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1.
The rice ( Oryza sativa L.) mutant of glu4a, lacking the glutelin alpha-2 subunit while the alpha-1 subunit increased (alpha-1H/alpha-2L), was used in this study. Two-dimensional electrophoresis analysis revealed that the mutant lacked the polypeptide pI6.71/alpha-2 encoded by glu4 while forming a new polypeptide of pI6.50/alpha-1. Experiments were conducted to identify the relationships between the mutated polypeptides of the mutant and to illustrate the mutation mechanism of the allele. Peptide mapping and amino-acid sequence analyses revealed that the newly formed glu4a encoded polypeptide pI6.50/alpha-1 of high homology with the deleted pI6.71/alpha-2 polypeptide which was encoded by glu4 (GluA-1). The nucleotide sequence revealed that the iso-electric point variation of the pI6.50/alpha-1 polypeptide was caused by a point mutation with nucleotide replacement at the variable region of the gene. These results suggested the possibility of altering glutelin quality by using single gene mutation.  相似文献   

2.
Nine rice Oryza sativa L.) mutant lines lacking the seed storage glutelin α-2 subunit were obtained from the progenies of fertilized egg cells treated with N-methy-N-nitrosourea (MNU). The mutants could be classified into three types: the α-1 subunit increased type (α-1H/α-2L), decreased the β-2 subunit decreased type (β-2L/α-2L) and the α-3 subunit increased type (α-3H/α-2L) according to their SDS-PAGE profiles. Two-dimensional electrophoresis analysis revealed that all of the mutants lacked a polypeptide of pI 6.71/α-2, while new polypeptides of pI 6.50/α-1 and pI 6.90/α-3 formed in α-1H/α-2L and α-3H/α-2L mutants respectively. Although the β-2L/α-2L mutants did not form new polypeptide, their pI 8.74/β-2 polypeptide was also decreased, suggesting that the two polypeptides decreased in β-2L/α-2L mutants might derive from the same glutelin precursor. These mutant lines are very useful in studying genetic characterisation,the mechanism of genetic regulation on biosynthesis, gene function and proteomics of rice seed storage glutelin.  相似文献   

3.
Nine rice Oryza sativa L.) mutant lines lacking the seed storage glutelin α-2 subunit were obtained from the progenies of fertilized egg cells treated with N-methy-N-nitrosourea (MNU). The mutants could be classified into three types: the α-1 subunit increased type (α-1H/α-2L), decreased the β-2 subunit decreased type (β-2L/α-2L) and the α-3 subunit increased type (α-3H/α-2L) according to their SDS-PAGE profiles. Two-dimensional electrophoresis analysis revealed that all of the mutants lacked a polypeptide of pI 6.71/α-2, while new polypeptides of pI 6.50/α-1 and pI 6.90/α-3 formed in α-1H/α-2L and α-3H/α-2L mutants respectively. Although the β-2L/α-2L mutants did not form new polypeptide, their pI 8.74/β-2 polypeptide was also decreased, suggesting that the two polypeptides decreased in β-2L/α-2L mutants might derive from the same glutelin precursor. These mutant lines are very useful in studying genetic characterisation,the mechanism of genetic regulation on biosynthesis, gene function and proteomics of rice seed storage glutelin.  相似文献   

4.
新的水稻谷蛋白α—1亚基缺失突变体   总被引:8,自引:2,他引:8  
从水稻受精卵MNU处理后代中获得4个谷蛋白α-1亚基缺失突变品系。SDS-PAGE和IEF分析表明这些突变体在共同缺失1条pI6.82多肽的同时,或形成新的多肽,或其他多肽表现量增加,这些突变体是由结构基因控制的,IEF分析同时显示2条多肽pI6.82和pI8.58源自同一条谷蛋白前驱体。这4个突变体对于改良水稻谷蛋白品质、研究谷蛋白生物合成遗传调控机制以及揭示谷蛋白基因功能是不可多得的研究材料。  相似文献   

