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1.
Purification and characterization of minor brain proteolipids: use of fast atom bombardment-mass spectrometry for peptide sequencing 总被引:2,自引:0,他引:2
P Lepage G Helynck J Y Chu B Luu O Sorokine E Trifilieff A Van Dorsselaer 《Biochimie》1986,68(5):669-686
A combination of lipophilic gel permeation chromatography and ion-exchange chromatography in organic solvents was used to purify low molecular weight proteolipids from bovine brain. Cleavage peptides were purified by HPLC and studied mainly by the fast atom bombardment--mass spectrometry technique. A proteolipid of Mr 14 000 contains several peptides from the first 113 amino acids of the major myelin proteolipid (MMPL) plus an extra unknown blocked N-terminal peptide. A proteolipid of Mr 16 000 contains smaller peptides belonging to a C-terminal fragment of MMPL of about 160 residues. These two proteolipids do not seem to be artifacts from MMPL. 相似文献
2.
C Ruppert H Kavermann S Wimmers R Schmid J Kellermann F Lottspeich H Huber K O Stetter V Müller 《The Journal of biological chemistry》1999,274(36):25281-25284
The proteolipid, a hydrophobic ATPase subunit essential for ion translocation, was purified from membranes of Methanococcus jannaschii by chloroform/methanol extraction and gel chromatography and was studied using molecular and biochemical techniques. Its apparent molecular mass as determined in SDS-polyacrylamide gel electrophoresis varied considerably with the conditions applied. The N-terminal sequence analysis made it possible to define the open reading frame and revealed that the gene is a triplication of the gene present in bacteria. In some of the proteolipids, the N-terminal methionine is excised. Consequently, two forms with molecular masses of 21,316 and 21,183 Da were determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The molecular and biochemical data gave clear evidence that the mature proteolipid from M. jannaschii is a triplication of the 8-kDa proteolipid present in bacterial F(1)F(0) ATPases and most archaeal A(1)A(0) ATPases. Moreover, the triplicated form lacks a proton-translocating carboxyl group in the first of three pairs of transmembrane helices. This finding puts in question the current view of the evolution of H(+) ATPases and has important mechanistic consequences for the structure and function of H(+) ATPases in general. 相似文献
3.
Purification and characterization of dicyclohexylcarbodiimide binding protein from mouse liver mitochondria 总被引:1,自引:0,他引:1
The DCCD-binding protein from mouse liver Mt has been purified by chloroform: methanol method. The DCCD binding is drastically reduced when lipids are extracted from the proteolipid. The proteolipid as well as the lipid extracted protein migrate as single component with 7.8 K daltons molecular weight. The protein fraction yields a single band (pI 5.8) on isoelectric focusing gels. The DCCD binding protein is a product of Mt translation and contains Val as the N-terminal residue. 相似文献
4.
The amino- and carboxyl-terminal amino acids of proteolipids from neural and non-neural sources were investigated. Amino-terminal amino acids were identified and quantitated by the dansyiation procedure. Carboxyl-terminal amino acids were determined after hydrazinolysis or enzymatic hydrolysis with carboxypeptidases. Proteolipid from white matter showed two terminal amino acids, regardless of the method of preparation. The major N-terminal amino acid was glycine and the minor one was glutamic acid or glutamine. The corresponding C-terminal amino acids were phenylalanine and glycine. Preparations of white matter proteolipid, therefore, contained more than one protein or protein chain. Proteolipids from brain mitochondria, heart, liver and kidney were characterized by N-terminal aspartic acid or asparagine and C-terminal lysine residues and they exhibited an amino acid composition which differed from white matter proteolipid. Our results suggest the existence of two classes of proteolipids, a myelin type and a non-myelin type. Synaptic membrane and grey matter proteolipids exhibited characteristics of both classes. 相似文献
5.
Yeast mitochondrial ATPase subunit 8, normally a mitochondrial gene product, expressed in vitro and imported back into the organelle. 总被引:4,自引:1,他引:3
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Subunit 8 of yeast mitochondrial F1F0-ATPase is a proteolipid made on mitochondrial ribosomes and inserted directly into the inner membrane for assembly with the other F0 membrane-sector components. We have investigated the possibility of expressing this extremely hydrophobic, mitochondrially encoded protein outside the organelle and directing its import back into mitochondria using a suitable N-terminal targeting presequence. This report describes the successful import in vitro of ATPase subunit 8 proteolipid into yeast mitochondria when fused to the targeting sequence derived from the precursor of Neurospora crassa ATPase subunit 9. The predicted cleavage site of matrix protease was correctly recognized in the fusion protein. A targeting sequence from the precursor of yeast cytochrome oxidase subunit VI was unable to direct the subunit 8 proteolipid into mitochondria. The proteolipid subunit 8 exhibited a strong tendency to embed itself in mitochondrial membranes, which interfered with its ability to be properly imported when part of a synthetic precursor. 相似文献
6.
