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1.
2.
Protein GLPT, a periplasmic protein previously recognized as closely related to the active transport of sn-glycerol-3-phosphate in Escherichia coli was isolated by the cold osmotic shock procedure. It was purified by Sephadex chromatography and isoelectric focussing. The purified protein does not exhibit any detectable binding activity toward sn-glycerol-3-phosphate. It has no activity as a glycerol phosphatase nor as a glycerol kinase. Polyacrylamide gel electrophoresis in the presence of dodecylsulfate of the protein subsequent to treatment in urea, boiling in dodecylsulfate and crosslinking indicates that it occurs as an oligomeric protein composed of four identical subunits of 40 000 molecular weight. Membrane vesicles of wild-type strains that contain protein GLPT in whole cells loose it during vesicle preparation. However, they still exhibit high transport activity toward sn-glycerol-3-phosphate. Membrane vesicles prepared from glp T mutants that may or may not contain protein GLPT do not transport sn-glycerol-3-phospahte. We conclude from these results that protein GLPT does not participate in the energy-dependent active transport through the cytoplasmic membrane but could be involved in facilitating the diffusion of sn-glycerol-3-phosphate through the outer layers of E. coli. 相似文献
3.
About fourfold purification of the stearyl-CoA: sn-glycerol-3-phosphate acyltransferases (GPAT) was achieved from liver microsomes by extraction with 1M phosphate buffer (pH 7–4) followed by gel filtration. The partially purified enzyme synthesized mainly diacylgly-cerophosphate and a small amount (5%) of monoacylglycerophosphate. Patty aoyl-CoA synthetase and to some extent stearyl-CoA desaturase were copurified along with the transferases. Data obtained from molecular sieve chromatography, polyacrylamide gel electrophoresis and electron microscopy showed that GPAT activity was tightly bound to a high molecular weight protein fraction with vesicular structure. 相似文献
4.
Poly-A RNA extracted from the rat liver was translated in a cell-free wheat germ system and a rabbit reticulocyte lysate. The subunit of tryptophan pyrrolase precipitated by specific antiserum after synthesis in vitro has the same molecular weight as the corresponding subunit derived from the rat liver. With specific antiserum prepared against tyrosine aminotransferase, however, a radioactive protein from both the in vitro assays was precipitated with an about 5% higher molecular weight than the tyrosine aminotransferase subunit precipitated from rat liver. The immunological evidence and the comparison of the specific peptide patterns prepared by cyanogen bromide treatment showed that the in vitro product corresponds to tyrosine aminotransferase. Various concentrations of potassium or spermidine used in the wheat germ translation system did not alter the size of the enzyme subunit synthesized. The run of the tyrosine aminotransferase purified form the rat liver in the SDS-polyacrylamide gel electrophoresis was not influenced by treatment with Escherichia coli alkaline phosphatase. The possibility is discussed that the larger enzyme synthesized in vitro represents a precursor molecule which is cleaved proteolytically in vivo. 相似文献
5.
Immunoglobulin heavy chain mRNA was purified from immunoprecipitated polysomes derived from the mouse myeloma tumor, MOPC-31C. The purified mRNA migrated predominantly as a single band upon polyacrylamide gel electrophoresis in 98% formamide and the molecular weight of this mRNA was calculated to be 700,000. This mRNA was as active as the purified light chain mRNA when it was employed as a template in a cell-free protein synthesizing system from wheat germ. The translation product had a molecular weight of 55,000 daltons, and migrated slightly faster than mature heavy chain upon polyacrylamide gel electrophoresis in sodium dodecylsulfate. The protein synthesized by the direction of this mRNA was shown to yield tryptic peptides corresponding to those derived from the mature heavy chain protein except that one missing peptide was replaced by another additional peptide. DNA complementary to the mRNA was synthesized by RNA-dependent DNA polymerase from avian myeloblastosis virus. Hybridization kinetic analysis between the heavy chain mRNA and its complementary DNA indicated that the RNA was essentially homogenous with rabbit globin mRNA as a standard. 相似文献
6.
