RESUMO
Aspergillopepsin II from Aspergillus niger var. macrosporus is a non-pepsin type or pepstatin-insensitive acid proteinase. To identify the catalytic residues of the enzyme, all acidic residues that are conserved in the homologous proteinases of family A4 were replaced with Asn, Gln, or Ala using site-directed mutagenesis. The wild-type and mutant pro-enzymes were heterologously expressed in Escherichia coli and refolded in vitro. The wild-type pro-enzyme was shown to be processed into a two-chain active enzyme under acidic conditions. Most of the recombinant mutant pro-enzymes showed significant activity under acidic conditions because of autocatalytic activation except for the D123N, D123A, E219Q, and E219A mutants. The D123A, E219Q, and E219A mutants showed neither enzymatic activity nor autoprocessing activity under acidic conditions. The circular dichroism spectra of the mutant pro- and mature enzymes were essentially the same as those of the wild-type pro- and mature enzyme, respectively, indicating that the mutant pro-enzymes were correctly folded. In addition, two single and one double mutant pro-enzyme, D123E, E219D, and D123E/E219D, did not show enzymatic activity under acidic conditions. Taken together, Glu-219 and Asp-123 are deduced to be the catalytic residues of aspergillopepsin II.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Ácido Aspártico/análise , Ácido Glutâmico/análise , Sequência de Aminoácidos , Substituição de Aminoácidos , Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/genética , Domínio Catalítico , Dicroísmo Circular , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Dobramento de Proteína , Proteínas Recombinantes/química , Relação Estrutura-AtividadeRESUMO
A cDNA for proctase B from Aspergillus niger var. macrosporus was isolated and sequenced. The deduced amino acid sequence (394 residues) of the preproform of the enzyme was highly homologous (98% identify) with those of aspergillopepsins I from A. awamori and A. saitoi, and moderately homologous (68% identity) with that of A. oryzae. Most of the sequence differences were found in the carboxyl-terminal domain.
Assuntos
Ácido Aspártico Endopeptidases/genética , Aspergillus niger/enzimologia , Clonagem Molecular , Sequência de Aminoácidos , Ácido Aspártico Endopeptidases/química , Aspergillus niger/genética , Sequência de Bases , DNA Complementar/química , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Relação Estrutura-AtividadeAssuntos
Ácido Aspártico Endopeptidases/química , Aspergillus niger/enzimologia , Precursores Enzimáticos/metabolismo , Serina Endopeptidases/biossíntese , Sequência de Aminoácidos , Animais , Aspergillus niger/genética , Bovinos , Clonagem Molecular , DNA Complementar , Ativação Enzimática , Escherichia coli , Expressão Gênica , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação Puntual , Desnaturação Proteica , Dobramento de Proteína , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/química , Serina Endopeptidases/genéticaAssuntos
Ácido Aspártico Endopeptidases/biossíntese , Ácido Aspártico Endopeptidases/química , Aspergillus niger/enzimologia , Sequência de Aminoácidos , Ácido Aspártico Endopeptidases/metabolismo , Clonagem Molecular/métodos , Precursores Enzimáticos/química , Precursores Enzimáticos/metabolismo , Escherichia coli , Expressão Gênica , Concentração de Íons de Hidrogênio , Cinética , Dados de Sequência Molecular , Pepsina A/metabolismo , Sinais Direcionadores de Proteínas/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , TermodinâmicaRESUMO
1. The Brown Norway (B/N) Katholiek rat is a mutant strain of plasma kininogen deficiency. The plasma of B/N-Katholiek rats was shown to contain only 3-5% of high-molecular-weight and low-molecular-weight kininogens (HK and LK) of the normal level by specific RIA, and 30% of prekallikrein was detected by amidase activity. However, HK antigen in the liver microsomal fraction of B/N-Katholiek rats was about 60% of that of normal rats. 2. In this paper we compare and discuss synthesis and secretion of HK and LK by primary cultures of livers of deficient and normal rats. The deficient hepatocytes could synthesize HK and LK in the same way as normal cells but could not secrete mature forms of HK and LK in the medium. Examination of the subcellular localization of the mutant HK in the hepatocytes showed that a larger amount of mutant HK antigen, compared to normal rats, was found in the 10,000 g fraction, which is rich in lysosomes, suggesting that the mutant HK may be transported to the lysosomes. 3. We also analyzed sequence of the HK cDNA of B/N-Katholiek and B/N-Kitasato rats and found a point mutation of G to A at nucleotide 487, which locates at the heavy chain region of HK and LK. 4. We constructed five expression plasmids to transfect COS-1 cells to examine HK secretion. COS-1 cells transfected with the plasmids containing the G to A transition could not secrete and retained HK, while those cells transfected with the plasmids containing normal G released HK into the medium. 5. These results indicate that a point mutation G to A at nucleotide 487, resulting in an amino acid transition from alanine (163) to threonine, is responsible for the defective secretion of HK and LK by the liver of B/N-Katholiek rats. We also discuss other cases of secretion defect of plasma proteins reported in the literature.