Isolation and terminal sequence determination of the major rat brain myelin proteolipid P7 apoprotein 总被引:5,自引:0,他引:5
J L Nussbaum J F Rouayrenc P Mandel J Jollès P Jollès 《Biochemical and biophysical research communications》1974,57(4):1240-1247
The major rat brain myelin proteolipid P7 apoprotein has been isolated in pure form by a preparative sodium dodecylsulphate gel electrophoresis system. Automated Edman degradation permitted the establishment of the N-terminal sequence up to the 20th amino acid. The C-terminal sequence was determined by the action of carboxypeptidase A. 相似文献
7.
Patrick Schindler Bang Luu Odile Sorokine Elisabeth Trifilieff Alain Van Dorsselaer 《Journal of neurochemistry》1990,55(6):2079-2085
In a developmental study, we have shown that DM-20 is present before proteolipid protein (PLP) in the fetal bovine cerebral hemispheres. When the white matter appears (27-30 weeks of gestation), the amount of DM-20 drastically increases. DM-20 remains the major proteolipid until birth. PLP is detected only 2-4 weeks after the appearance of white matter, that is, more than 4 weeks after the appearance of DM-20. The early appearance of DM-20 at the beginning of myelination raises the question of its particular function. In the adult bovine cerebral hemispheres, PLP is the major proteolipid but DM-20 remains quantitatively important because the PLP/DM-20 ratio ranges from 1.5 to 1.7. In the same developmental study we have, in the fetal cerebral hemispheres, isolated and characterized a novel proteolipid (apparent Mr 20,000), which appears even before DM-20 and is not detected in the adult brain. It is structurally related to PLP and DM-20 because the first 31 N-terminal amino acid residues are the same. However, in immunoblot, it did not react either with the antitridecapeptide 117-129 antiserum of PLP or with the anti-C-terminal hexapeptide antiserum of PLP. 相似文献
8.
The vacuolar (H+)-ATPases (V-ATPases) are ATP-dependent proton pumps that operate by a rotary mechanism in which ATP hydrolysis drives rotation of a ring of proteolipid subunits relative to subunit a within the integral V(0) domain. In vivo dissociation of the V-ATPase (an important regulatory mechanism) generates a V(0) domain that does not passively conduct protons. EM analysis indicates that the N-terminal domain of subunit a approaches the rotary subunits in free V(0), suggesting a possible mechanism of silencing passive proton transport. To test the hypothesis that the N-terminal domain inhibits passive proton flux by preventing rotation of the proteolipid ring in free V(0), factor Xa cleavage sites were introduced between the N- and C-terminal domains of subunit a (the Vph1p isoform in yeast) to allow its removal in vitro after isolation of vacuolar membranes. The mutant Vph1p gave rise to a partially uncoupled V-ATPase complex. Cleavage with factor Xa led to further loss of coupling of proton transport and ATP hydrolysis. Removal of the N-terminal domain by cleavage with factor Xa and treatment with KNO3 and MgATP did not, however, lead to an increase in passive proton conductance by free V(0), suggesting that removal of the N-terminal domain is not sufficient to facilitate passive proton conductance through V(0). Photoactivated cross-linking using the cysteine reagent maleimido benzophenone and single cysteine mutants of subunit a demonstrated the proximity of specific sites within the N-terminal domain and subunits E and G of the peripheral stalk. These results suggest that a localized region of the N-terminal domain (residues 347-369) is important in anchoring the peripheral stator in V1V0. 相似文献
9.