5.
Lamprey 48-kDa lens protein represents a novel class of crystallins   总被引:2,自引:0,他引:2  
S O Stapel  W W de Jong 《FEBS letters》1983,162(2):305-309
SDS-PAGE revealed a major Mr 48 000 polypeptide of pI around 8 in the water-soluble fraction of lamprey lenses. It occurs as a monomeric protein, and its amino acid composition and tryptic peptides show no resemblances to alpha-, beta-, gamma- or delta-crystallin. Immunoblotting with antiserum against the 48-kDa protein revealed an immunologically related polypeptide of similar Mr in reptiles, several birds and a fish, but showed no cross-reactivity with any other water-soluble lens component. The 48-kDa protein is not detected in many birds and fishes, and in the investigated mammals and amphibians.  相似文献   

6.
Purification and properties of eIF-2 phosphatase   总被引:2,自引:0,他引:2  
Eukaryotic initiation factor 2 (eIF-2) phosphatase has been purified 840-fold to apparent homogeneity from rabbit reticulocyte lysate. Native eIF-2 phosphatase has a Mr = 98,000, pI = 6.1, s20,w = 5.1, and a Stokes radius = 38 A. A subunit composition of one 60,000-dalton polypeptide and one 38,000-dalton polypeptide is indicated. The Km for [32P]eIF-2 is 30 microM and the Vmax = 1.1 nmol of phosphate released/min/microgram. The 38,000-dalton subunit of eIF-2 phosphatase does not co-electrophorese with the catalytic subunit of liver phosphorylase phosphatase, a type 1 protein phosphatase. The specificity of eIF-2 phosphatase for phosphorylation sites on th alpha- and beta-subunits of eIF-2 appears to be determined by the environment of the phosphatase and substrate. Both the alpha- and beta-subunits of [32P]eIF-2 are rapidly dephosphorylated by the purified phosphatase. In unfractionated lysate and in unfractionated lysate supplemented with an equivalent activity of the purified phosphatase, only the alpha-subunit of eIF-2 is dephosphorylated. This indicates other factors are present in the lysate which govern the dephosphorylation of eIF-2.  相似文献   

7.
Canine prostatic fluid, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing conditions, is characterized by the presence of a single diffuse band (Mr approximately 30,000) which accounts for over 90% of the total protein. The biosynthesis of this protein is under androgen control. Castration results in the disappearance of this protein, whereas its presence in the prostate can be maintained in the castrated animal with exogenous androgen. Analysis of the native protein by isoelectric focusing revealed the presence of 10-13 charged variants with pI values in the range of 6.5 to 8.4. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions revealed that each isoform is constructed of two dissimilar polypeptide subunits covalently linked through disulfide bonds. One subunit has a molecular weight of 15,000 (H chain); the second subunit (L chain) has a variable molecular weight in the 12,000-14,000 range. The H and L subunits have been purified by preparative isoelectric focusing and chemically characterized. Based on tryptic peptide mapping, NH2-terminal analysis, amino acid and carbohydrate composition, the H and L subunits are structurally unrelated and consequently appear to be unique gene products. Furthermore, the L subunit is glycosylated which potentially accounts for its size heterogeneity. Quantitative NH2-terminal analysis indicated that the H and L subunits are present in the native molecule in a ratio of 2:1, suggesting that the native molecule is a trimer with an apparent molecular weight of 43,000. Based on electrophoretic data, the glycoprotein also constitutes the major fraction of the soluble protein in canine prostatic tissue; its presence is organ specific. This glycoprotein should prove useful as a marker for prostatic function under varying hormonal and environmental conditions.  相似文献   