Assuntos
Cininogênios/deficiência , Animais , DNA Complementar , Cininogênios/biossíntese , Cininogênios/genética , Cininogênios/metabolismo , Fígado/citologia , Fígado/metabolismo , Lisossomos/metabolismo , Peso Molecular , Plasmídeos , Mutação Puntual , Ratos , Ratos Endogâmicos BN , TransfecçãoRESUMO
To clarify the mechanism of the secretion defect of high molecular weight kininogen (HK) and low molecular weight kininogen (LK) by the liver of Brown Norway (B/N) Katholiek rats causing plasma kininogen deficiency, we cloned cDNAs for HK from cDNA libraries of the livers of B/N Katholiek and B/N Kitasato rats. A point mutation of G to A at nucleotide 487 was found in the cDNA of B/N Katholiek rats by sequence analysis of the cDNAs (including the entire HK-coding region) obtained from both strains. Both B/N Katholiek and B/N Kitasato rat cDNA fragments were introduced into a eukaryotic vector, pRc/CMV, to construct their respective expression plasmid, which was used to transfect COS-1 cells. At 24 h of incubation, the culture medium of COS-1 cells transfected with the B/N Katholiek rat cDNA contained only 10% of the HK antigen that was found in COS-1 cells transfected with the B/N Kitasato rat cDNA. More HK antigen was retained in the former cells. Moreover, cells transfected with B/N Katholiek rat cDNA, in which the A at nucleotide 487 was artificially replaced by G, secreted a significant amount of HK into the medium. These results suggest that a point mutation of G to A at nucleotide 487, which causes a substitution of Ala163 to Thr in the heavy chain of HK and LK, is responsible for the defective secretion of HK and LK by the liver of B/N Katholiek rats.
Assuntos
Cininogênios/metabolismo , Fígado/enzimologia , Mutação Puntual , Alanina/metabolismo , Animais , Sequência de Bases , Northern Blotting , Linhagem Celular , Clonagem Molecular , DNA , Sondas de DNA , Cininogênios/genética , Fígado/metabolismo , Dados de Sequência Molecular , Plasmídeos , Ratos , Ratos Endogâmicos BN , Treonina/metabolismo , TransfecçãoRESUMO
Proteinase A obtained from the culture medium of Aspergillus niger var. macrosporus is a unique acid endopeptidase that is insensitive (or less sensitive) to specific inhibitors of ordinary acid or aspartic proteinases, such as pepstatin, diazoacetyl-DL-norleucine methyl ester, and 1,2-epoxy-3-(p-nitrophenoxy)-propane. In the preceding paper (Takahashi, K., Inoue, H., Sakai, K., Kohama, T., Kitahara, S., Takishima, K., Tanji, M., Athauda, S. B. P., Takahashi, T., Akanuma, H., Mamiya, G., Yamasaki, M. J. Biol. Chem. 266, 19480-19483), we reported the complete primary structure of the mature enzyme determined at the protein level. The enzyme has a unique two-chain structure with a 39-residue light (L) chain and a 173-residue heavy (H) chain linked noncovalently. As an extension of this study, we isolated genomic and cDNA clones encoding this proteinase and determined their nucleotide sequences. To isolate a genomic clone, the genomic DNA was selectively amplified by polymerase chain reaction using mixed oligonucleotide primers designed from the amino acid sequence of the H chain, and a specific probe thus generated was used for screening a lambda gt10 genomic library. A cDNA for the enzyme was also selectively amplified by polymerase chain reaction using primers synthesized based on the sequence of the genomic DNA. Sequencing of the cloned genomic DNA and cDNA revealed the nucleotide sequence of the structural gene for the enzyme of 846 base pairs without introns. It encodes the precursor form of proteinase A, including an NH2-terminal preprosequence of 59 residues, the L chain of 39 residues, an intervening sequence of 11 residues, and the H chain of 173 residues linked in that order. Thus, proteinase A is thought to be synthesized as a single peptide chain preproenzyme of 282 residues, which is processed to generate the mature two-chain form.
