-
European Journal of Biochemistry Jul 1983Proteolipid apoproteins have been isolated from a whole bovine brain homogenate by chloroform/methanol extraction, and fractionated by chromatography on modified...
Proteolipid apoproteins have been isolated from a whole bovine brain homogenate by chloroform/methanol extraction, and fractionated by chromatography on modified (lipophilic) Sephadex, followed by ion-exchange chromatography on CM-Trisacryl. The various final, highly hydrophobic, fractions are homogeneous (sodium dodecyl sulfate/polyacrylamide gel electrophoresis). Transmembrane ion transfers were studied by 22Na + flux and electrical conductance measurements. Single channel events were observed at low protein concentrations, in particular with one of the final homogeneous apoproteolipids of molecular mass 24 kDa.
Topics: Amino Acids; Animals; Brain Chemistry; Cattle; Cell Membrane Permeability; Chemical Phenomena; Chemistry; Electrophoresis, Polyacrylamide Gel; Membrane Lipids; Proteolipids
PubMed: 6861750
DOI: 10.1111/j.1432-1033.1983.tb07518.x -
Biophysical Journal Jan 2020Membrane proteins are embedded in a complex lipid environment that influences their structure and function. One key feature of nearly all biological membranes is a...
Membrane proteins are embedded in a complex lipid environment that influences their structure and function. One key feature of nearly all biological membranes is a distinct lipid asymmetry. However, the influence of membrane asymmetry on proteins is poorly understood, and novel asymmetric proteoliposome systems are beneficial. To our knowledge, we present the first study on a multispanning protein incorporated in large unilamellar liposomes showing a stable lipid asymmetry. These asymmetric proteoliposomes contain the Na/H antiporter NhaA from Salmonella Typhimurium. Asymmetry was introduced by partial, outside-only exchange of anionic phosphatidylglycerol (PG), mimicking this key asymmetry of bacterial membranes. Outer-leaflet and total fractions of PG were determined via ζ-potential (ζ) measurements after lipid exchange and after scrambling of asymmetry. ζ-Values were in good agreement with exclusive outside localization of PG. The electrogenic Na/H antiporter was active in asymmetric liposomes, and it can be concluded that reconstitution and generation of asymmetry were successful. Lipid asymmetry was stable for more than 7 days at 23°C and thus enabled characterization of the Na/H antiporter in an asymmetric lipid environment. We present and validate a simple five-step protocol that addresses key steps to be taken and pitfalls to be avoided for the preparation of asymmetric proteoliposomes: 1) optimization of desired lipid composition, 2) detergent-mediated protein reconstitution with subsequent detergent removal, 3) generation of lipid asymmetry by partial exchange of outer-leaflet lipid, 4) verification of lipid asymmetry and stability, and 5) determination of protein activity in the asymmetric lipid environment. This work offers guidance in designing asymmetric proteoliposomes that will enable researchers to compare functional and structural properties of membrane proteins in symmetric and asymmetric lipid environments.
Topics: Lipids; Proteolipids; Salmonella typhimurium; Unilamellar Liposomes
PubMed: 31843262
DOI: 10.1016/j.bpj.2019.10.043 -
Genes Oct 2022As an antimicrobial peptide, NK-lysin () plays an important role in the innate immune system of organisms. In this study, 300 piglets (68 Landrace pigs, 158 Large White...
As an antimicrobial peptide, NK-lysin () plays an important role in the innate immune system of organisms. In this study, 300 piglets (68 Landrace pigs, 158 Large White pigs and 74 Songliao Black pigs) were used to further explore the function of gene in porcine immune system. The quantitative real-time PCR analysis detected the gene's expression, and the result demonstrated that mRNA was expressed in lung, spleen, stomach, kidney, liver and heart, and the expression level decreased sequentially. A single-nucleotide polymorphism (SNP, g.59070355 G > A) in intron 3 of the gene was detected by PCR amplification and sequencing. The results of the Chi-square (χ2) test showed that the genotype of the SNP was consistent with the Hardy-Weinberg equilibrium. What's more, association analysis results showed the SNP in gene was significantly associated with T lymphocyte subpopulations. Different genotypes had significant effects on the proportion of CD4CD8, CD4CD8, CD4CD8, CD8, CD4/CD8 in peripheral blood ( < 0.05). These results further suggested that could be recognized as a promising immune gene for swine disease resistance breeding.
