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1.
The structural organization of Bordetella pertussis adenylate cyclase was examined by limited proteolysis with trypsin and/or cross-linking with azido-calmodulin a photoactivable derivative of its activator, calmodulin (CaM). Adenylate cyclase (which consists of three structurally related peptides of 50, 45, and 43 kDa as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) formed a 1:1 complex with CaM or azido-CaM. CaM-bound adenylate cyclase was cleaved by trypsin into two separate trypsin-resistant fragments of 25 and 18 kDa which both interacted with CaM as judged by their ability to be cross-linked with azido-CaM. These two fragments remained associated with CaM in a catalytically active conformation resembling that of the undigested complex. When proteolysis was carried out in the absence of CaM, the adenylate cyclase was completely inactivated in less than 3 min. Sodium dodecyl sulfate-polyacrylamide gel revealed a single 24-kDa trypsin-resistant fragment. Since this fragment cannot be cross-linked with azido-CaM we suggest that the CaM-binding site on the 25-kDa moiety of the adenylate cyclase is located on a short segment of 1 kDa.  相似文献   

2.
In order to identify molecular features of the calmodulin (CaM) activated adenylate cyclase of Bordetella pertussis, a truncated cya gene was fused after the 459th codon in frame with the alpha-lacZ' gene fragment and expressed in Escherichia coli. The recombinant, 604 residue long protein was purified to homogeneity by ion-exchange and affinity chromatography. The kinetic parameters of the recombinant protein are very similar to that of adenylate cyclase purified from B.pertussis culture supernatants, i.e. a specific activity greater than 2000 mumol/min mg of protein at 30 degrees C and pH 8, a KmATP of 0.6 mM and a Kd for its activator, CaM, of 0.2 nM. Proteolysis with trypsin in the presence of CaM converted the recombinant protein to a 43 kd protein with no loss of activity; the latter corresponds to the secreted form of B.pertussis adenylate cyclase. Site-directed mutagenesis of residue Trp-242 in the recombinant protein yielded mutants expressing full catalytic activity but having altered affinity for CaM. Thus, substitution of an aspartic acid residue for Trp-242 reduced the affinity of adenylate cyclase for CaM greater than 1000-fold. Substitution of a Gln residue for Lys-58 or Lys-65 yielded mutants with a drastically reduced catalytic activity (approximately 0.1% of that of wild-type protein) but with little alteration of CaM-binding. These results substantiated, at the molecular level, our previous genetic and biochemical studies according to which the N-terminal tryptic fragment of secreted B.pertussis adenylate cyclase (residues 1-235/237) harbours the catalytic site, whereas the C-terminal tryptic fragment (residues 235/237-399) corresponds to the main CaM-binding domain of the enzyme.  相似文献   

3.
D C Au  H R Masure  D R Storm 《Biochemistry》1989,28(7):2772-2776
A 2.7-kb cya A gene fragment encoding the amino-terminal end of the calmodulin-sensitive adenylate cyclase from Bordetella pertussis has been placed under the control of the lac promoter for expression in Escherichia coli. Following induction with isopropyl beta-D-thiogalactoside, calmodulin-sensitive adenylate cyclase activity was detected in a cell extract from E. coli. The expression vector directed the synthesis of a 90-kDa polypeptide that was recognized by rabbit polyclonal antibodies raised against the catalytic subunit of B. pertussis adenylate cyclase. Inspection of the deduced amino acid sequence of the cya A gene product revealed a sequence with homology to consensus sequences for an ATP-binding domain found in many ATP-binding proteins. On the basis of the analysis of nucleotide binding proteins, a conserved lysine residue has been implicated in the binding of ATP. A putative ATP-binding domain in the B. pertussis adenylate cyclase possesses an analogous lysine residue at position 58. To test whether lysine 58 of the B. pertussis adenylate cyclase is a crucial residue for enzyme activity, it was replaced with methionine by oligonucleotide-directed mutagenesis. E. coli cells were transformed with the mutant cya A gene, and the expressed gene product was characterized. The mutant protein exhibited neither basal nor calmodulin-stimulated enzyme activity, indicating that lysine 58 plays a critical role in enzyme catalysis.  相似文献   

