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1.
The acetylcholine receptor as part of a protein complex in receptor-enriched membrane fragments from Torpedo californica electric tissue 总被引:6,自引:0,他引:6
The acetylcholine receptor from Torpedo californica electric tissue consisting of polypeptide chains of molecular weight 42000 (+/- 2000) is part of a protein complex. Cross-linking experiments with bifunctional reagents have shown that this complex has possibly a pentameric structure with a molecular weight of 270000 (+/- 30000). Besides the receptor subunit (alpha-chain), at least three further classes of polypeptide chains are part of the complex: beta (Mr 48000), gamma (Mr 62000) and delta (Mr 68000). This can be shown by cross-linking the proteins extracted from receptor-enriched membrane fractions with a cleavable reagent: From the 270000 molecular weight particle the four predominant polypeptide chains of the membrane, alpha, beta, gamma, and delta, can be obtained. The gamma-polypeptide chains appear to form a dimer connected by an inter-chain disulphide bridge. 相似文献
2.
Chimeric immunoglobulin-T cell receptor proteins form functional receptors: implications for T cell receptor complex formation and activation 总被引:10,自引:0,他引:10
We constructed chimeric receptor chains in which an immunoglobulin heavy chain variable region (VH) from a phosphorylcholine-specific antibody is substituted for T cell receptor (Tcr) alpha and beta V regions. We demonstrate that the VH region joined to either the C alpha or the C beta region can form stable chimeric proteins in EL4 T cells. Both chimeric receptor chains associate with CD3 polypeptides in functional receptor complexes and respond to phosphorylcholine coupled to Sepharose beads. The VH-C alpha chimeric chain associates with the EL4 beta chain, while the VH-C beta chimeric protein appears to form either a homodimer or a heterodimer with the native EL4 beta chain. Thus, functional receptor complexes can be formed using two C beta regions, and the C alpha region may not be required for CD3 association and surface expression of Tcr complexes. 相似文献
3.
The signal sequence receptor has a second subunit and is part of a translocation complex in the endoplasmic reticulum as probed by bifunctional reagents 总被引:14,自引:7,他引:7
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《The Journal of cell biology》1990,111(6):2283-2294
Bifunctional cross-linking reagents were used to probe the protein environment in the ER membrane of the signal sequence receptor (SSR), a 24-kD integral membrane glycoprotein (Wiedmann, M., T. V. Kurzchalia, E. Hartmann, and T. A. Rapoport. 1987. Nature [Lond.]. 328:830-833). The proximity of several polypeptides was demonstrated. A 22-kD glycoprotein was identified tightly bound to the 34-kD SSR even after membrane solubilization. The 34-kD polypeptide, now termed alpha SSR, and the 22-kD polypeptide, the beta SSR, represent a heterodimer. We report on the sequence of the beta SSR, its membrane topology, and on the mechanism of its integration into the membrane. Cross-linking also produced dimers of the alpha-subunit of the SSR indicating that oligomers of the SSR exist in the ER membrane. Various bifunctional cross-linking reagents were used to study the relation to ER membrane proteins of nascent chains of preprolactin and beta-lactamase at different stages of their translocation through the membrane. The predominant cross-linked products obtained in high yields contained the alpha SSR, indicating in conjunction with previous results that it is a major membrane protein in the neighborhood of translocating nascent chains of secretory proteins. The results support the existence of a translocon, a translocation complex involving the SSR, which constitutes the specific site of protein translocation across the ER membrane. 相似文献
4.