Assuntos
Ácido Aspártico Endopeptidases/genética , Aspergillus niger/enzimologia , Precursores Enzimáticos/genética , Sequência de Aminoácidos , Sequência de Bases , Southern Blotting , Clonagem Molecular , DNA/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Homologia de Sequência do Ácido Nucleico , TATA BoxAssuntos
Ácido Aspártico Endopeptidases/antagonistas & inibidores , Aspergillus niger/enzimologia , Pepstatinas/farmacologia , Sequência de Aminoácidos , Ácido Aspártico Endopeptidases/química , Precursores Enzimáticos/química , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Estrutura Molecular , Conformação Proteica , Relação Estrutura-AtividadeAssuntos
Adenocarcinoma/patologia , Antibióticos Antineoplásicos/farmacologia , Linhagem Celular Transformada/efeitos dos fármacos , Oncogenes , Neoplasias Pancreáticas/patologia , Células Tumorais Cultivadas/efeitos dos fármacos , Adenocarcinoma/genética , Alanina/análogos & derivados , Alanina/farmacologia , Animais , Divisão Celular/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/genéticaRESUMO
Azatyrosine [L-beta-(5-hydroxy-2-pyridyl)-alanine], an antibiotic isolated from Streptomyces chibanensis, inhibited the growth of NIH 3T3 cells transformed by the activated human c-Ha-ras gene but did not significantly inhibit the growth of normal NIH 3T3 cells. Surprisingly, upon treatment with azatyrosine most of the transformed cells apparently became normal. These apparently normal cells, named revertant cells, grew in the presence of azatyrosine and stopped growing when they reached confluency, and their normal phenotype persisted during prolonged culture in the absence of azatyrosine. The revertant cells did not grow in soft agar and scarcely proliferated in nude mice. The human c-Ha-ras gene present in transformed NIH 3T3 cells was still present in the revertant cells and was expressed to the same extent as in the original transformed cells, producing the same amount of activated p21. Treatment with azatyrosine caused similar conversion of NIH 3T3 cells transformed by activated c-Ki-ras, N-ras, or c-raf to apparently normal cells, but NIH 3T3 cells transformed by hst or ret were not exclusively converted by azatyrosine. Human pancreatic adenocarcinoma cells, which are known to contain an amplified activated c-Ki-ras gene and an amplified c-myc gene, were also converted to flat and giant revertant cells by treatment with azatyrosine.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Transformação Celular Neoplásica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes ras/efeitos dos fármacos , Proto-Oncogenes/efeitos dos fármacos , Alanina/farmacologia , Animais , Southern Blotting , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Humanos , Cinética , Leucemia Promielocítica Aguda , Camundongos , Proteína Oncogênica p21(ras)/análiseRESUMO
A mixed probe consisting of two synthetic deoxynucleotides (52 and 54 mers referred to as 50-mer) with arbitrarily chosen C or G for the third letters was prepared based on the amino acid sequences No. 31-48 and No. 72-90 of macromomycin (MCM) apoprotein and successfully used to clone the MCM apoprotein gene. Digestion with Sph I of total DNA of MCM-producing Streptomyces macromomyceticus M480-M1 yielded a 2.6-kb fragment that hybridized strongly to the probes. The hybridized probe was stable to washing with 3 x SSC at 75 degrees C. Radioactivity derived from the hybridized probe was comparable to that expected theoretically from hybridization between the probe and the true target sequence. The 2.6-kb fragment was cloned into Escherichia coli RR1 with pBR322 and subsequently subcloned into Streptomyces lividans TK21 with pIJ702. Nucleotide sequence analysis of the cloned fragment verified the existence of the sequence corresponding to the amino acid sequence of MCM apoprotein and about 90% homologies with the probes. Thus, the use of relatively long deoxynucleotide probes with arbitrarily chosen C or G for the third letters will be advantageous in cloning Streptomyces protein genes where more than 90% of the third letters have been known to be C or G. In addition, theoretical diagnosis of hybridization should be a great help to distinguish true positives from false ones.