sn-Glycerol-3-phosphate transport in Salmonella typhimurium 总被引:7,自引:5,他引:2
Salmonella typhimurium contains a transport system for sn-glycerol-3-phosphate that is inducible by growth on glycerol and sn-glycerol-3-phosphate. In fully induced cells, the system exhibited an apparent Km of 50 microM and a Vmax of 2.2 nmol/min . 10(8) cells. The corresponding system in Escherichia coli exhibits, under comparable conditions, a Km of 14 microM and a Vmax of 2.2 nmol/min . 10(8) cells. Transport-defective mutants were isolated by selecting for resistance against the antibiotic fosfomycin. They mapped in glpT at 47 min in the S. typhimurium linkage map, 37% cotransducible with gyrA. In addition to the glpT-dependent system, S. typhimurium LT2 contains, like E. coli, a second, ugp-dependent transport system for sn-glycerol-3-phosphate that was derepressed by phosphate starvation. A S. typhimurium DNA bank containing EcoRI restriction fragments in phage lambda gt7 was used to clone the glpT gene in E. coli. Lysogens that were fully active in the transport of sn-glycerol-3-phosphate with a Km of 33 microM and a Vmax of 2.0 nmol/min . 10(8) cells were isolated in a delta glpT mutant of E. coli. The EcoRI fragment harboring glpT was 3.5 kilobases long and carried only part of glpQ, a gene distal to glpT but on the same operon. The fragment was subcloned in multicopy plasmid pACYC184. Strains carrying this hybrid plasmid produced large amounts of cytoplasmic membrane protein with an apparent molecular weight of 33,000, which was identified as the sn-glycerol-3-phosphate permease. Its properties were similar to the corresponding E. coli permease. The presence of the multicopy glpT hybrid plasmid had a strong influence on the synthesis or assembly of other cell envelope proteins of E. coli. For instance, the periplasmic ribose-binding protein was nearly absent. On the other hand, the quantity of an unidentified E. coli outer membrane protein usually present only in small amounts increased. 相似文献
7.
J A Thomson 《Gene》1977,1(5-6):347-356
A hybrid plasmid, pLC 16-4, from the ColE1-DNA (E. coli) bank of Clarke and Carbon (1976) carrying pfkA was used to program an in vitro protein synthesis system from E. coli. Phosphofructokinase was the main product, as determined by enzyme assay, immunoprecipitation and gel electrophoresis. The enzyme was synthesized in vitro without added cAMP at a rate (enzyme/genome/h) ca. 30% the in vivo value, a higher efficiency than usually found in cell free systems. The plasmic molecular weight is ca. 16.10(6) daltons. 相似文献
8.
Periplasmic protein related to the sn-glycerol-3-phosphate transport system of Escherichia coli. 总被引:16,自引:12,他引:4
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Two-dimensional gel electrophoresis of shock fluids of Escherichia coli K-12 revealed the presence of a periplasmic protein related to sn-glycerol-3-phosphate transport (GLPT) that is under the regulation of glpR, the regulatory gene of the glp regulon. Mutants selected for their resistance to phosphonomycin and found to be defective in sn-glycerol-3-phosphate transport either did not produce GLPT or produced it in reduced amounts. Other mutations exhibited no apparent effect of GLPT. Transductions of glpT+ nalA phage P1 into these mutants and selection for growth on sn-glycerol-3-phosphate revealed a 50% cotransduction frequency to nalA. Reversion of mutants taht did not produce GLPT to growth on sn-glycerol-3-phosphate resulted in strains that produce GLPT. This suggests a close relationship of GLPT to the glpT gene and to sn-glycerol-3-phosphate transport. Attempts to demonstrate binding activity of GLPT in crude shock fluid towards sn-glycerol-3-phosphate have failed so far. However, all shock fluids, independent of their GLPT content, exhibited an enzymatic activity that hydrolyzes under the conditions of the binding assay, 30 to 60% of the sn-glycerol-3-phosphate to glycerol and inorganic orthophosphate. 相似文献
9.
A protein has been purified from the membranes of bloodstream forms of Trypanosoma brucei brucei. The purified material contained a single polypeptide chain of molecular mass 67 kilodaltons as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; under "native" conditions it migrated through a Sephacryl S-300 column with a similar molecular mass. The purified protein catalysed electron transfer from sn-glycerol 3-phosphate to oxygen with the subsequent formation of water. Electron transfer by the purified enzyme to O2 was dependent on the presence of low concentrations of the mediator phenazine methosulfate. This protein is clearly the major membrane-bound sn-glycerol-3-phosphate dehydrogenase, but it also has some characteristics suggestive of the trypanosome alternative oxidase activities. 相似文献
10.