Makoto Miura Fumio Yamauchi Yasushi Ogawa Kazuo Shibasaki 《Bioscience, biotechnology, and biochemistry》2013,77(6):1631-1637
A proteolipid was isolated from the chloroform–methanol (2:1, by vol.) extract of defatted soybean meals by a modified Folch method. The proteolipid gave a yield of 0.05% of the defatted meals, and the ratio of protein and lipid was neary 3:4. The complex gave a single band containing both protein and lipid on polyacrylamide gel electrophoresis. TLC analysis of the lipid moiety showed that the major components were glycolipids and phospholipids. The protein moiety contained more hydrophobic amino acids and less acidic amino acids in comparison with the amino acid composition of soybean globulin. The protein moiety contained two kinds of protein component (I and II) which have molecular weights of 13,000 (I) and 15,000 (II) on SDS-urea polyacrylamide gel electrophoresis, and N-terminal amino acids of alanine (I) and glutamic acid (II). The apoprotein is a new protein and different from the whey proteins or globulins of soybean. 相似文献
10.
Marie-Christine Birling Guy Roussel Françoise Nussbaum Jean-Louis Nussbaum 《Neurochemical research》1993,18(8):937-945
Bovine myelin/oligodendrocyte glycoprotein (MOG) was purified from a Wolfgram protein fraction of brain myelin by molecular sieving and preparative gel electrophoresis. The N-terminal sequence of this wheat germ agglutinin reacting glycoprotein was determined. Antibodies against purified MOG and synthetic N-terminal octapeptide of MOG were produced in rabbits. Respective affinity purified antibody preparations gave identical results on Western blots. Treatment with specific glycosidases indicated that the oligosaccharide chains of MOG are only of N-chain type. This glycoprotein seems to be restricted to mammalian species since it was not detected in other animal species, ranging from fish up to reptiles. Immunohistochemical investigations on rat brain sections revealed that MOG is restricted to myelin sheaths and oligodendrocytes, thus corroborating previous results obtained with the MOG 8-18C5 monoclonal antibody. Decreased staining pattern in Jimpy brain further attested its specific localization in myelin-related structures. The octapeptide site-specific antibodies were not reactive on brain sections which may be attributed to the burying of this N-terminal sequence in the membrane. These MOG polyclonal antibodies appear to be valuable tools for further studies concerning this minor glycoprotein.Abbreviations BSA
bovine serum albumin
- CNS
central nervous system
- DM-20
minor myelin proteolipid protein
- MAG
Myelin-associated glycoprotein
- MBP
myelin basic proteins
- MOG
Myelin/oligodendrocyte glycoprotein
- OMgp
Oligodendrocyte/Myelin glycoprotein
- PAGE
polyacrylamide gel electrophoresis
- PBS
phosphate buffered saline
- PeptMOG
n-terminal octapeptide of MOG
- PLP
major myelin proteolipid protein
- PMSF
phenylmethylsulfonylfluoride
- SDS
sodium dodecylsulphate
- TBS
Tris buffered saline
- WPF
Wolfgram protein fraction
- WGA
Wheat germ agglutinin 相似文献
11.
Subunit a of the vacuolar H(+)-ATPases plays an important role in proton transport. This membrane-integral 100-kDa subunit is thought to form or contribute to proton-conducting hemichannels that allow protons to gain access to and leave buried carboxyl groups on the proteolipid subunits (c, c', and c″) during proton translocation. We previously demonstrated that subunit a contains a large N-terminal cytoplasmic domain followed by a C-terminal domain containing eight transmembrane (TM) helices. TM7 contains a buried arginine residue (Arg-735) that is essential for proton transport and is located on a helical face that interacts with the proteolipid ring. To further define the topology of the C-terminal domain, the accessibility of 30 unique cysteine residues to the membrane-permeant reagent N-ethylmaleimide and the membrane-impermeant reagent polyethyleneglycol maleimide was determined. The results further define the borders of transmembrane segments in subunit a. To identify additional buried polar and charged residues important in proton transport, 25 sites were individually mutated to hydrophobic amino acids, and the effect on proton transport was determined. These and previous results identify a set of residues important for proton transport located on the cytoplasmic half of TM7 and TM8 and the lumenal half of TM3, TM4, and TM7. Based upon these data, we propose a tentative model in which the cytoplasmic hemichannel is located at the interface of TM7 and TM8 of subunit a and the proteolipid ring, whereas the lumenal hemichannel is located within subunit a at the interface of TM3, TM4, and TM7. 相似文献
12.
