Localization of the basic protein and lipophilin in the myelin membrane with a non-penetrating reagent

The localization of proteins in myelin was studied by the use of a non-penetrating penetrating reagent. Tritiated 4,4′-diisothiocyano-2,2′-ditritiostilbene disulfonic acid was used to label the isolated myelin membrane. The membrane was labelled, the basic protein and the hydrophobic protein, lipophilin, were isolated. After 10 min of exposure to the reagent, the specific activity of lipophilin was found to be 10 times greater than that of the basic protein. Water shock did not alter the specific activities. However, sonication increased the specific activity of lipophilin but not that of basic protein. When the isolated proteins were labelled with ³H-labelled, 4,4′-diisothiocyano-2,2′-ditritiostilbene disulfonic acid, the specific activity of the basic protein was 10 times that of lipophilin. We concluded that the low specific activity of basic protein isolated from the labelled membrane was due to the inaccessible position of this protein in the membrane bilayer.     

Localization of the basic protein and lipophilin in the myelin membrane with a non-penetrating reagent.

The localization of proteins in myelin was studied by the use of a non-penetrating reagent. Tritiated 4,4'-diisothiocyano-2,2'-ditritiostilbene disulfonic acid was used to label the isolated myelin membrane. The membrane was labelled, the basic protein and the hydrophobic protein, lipophilin, were isolated. After 10 min of exposure to the reagent, the specific activity of lipophilin was found to be 10 times greater than that of the basic protein. Water shock did not alter the specific activities. However, sonication increased the specific activity of lipophilin but not that of basic protein. When the isolated proteins were labelled with 3H-labelled 4,4'-diisothiocyano-2,2'-ditritiostilbene disulfonic acid, the specific activity of the basic protein was 10 times that of lipophilin. We concluded that the low specific activity of basic protein isolated from the labelled membrane was due to the inaccessible position of this protein in the membrane bilayer.     

Structure of cytochrome b5 and its topology in the microsomal membrane

The complete amino acid sequence of human and chicken liver microsomal cytochrome b5 was determined. The amino termini of cytochrome b5 from four other mammalian species were examined in order to determine their complete covalent structure. As in the rat species, cytochrome b5 preparations from man, rabbit, calf and horse had an acetylated alanine as the first residue. In contrast, the pig cytochrome had alanine at the amino terminus. The amino terminus of the chicken cytochrome b5 was also unmodified, and extended three residues absent in the mammalian species. In order to investigate whether the carboxy-terminal segment of cytochrome b5 is located on the cytosolic or the luminal side of the microsomal membrane, rabbit liver microsomes were treated with trypsin and subjected to gel filtration and high-pressure liquid chromatography. The nonpolar peptide isolated from these microsomes lacked the terminal hexapeptide, indicating that when cytochrome b5 is bound to intact microsomes, the carboxy terminus is located on the cytosolic side of the membrane and does not extend in the lumen of the endoplasmic reticulum.     

Chemical modification of rat liver microsomal cytochrome P-450: study of enzymic properties and membrane topology

Isolated rat liver cytochrome P-450IIB1 was alkylated and acetylated at primary amino groups, and the position of the modified amino acids in the protein was identified. Alkylation of up to nine amino groups did not disturb the interaction of reconstituted P-450 and NADPH-cytochrome-P-450 reductase in a way that hydroxylation of benzphetamine was altered, whereas deethylation of 7-ethoxycoumarin was gradually reduced in parallel with impaired 7-ethoxycoumarin binding. Acetylation of four lysine residues completely inhibited binding and metabolism of 7-ethoxycoumarin but not of benzphetamine. These results suggest the presence of different substrate binding sites on P-450. Exhaustive proteolysis of modified P-450 in proteoliposomes liberated all but the N-terminal modified peptide and 85 to 90% of the cytochrome's mass from intact proteoliposomes. These findings further support our previously proposed model of P-450 topology (Vergères, G., Winterhalter, K.H. and Richter, C. (1989) Biochemistry 28, 3650-3655), in which P-450 is anchored to the membrane with the N-terminal peptide only, the N-terminal methionine facing the lumenal interior.     