8.
Determination of the complete amino-acid sequence of the subunits of B-phycoerythrin from Porphyridium cruentum has shown that the alpha subunit contains 164 amino-acid residues and the beta subunit contains 177 residues. When the sequences of B- and C-phycoerythrins are aligned with those of other phycobiliproteins, it is obvious that B-phycoerythrin lacks a deletion at beta-21-22 present in C-phycoerythrin. However, relative to C-phycoerythrin from Fremyella diplosiphon (Calothrix) (Sidler, W., Kumpf, B., Rüdiger, W. and Zuber, H. (1986) Biol. Chem. Hoppe-Seyler 367, 627-642), B-phycoerythrin has deletions at beta-141k-o, beta-142, beta-143, beta-147 and beta-148. The four singly-linked phycoerythrobilins at positions alpha-84, alpha-143a, beta-84 and beta-155, and the doubly-linked phycoerythrobilin at position beta-50/61 are at sites homologous to the attachment sites in C-phycoerythrin. The aspartyl residues (alpha-87, beta-87, and beta-39), that interact with the bilins at alpha-84, beta-84, and beta-155 in C-phycocyanin, are found in the homologous positions in B-phycoerythrin. B-Phycoerythrin, in common with other phycobiliproteins, contains a N gamma-methylasparagine residue at position beta-72.  相似文献   

9.
The basidiolipids of six mushroom species, i.e. the basidiomycetes Amanita virosa (engl., death cup), Calvatia exipuliformis (engl., puffball), Cantharellus cibarius (engl., chanterelle), Leccinum scabrum (engl., red birch boletus), Lentinus edodes (jap., Shiitake), and Pleurotus ostreatus (engl., oystermushroom), were isolated, and their chemical structures investigated. All glycolipids are structurally related to those of the Agaricales (engl., field mushroom). They are glycoinositolphosphosphingolipids, their ceramide moiety consisting of t18:0-trihydroxysphinganine and an alpha-hydroxy long-chain fatty acid. In contrast to a previous study [Jennemann, R., Bauer, B.L., Bertalanffy, H., Geyer, R., Gschwind, R.M., Selmer, T. & Wiegandt, H. (1999) Eur. J. Biochem. 259, 331--338], the glycoside anomery of the hexose (mannose) connected to the inositol of all investigated basidiomycete glycolipids, including the basidiolipids of Agaricus bisporus, was determined unequivocally to be alpha. Therefore, the root structure of all basidiolipids consists of alpha-DManp-2Ins1-[PO(4)]-Cer. In addition, for some mushroom species, the occurrence of an inositol substitution position variant, alpha-Manp-4Ins1-[PO(40]-Cer, is shown. The carbohydrate of chanterelle basidiolipids consists solely of mannose, i.e. Cc1, Man alpha-3 or -6Man alpha; Cc2, Man alpha-3(Man alpha-6)Man alpha-. All other species investigated show extension of the alpha-mannoside in the 6-position by beta-galactoside, which, in some instances, is alpha-fucosylated in 2-position (Fuc alpha-2)Gal beta-6Man alpha-. Further sugar chain elongation at the beta-galactoside may be in 3- and/or 6-position by alpha-galactoside, e.g. Ce4, Po2, Gal alpha-3-(Gal alpha-6)(Fuc alpha-2)Gal beta-6Man alpha-, whereas A. virosa, Av-3, has a more complex, highly alpha-fucosylated terminus, Gal alpha-3 (Fuc alpha-2)(Fuc alpha-6)Gal alpha-2(Gal alpha-3)Gal beta-6Man alpha-. L. edodes basidiolipids show further elongation by alpha-mannoside, e.g. Le3, Man alpha-2Man alpha-6Gal alpha-3(Fuc alpha-2)Gal beta-6Man alpha-, C. exipuliformis glycolipid by alpha-glucoside, i.e. Ce3, Glc alpha-6Gal beta-6Man alpha-. Basidiolipid Ls1 from L. scabrum, notably, has a 3-alpha-mannosylated alpha-fucose, i.e. Gal alpha-6(Man alpha-3Fuc alpha-2)Gal alpha-6Gal beta-6Man alpha-. In conclusion, basidiolipids, though identical in their ceramide constitution, display wide and systematic mushroom species dependent variabilities of their chemical structures.  相似文献   