A Van Dorsselaer R Nebhi O Sorokine P Schindler B Luu 《Comptes rendus de l'Académie des sciences. Série III, Sciences de la vie》1987,305(14):555-560
By studying highly purified CNS proteolipids, we have shown that DM-20 proteolipid, which was considered, until now, to be a minor brain proteolipid is, in fact, almost as abundant as the Major Myelin Proteolipid known also as Proteolipid Protein (PLP). DM-20 proteolipid is even the major brain proteolipid in young foetuses. It is only during myelinisation that the "Proteolipid Protein" increases rapidly and becomes equivalent in weight to DM-20 proteolipid. This study raises the question of the particular function of DM-20 proteolipid. 相似文献
13.
An AG----GG transition at a splice site in the myelin proteolipid protein gene in jimpy mice results in the removal of an exon 总被引:11,自引:0,他引:11
The myelin proteolipid protein gene was characterized in jimpy mice to identify the specific mutation that produces dysmyelination, oligodendrocyte cell death, and death of the animal by 30 days of age. Exon 5 and flanking intron segments were isolated from jimpy proteolipid protein genomic clones and sequenced. A single nucleotide difference was noted between the normal and jimpy proteolipid protein genes: the conversion of an AG/GT to a GG/GT in the splice acceptor signal preceding exon 5, which apparently destroys the splice signal. Thus, exon 5 of the mouse myelin proteolipid protein gene is skipped during the processing of mRNA, producing a shortened proteolipid protein mRNA. 相似文献
14.
Anthony T. Campagnoni Celia W. Campagnoni Gary R. Dutton James Cohen 《Developmental neurobiology》1976,7(4):313-324
The accumulation and distribution of proteolipid proteins in rat brain and selected brain regions (cerebellum, cerebral cortex, basal ganglia, and hippocampus) were studied during early postnatal development. In whole brain an eightfold increase of proteolipid was observed between ten and 33 days after birth. This was reflected in the separate regions examined where the proteolipid protein content increased six- to ten-fold during the same period. The basal ganglia and cerebral cortex contributed the greatest amount to the total proteolipid present. However, at 28–33 days the greatest concentration (mg/g tissue) was observed in the basal ganglia and hippocampus. When the proteolipid protein preparations were examined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, distinctive, heterogeneous patterns for each brain region were obtained. Proteolipid from basal ganglia (the region richest in white matter) consisted primarily of two major protein bands with apparent molecular weights of approximately 21,500 and 26,000. Both of these bands dramatically increased in quantity during myelination, and the larger protein coelectrophoresed with isolated myelin proteolipid protein. Both bands were also found present in proteolipid preparations from the other brain regions but in varying amounts relative to the total. The data suggest that the increase in proteolipid observed during this developmental period was due in large measure to the accumulation of myelin-specific proteolipids, but also that a significant proportion of the increase was due to the accumulation of nonmyelin components. 相似文献
15.
Amino acid composition of a new mitochondrially translated proteolipid isolated from yeast mitochondria and from the OSATPase complex 总被引:2,自引:0,他引:2
J Velours M Esparza B Guerin 《Biochemical and biophysical research communications》1982,109(4):1192-1199
A new mitochondrially translated 10000 Mr proteolipid was isolated from yeast mitochondria. This proteolipid was purified by phosphocellulose chromatography, followed by reverse phase HPLC. This proteolipid was also extracted from the oligomycin sensitive ATPase complex and purified by HPLC. Its amino acid composition is different from the Dicyclohexylcarbodiimide binding protein. 相似文献
16.
Abstract: A frozen mixture of solubilized brain proteolipid proteins in chloroform-methanol is not sublimable in a vacuum. However, when 7 to 10 volumes of benzene were added to a chloroform-methanol solution containing 5 mg of proteolipid protein per ml, the proteolipid proteins remained in solution for a while and the frozen mixture was easily sublimated at 2 mm Hg. Before the addition of benzene, higher concentrations of protein required the acidification of the medium to avoid precipitation of proteolipid proteins. In contrast to what happens when proteolipid proteins are obtained by the evaporation of the organic mixture at room temperature, the protein obtained by lyophilization was soluble in aqueous solutions of ionic and nonionic detergents. Sodium dodecyl sulfate at 0.6 to 0.7% concentration completely solubilized the proteolipid protein obtained by lyophilization. With the nonionic detergents Lubrol WX and Triton X-100, a solubilization between 50 and 65% was achieved. Sodium deoxycholate was practically ineffective. Triton X-100 showed selectivity in solubilizing certain proteins. The role of lipids in the solubilization of proteolipid proteins with detergents is discussed. 相似文献
17.