4.
The trifunctional beta-subunit of anthranilate synthase complex of Neurospora crassa has been purified from a mutant which produces no detectable alpha-subunit. The isolated beta-subunit appeared to be a highly asymmetric dimer with a s20,w of 7.35 and an apparent molecular weight of 200,000 as determined by gel filtration on Sephacryl S-300 compared with a monomer molecular weight of approximately 84,000 Da as determined by sodium dodecyl sulfate-gel electrophoresis. The purified subunit was cleaved by elastase, trypsin, or chymotrypsin into fragments which retained the three enzyme activities. After elastase digestion, two active fragments were separated by gel filtration and ion exchange chromatography. A 30,000-Da fragment, which behaved as a monomer on gel filtration, interacted with free alpha-subunit to produce glutamine-dependent anthranilate synthase activity. A second 56,000-Da fragment, which behaved as an asymmetric dimer (apparent molecular weight 140,000) on gel filtration, retained both N-(5'-phosphoribosyl)anthranilate isomerase and indole-3-glycerol phosphate synthase activity. The failure to detect an NH2-terminal amino acid residue on either the intact beta-subunit or the 30,000-Da complementing fragment, while the 56,000-Da fragment possessed an NH2-terminal histidine residue, indicated that the complementing fragment was derived from the NH2-terminal sequence of the beta-subunit.  相似文献   

5.
The catalytic component of calmodulin-independent adenylate cyclase of cattle cerebral cortex was solubilized and purified to the homogeneous state. The conditions for preparative obtaining of the enzyme on the column with immobilized antibodies to adenylate cyclase were found. These antibodies were proved to interact with the calmodulin-independent rather than the calmodulin-dependent form of the enzyme. Molecular mass of the calmodulin-independent adenylate cyclase determined electrophoretically is 140 +/- 10 kDa. Amino acid composition of the enzyme and sequences of its fragments (in total 300 amino acid residues) obtained upon treatment with lysyl-specific proteinase from Achromobacter liticus were determined. Clone containing a cDNA 605 bp insertion coding for the 183 amino acid residue fragment of adenylate cyclase was isolated from the bovine brain cDNA library. Homology of this fragment to the known sequences of Escherichia coli and Bordetella pertussis adenylate cyclases was revealed.  相似文献   

6.
The cytoplasmic nitrate reductase in heme mutant H-14 of Staphylococcus aureus was partially purified by steps which included ammonium sulfate fractionation and chromatography on Bio-Gel A 1.5m and ion-exchange columns. The active fractions from the ion-exchange columns showed two forms of the enzyme upon electrophoresis in nondenaturing gels of polyacrylamide; these corresponded to proteins of R(f) 0.16 and 0.28. Each form contained a predominant polypeptide of molecular weight 140,000, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The R(f) 0.16 form contained another major polypeptide of molecular weight 57,000, but the R(f) 0.28 form contained several other polypeptides. The sedimentation properties of the enzyme were examined after partial purification on Bio-Gel A 1.5m. In sucrose gradients containing Triton X-100 the enzyme sedimented as a homogeneous peak with an estimated molecular weight of 225,000; without detergent a heterogeneous profile was observed of molecular weight greater than 250,000. Treatment of the enzyme with trypsin increased the specific activity, and the enzyme sedimented as a homogeneous peak in sucrose gradients without Triton X-100, with an estimated molecular weight of 202,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that trypsin treatment converted the polypeptide of molecular weight 140,000 to a polypeptide of molecular weight 112,000. We conclude that the cytoplasmic nitrate reductase of S. aureus has a large subunit of molecular weight 140,000, which can be modified by trypsin to a polypeptide of molecular weight 112,000 without loss of catalytic activity.  相似文献   

7.
Soluble guanylate cyclase from rat lung exists as a heterodimer   总被引:8,自引:0,他引:8  
The soluble form of guanylate cyclase (EC 4.6.1.2) from rat lung has been purified to homogeneity by a one-step immunoaffinity chromatographic procedure. The purified soluble guanylate cyclase has specific activities of 432 and 49.1 nmol of cyclic GMP formed per min/mg protein with manganese and magnesium ions as a cofactor, respectively. This represents a purification of approximately 2,000-fold with a 50% recovery. The native enzyme has a molecular weight of 150,000 and a Stokes radius of 4.8 nm as determined on Spherogel TSK-G3000SW gel permeation chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis results in two protein-staining bands with molecular weights of 82,000 and 70,000. The purified soluble guanylate cyclase was also subjected to native polyacrylamide gel electrophoresis, isoelectric focusing electrophoresis, ion exchange chromatography, and GTP-agarose affinity chromatography. These additional purification procedures confirmed the presence of a single protein peak coincident with enzyme activity. The two subunits separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis were shown to have different primary structures by immunoblotting with monoclonal and polyclonal antibodies prepared against purified soluble guanylate cyclase and by peptide mapping with papain or Staphylococcus aureus V8 protease treatment. These data demonstrate that soluble guanylate cyclase purified from rat lung is a heterodimer composed of 82,000- and 70,000-dalton subunits with different primary structures.  相似文献   