G J Giotta 《The Journal of biological chemistry》1976,251(5):1247-1252
(Na+ + K+)-dependent adenosine triphosphatase (NaK-ATPase) consists of two polypeptide chains, a large polypeptide with a molecular weight of about 100,000, and a sialoglycoprotein with a molecular weight of about 40,000. Cross-linking of purified NaK-ATPase with the (o-phenanthroline)2-cupric ion complex (CP) results in the reversible formation of dimers, trimers, tetramers, and pentamers of the large polypeptide and loss of NaK-ATPase activity. ATPase activity is partially recovered if NaK-ATPase is incubated with beta-mercaptoethanol after treatment with CP. In contrast to these results, if NaK-ATPase is cross-linked in crude canine kidney microsomes, only a dimer of the large polypeptide is formed. No cross-linking of the sialoglycoprotein to the large polypeptide is detected when NaK-ATPase is cross-linked in purified form. However, when NaK-ATPase is reacted with CP in either purified or microsomal form, the sialoglycoprotein cross-links to itself yielding a high molecular weight aggregate. The results show that the functional subunit structure of NaK-ATPase consists of at least two large polypeptides. 相似文献
5.
The guanine nucleotide binding proteins (G proteins) that couple hormone and other receptors to a variety of intracellular effector enzymes and ion channels are heterotrimers of alpha, beta, and gamma subunits. One way to study the interfaces between subunits is to analyze the consequences of chemically cross-linking them. We have used 1,6-bismaleimidohexane (BMH), a homobifunctional cross-linking reagent that reacts with sulfhydryl groups, to cross-link alpha to beta subunits of Go and Gi-1. Two cross-linked products are formed from each G protein with apparent molecular masses of 140 and 122 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both bands formed from Go reacted with anti-alpha o and anti-beta antibody. The mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is anomalous since the undenatured, cross-linked proteins have the same Stokes radius as the native, uncross-linked alpha beta gamma heterotrimer. Therefore, each cross-linked product contains one alpha and one beta subunit. Activation of Go by guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) does not prevent cross-linking of alpha to beta gamma, consistent with an equilibrium between associated and dissociated subunits even in the presence of GTP gamma S. The same cross-linked products of Go are formed in brain membranes reacted with BMH as are formed in solution, indicating that the residues cross-linked by BMH in the pure protein are accessible when Go is membrane bound. Analysis of tryptic peptides formed from the cross-linked products indicates that the alpha subunit is cross-linked to the 26-kDa carboxyl-terminal portion of the beta subunit. The cross-linked G protein is functional, and its alpha subunit can change conformation upon binding GTP gamma S. GTP gamma S stabilizes alpha o to digestion by trypsin (Winslow, J.W., Van Amsterdam, J.R., and Neer, E.J. (1986) J. Biol. Chem. 261, 7571-7579) and also stabilizes the alpha subunit in the cross-linked product. Cross-linked G o can be ADP-ribosylated by pertussis toxin. This ADP-ribosylation is inhibited by GTP gamma S with a concentration dependence that is indistinguishable from that of the control, uncross-linked G o. These two kinds of experiments indicate that alpha o is able to change its conformation even though it cannot separate completely from beta gamma. Thus, although dissociation of the subunits accompanies activation of G o in solution, it is not obligatory for a conformational change to occur in the alpha subunit. 相似文献
6.
Physical-chemical studies of pyruvate carboxylase from Pseudomonas citronellolis demonstrate that the enzyme has an alpha 4 beta 4 structure. The individual polypeptides, alpha (Mr = 65,000) and beta (Mr = 54,000), were separated and isolated by preparative gel electrophoresis. Analysis of the relationship between Coomassie blue staining and protein quantity for each polypeptide indicated that the alpha and beta subunits are present in a 1:1 stoichiometry in the native enzyme. Determinations of the molecular weight of the protein by sedimentation equilibrium (Mr = 454,000), gel filtration analysis (Mr = 510,000), disc gel electrophoresis (Mr = 530,000), and mass measurement from the Scanning Transmission Electron Microscope (Mr = 530,000) are consistent with the proposed alpha 4 beta 4 structure. Disc gel electrophoresis studies revealed that under certain circumstances the enzyme may dissociate to a smaller molecular weight species (Mr = 228,000). This dissociation phenomenon could explain the earlier reported observation of Taylor et al. ((1972) J. Biol. Chem 22, 7388-8390) that the enzyme had a molecular weight of 265,000. Evidence from electron microscopic studies shows that the three-dimensional structure of this enzyme is quite distinct from other species of pyruvate carboxylase. The enzyme does not show the typical rhombic appearance which has been noted for chicken liver, sheep liver, and yeast pyruvate carboxylase. 相似文献
7.