Topics: Swine; Animals; Proteolipids; Lymphocyte Subsets; Genomics
PubMed: 36360222
DOI: 10.3390/genes13111985 -
Methods in Molecular Biology (Clifton,... 2020Proteoliposome reconstitution is a method of choice for the investigation of membrane proteins as it allows their manipulation in the desired hydrophobic environment and...
Proteoliposome reconstitution is a method of choice for the investigation of membrane proteins as it allows their manipulation in the desired hydrophobic environment and allows one to tackle their study from both functional and structural points of view. Methods for their rapid and efficient reconstitution have been known for a long time but the quality and dispersity of the resulting suspensions is often overlooked. Here we describe our routine for the obtention of monodisperse populations of proteoliposomes as well as for the quantitation of protein per liposome.
Topics: Detergents; Membrane Proteins; Micelles; Proteolipids
PubMed: 33582987
DOI: 10.1007/978-1-0716-0724-4_3 -
Biochimica Et Biophysica Acta Oct 1982Proteolipid proteins were extracted from adult rat brain subcellular fractions and purified by chromatography on Sephadex LH-60. Polyacrylamide gel electrophoresis of...
Proteolipid proteins were extracted from adult rat brain subcellular fractions and purified by chromatography on Sephadex LH-60. Polyacrylamide gel electrophoresis of the delipidized proteins, in the presence or absence of 8 M urea, was carried out with all fractions. The distribution of the various types of proteolipid proteins was studied and their molecular weight calculated by the Ferguson relationship. Several bands of proteolipid proteins were found in the five membrane fractions analyzed. Some of them, such as the 17.5 K and 37 K components were very prominent in mitochondria and synaptosomes. The 30 K component was found in myelin-derived membranes and in microsomes, while the 20 K and 25 K proteolipid proteins were present in all subcellular fractions. The 30 K component (proteolipid protein (PLP)), typical of the purified myelin membranes, showed a similar distribution to that of 2',3'-cyclic-nucleotide 3'-phosphohydrolase (EC 3.1.4.37) activity, while the other major proteolipid protein present in all subcellular fractions (25 K) did not show such parallelism, indicating that it might not be an exclusive component of myelin. The electrophoretic pattern of microsomal proteolipid proteins did not show the high molecular weight components (aggregates of PLP) which are found in myelin. Furthermore, the 30 K component showed a smaller Y0 value than that of the 30 K found in myelin. Thus the presence of 30 K proteolipid protein in microsomes should not be considered as being due to myelin contamination.
Topics: 2',3'-Cyclic-Nucleotide Phosphodiesterases; Animals; Brain; Electrophoresis, Polyacrylamide Gel; Female; Male; Microsomes; Mitochondria; Molecular Weight; Myelin Sheath; Proteolipids; Rats; Rats, Inbred Strains; Synaptosomes; Urea
PubMed: 6291609
DOI: 10.1016/0005-2736(82)90417-5 -
Biochimica Et Biophysica Acta Nov 1998Surfactant protein B is a small homodimeric protein that is found tightly associated with surfactant lipids in the alveolar space. In this review, we discuss the actions... (Review)
Review
Surfactant protein B is a small homodimeric protein that is found tightly associated with surfactant lipids in the alveolar space. In this review, we discuss the actions of SP-B on phospholipid membranes using information predominantly obtained from model membrane systems. We try to correlate these model actions with current concepts of SP-B structure and proposed biological functions. These functions may include critical roles in the intracellular assembly of surfactant through a role in lamellar body organogenesis, the structural rearrangement of secreted surfactant lipids into tubular myelin, and the subsequent rapid insertion of secreted surfactant phospholipids into the surface film itself. The relevance of SP-B to human biology is emphasized by the fatal respiratory distress that is associated with a genetic deficiency of SP-B and the important role of SP-B in certain exogenous surfactant formulations in wide clinical use.
Topics: Lipid Bilayers; Liposomes; Protein Binding; Protein Precursors; Protein Processing, Post-Translational; Protein Structure, Secondary; Protein Structure, Tertiary; Proteolipids; Pulmonary Surfactants
PubMed: 9813296
DOI: 10.1016/s0925-4439(98)00064-7 -
Journal of Biochemistry Aug 1992A cDNA (T3-L) encoding the 16 kDa subunit of vacuolar H(+)-ATPase was cloned from a cDNA library of rat liver. A polypeptide of 155 amino acids with a molecular mass of...