Cell evolution and the problem of membrane topology

Cells somehow evolved from primordial chemistry and their emergence depended on the co-evolution of the cytoplasm, a genetic system and the cell membrane. It is widely believed that the cytoplasm evolved inside a primordial lipid vesicle, but here I argue that the earliest cytoplasm could have co-evolved to high complexity outside a vesicle on the membrane surface. An invagination of the membrane, aided by an early cytoskeletal system, may have formed the first cells--initially within primordial vesicles.     

Insertion mutagenesis and membrane topology model of the Pseudomonas aeruginosa outer membrane protein OprM

Pseudomonas aeruginosa OprM is a protein involved in multiple-antibiotic resistance as the outer membrane component for the MexA-MexB-OprM efflux system. Planar lipid bilayer experiments showed that OprM had channel-forming activity with an average single-channel conductance of only about 80 pS in 1 M KCl. The gene encoding OprM was subjected to insertion mutagenesis by cloning of a foreign epitope from the circumsporozoite form of the malarial parasite Plasmodium falciparum into 11 sites. In Escherichia coli, 8 of the 11 insertion mutant genes expressed proteins at levels comparable to those obtained with the wild-type gene and the inserted malarial epitopes were surface accessible as assessed by indirect immunofluorescence. When moved to a P. aeruginosa OprM-deficient strain, seven of the insertion mutant genes expressed proteins at variable levels comparable to that of wild-type OprM and three of these reconstituted MIC profiles resembling those of the wild-type protein, while the other mutant forms showed variable MIC results. Utilizing the data from these experiments, in conjunction with multiple sequence alignments and structure predictions, an OprM topology model with 16 beta strands was proposed.     

Sphingolipid topology and the dynamic organization and function of membrane proteins

When acquiring internal membranes and vesicular transport, eukaryotic cells started to synthesize sphingolipids and sterols. The physical differences between these and the glycerophospholipids must have enabled the cells to segregate lipids in the membrane plane. Localizing this event to the Golgi then allowed them to create membranes of different lipid composition, notably a thin, flexible ER membrane, consisting of glycerolipids, and a sturdy plasma membrane containing at least 50% sphingolipids and sterols. Besides sorting membrane proteins, in the course of evolution the simple sphingolipids obtained key positions in cellular physiology by developing specific interactions with (membrane) proteins involved in the execution and control of signaling. The few signaling sphingolipids in mammals must provide basic transmission principles that evolution has built upon for organizing the specific regulatory pathways tuned to the needs of the different cell types in the body.     

S79株腮腺炎病毒制备纯化抗原用于诊断试剂的研究

以S79株腮腺炎病毒制备纯抗原,用于制备检测腮腺炎抗体(IgG)的ELISA试剂盒。将腮腺炎病毒S79株培养液用中空纤维超滤器进行浓缩,经PEG沉淀后,采用蔗糖密度梯度离心法纯化抗原;以纯化的S79株腮腺炎病毒为包被抗原,制备成ELISA腮腺炎病毒抗体(IgG)诊断试剂,并与SIGMA同类产品进行比较。结果显示,经纯化的S79株抗原的比活力为412.9HAU/mg,杂蛋白清除率为99.5%。应用纯化的S79株腮腺炎病毒抗原制备的ELISA试剂,与SIGMA试剂比较,敏感度为94.4%,特异度为91.7%,一致性为93.3%。结论,以纯化的S79株腮腺炎病毒为包被抗原,可用于大批量ELISA腮腺炎抗体诊断试剂盒的制备。     

Studies on membrane topology, N-glycosylation and functionality of SARS-CoV membrane protein

Background

Dengue is the most important arbovirus disease in tropical and subtropical countries. The viral envelope (E) protein is responsible for cell receptor binding and is the main target of neutralizing antibodies. The aim of this study was to analyze the diversity of the E protein gene of DENV-3. E protein gene sequences of 20 new viruses isolated in Ribeirao Preto, Brazil, and 427 sequences retrieved from GenBank were aligned for diversity and phylogenetic analysis.

Results

Comparison of the E protein gene sequences revealed the presence of 47 variable sites distributed in the protein; most of those amino acids changes are located on the viral surface. The phylogenetic analysis showed the distribution of DENV-3 in four genotypes. Genotypes I, II and III revealed internal groups that we have called lineages and sub-lineages. All amino acids that characterize a group (genotype, lineage, or sub-lineage) are located in the 47 variable sites of the E protein.

