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1.
Protein Sci ; 2(9): 1441-51, 1993 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-8401229

RESUMO

Several amino groups of recombinant human erythropoietin are selectively cross-linked by specific cross-linkers including disuccinimidyl suberate or dithiobis(succinimidyl propionate). Intramolecular cross-linkings are obtained without significant change of the protein conformation using appropriate concentrations (0.2 mM) of the cross-linkers, which possess an 11-12-A length of a spacer between two reacting groups. Intramolecularly cross-linked peptides obtained suggest that several amino groups in erythropoietin (EPO) are positioned at a distance of near 12 A in the solution state. These interfacing amino groups include Lys 20-Lys 154, Lys 45-Lys 140, Lys 52-Lys 154, Lys 52-Lys 140, and Ala 1-Lys 116. A comparison of the cross-linking results between nonglycosylated EPO and glycosylated EPO suggests that both proteins retain high similarity regarding protein conformation. These results fit a structural model similar to that of human growth hormone, in which four alpha-helical bundles and a long stretch of beta-sheet structure are involved in the active protein.


Assuntos
Reagentes de Ligações Cruzadas , Eritropoetina/química , Proteínas Recombinantes/química , Sequência de Aminoácidos , Animais , Células CHO , Dicroísmo Circular , Cricetinae , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Glicosilação , Humanos , Substâncias Macromoleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Mapeamento de Peptídeos , Conformação Proteica , Estrutura Secundária de Proteína
2.
Protein Sci ; 2(10): 1664-74, 1993 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-8251941

RESUMO

Recombinant human brain-derived neurotrophic factor (BDNF) is now under extensive investigation because of its potential clinical applications. Radioactively labeled proteins are usually required to study receptor binding and pharmacokinetic properties of proteins. This study was undertaken to see if iodination affects the biological and conformational properties of a recombinant BDNF. BDNF was iodinated using a stoichiometric amount of nonradioactive cold NaI to minimize multiple iodinations. Of the four tyrosines present in BDNF--Tyr-52, Tyr-54, Tyr-63, and Tyr-86--only Tyr-63 and Tyr-86 were iodinated under the experimental conditions used. Iodination of Tyr-63 resulted in modification without alteration of the biological activity, whereas iodination of Tyr-86 resulted in a molecule with highly compromised biological activity. Similar inactivation was observed if both Tyr-63 and Tyr-86 were iodinated. These modified proteins exhibited conformation and dimerization apparently identical to those of the native protein, as demonstrated by analytical ultracentrifugation, gel filtration, light scattering, and circular dichroism. From these results, we concluded that Tyr-52 and Tyr-54 are not accessible to the reagent and are probably buried in the hydrophobic core, whereas Tyr-63 and Tyr-86 are exposed on the surface of the molecule; of the two exposed residues, only Tyr-86 contributes to the biological activity.


Assuntos
Iodo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Fator Neurotrófico Derivado do Encéfalo , Galinhas , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Gânglios/efeitos dos fármacos , Gânglios/ultraestrutura , Humanos , Substâncias Macromoleculares , Espectrometria de Massas , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/farmacologia , Pepsina A/metabolismo , Mapeamento de Peptídeos , Conformação Proteica , Proteínas Recombinantes/metabolismo , Iodeto de Sódio/metabolismo , Relação Estrutura-Atividade , Tirosina/metabolismo , Ultracentrifugação
5.
Anal Biochem ; 173(2): 296-306, 1988 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-3189811

RESUMO

A novel apparatus for performing manual gas-phase Edman chemistry on protein and peptide samples is described. Edman chemistry is performed in 6 to 10 Teflon continuous flow reactors (CFR), previously described by J.E. Shively et al. (1987) Anal. Biochem. 163, 517-529). The CFRs are packed with 10-15 mg of Polybrene-coated spherical silica (Porasil B, Waters Associates). The gas-phase coupling reagent and cleavage reagent are 5% aqueous triethylamine and anhydrous trifluoroacetic acid, respectively, delivered by a stream of argon gas. The delivery of the gas-phase reagents is manually controlled with Hamilton 3-way valves and 2-way valves, and that of the solvents, ethyl acetate and butyl chloride, by syringe pipetting. The average cycle time is 15-20 min for 6 to 10 samples run simultaneously. Conversion of the anilinothiazolinone to phenylthiohydantoin (PTH) amino acid derivatives is accomplished manually with 25% aqueous trifluoroacetic acid. The PTH amino acids are analyzed by reversed-phase HPLC using an autosampler for handling multiple samples. Excellent results were obtained in the 100-200 pmol range. Protein samples can be sequenced from 15-20 cycles, and peptide samples usually to the COOH terminus. Initial yields ranged from 30 to 60% and repetitive yields ranged from 90 to 96%. The sample washout and size of background peaks are significantly reduced, compared to older methods of manual sequence analysis. The yields and background signal to noise are comparable to automated gas-phase Edman chemistry. The improved manual Edman described represents a low cost alternative to automated sequence analysis, and has the advantage being able to process multiple samples simultaneously.