Polysomes producing IgGl(kappa) myeloma protein were specifically selected by an immunoprecipitation method, and immunoglobulin light chain mRNA was purified from the precipitated polysomes. The purified mRNA migrated predominantly as a single band and the molecular weight of this mRNA was calculated to be 410.000 by polyacrylamide gel electrophoresis in 98% formamide. A protein possessing a molecular weight of 25,000, which is the size of the light chain precursor, was synthesized as a major product of translation in a wheat germ cell-free system. DNA complementary to the mRNA (cDNA) was prepared with avian myeloblastosis virus RNA-dependent DNA polymerase. This cDNA had an average size of 8.3S as determined by sedimentation through an alkaline sucrose gradient. Using this cDNA, Crt 1/2 values of template RNA and RNA from various preparations were calculated from the results of molecular hybridization. The relative content of the mRNA increased 4,4-fold during the immunoprecipitation of polysomes. 相似文献
11.
Cell-free synthesis of the mitochondrial ADP/ATP carrier protein of Neurospora crassa. 总被引:9,自引:0,他引:9
ADP/ATP carrier protein was synthesized in heterologous cell-free systems programmed with Neurospora poly(A)-containing RNA and homologous cell-free systems from Neurospora. The apparent molecular weight of the product obtained in vitro was the same as that of the authentic mitochondrial protein. The primary translation product obtained in reticulocyte lysates starts with formylmethionine when formylated initiator methionyl-tRNA (fMet-tRNAfMet) was present. The product synthesized in vitro was released from the ribosomes into the postribosomal supernatant. The evidence presented indicates that the ADP/ATP carrier is synthesized as a polypeptide with the same molecular weight as the mature monomeric protein and does not carry an additional sequence. 相似文献
12.
Spore coat protein synthesis in cell-free systems from sporulating cells of Bacillus subtilis.
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Cell-free systems for protein synthesis were prepared from Bacillus subtilis 168 cells at several stages of sporulation. Immunological methods were used to determine whether spore coat protein could be synthesized in the cell-free systems prepared from sporulating cells. Spore coat protein synthesis first occurred in extracts from stage t2 cells. The proportion of spore coat protein to total proteins synthesized in the cell-free systems was 2.4 and 3.9% at stages t2 and t4, respectively. The sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis patterns of immunoprecipitates from the cell-free systems showed the complete synthesis of an apparent spore coat protein precursor (molecular weight, 25,000). A polypeptide of this weight was previously identified in studies in vivo (L.E. Munoz, Y. Sadaie, and R.H. Doi, J. Biol. Chem., in press). The synthesis in vitro of polysome-associated nascent spore coat polypeptides with varying molecular weights up to 23,000 was also detected. These results indicate that the spore coat protein may be synthesized as a precursor protein. The removal of proteases in the crude extracts by treatment with hemoglobin-Sepharose affinity techniques may be preventing the conversion of the large 25,000-dalton precursor to the 12,500-dalton mature spore coat protein. 相似文献
13.
Identification of an actin-like protein and of its messenger ribonucleic acid in Saccharomyces cerevisiae. 总被引:10,自引:0,他引:10
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We have identified a yeast protein that resembles actins from other eucaryotes in its tight binding to pancreatic deoxyribonuclease I, its copolymerizaton with purified muscle actin, its one-dimensional peptide map, and its apparent polymerization into 7-nm filaments. The yeast actin-like protein yielded a single spot on two-dimensional polyacrylamide gel electrophoresis, suggesting that a single protein species was present. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the actin-like protein had an apparent molecular weight of 45,000 compared with 42,000 for muscle actin. In an attempt to identify the messenger ribonucleic acid coding for the actin-like protein, yeast polyadenylic acid-rich ribonucleic acid was translated in wheat germ and reticulocyte cell-free protein-synthesizing systems. The actin-like protein was identified among the translation products of the reticulocyte system by its tight binding to deoxyribonuclease I, its comigration with the in vivo-synthesized actin-like protein during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, an the similarity of its peptide map to that of the in vivo-synthesized protein. A yeast protein synthesized in the wheat-germ system was also found to bind to deoxyribonuclease I and to copolymerize with muscle actin. However, its apparent molecular weight was about 35,000, suggesting that it was a product either of incomplete translation or of proteolytic cleavage of the actin-like protein. 相似文献
14.