Mammalian fatty acid synthetase is a structurally and functionally symmetrical dimer 总被引:2,自引:0,他引:2
We have explored a comprehensive experimental approach to determine whether the two condensing-enzyme active centers of the mammalian fatty acid synthetase are simultaneously functional. Our strategy involved utilization of trypsinized fatty acid synthetase, which is a nicked homodimer composed of two pairs of 125 + 95-kDa polypeptides. These core polypeptides lack the chain-terminating thioesterase domains but retain all other functional domains of the native enzyme and can assemble long-chain acyl moieties at a rate equal to that of the native enzyme. The 4'-phosphopantetheine content of these enzyme preparations, estimated from the amount of beta-alanine present, from the amount of taurine formed by performic acid oxidation and from the amount of carboxymethylcysteamine formed by alkylation with iodo[2-14C]acetate, was typically 0.86 mol/mol 95-kDa polypeptide. The stoichiometry of long-chain acyl-enzyme synthesis, measured with radiolabeled precursors, indicated that 0.84 mol acyl-chains were assembled/mol 95-kDa polypeptide. When the small amount of apoenzyme present is taken into account, this stoichiometry translates to 1.94 acyl chains per holoenzyme dimer. The 125-kDa polypeptide of one subunit could be cross-linked to the 95-kDa polypeptide of the other subunit by 1,3-dibromo-2-propanone yielding a single molecular species of 220 kDa. Cross-linking was accompanied by a loss of condensing-enzyme activity. This result is consistent with a structurally symmetrical model for the animal fatty acid synthetase [J.K. Stoops and S.J. Wakil (1981) J. Biol. Chem. 256, 5128-5133] in which the juxtaposed 4'-phosphopantetheine and cysteine thiols of opposing subunits that form the two potential catalytic centers for condensing activity are readily susceptible to cross-linking. Both half-maximal cross-linking and 50% inhibition of activity were observed with 1 mol 1,3-dibromo-2-propanone bound/mol enzyme. After assembly of long-chain acyl moieties on the 4'-phosphopantetheine residues, no vacant condensing-enzyme active sites were demonstrable either by cross-linking with 1,3-dibromo-2-propanone or by formation of carboxymethylcysteamine on treatment with iodoacetate. These results are consistent with a structurally and functionally symmetrical model for the mammalian fatty acid synthetase in which the two condensation sites are simultaneously active. 相似文献
8.
Further characterization of the Friend murine leukemia virus reverse transcriptase-RNase H complex. 总被引:14,自引:14,他引:0
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K Moelling 《Journal of virology》1976,18(2):418-425
9.
ATPase of Escherichia coli: purification, dissociation, and reconstitution of the active complex from the isolated subunits. 总被引:5,自引:0,他引:5
A simple procedure for the purification of Mg2+-stimulated ATPase of Escherichia coli by fractionation with poly(ethylene glycols) and gel filtration is described. The enzyme restores ATPase-linked reactions to membrane preparations lacking these activities. Five different polypeptides (alpha, beta, gamma, delta, epsilon) are observed in sodium dodecyl sulfate electrophoresis. Freezing in salt solutions splits the enzyme complex into subunits which do not possess any catalytic activity. The presence of different subunits is confirmed by electrophoretic and immunological methods. The active enzyme complex can be reconstituted by decreasing the ionic strength in the dissociated sample. Temperature, pH, protein concentration, and the presence of substrate are each important determinants of the rate and extent of reconstitution. The dissociated enzyme has been separated by ion-exchange chromatography into two major fragments. Fragment IA has a molecular weight of about 100000 and contains the alpha, gamma, and epsilon polypeptides. The minor fragment, IB, has about the same molecular weight but contains, besides alpha, gamma, and epsilon, the delta polypeptide. Fragment II, with a molecular weight of about 52000, appears to be identical with the beta polypeptide. ATPase activity can be reconstituted from fragments IA and II, whereas the capacity of the ATPase to drive energy-dependent processes in depleted membrane vesicles is only restored after incubation of these two fractions with fraction IB, which contains the delta subunit. 相似文献
10.
