption (EBA) chromatography procedure was used to purify pig immunoglobulin G from slaughterhouse pig plasma (more than 100 litres), resulting in >85% pure pig IgG (ppIgG). The ppIgG thus comprised natural pig immunoglobulins and was subsequently shown to contain activity towards four pig-relevant bacterial strains (three different types of Escherichia coli and one type of Salmonella enterica) but not towards a fish pathogen (Yersinia ruckeri), and was demonstrated to inhibit the binding of the four pig relevant bacteria to a pig intestinal cell line (IPEC-J2). Finally it was demonstrated in an in vivo weaning piglet model for intestinal colonization with an E. coli F4+ challenge strain that ppIgG given in the feed significantly reduced shedding of the challenge strain, reduced the proportion of the bacterial family Enterobacteriaceae, increased the proportion of families Enterococcoceae and Streptococcaceae and generally increased ileal microbiota diversity. Conclusively, our data support the idea that natural IgG directly purified from pig plasma and given as a feed supplement can be used in modern swine production as an efficient and cost-effective means for reducing both occurrence of PWD and antibiotics usage and with a potential for the prevention and treatment of other intestinal infectious diseases even if the causative agent might not be known.
| S-EPMC4744083 | biostudies-literature
Project description:Diverse cellular signaling events, including B cell receptor (BCR) activation, are hypothesized to be facilitated by domains enriched in specific plasma membrane lipids and proteins that resemble liquid-ordered phase-separated domains in model membranes. This concept remains controversial and lacks direct experimental support in intact cells. Here, we visualize ordered and disordered domains in mouse B lymphoma cell membranes using super-resolution fluorescence localization microscopy, demonstrate that clustered BCR resides within ordered phase-like domains capable of sorting key regulators of BCR activation, and present a minimal, predictive model where clustering receptors leads to their collective activation by stabilizing an extended ordered domain. These results provide evidence for the role of membrane domains in BCR signaling and a plausible mechanism of BCR activation via receptor clustering that could be generalized to other signaling pathways. Overall, these studies demonstrate that lipid mediated forces can bias biochemical networks in ways that broadly impact signal transduction.
| S-EPMC5373823 | biostudies-literature
Project description:1. Rat and rabbit erythrocyte plasma-membrane proteins were solubilized with decanoyl-N-methylglucamide and reconstituted into liposomes. The procedure includes detergent removal by gel filtration, followed by a freeze-thaw step. 2. The rate of [1-14C]pyruvate uptake into these vesicles was inhibited by approx. 70% by alpha-cyano-4-hydroxycinnamate and p-chloromercuribenzenesulphonate. The extent of uptake at equilibrium was not affected by the presence of these inhibitors, but was dependent on the osmolarity of the suspending medium. 3. Reconstituted bovine erythrocyte membranes, which have no lactate carrier, showed a much slower time course of pyruvate uptake, with no inhibitor-sensitive component. 4. L- but not D-lactate competed for alpha-cyano-4-hydroxycinnamate-sensitive [1-14C]pyruvate uptake.
| S-EPMC1135089 | biostudies-other
Project description:BackgroundSeeds of Paeonia ostii have been proposed as a source of raw material for the production of edible oil; however, lack of information about the developmental biology of the seeds hampers our ability to use them. Our aim was to investigate development of the seed coat, endosperm and embryo of P. ostii in relation to timing of accumulation of nutrient reserves from pollination to seed maturity. Ovules and developing seeds of P. ostii were collected at various stages of development from zygote to maturity. Seed fresh mass, dry mass, germination, moisture, soluble sugars, starch, protein and oil content were determined. Ontogeny of seeds including embryo, endosperm and seed coat were analyzed histologically.ResultsThe ovule of P. ostii is anatropous, crassinucellate and bitegmic. The zygote begins to divide at about 5 days after pollination (DAP), and the division is not accompanied by cell wall formation. By 25 DAP, the proembryo begins to cellularize. Thereafter, several embryo primordia appear at the surface of the cellularized proembryo, but only one matures. Endosperm development follows the typical nuclear type. The seed coat is derived from the outer integument. During seed development, soluble sugars, starch and crude fat content increased and then decreased, with maximum contents at 60, 80 and 100 DAP, respectively. Protein content was relatively low compared with soluble sugars and crude fat, but it increased throughout seed development.ConclusionsDuring seed development in P. ostii, the seed coat acts as a temporary storage tissue. Embryo development of P. ostii can be divided into two stages: a coenocytic proembryo from zygote (n + n) that degenerates and a somatic embryo from peripheral cells of the proembryo (2n → 2n). This pattern of embryogeny differs from that of all other angiosperms, but it is similar to that of gymnosperms.
| S-EPMC8684281 | biostudies-literature