10.
11.
These studies describe the properties of three mit- mutants designated EM17, EM25, and PZ1, all mapping at two closely linked sites near one of the boundaries of the region of the mitochondrial genome concerned with the specification of cytochrome b. They all exhibit complex phenotypes affecting cytochrome b, cytochrome aa3, and additional polypeptides not found in the wild type. In the case of EM 17 this complexity can be ascribed to the presence of two mutations induced in the course of the initial mutagenic treatment: one, the cob2 mutation proper, is responsible for the loss of cytochrome b which is replaced by an altered, functionally inactive polypeptide, cytochrome b. This polypeptide can be further modified, or even eliminated, by the controlled introduction of another mutation in the cob1 segment of the cob region. The reduction in cytochrome oxidase subunit I, responsible for the effects on cytochrome aa3 and enzymatic activity in EM17, is due to a second (not mit-) mutation that has been located in the par1-proximal segment of the oxi3 region. This second mutation as well as the cob mutation can be overcome, and the respective aspect of wild type function restored to EM17, by recombination with rho- strains retaining the appropriate segment(s) of the wild type genome. The phenotype of the other two mutants is due to a single mutagenic event. This conclusion is confirmed by their ability to restore wild type functions by reversion. The mutation in EM25 appears to be due to a frameshift, which has led to premature chain termination, producing a polypeptide of Mr = 15,000 related to apocytochrome b. This change is accompanied by a decrease in the amount of subunit I of cytochrome oxidase. Revertants fall into three classes: on galactose two produce a polypeptide indistinguishable from apocytochrome b, but vary in its amount, while the third fails to increase apocytochrome b above mutant levels. Production of subunit I is increased but fails to reach wild type levels. Complete restoration of wild type functions can, however, be obtained by recombination of EM25 with rho- (cob2+) strains. Mutation PZ1 results in a complete absence of any polypeptide related to apocytochrome b and of cytochrome oxidase subunit I. These cells produce a novel polypeptide with a Mr = 45,000 not found in the wild type, and unrelated to all its normal polypeptides. Reversion or recombination with rho- (cob2+) strains results in virtually complete restoration of all wild type functions and the elimination of the novel polypeptide.  相似文献   

12.
In fetal pneumocytes, increasing P(O(2)) can raise apical Na(+) conductance (G(Na(+))) and increase the abundance of epithelial Na(+) channel subunit (alpha-, beta-, and gamma-ENaC) mRNA, suggesting that the rise in G(Na(+)), which may be important to the perinatal maturation of the lung, reflects O(2)-evoked ENaC gene expression. However, we now show that physiologically relevant increases in P(O(2)) do not affect alpha-, beta-, and gamma-ENaC mRNA abundance in pneumocytes maintained (approximately 48 h) in hormone-free medium or in medium supplemented with dexamethasone and tri-iodothyronine, although the response does persist in cells maintained in medium containing a complex mixture of hormones/growth factors. However, parallel electrometric studies revealed clear increases in G(Na(+)) under all tested conditions and so it is now clear that O(2)-evoked increases in G(Na(+)) can occur without corresponding increases in ENaC mRNA abundance. It is therefore unlikely that this rise in G(Na(+)) is secondary to O(2)-evoked ENaC gene expression.  相似文献   

13.
Cultured smooth muscle A7r5 cells were able to take up alpha-tocopherol (32 +/- 1.2 nmol/mg protein) the largest part of which (60%) was present in the cytosolic fraction. Using a tocopherol-based affinity chromatography and alpha-, beta-, gamma-, and delta-tocopherols as eluants, three polypeptides of molecular masses 81, 58 and 31 kDa were eluted. This preparation had alpha-[3H]tocopherol binding capability. The 58-kDa polypeptide could also be eluted by chromanol and the 81-kDa polypeptide could be eluted also by phytol. The 81-kDa polypeptide had the unique P-E-E-D-Q-X-Q-Y N-terminal sequence.  相似文献   