The integrin alpha 3 beta 1 is a multiligand extracellular matrix receptor found on many cell types. Immunoprecipitations of 125I-surface-labeled prostate carcinoma cell lines, DU145 and PC-3, with the anti-alpha 3 integrin monoclonal antibodies J143 or PIB5, resulted in the coimmunoprecipitation, along with the expected alpha 3 beta 1 heterodimer, of a polypeptide with a molecular mass of 225 kDa. This protein could also be copurified with the 155-kDa alpha 3 and 115-kDa beta 1 subunits upon affinity chromatography of 125I-surface-labeled cell extracts on anti-alpha 3 antibody-Sepharose columns. Upon reduction, this 225-kDa protein generated 130- and 95-kDa polypeptides, while the 155-kDa alpha 3 subunit generated 130- and 25-kDa polypeptides. The 225-kDa protein did not generate a 25-kDa polypeptide. Deglycosylation and reduction of the 225-kDa protein resulted in the generation of 110- and 95-kDa polypeptides, while deglycosylation and reduction of the 155-kDa alpha 3 resulted in a 110-kDa polypeptide identical in size to the 110-kDa polypeptide generated from the 225-kDa protein. Peptide maps generated from the 110-kDa components of the 225-kDa polypeptide and the 155-kDa alpha 3 integrin subunit were identical, as were their N-terminal amino acid sequences. An antibody directed against the cytoplasmic domain of the alpha 3 subunit immunoprecipitated the 225-kDa polypeptide in addition to the 155-kDa alpha 3 subunit. Furthermore, Northern blot analysis of RNA from DU145 and PC-3 cells with a human alpha 3 cDNA probe identified an mRNA species of 6.2 kb in addition to a major mRNA species of 4.3 kb. The larger mRNA species, which is of an appropriate size for encoding a polypeptide of approximately 220-kDa, was not detectable in cells which did not express the 225-kDa protein. These data demonstrate that the 225-kDa polypeptide represents a novel integrin alpha 3 subunit consisting of the alpha 3 integrin heavy chain disulfide-bonded to a 95-kDa polypeptide which may represent an alternative "light" chain to the 25-kDa light chain of the alpha 3 subunit. 相似文献
11.
Geberhiwot T Ingerpuu S Pedraza C Neira M Lehto U Virtanen I Kortesmaa J Tryggvason K Engvall E Patarroyo M 《Experimental cell research》1999,253(2):723-732
Laminins, a family of heterotrimeric proteins with cell adhesive/signaling properties, are characteristic components of basement membranes of vasculature and tissues. In the present study, permeabilized platelets were found to react with a monoclonal antibody to laminin gamma1 chain by immunofluorescence. In Western blot analysis of platelet lysates, several monoclonal antibodies to gamma1 and beta1 laminin chains recognized 220- to 230-kDa polypeptides, under reducing conditions, and a structure with much slower electrophoretic mobility under nonreducing conditions. Immunoaffinity purification on a laminin beta1 antibody-Sepharose column yielded polypeptides of 230, 220, 200, and 180 kDa from platelet lysates. In the purified material, mAbs to beta1 and gamma1 reacted with the two larger polypeptides, while affinity-purified rabbit antibodies to laminin alpha4 chain recognized the smallest polypeptide. Identity of the polypeptides was confirmed by microsequencing. One million platelets contained on average 1 ng of laminin (approximately 700 molecules per cell), of which 20-35% was secreted within minutes after stimulation with either thrombin or phorbol ester. Platelets adhered to plastic surfaces coated with the purified platelet laminin, and this process was largely inhibited by antibodies to beta1 and alpha6 integrin chains. We conclude that platelets contain and, following activation, secrete laminin-8 (alpha4beta1gamma1) and that the cells adhere to the protein by using alpha6beta1 integrin. 相似文献
12.