8.
We have isolated protransglutaminase E, the zymogen form of epidermal transglutaminase E, from the skin of the adult guinea pig. This zymogen is the source of the large majority of soluble transglutaminase activity of skin. A molecular weight value for protransglutaminase E of 77,800 +/- 700, estimated by sedimentation equilibrium, is in close agreement with the apparent values determined by exclusion chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment of the proenzyme with dispase, proteinase K, trypsin, or thrombin produces active enzyme. The enzyme, transglutaminase E, formed by the action of dispase, was observed to exist in the native state as a molecule indistinguishable in size from the zymogen. Under denaturing conditions, however, the enzyme dissociates into two fragments with molecular weights of 50,000 and 27,000. The observation that reducing agents are not needed for this dissociation suggests a noncovalent association of the two peptide chains in the native enzyme. Evidence that the catalytically essential -SH group of the enzyme residues in the Mr 50,000 fragment and that only the Mr 27,000 fragment possesses an unmasked amino terminus provides the basis for a proposed model of zymogen activation. Whether the noncatalytic fragment plays a role in catalysis is not known because separation of the fragments of native enzyme was not achieved.  相似文献   

9.
The gene for 10-formyltetrahydrofolate synthetase (EC 6.3.4.3) from the purinolytic anaerobic bacterium Clostridium acidiurici ("Clostridium acidi-urici") was cloned into Escherichia coli JM83 with plasmid pUC8. A C. acidiurici genomic library was prepared in E. coli from a partial Sau3A digest and screened with antibody against the synthetase. Of 10 antibody-positive clones, 1 expressed a high level of synthetase activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis demonstrated that the protein synthesized in E. coli had the same subunit molecular weight as the C. acidiurici enzyme. The gene was located on an 8.3-kilobase genomic insert and appeared to be transcribed from its own promoter. Analysis of genomic digests with a fragment of the synthetase gene indicated that one copy of the gene was present in the C. acidiurici chromosome.  相似文献   

10.
A novel photoreactive calmodulin (CaM) derivative was developed and used to label the purified CaM-sensitive adenylate cyclase from bovine cortex. 125I-CaM was conjugated with the heterobifunctional cross-linking agent p-nitrophenyl 3-diazopyruvate (DAPpNP). Spectral data indicated that diazopyruvoyl (DAP) groups were incorporated into the CaM molecule. Iodo-CaM-DAPs behaved like native CaM with respect to (1) Ca2+-dependent enhanced mobility on sodium dodecyl sulfate-polyacrylamide gels and (2) Ca2+-dependent stimulation of adenylate cyclase activity. 125I-CaM-DAP photochemically cross-linked to CaM-binding proteins in a manner that was both Ca2+ dependent and CaM specific. Photolysis of forskolin-agarose-purified adenylate cyclase from bovine cortex with 125I-CaM-DAP produced a single cross-linked product which migrates on sodium dodecyl sulfate-polyacrylamide gels with an apparent molecular weight of approximately 140,000.  相似文献   

11.
Guanylate cyclase has been purified from extracts of Escherichia coli. After a 1000-fold purification, the enzyme contains only minor contaminants as judged by disc gel electrophoresis. The Km for GTP is approximately 7 times 10(-5) M and the optimal pH is 8.0. More activity is observed with Mn2+ than with Mg2+, and maximal activity is observed at 0.14 mM Mn2+ and 1.4 mM Mg2+. Based on its behavior on Sephadex G-100, the molecular weight of E. coli guanylate cyclase is about 30,000. Disc gel electrophoretic analysis indicates that the enzyme consists of a single polypeptide chain. Guanylate cyclase does not form 3':5'-AMP from ATP, and therefore, is distinct from adenylate cyclase.  相似文献   