14.
15.
The discovery that the phycocyanobilin group attached to Cys-155 of the beta subunit of C-phycocyanin is D-ring linked (Bishop, J. E., Lagarias, J. C., Nagy, J. O., Schoenleber, R. W., Rapoport, H., Klotz, A. V., and Glazer, A. N. (1986) J. Biol. Chem. 261, 6790-6796) prompted examination of the linkage mode for phycoerythrobilin (PEB) groups attached at the corresponding position in other biliproteins. Appropriate small peptides were obtained by exhaustive enzymatic digestion of Porphyridium cruentum R-phycocyanin (peptide R-PC beta-2TP PEB) and B-phycoerythrin (peptide B-PE beta-2TP PEB). These peptides had the following structures R-PC beta-2TP PEB Gly-Asp-Cys(PEB)-Ser-Ser B-PE beta-2TP PEB Cys(PEB)-Thr-Ser. The spectroscopic and chemical properties of these peptides were compared with those of P. cruentum B-phycoerythrin peptide alpha-1 PEB, Cys(PEB)-Tyr-Arg, in which the bilin is A-ring linked (Schoenleber, R. W., Leung, S.-L., Lundell, D. J., Glazer, A. N., and Rapoport, H. (1983) J. Am. Chem. Soc. 105, 4072-4076). The PEB groups in peptides R-PC beta-2TP PEB and B-PE beta-2TP PEB were shown to be D-ring linked on the basis of the following criteria. Secondary ion mass spectrometry showed the bilins in these peptides and in alpha-1 PEB to have the same mass. The 18'-CH3, 18'-H, and 15-H resonances in the 1H NMR spectra of R-PC beta-2TP PEB and B-PE beta-2TP PEB appear significantly upfield from the corresponding thioether-linked ring A resonances seen in the spectrum of peptide alpha-1 PEB. The CD spectra of the two former peptides showed a strong positive Cotton effect at 300 nm. Such a Cotton effect is absent from the CD spectrum of peptide alpha-1 PEB and those of other A-ring-linked PEB peptides. Refluxing in methanol led to a near-quantitative release of PEB from alpha-1 PEB but no release from R-PC beta-2TP PEB and less than 20% release from B-PE beta-2TP PEB. In conjunction with earlier studies, these results show that distinctive amino acid sequences are found about the attachment sites for A-ring-linked, D-ring-linked, and dilinked (A- and D-ring-linked) bilins on the alpha and beta subunits of cyanobacterial and red algal phycobiliproteins and that the mode of linkage can be correctly predicted from inspection of the amino acid sequence.  相似文献   

16.
We have examined 11 previously described cultured rat hepatoma mutants with absent or reduced phenylalanine hydroxylase activity (Choo and Cotton, 1977). Immunological and electrophoretic methods failed to detect any structurally altered protein in these mutants. In nine independently isolated revertants from four different mutants, wild-type protein was regained (or accentuated). This evidence suggests that the mutation involved in these mutants is most likely to be regulatory in nature. These studies have provided three reasons for believing that in cultured rat hepatoma cells one gene codes for a single polypeptide chain, a number of which combine to form the active phenylalanine hydroxylase multimer: (1) Analysis of the purified protein by two-dimensional electrophoresis revealed only a single polypeptide chain. (2) This polypeptide was diminished or undetectable in crude extracts of 11 independently isolated mutants with absent of reduced activity. (3) In none of these 11 mutants was the polypeptide we have designated to be phenylalanine hydroxylase present at normal levels, as would be expected if the mutation were at another locus responsible for a possible second subunit.  相似文献   