The human alpha 2-macroglobulin receptor: identification of a 420-kD cell surface glycoprotein specific for the activated conformation of alpha 2-macroglobulin 总被引:4,自引:0,他引:4
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J D Ashcom S E Tiller K Dickerson J L Cravens W S Argraves D K Strickland 《The Journal of cell biology》1990,110(4):1041-1048
Ligand affinity chromatography was used to purify a cell surface alpha 2-macroglobulin (alpha 2M) receptor. Detergent extracts of human placenta were applied to an affinity matrix consisting of alpha 2M, previously reacted with methylamine, coupled to Sepharose. Elution with EDTA specifically released polypeptides with apparent molecular masses of 420 and 39 kD. In some preparations, small amounts of a 90-kD polypeptide were observed. The 420- and 39-kD polypeptides appear specific for the forms of alpha 2M activated by reaction with proteinases or methylamine and do not bind to an affinity matrix consisting of native alpha 2M coupled to Sepharose. Separation of these two polypeptides was accomplished by anion exchange chromatography, and binding activity was exclusively associated with the 420-kD polypeptide. The purified 420-kD protein binds to the conformationally altered forms of alpha 2M that are known to specifically interact with alpha 2M receptors and does not bind to native alpha 2M. Binding of the 420-kD polypeptide to immobilized wheat germ agglutinin indicates that this polypeptide is a glycoprotein. The cell surface localization of the 420-kD glycoprotein was confirmed by affinity chromatography of extracts from surface radioiodinated fibroblasts. These properties suggest that the 420-kD polypeptide is a cell surface receptor for the activated forms of alpha 2M. 相似文献
13.
Enrichment and characterization of the mRNAs of four aminoacyl-tRNA synthetases from yeast.
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We have partially purified the messenger RNAs for yeast arginyl-, aspartyl-, valyl-, alpha and beta subunits of phenylalanyl-tRNA synthetases in order to study their biosynthesis and ultimately, to isolate their genes. Sucrose gradient fractionation of poly U-Sepharose selected mRNAs resulted in a ten fold enrichment of the in vitro translation activity of these mRNAs. The translation products of messenger RNAs for arginyl- and valyl-tRNA synthetases have the same molecular weight as the purified enzymes; translation of aspartyl-tRNA synthetase messenger RNA yielded a 68 kD molecular weight polypeptide (while the purified cristallisable enzyme appears as a 64-66 kD doublet, which, as we showed is a proteolysis product). The translation of the mRNAs for alpha and beta phenylalanyl-tRNA synthetase gave polypeptides having the same molecular weight as those obtained from the purified enzyme, but the major translation products are slightly heavier, indicating that they may be translated as precursors. As estimated from centrifugation experiments mRNAs of arginyl-, aspartyl-, alpha and beta subunits of phenylalanyl-tRNA synthetase were 1700-2000 nucleotides long, indicating that alpha and beta are translated from two different mRNAs. 相似文献
14.