Conclusion

Our results provide information about the most frequent amino acid changes and diversity of the E protein of DENV-3.     

Peptide-specific antibodies and proteases as probes of the transmembrane topology of the bovine heart mitochondrial porin.

We have investigated the transmembrane topology of the bovine heart mitochondrial porin by means of proteases and antibodies raised against the amino-terminal region of the protein. The antisera against the human N-terminus reacted with porin in Western blots of NaDodSO4-solubilized bovine heart mitochondria and with the membrane-bound porin in enzyme-linked immunosorbent assay (ELISA). The immunoreaction with mitochondria coated on microtiter wells showed that the amino-terminal region of the protein is not embedded in the lipid bilayer but is exposed to the cytosol. Back-titration of unreacted anti-N-terminal antibodies after their incubation with intact mitochondria demonstrated that the porin N-terminus is also exposed in "noncoated" mitochondria. No difference in antisera reactivity was observed between intact and broken mitochondria. Intact and broken mitochondria were subjected to proteolysis by specific proteases. The membrane-bound bovine heart porin was strongly resistant to proteolysis, but a few specific cleavage sites were observed. Staphylococcus aureus V8 protease gave a large 24K N-terminal peptide, trypsin produced a 12K N-terminal and an 18K C-terminal peptide, and chymotrypsin gave two peptides of Mr 19.5K and 12.5K, which were both recognized by the antiserum against the human N-terminus. Carboxypeptidase A was ineffective in cleaving the membrane-bound porin in both intact and broken mitochondria. Thus, the carboxy-terminal part of the protein is probably not exposed to the water phase. The cleavage patterns of membrane-bound porin, obtained with S. aureus V8 protease, trypsin, and chymotrypsin, showed no difference between intact and broken mitochondria, thus indicating that all porin molecules have the same orientation in the membrane. The computer analysis of the sequence of human B-lymphocyte porin suggested that 16 beta-strands can span the phospholipid bilayer. This result, together with the overall information presented, allowed us to draw a possible scheme of the transmembrane arrangement of mammalian mitochondrial porin.     

Determining the membrane topology of peptides by fluorescence quenching

Determination of the topology of peptides in membranes is important for characterizing and understanding the interactions of peptides with membranes. We describe a method that uses fluorescence quenching arising from resonance energy transfer ("FRET") for determining the topology of the tryptophan residues of peptides partitioned into phospholipid bilayer vesicles. This is accomplished through the use of a novel lyso-phospholipid quencher (lysoMC), N-(7-hydroxyl-4-methylcoumarin-3-acetyl)-1-palmitoyl-2-hydroxy-sn-gly cero-3-phosphoethanolamine. The design principle was to anchor the methylcoumarin quencher in the membrane interface by attaching it to the headgroup of lyso-phosphoethanolamine. We show that lysoMC can be incorporated readily into large unilamellar phospholipid vesicles to yield either symmetrically (both leaflets) or asymmetrically (outer leaflet only) labeled bilayers. LysoMC quenches the fluorescence of membrane-bound tryptophan by the F?rster mechanism with an apparent R(0) that is comparable to the thickness of the hydrocarbon core of a lipid bilayer (approximately 25 A). Consequently, the methylcoumarin acceptor predominantly quenches tryptophans that reside in the same monolayer as the probe. The topology of a peptide's tryptophan in membranes can be determined by comparing the quenching in symmetric and asymmetric lysoMC-labeled vesicles. Because it is essential to know that asymmetrically incorporated lysoMC remains so under all conditions, we also developed a second type of FRET experiment for assessing the rate of transbilayer diffusion (flip-flop) of lysoMC. Except in the presence of pore-forming peptides, there was no measurable flip-flop of lysoMC, indicating that asymmetric distributions of quencher are stable. We used these methods to show that N-acetyl-tryptophan-octylamide and tryptophan-octylester rapidly equilibrate across phosphatidylcholine (POPC) and phosphatidylglycerol (POPG) bilayers, while four amphipathic model peptides remain exclusively on the outer monolayer. The topology of the amphipathic peptide melittin bound to POPC could not be determined because it induced rapid flip-flop of lysoMC. Interestingly, melittin did not induce lysoMC flip-flop in POPG vesicles and was found to remain stably on the external monolayer.     