12.
The Pasteurella multocida adenylate cyclase gene has been cloned and expressed in Escherichia coli. The primary structure of the protein (838 amino acids) deduced from the corresponding nucleotide sequence was compared with that of E. coli. The two enzymes have similar molecular sizes and, based on sequence conservation at the protein level, are likely to be organized in two functional domains: the amino-terminal catalytic domain and the carboxy-terminal regulatory domain. It was shown that P. multocida adenylate cyclase synthesizes increased levels of cyclic AMP in E. coli strains deficient in the catabolite gene activator protein compared with wild-type strains. This increase does not occur in strains deficient in both the catabolite gene activator protein and enzyme III-glucose, indicating that a protein similar to E. coli enzyme III-glucose is involved in the regulation of P. multocida adenylate cyclase. It also indicates that the underlying process leading to enterobacterial adenylate cyclase activation has been conserved through evolution.  相似文献   

13.
A calmodulin-sensitive adenylate cyclase has been purified to apparent homogeneity from bovine cerebral cortex using calmodulin-Sepharose followed by forskolin-Sepharose and wheat germ agglutinin-Sepharose. The final product appeared as one major polypeptide of approximately 135,000 daltons on sodium dodecyl sulfate-polyacrylamide gels. This polypeptide was a major component of the protein purified through calmodulin-Sepharose. The catalytic subunit was stimulated 3-4-fold by calmodulin (CaM) with a turnover number greater than 1000 min-1 and was directly inhibited by adenosine. The catalytic subunit of the enzyme interacted directly with 125I-CaM on a sodium dodecyl sulfate-polyacrylamide gel overlay system, and this interaction was Ca2+ concentration dependent. In addition, the catalytic subunit was shown to directly bind 125I-labeled wheat germ agglutinin using a sodium dodecyl sulfate-polyacrylamide gel overlay technique, and N-acetylglucosamine inhibited binding of the lectin to the catalytic subunit. Calmodulin did not inhibit binding of wheat germ agglutinin to the catalytic subunit, and the binding of calmodulin was unaffected by wheat germ agglutinin. These data illustrate that the catalytic subunit of the calmodulin-sensitive adenylate cyclase is a glycoprotein which interacts directly with calmodulin and that adenosine can inhibit the enzyme without intervening receptors or G coupling proteins. It is concluded that the catalytic subunit of adenylate cyclase is a transmembrane protein with a domain accessible from the outer surface of the cell.  相似文献   

14.
Purification of Epstein-Barr virus DNA polymerase from P3HR-1 cells.   总被引:17,自引:12,他引:5       下载免费PDF全文
The Epstein-Barr virus DNA polymerase was purified from extracts of P3HR-1 cells treated with n-butyrate for induction of the viral cycle. Sequential chromatography on DNA cellulose, phosphocellulose, and blue Sepharose yielded an enzyme preparation purified more than 1,300-fold. The purified enzyme was distinct from cellular enzymes but resembled the viral DNA polymerase in cells infected with herpes simplex virus type 1 or 2. The active enzyme had an apparent molecular weight of 185,000 as estimated by gel filtration on Sephacryl S-300. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a major polypeptide corresponding to a molecular weight of ca. 110,000. This polypeptide correlated with the catalytic function of the purified enzyme, whereas the other, less abundant polypeptides did not. By immunoblotting, the 110,000-molecular-weight polypeptide could be identified as a viral polypeptide. It could not be determined whether the native enzyme was composed of more than one polypeptide.  相似文献   

15.
A trypsin inhibitor (ACTI) was isolated and purified from the seeds of Acacia confusa by gel filtration, and trypsin-Sepharose 4B column affinity chromatography. The molecular weight of ACTI was found to be 21,000 +/- 1,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid composition analysis. ACTI contained four half-cystine and no methionine residues, and was rich in aspartic acid, glutamic acid, glycine, leucine, and lysine residues. The native trypsin inhibitor was composed of two polypeptide chains, and it inhibited trypsin and alpha-chymotrypsin stoichiometrically at the molar ratio of 1:1 and 2:1, respectively. The amino-terminal sequence analysis of the A. confusa trypsin inhibitor A and B chains revealed a more extensive homology with Acacia elata and silk tree trypsin inhibitors, and a less extensive homology with Kunitz soybean trypsin inhibitor.  相似文献   

16.
The Mo-Fe protein of Azotobacter vinelandii nitrogenase was fractionated on 9.5 M urea isoelectric focusing gels and gave three distinct bands (alpha', alpha", beta'). Protein focused on nondenaturing gels gave a single brown band, which when excised and refocused on a denaturing gel gave the three-band pattern. Partial trypsin digestion of the subunits and fractionation of the peptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the alpha' and alpha" polypeptide moieties were the same. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the alpha' and beta' proteins with appropriate molecular weight standards indicated Mr = 61,000 and 57,000, respectively. This is consistent with an overall alpha 2 beta 2 mass of 236,000 daltons.  相似文献   