Proteolipids were isolated from 20 day old normal andjimpy mouse brain by extraction into chloroform-methanol (21, w/v), delipidated by size-exclusion HPLC, and analyzed by SDS-PAGE, Western blots, amino acid analyses, and N-terminal sequencing. SDS-PAGE showed that a major proteolipid fromjimpy mouse brain had an apparent molecular weight of 23 kDa, intermediate to that of PLP and DM-20 from normal mouse brain. Western blots with 3 different antibodies which recognize residues 200–224, 116–150, and 270–276 respectively recognized immunoreactive material in normal andjimpy PLP. Since antibody reactive with 270–276 did not recognizejimpy PLP, an altered C-terminus of thejimpy protein is suggested. These results demonstrated that a PLP can be partially purified fromjimpy mouse brain. Amino acid analyses failed to show the predicted increase in cysteinyl residues (predicted from cDNA) injimpy PLP. However, whenjimpy brain proteolipids were subjected to N-terminal sequencing, Gly, Leu, Leu, Gly the first four amino acids of PLP were detected. Thus, the partial purification of a proteolipid fromjimpy mouse brain, whose characteristics (apparent molecular weight, immunoreactivity, N-terminal sequence and relative net charge) strongly suggested that PLP of altered size is present injimpy mouse brain.Abbreviations BCIP
5-bromo-4-chloro-3-indolyl phosphate toluidine salt
- MBP
myelin basic protein
- NBT
-nitro blue tetrazolium chloride
- PITC
phenylisothiocyanate
- PLP
myelin proteolipid protein
- PVDF
polyvinylidene difluoride
- SDS-PAGE
sodium dodecyl sulfate polyacrylamide gel electrophoresis
Special issue dedicated to Dr. Marjorie B. Lees. 相似文献
18.
Production and Purification of Antibody to Bovine White Matter Proteolipid Apoprotein 总被引:1,自引:1,他引:0
Wendy B. Macklin Gerald A. Schwarting Marjorie B. Lees Steven R. Cohen 《Journal of neurochemistry》1981,36(1):101-106
Circulating antibody to the bovine white matter proteolipid apoprotein was detected in rabbits 1 month after a single injection of the water-soluble form of the apoprotein. By double immunodiffusion, the antiserum reacted specifically with the delipidated proteolipid apoprotein and the crude proteolipid fraction containing complex lipids; after exposure of the proteolipid apoprotein to sodium dodecyl sulfate (SDS), no reactivity was observed. The antiserum did not react with other myelin components, i.e., basic protein, cerebroside or GM1 ganglioside, nor was there reactivity with non-neural proteolipids. The anti-apoprotein antibody was purified by affinity chromatography. The antibody-antigen interaction is apparently very hydrophobic, since elution of the antibody from the affinity column requires buffer containing 0.5% Triton X-100-4 M-urea. 相似文献
19.
Sequence studies of the apoprotein of the major rat brain myelin proteolipid are made difficult by the aggregation of the water-soluble form of the protein and of its split peptides. By digestion with Staphylococcus aureus V8 protease and Lysobacter enzymogenes Lys-C endoproteinase or after partial acid hydrolysis, a new series of fragments has been obtained. Sequence data from these fragments permitted the alignment of tryptic peptides and BNPS-skatole fragments analyzed in previous studies. A nearly complete sequence of the C-terminal moiety of the apoprotein is described; in addition, large segments of the N-terminal part have been characterized. 相似文献
20.
A protein, the mediatophore, has been purified from Torpedo electric organ presynaptic plasma membranes. This protein mediates the release of acetylcholine through artificial membranes when activated by calcium and is made up of 15-kDa proteolipid subunits. After immunization with purified delipidated mediatophore, monoclonal antibodies binding to the 15-kDa proteolipid band on Western blots of purified mediatophore were selected. A 15-kDa proteolipid antigen was also detected in cholinergic synaptic vesicles. Using an immunological assay, it was estimated that presynaptic plasma membranes and synaptic vesicles contain similar proportions of 15-kDa proteolipid antigen. Detection by immunofluorescence in the electric organ showed that only nerve endings were labeled. In electric lobes, the staining was associated with intracellular membranes of the electroneuron cell bodies and in axons. Nerve endings at Torpedo neuromuscular junctions were also labeled with anti-15-kDa proteolipid monoclonal antibodies. 相似文献