Enhanced membrane protein topology prediction using a hierarchical classification method and a new scoring function

The prediction of transmembrane (TM) helix and topology provides important information about the structure and function of a membrane protein. Due to the experimental difficulties in obtaining a high-resolution model, computational methods are highly desirable. In this paper, we present a hierarchical classification method using support vector machines (SVMs) that integrates selected features by capturing the sequence-to-structure relationship and developing a new scoring function based on membrane protein folding. The proposed approach is evaluated on low- and high-resolution data sets with cross-validation, and the topology (sidedness) prediction accuracy reaches as high as 90%. Our method is also found to correctly predict both the location of TM helices and the topology for 69% of the low-resolution benchmark set. We also test our method for discrimination between soluble and membrane proteins and achieve very low overall false positive (0.5%) and false negative rates (0 to approximately 1.2%). Lastly, the analysis of the scoring function suggests that the topogeneses of single-spanning and multispanning TM proteins have different levels of complexity, and the consideration of interloop topogenic interactions for the latter is the key to achieving better predictions. This method can facilitate the annotation of membrane proteomes to extract useful structural and functional information. It is publicly available at http://bio-cluster.iis.sinica.edu.tw/~bioapp/SVMtop.     

Predicted secondary structure and membrane topology of the scrapie prion protein

The integral membrane sialoglycoprotein PrPSc is the only identifiable component of the scrapie prion. Scrapie in animals and Creutzfeldt-Jakob disease in humans are transmissible, degenerative neurological diseases caused by prions. Standard predictive strategies have been used to analyze the secondary structure of the prion protein in conjunction with Fourier analysis of the primary sequence hydrophobicities to detect potential amphipathic regions. Several hydrophobic segments, a proline- and glycine-rich repeat region and putative glycosylation sites are incorporated into a model for the integral membrane topology of PrP. The complete amino acid sequences of the hamster, human and mouse prion proteins are compared and the effects of residue substitutions upon the predicted conformation of the polypeptide chain are discussed. While PrP has a unique primary structure, its predicted secondary structure shares some interesting features with the serum amyloid A proteins. These proteins undergo a post-translational modification to yield amyloid A, molecules that share with PrP the ability to polymerize into birefringent filaments. Our analyses may explain some experimental observations on PrP, and suggest further studies on the properties of the scrapie and cellular PrP isoforms.     

Short-chain aliphatic alcohols increase rat-liver microsomal membrane fluidity and affect the activities of some microsomal membrane-bound enzymes

n-Butyl and isoamyl alcohols decrease the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene and enhance the efficiency of pyrene excimer formation when these probes are incorporated in rat-liver microsomal membrane, suggesting an increase in rotational and translational mobilities. Neither alcohol modifies NADH-ferricyanide reductase activity but both increase NADH-cytochrome c reductase activity. This was interpreted as an increase in the rate of lateral diffusion of the proteins cytochrome b5 and cytochrome b5 reductase as a consequence of the enhanced membrane lipid phase fluidity. Microsomal delta 9 and delta 6 desaturase activities in the presence of isoamyl alcohol were also studied. This alcohol decreases delta 9 desaturation when it is measured at a low substrate concentration (13 microM palmitic acid), but it is not modified when it is measured at a high substrate concentration (66 microM palmitic acid). delta 6 desaturation is diminished by isoamyl alcohol when it is measured with both 13 microM and 66 microM linoleic acid. The influence of isoamyl alcohol on the glucose-6-phosphatase system activity was also studied. In non-detergent-treated microsomes, isoamyl alcohol enhances glucose-6-phosphatase activity. However, if microsomes are previously treated with 0.1% Triton X-100 isoamyl alcohol does not modify this activity. The enhancement of the glucose 6-phosphate transport rate is not due to membrane permeability barrier disruption, since isoamyl alcohol does not modify mannose-6-phosphohydrolase latency. This would suggest that an increase in membrane lipid phase fluidity specifically activates glucose 6-phosphate transport across the membrane.