Identification and partial characterization of a novel bipartite protein antigen associated with the outer membrane of Escherichia coli. 总被引:1,自引:1,他引:0
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A study by crossed immunoelectrophoresis performed in conjunction with precipitate excision and polypeptide analysis identified a new antigen complex in the envelope of Escherichia coli ML308-225. This antigen corresponds to antigen 43 in the crossed immunoelectrophoresis profile of membrane vesicles (P. Owen and H. R. Kaback, Proc. Natl. Acad. Sci. USA 75:3148-3152, 1978). Immunoprecipitation experiments conducted with specific antiserum revealed that the complex was expressed on the cell surface and that it contained, in equal stoichiometry, two chemically distinct polypeptides termed alpha and beta (Mrs of 60,000 and 53,000, respectively). The beta polypeptide was heat modifiable, displaying an apparent Mr of 37,000 when solubilized at temperatures below 70 degrees C. Analysis of fractions obtained following cell disruption, isopycnic centrifugation, and detergent extraction indicated that both alpha and beta polypeptides were components of the outer membrane. The two polypeptides were not linked by disulfide bonds, and neither was peptidoglycan associated. The complex contained no detectable lipopolysaccharide, enzyme activity, fatty acyl groups, or other cofactors. Neither correlated with E. coli proteins of similar molecular weight which had previously been shown to be associated with the outer membrane. Antibodies were raised to individual alpha and beta polypeptides. Each of these sera was shown to be subunit specific when tested against denatured membrane proteins. In contrast, each immunoglobulin preparation coprecipitated both alpha and beta polypeptides when tested against undenatured proteins derived from Triton X-100-treated membranes. The results reveal the presence of a novel bipartite protein antigen in the outer membrane of E. coli. 相似文献
15.
16.
17.
The soluble domains of the b and b' subunits of the ATP synthase of the cyanobacterium Synechocystis PCC 6803 were expressed with His tags attached to their N-termini. Following purification, the polypeptides were characterized by chemical cross-linking, analytical ultracentrifugation, and circular dichroism spectroscopy. Treatment of a mixture of the soluble b and b' domains with a chemical cross-linking agent led to substantial formation of cross-linked dimers, whereas similar treatment of either domain by itself resulted in only trace formation of cross-linked species. The molecular weights of the domains of b and b' in solution at 20 degrees C, measured by sedimentation equilibrium, were 17 800+/-700 and 16 300+/-400, respectively, compared to calculated polypeptide molecular weights of 16 635 and 15 422, whereas a mixture of b and b' gave a molecular weight of 29 800+/-800. The sedimentation coefficient of an equimolar mixture was 1.73+/-0.03. The circular dichroism spectra of the individual polypeptides indicated helical contents in the range of 40-50%; the spectrum of the mixture revealed changes indicative of coiled-coil formation and a helical content of 60%. The results indicate that the cytosolic domains of the b and b' subunits exist individually as monomers but form a highly extended heterodimer when they are mixed together. 相似文献
18.
All-trans-retinoic acid, an endogenous morphogen, induced neuronal differentiation of P19 murine embryonal carcinoma cells. Peak differentiation, as judged by the elaboration of neuronal processes, occurred 8 days after exposure of the cells to 0.5 mM retinoic acid, a concentration known to induce neuronal differentiation. An examination of the expression of the extracellular matrix receptors, integrins, during this retinoic acid-induced differentiation period, demonstrated a specific and strong induction of expression of two polypeptides (130 and 115 kDa) immunoprecipitated with an anti-human vitronectin receptor antiserum. The expression of a 90-kDa polypeptide, also immunoprecipitating with this antiserum was induced as well, but to a much smaller extent. The expression of a 96-kDa polypeptide immunoprecipitated by this antiserum and present in the untreated cells was not induced by retinoic acid. The increase in the expression of these polypeptides paralleled the neuronal differentiation of the P19 embryonal carcinoma cells. The expression of these integrins was not induced in a variant of the P19 cells, P19RAC65, which are resistant to differentiation induction by retinoic acid. Utilizing integrin subunit-specific anti-cytoplasmic peptide antibodies together with immunoprecipitation and Western blot analysis, the 130- and 115-kDa polypeptides were identified as the integrin alpha v and beta 1 subunits, respectively. The 90-kDa polypeptide, also induced by retinoic acid, was identified as beta 3, whereas the identity of the uninduced 96-kDa polypeptide remains unclear as yet. Peptide map analysis of deglycosylated polypeptides demonstrated that the 90- and 96-kDa polypeptides are distinct proteins and that the 115-kDa polypeptides immunoprecipitated with either anti-alpha v or anti-beta 1 antibodies are identical, further establishing that the 115-kDa polypeptide associating with alpha v is beta 1. The retinoic acid-induced expression of beta 1 occurred at the level of mRNA expression which also paralleled neuronal differentiation, but peaked slightly ahead of the cell surface expression of beta 1. The expression of other beta 1-associated alpha subunits was not induced by retinoic acid in these cells. These data demonstrate that retinoic acid strongly induces the expression of the integrin heterodimer alpha v beta 1 and also, to a smaller extent, the expression of alpha v beta 3. The retinoic acid-induced, high level surface expression of the alpha v beta 1 heterodimer is tightly correlated with the induction of neuronal differentiation by retinoic acid. This finding suggests an important role for the alpha v beta 1 heterodimer in the neuronal differentiation process. 相似文献
19.