A cDNA (T3-L) encoding the 16 kDa subunit of vacuolar H(+)-ATPase was cloned from a cDNA library of rat liver. A polypeptide of 155 amino acids with a molecular mass of 15,807 Da (pI = 9.5) having four hydrophobic stretches was predicted. T3-L polypeptide was 92% and 100% identical with the 16 kDa proteolipid of bovine chromaffin granule and that of mouse, respectively. Antisera raised against the NH2-terminal of the T3-L polypeptide reacted positively with the membrane ghosts of rat liver tritosomes and the partially purified H(+)-ATPase thereof. Western blotting of subcellular fractions with the antisera showed high abundance of 16 kDa protein in the lysosomes, although a significant amount was also detected in the Golgi apparatus. Western blotting of rat tissues revealed high levels of 16 kDa proteolipid in the brain and the kidney. Northern blots with T3-L similarly showed considerably high expression of T3-L mRNA in the brain and the kidney. Southern hybridization of rat genomic DNA with T3-L showed at most three distinct bands, regardless of the stringency of hybridization and whether hybridization was performed with its subfragments. This suggests the possibility of multiple (at least three) homologous/identical genes encoding 16 kDa proteolipid. The possible presence and significance of isoforms of 16 kDa proteolipid in rats are discussed.
Topics: Amino Acid Sequence; Animals; Base Sequence; Blotting, Southern; Blotting, Western; Brain; Cloning, Molecular; DNA; Golgi Apparatus; Kidney; Lysosomes; Molecular Sequence Data; Organ Specificity; Organelles; Proteolipids; Proton-Translocating ATPases; Rats; Sequence Alignment; Vacuoles
PubMed: 1400263
DOI: 10.1093/oxfordjournals.jbchem.a123879 -
Journal of Neuroscience Research Jan 1991Plasmolipin is a plasma membrane proteolipid which has recently been described as a component of myelin (Cochary et al.: Journal of Neurochemistry 55:602-610, 1990). The...
Plasmolipin is a plasma membrane proteolipid which has recently been described as a component of myelin (Cochary et al.: Journal of Neurochemistry 55:602-610, 1990). The present study reports the expression and localization of plasmolipin in primary glial cultures and secondary oligodendrocyte cultures. Double-label immunofluorescence showed that plasmolipin was expressed by galactocerebroside (GC)-positive oligodendrocytes, but was absent from astrocytes, characterized by their positive staining for glial fibrillary acidic protein (GFAP). At 1 week in culture plasmolipin staining was relatively weak in the cell body of some of the GC-positive cells. During the following 3 weeks in culture plasmolipin staining of oligodendrocytes gradually increased and was present in the cell body, its plasma membrane, and all the processes. However, the plasmolipin antibodies did not stain regions of the flat membrane sheets. Western blot analysis of homogenates from primary glial cultures showed that plasmolipin levels gradually increased during the first 5 weeks in culture. We conclude that the presence of plasmolipin in myelin is a result of its expression by oligodendrocytes.
Topics: Animals; Astrocytes; Cells, Cultured; Fluorescent Antibody Technique; Galactosylceramides; Gene Expression Regulation; Membrane Proteins; Mice; Myelin Basic Protein; Myelin and Lymphocyte-Associated Proteolipid Proteins; Nerve Tissue Proteins; Oligodendroglia; Proteolipids; Rats
PubMed: 1710283
DOI: 10.1002/jnr.490280108 -
Nature Immunology Feb 2006
Topics: Animals; Carrier Proteins; Immunity, Innate; Membrane Transport Proteins; Mice; Mice, Knockout; Models, Immunological; Myelin Proteins; Myelin and Lymphocyte-Associated Proteolipid Proteins; Proteolipids; Repressor Proteins; Signal Transduction; Suppressor of Cytokine Signaling 1 Protein; Suppressor of Cytokine Signaling Proteins; Toll-Like Receptors
PubMed: 16424886
DOI: 10.1038/ni0206-123 -
Annual Review of Physiology 1991
Review
Topics: Animals; Humans; Protein Conformation; Proteolipids; Pulmonary Surfactant-Associated Proteins; Pulmonary Surfactants
PubMed: 2042965
DOI: 10.1146/annurev.ph.53.030191.002111