17.
The gene coding for proline iminopeptidase in Bacillus coagulans was cloned and expressed in Escherichia coli. Nucleotide sequencing revealed an 861-bp open reading frame with an unusual TTG initiation codon, encoding a 287-amino-acid protein. The calculated molecular weight of the product was 32,415. The amino acid sequences of the amino-terminal region and those of some peptide fragments obtained by endoproteinase Asp-N digestion of the purified enzyme completely coincided with those deduced from the nucleotide sequence. The rare TTG initiation codon that normally codes for leucine was translated as a formal initiation codon; a methionine residue was found at the amino terminus of the enzyme. By using a vector bearing the strong tac promoter, an expression level as high as 200-fold that of the first clone was achieved. The replacement of the TTG initiation codon with ATG and a simultaneous reduction of the distance to the tac promoter resulted in a further increase of 2.5-fold. The expressed enzyme was easily purified to homogeneity by hydrophobic chromatography on a Toyopearl HW-65C column and crystallization, with a recovery of activity of 36%. The molecular weight was found to be 33,000 by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration on a Hi-Load 16/60 Superdex 200 fast protein liquid chromatography column. The expressed enzyme showed the same catalytic and physicochemical properties as those of the wild type, specifically cleaving the N-terminal proline from small substrates.  相似文献   

18.
The gene coding for the Escherichia coli enzyme 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase has been cloned and sequenced. This gene, designated folK, codes for a protein of 159 amino acids, including an amino-terminal methionine. The protein was overexpressed in E. coli MC4100 by cloning the gene behind the lacUV5 promoter in a high-copy-number plasmid. The enzyme was purified to homogeneity. Amino-terminal analysis of the purified protein showed that the amino-terminal methionine had been removed. The compositional molecular mass (17,945 Da) was identical to the molecular mass determined by mass spectrometry. The enzyme was observed to have a large number of proline residues and migrated anomalously in sodium dodecyl sulfate-polyacrylamide gels, with an apparent molecular mass of 23,000 Da.  相似文献   

19.
Keratin-agarose plate and keratin-polyacrylamide enzymography methods were developed to demonstrate proteolytic digestion of epidermal keratin. By applying these methods, keratin hydrolase was purified from Tris-buffered saline extract of psoriatic scales by 50% ammonium sulfate precipitation, passage through a lysine-Sepharose column, DEAE-Sepharose, Sephacryl S-200, high-performance cation-exchange chromatography on Mono S, and aprotinin-Sepharose affinity chromatography. The final preparation demonstrated a single protein band at molecular weight 30,000 judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Furthermore, in keratin-polyacrylamide slab gels, the purified enzyme preparation showed a translucent band at molecular weight 30,000, indicating keratin digestion. Keratin hydrolase digested reassembled epidermal keratin as well, whereas it had no effect on guinea pig hair keratin. The enzyme demonstrated a high level of hydrolytic activity on Ile-Pro-Arg-p-nitroanilide and other peptidyl arginine substrates, while it showed a low level of activity on Val-Leu-Lys-p-nitroanilide, and no activity on Arg-Pro-Tyr-p-nitroanilide, Glu-Pro-Val-p-nitroanilide, or Ala-Ala-Ala-p-nitroanilide. The keratin hydrolase was a serine proteinase, inactivated by diisopropylfluorophosphate, phenylmethylsulfonyl fluoride, tosyl-lysyl-chloromethyl ketone, antipain, leupeptin, soybean trypsin inhibitor, aprotinin, and p-aminobenzamidine. The keratinolytic activity was not detected in normal epidermal extract.  相似文献   

20.
Three forms of adenylate cyclase have been detected in Y. pestis: membrane-bound, cytoplasmic and extracellular. Extracellular adenylate cyclase has been purified so as to achieve a homogeneous state, and some of its physicochemical parameters have been investigated. In the process of purification the initial preparation of this enzyme has been subjected to heating at 100 degrees C for 15 minutes, fractionation with ammonium sulfate, and gel filtration on Sephadex G-100. The homogeneity of adenylate cyclase has been confirmed by electrophoresis in 7.5% polyacrylamide gel and precipitation by the plague agglutinating serum. The enzyme has been found to have a molecular weight of 30,000 daltons and to show the optimum activity at pH 7.0-7.2 and at a temperature between 37 and 40 degrees C. Monospecific rabbit serum to the homogeneous preparation of adenylate cyclase has been obtained.  相似文献   

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