Koga Y Ohga M Tsujimura M Morii H Kawarabayasi Y 《Bioscience, biotechnology, and biochemistry》2006,70(1):282-285
sn-Glycerol-1-phosphate dehydrogenase is responsible for the formation of sn-glycerol-1-phosphate, the backbone of membrane phospholipids of Archaea. This activity had never been detected in cell-free extract of Sulfolobus sp. Here we report the detection of this activity on the thermostable ST0344 protein of Sulfolobus tokodaii expressed in Escherichia coli, which was predicted from genomic information on S. tokodaii. This is another line of evidence for the general mechanism of sn-glycerol-1-phosphate formation by the enzyme. 相似文献
15.
Characteristics of a ugp-encoded and phoB-dependent glycerophosphoryl diester phosphodiesterase which is physically dependent on the ugp transport system of Escherichia coli. 总被引:8,自引:6,他引:2
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The ugp-encoded transport system of Escherichia coli accumulates sn-glycerol-3-phosphate with high affinity; it is binding protein mediated and part of the pho regulon. Here, we report that glycerophosphoryl diesters (deacylated phospholipids) are also high-affinity substrates for the ugp-encoded system. The diesters are not taken up in an unaltered form but are hydrolyzed during transport to sn-glycerol-3-phosphate plus the corresponding alcohols. The enzyme responsible for this reaction is not essential for the translocation of sn-glycerol-3-phosphate or for the glycerophosphoryl diesters but can only hydrolyze diesters that are in the process of being transported. Diesters in the periplasm or in the cytoplasm were not recognized, and no enzymatic activity could be detected in cellular extracts. The enzyme is encoded by the last gene in the ugp operon, termed ugpQ. The product of the ugpQ gene, expressed in minicells, has an apparent molecular weight of 17,500. We present evidence that only one major phoB-dependent promoter controls all ugp genes. 相似文献
16.
The cold osmotic shock procedure releases a protein (GLPT) from the cell envelope of Escherichia coli that is related to the transport of sn-glycerol-3-phosphate in this organism. The evidence for this correlation is as follows: (1) GLPT is under the regulatory control of the glpR gene. (2) Some glpT mutants that were isolated as phosphonomycin resistant clones do not synthesize GLPT. Revertants of these mutants (growth on sn-glycerol 3-phosphate) again synthesize GLPT. (3) Some amber mutations in glpT reduce the amount of GLPT while suppressed strains produce normal amounts. (4) Transfer of a plasmid carrying the glpT genes into a strain lacking GLPT and sn-glycerol-3-phosphate transport restores both functions in the recipient. Transport and GLPT synthesis in the plasmid carrying strain are increased 2- to 3-fold over a fully induced wild-type strain, but appear to be constitutive. GLPT is a soluble protein of molecular weight 160,000 composed of 4 identical subunits. The 160,000 molecular weight complex is stable in 1% sodium dodecylsulfate at room temperature. Upon boiling in 1% sodium dodecylsulfate GLPT dissociates into its subunits. Likewise, 8 M urea at room temperature dissociates GLPT into its subunits. Dialysis of dissociated GLPT against phosphate or Tris-HCl buffer, pH 7.0, allows renaturation to the tetrameric form. The protein is acidic in nature (isoelectric point 4.4). In contrast to the typical transport-related periplasmic-binding proteins, no conditions could be found where pure GLPT exhibited binding activity toward its supposed substrate, sn-glycerol-3-phosphate. In vivo new appearance of transport activity for sn-glycerol-3-phosphate transport occurs only shortly before cell division. However, GLPT synthesis does not fluctuate during the cell cycle. The available evidence indicates a cell-division-dependent processing of GLPT in the cell envelope as a reason for the alteration in transport activity. Transport in whole cells is sensitive to the cold osmotic shock procedure, demonstrating the participation of an essential periplasmic component. However, isolated membrane vesicles that are devoid of periplasmic components, including GLPT, are fully active in sn-glycerol-3-phosphate transport. Therefore, we conclude that GLPT is essential in overcoming a diffusion barrier for sn-glycerol-3-phosphate established by the outer membrane. Attempts to isolate mutants that are transport negative in whole cells due to a defect in GLPT but are active in isolated membrane vesicles have failed so far. All GLPT mutants tested, whether or not they synthesize GLPT, are not active in isolated membrane vesicles. Iodination of whole cells with [125I] followed by osmotic shock reveals that several shock-releasable proteins including GLPT become radioactively labeled. This indicates that some portions of GLPT are accessible to the external medium. 相似文献
17.