Genes for the alpha and beta subunits of the phenylalanyl-transfer ribonucleic acid synthetase of Escherichia coli. 总被引:10,自引:6,他引:4
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The phenylalanyl-transfer ribonucleic acid synthetase of Escherichia coli is a tetramer that contains two different kinds of polypeptide chains. To locate the genes for the two polypeptides, we analyzed temperature-sensitive mutants with defective phenylalanyl-transfer ribonucleic acid synthetases to see which subunit was altered. The method was in vitro complementation; mutant cell extracts were mixed with purified separated alpha or beta subunits of the wild-type enzyme to generate an active hybrid enzyme. With three mutants, enzyme activity appeared when alpha was added, but not when beta was added: these are, therefore, assumed to carry lesions in the gene for the alpha subunit. Two other mutants gave the opposite response and are presumably beta mutants. Enzyme activity is also generated when alpha and beta mutant extracts are mixed, but not when two alpha or two beta mutant extracts are mixed. The inactive mutant enzymes appear to be dissociated, as judged by their sedimentation in sucrose density gradients, but the dissociation may be only partial. The active enzyme generated by complementation occurred in two forms, one that resembled the native wild-type enzyme and one that sedimented more slowly. Both alpha and beta mutants are capable of generating the native form, although alpha mutants require prior urea denaturation of the defective enzyme. With the mutants thus characterized, the genes for the alpha and beta subunits (designated pheS and heT, respectively) were mapped. The gene order, as determined by transduction is aroD-pps-pheT-pheS. The pheS and pheT genes are close together and may be immediately adjacent. 相似文献
20.
S-Adenosylmethionine synthetase from human lymphocytes. Purification and characterization 总被引:8,自引:0,他引:8
S-Adenosylmethionine synthetase has been purified to apparent homogeneity from human chronic lymphocytic leukemia cells. Equilibrium sedimentation studies and denaturing polyacrylamide gel electrophoresis indicate that the native enzyme has a molecular weight of 185,000 and a subunit composition of either alpha alpha' beta 2, alpha 2 beta 2, or alpha' 2 beta 2, where alpha, alpha', and beta are polypeptide chains of molecular weight 53,000, 51,000, and 38,000. The alpha and alpha' subunits appear to be the same polypeptide and presumably differ by some kind of post-translational modification. Stoichiometric studies show that the expected products S-adenosylmethionine, pyrophosphate, and orthophosphate are generated in equimolar amounts. The enzyme exhibits linear kinetics with respect to substrate dependency and product inhibition, except for orthophosphate which shows parabolic noncompetitive inhibition with respect to ATP. Initial velocity studies of substrate dependence and product inhibition indicate a steady state mechanism that is ordered Bi Ter with ATP adding before L-methionine and S-adenosylmethionine as the first product released. Pyrophosphate and orthophosphate, however, appear to be released by a random mechanism. Free Mg2+ is an essential activator with a half-maximal effect at 1.0 mM. The Km and Kia for ATP are 31 microM and 84 microM, and the Km for L-methionine is 3.3 microM. The enzyme also has tripolyphosphatase activity which is stimulated by S-adenosylmethionine. 相似文献