17.
18.
The epithelial Na+ channel (ENaC) is a tetramer of two alpha-, one beta-, and one gamma-subunit, but little is known about its assembly and processing. Because co-expression of mouse ENaC subunits with three different carboxyl-terminal epitope tags produced an amiloride-sensitive sodium current in oocytes, these tagged subunits were expressed in both Chinese hamster ovary or Madin-Darby canine kidney type 1 epithelial cells for further study. When expressed alone alpha-(95 kDa), beta-(96 kDa), and gamma-subunits (93 kDa) each produced a single band on SDS gels by immunoblotting. However, co-expression of alphabetagammaENaC subunits revealed a second band for each subunit (65 kDa for alpha, 110 kDa for beta, and 75 kDa for gamma) that exhibited N-glycans that had been processed to complex type based on sensitivity to treatment with neuraminidase, resistance to cleavage by endoglycosidase H, and GalNAc-independent labeling with [3H]Gal in glycosylation-defective Chinese hamster ovary cells (ldlD). The smaller size of the processed alpha- and gamma-subunits is also consistent with proteolytic cleavage. By using alpha- and gamma-subunits with epitope tags at both the amino and carboxyl termini, proteolytic processing of the alpha- and gamma-subunits was confirmed by isolation of an additional epitope-tagged fragment from the amino terminus (30 kDa for alpha and 18 kDa for gamma) consistent with cleavage within the extracellular loop. The fragments remain stably associated with the channel as shown by immunoblotting of co-immunoprecipitates, suggesting that proteolytic cleavage represents maturation rather than degradation of the channel.  相似文献   

19.
Direct photoaffinity labeling of purified bovine heart NADH:ubiquinone oxidoreductase (complex I) with 32P-labeled NAD(H), NADP(H) and ADP has shown that five polypeptides become labeled, with molecular masses of 51, 42, 39, 30, and 18-20 kDa. The 51 and the 30-kDa polypeptides were labeled with either [32P]NAD(H), [32P]NADP(H) or [beta-32P]ADP. The 42-kDa polypeptide was labeled with [32P]NAD(H) and to a small extent with [beta-32P]ADP. It was not labeled with [32P]NADP(H). The 39-kDa polypeptide was labeled with [32P]NADPH and to a small extent with [beta-32P]ADP. Our previous studies had shown that this subunit also binds NADP, but not NAD(H) [Yamaguchi, M., Belogrudov, G.I. & Hatefi, Y. (1998) J. Biol. Chem. 273, 8094-8098]. The 18-20-kDa polypeptide was labeled only with [32P]NADPH. Among these polypeptides, the 51-kDa subunit is known to contain FMN and a [4Fe-4S] cluster, and is the NAD(P)H-binding subunit of the primary dehydrogenase domain of complex I. The possible roles of the other nucleotide-binding subunits of complex I have been discussed.  相似文献   

20.
A A Peixoto  J C Hall 《Genetics》1998,148(2):827-838
cacophony (cac), a mutation affecting the courtship song in Drosophila melanogaster, is revealed to cause temperature-sensitive (TS) abnormalities. When exposed to high temperatures (37 degrees), cac flies show frequent convulsions and pronounced locomotor defects. This TS phenotype seems consistent with the idea that cac is a mutation in a calcium-channel gene; it maps to the same X-chromosomal locus that encodes the polypeptide comprising the alpha-1 subunit of this membrane protein. Analysis of the courtship song of some TS physiological mutants showed that slowpoke mutations, which affect a calcium-activated potassium channel, cause severe song abnormalities. Certain additional TS mutants, in particular para(ts1) and nap(ts1), exhibit subtler song defects. The results therefore suggest that genes involved in ion-channel function are a potential source of intraspecific genetic variation for song parameters, such as the number of cycles present in "pulses" of tone or the rate at which pulses are produced by the male''s courtship wing vibrations. The implications of these findings from the perspective of interspecific lovesong variations in Drosophila are discussed.  相似文献   

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