Isolation and characterization of casein mRNAs from lactating ewe mammary glands. 总被引:1,自引:1,他引:0
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RNA from bound polysomers of lactating ewe's mammary gland directs the synthesis of the three major milk proteins (alphas, beta and kappa-caseins) in a cell-free system derived from rabbit reticuyocytes. The "in vitro" product was identified by immunoprecipitation with specific antibodies and by electrophoresis in SDS polyacrylamide gel. Each of these messengers was purified from 20 to 25 fold from total membrane-bound polysomal RNA using poly U-Sepharose chromatography. This purified fraction assayed in a reticulocyte cell-free system is able to direct also the synthesis of 2 minor secretory proteins (beta-lactoglobulin and alpha-lactalbumin). The messenger RNAs purified by hybridization to poly U-Sepharose have a sedimentation coefficient of about 12 S and an apparent molecular weight of approximatively 3.5 s 10-5 daltons was observed by polyacrylamide gel electrophoresis under denaturing contitions. This value which correspond to about 900 nucleotides is significantly greater than the number expected for coding milk proteins. 相似文献
18.
M A Haralson S J Fairfield W E Nicholson R W Harrison D N Orth 《The Journal of biological chemistry》1979,254(7):2172-2175
Polysomes or mRNA prepared from cultured AtT-20/D16v mouse pituitary adenocarcinoma cells direct the efficient incorporation of amino acid into newly synthesized material in the presence of wheat germ translational factors. A significant franction of the total cell-free product is specifically immunoprecipitable with corticotropin antibody purified by immune affinity chromatography. Analysis of the cell-free synthesized immunoreactive products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals that two high molecular weight corticotropin species (Mr congruent to 32,500 and 28,000) are synthesized in an approximate 2:1 ratio. Neither product contains carbohydrate based upon concanavalin A chromatography or exposure to polysaccharidases. The smaller molecular weight product does not appear to arise from proteolytic processing since both species are synthesized in approximately the same ratio in cell-free reaction mixtures directed by either polysomes or mRNA. These results suggest that AtT-20/D16v cells contain two distinct mRNA poluations specifying the synthesis of two different high molecular weight forms of mouse corticotropin. 相似文献
19.
Glycerophosphate acyltransferase (acyl-CoA:sn-glycerol-3-phosphate O-acyltransferase, EC 2.3.1.15) solubilized from Escherichia coli membranes was highly activated by phosphatidylglycerol. Phosphatidylethanolamine, cardiolipin and 1,2-diacyl-sn-glycerol 3-phosphate showed no effect. The Km of the enzyme for sn-glycerol 3-phosphate was increased 20-fold by solubilization. The value could not be restored by the addition of phospholipids. Temperature-sensitive regulation of the synthesis of either 1-palmitoyl- or cis-vaccenoyl-sn-glycerol 3-phosphate by the solubilized enzyme was identical with that by the membrane-bound enzyme in vivo and in vitro. The proportion of the molecular species of 1-acyl-sn-glycerol 3-phosphate varied when the ratios of palmitoyl-CoA and cis-vaccenoyl-CoA were changed, but changes in the sn-glycerol 3-phosphate concentration had no effect on selective acylation by both the solubilized and membrane-bound enzymes. 相似文献
20.
Venom glands of honeybees synthesize the peptide melittin via the precursor promelittin. Total RNA preparations from venom glands served as template in a cell-free system prepared from mammalian cells. The heterologous system translated the insect mRNA with approximately the same efficiency as hemoglobin mRNA. A polypeptide was synthesized which, as shown by acrylamide gel electrophoresis in the presence of detergent, has a higher molecular weight than promelittin. Analysis of peptic fragments as well as Edman degradation have demonstrated that sequences characteristic of venom gland promelittin are present in this product formed in vitro. Furthermore, a bacterial protease which specifically splits after acidic residues liberates from the cell-free product a fragment which closely resembles melittin. Evidence is presented that most of the extra amino acids are located at the amino terminus of the product formed in vitro. The larger polypeptide detected in vitro may represent a precursor of promelittin. 相似文献