Assuntos
Apoproteínas/genética , Proteínas de Bactérias , Sondas de Ácido Nucleico , Streptomyces/genética , Sequência de Aminoácidos , Apoproteínas/biossíntese , Sequência de Bases , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação da Expressão Gênica , Vetores Genéticos , Peptídeos e Proteínas de Sinalização Intercelular , Dados de Sequência Molecular , Peptídeos , Plasmídeos , Homologia de Sequência do Ácido Nucleico , Streptomyces/metabolismoRESUMO
We have isolated and sequenced a gene (amy) coding for alpha-amylase (EC 3.2.1.1.) from the Streptomyces hygroscopicus genome (H. Hidaka, Y. Koaze, K. Yoshida, T. Niwa, T. Shomura, and T. Niida, Die Stärke 26:413-416, 1974). Amylase was purified to obtain amino acid sequence information which was used to synthesize oligonucleotide probes. amy-containing Escherichia coli cosmids identified by hybridization did not express amylase activity. Subcloning experiments indicated that amy could be expressed from the lac promoter in E. coli or from its own promoter in S. lividans. The amy nucleotide sequence indicated that it coded for a protein of 52 kilodaltons (478 amino acids). Secreted alpha-amylase contained amino- and carboxy-terminal as well as internal amino acid sequences which were consistent with the nucleotide sequence. The 30-residue leader sequence showed similarities to those found in other procaryotes. The DNA sequence 5' to the amy structural gene contained a sequence complementary to the 3'-terminal sequence of 16S rRNA of S. lividans (M. J. Bibb and S. N. Cohen, Mol. Gen. Genet. 187:265-277, 1982). The transcriptional start points of amy were determined by mung bean nuclease mapping, but the promoter of amy was not similar to the consensus sequence found in other procaryotes.
Assuntos
Clonagem Molecular , Genes Bacterianos , Genes , Streptomyces/genética , alfa-Amilases/genética , Sequência de Aminoácidos , Sequência de Bases , Cosmídeos , Enzimas de Restrição do DNA , Escherichia coli/genética , Hibridização de Ácido Nucleico , Streptomyces/enzimologiaRESUMO
mRNA for bacteriorhodopsin from Halobacterium halobium has been partially purified. By using this mRNA as template in the presence of reverse transcriptase RNA-dependent DNA nucleotidyltransferase and a 5'-[32P] synthetic oligodeoxyribonucleotide corresponding to amino acids 9-12 of bacteriorhodopsin as primer, we have isolated the major 5'-[32P]cDNA product, approximately 80 nucleotides long, and determined its sequence. Based on the cDNA sequence, the 5'-proximal sequence of bacteriorhodopsin mRNA is G-C-A-U-G-U-U-G-G-A-G-U-U-A-U-U-G-C-C-A-A-C-A-G-C-A-G-U-G-G-A-G-G-G-G-G-U-A-U-C -G-C-A-G-G-C-C-C-A-G-A-U-C-A-C-C-G-G-A-C-G-U-C-C-G. This includes the expected sequence for amino acids 1-8 and shows that bacteriorhodopsin is synthesized as a precursor that is at least 13 amino acids longer (Met-Leu-Glu-Leu-Leu-Pro-Thr-Ala-Val-Glu-Gly-Val-Ser) at the NH2 terminus. Agarose/urea gel electrophoresis of the partially purified mRNA showed several bands; of these, a major one hybridized with 5'-[32P]cDNA. These results suggest that the bacteriorhodopsin mRNA in the partially purified preparation is homogeneous in size and that it constitutes a substantial portion of the RNA preparation subjected to electrophoresis.
Assuntos
Bacteriorodopsinas/genética , Carotenoides/genética , Precursores de Proteínas/genética , RNA Mensageiro/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Halobacterium/genéticaRESUMO
The syntheses of N1- and N2-isopropylformycin (10, 11), formycin 3',5'-cyclic and 2',3'-cyclic phosphate (3,7) and their N-methyl and N-isopropyl derivatives (13, 15, 19, 23) are described. It was observed that substitution at N1 or N2 with a bulky alkyl group or cyclic phosphorylation of the ribose moiety made formycin resistant to adenosine deaminase.