Assuntos
Peptídeos/análise , Proteínas/análise , Sequência de Aminoácidos , Equipamentos e Provisões , Indicadores e Reagentes , Solventes
6.
Eur J Biochem ; 169(3): 449-55, 1987 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-3691502

RESUMO

Using specific antibodies against adrenodoxin reductase (AR), we screened lambda gt11 cDNA expression libraries constructed from bovine adrenal cortex mRNA, and isolated several putative clones coding for this enzyme. Concurrently we determined the amino acid sequences of fragments from it. A deoxyinosine-containing oligonucleotide probe, generated for one of the sequences, reacted specifically with one of the cloned cDNAs of about 1600 base pairs. The codon sequence of this cDNA matched the peptide sequences, further confirming its identity as a copy of AR mRNA. RNA blot analysis indicates that in the adrenal cortex and corpus luteum there is only one major mRNA (approximately 2000 bp) for AR. The levels of this mRNA are at least 40-fold lower in the liver and kidney which are also known to contain in homologue of AR. As compared to adrenodoxin and cytochrome P-450scc mRNAs, AR mRNA levels in the adrenal cortex appear to be about 10-fold lower. Southern blot analysis of bovine and human genomic DNAs reveals that in both of these species there is only one gene for AR. These results indicate that only a single reductase serves the different mitochondrial P-450 systems in steroidogenic tissues.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , DNA/isolamento & purificação , Ferredoxina-NADP Redutase/genética , Mitocôndrias/enzimologia , NADH NADPH Oxirredutases/genética , Córtex Suprarrenal/enzimologia , Adrenodoxina/genética , Animais , Bovinos , Regulação da Expressão Gênica , Humanos , Isoenzimas/genética , Hibridização de Ácido Nucleico
7.
Biochem Biophys Res Commun ; 127(1): 94-8, 1985 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-3919729

RESUMO

We have determined the complete amino acid sequence of a 20K Da COOH-terminal fragment of porcine NADPH-cytochrome P-450 reductase. The 20K Da fragment is probably produced by a proteolytic cleavage of the intact protein in porcine liver microsomes, and since the cleavage does not affect enzymatic activity, the fragment has been studied as a distinct domain. The sequence comprises 175 amino acids including three cysteine residues, one of which has been previously identified as protected by NADPH from S-carboxymethylation. The NADPH-protected cysteine lies in a stretch of 12 residues with partial homology to glutathione reductase, and is adjacent to a hydrophobic region containing a glycine-rich stretch homologous to other FAD-containing proteins. The predicted secondary structure over this entire region is beta-sheet/beta-turn/beta-sheet/alpha-helix/beta-sheet/beta-turn/alpha-h elix corresponding to hydrophobic residues 21-28/glycine-rich residues 29-33/residues 34-38/residues 39-54/residues 56-61/NADPH-protected cysteine residues 62-78/residues 71-82. It is possible that the 20K Da domain provided a significant portion of the sequence responsible for binding FAD and NADPH in the intact enzyme. This data provides a basis for further active site studies.


Assuntos
Microssomos Hepáticos/enzimologia , NADPH-Ferri-Hemoproteína Redutase/análise , Fragmentos de Peptídeos/análise , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Relação Estrutura-Atividade , Suínos
8.
Biochemistry ; 32(9): 2431-7, 1993 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-8443183

RESUMO

Interchain cystines of PDGF-BB dimer were characterized by Edman reaction and by SDS-PAGE analysis on the protein which was chemically cleaved at Trp-40. It was found that Cys-43 has a key role in dimer formation, asymmetrically cross-linked to a cysteine residue of another identical subunit. The remaining cystines participate in the intramolecular disulfide linkages. Pepsin digestion of PDGF-BB dimer generated several small peptides and one ubiquitous Cys-containing peptide. Sequence analyses of several Cys-containing peptides indicated the existence of three intramolecular disulfide linkages including Cys-16--Cys-60, Cys-49--Cys-97, and Cys-53--Cys-99. Two interchain disulfide bonds of Cys-43--Cys-52 between two subunits were deduced from the partial reduction and alkylation of PDGF-BB. This study provides chemically determined disulfide linkages of PDGF-BB.


Assuntos
Dissulfetos/química , Fator de Crescimento Derivado de Plaquetas/química , Proteínas Recombinantes/química , Acetilação , Alquilação , Sequência de Aminoácidos , Becaplermina , Cisteína/análise , Humanos , Dados de Sequência Molecular , Oxirredução , Fragmentos de Peptídeos/química , Conformação Proteica , Proteínas Proto-Oncogênicas c-sis
9.
J Biol Chem ; 252(20): 7081-8, 1977 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-561781

RESUMO

A total of 25 tryptic peptides was isolated from the S-beta-carboxymethyl derivative of Clostridium pasteurianum iron protein (N2). In order to obtain the various peptides in pure state, a combination of gel permeation, cation and anion exchange column chromatographic methods, as well as various ascending paper chromatographic methods were adopted. Sequence studies of the tryptic peptides were carried out mainly by a modified manual Edman degradation procedure and also by automated analysis, carboxypeptidase digestion, and by hydrazinolysis. Thus, 242 residues (88.6%) out of a total of 273 amino acid residues were sequenced in the present study. The sum of the amino acid residues in the tryptic peptides isolated from iron protein (N2) accounted for the 273 amino acid residues present in the iron protein.


Assuntos
Clostridium/enzimologia , Ferredoxinas , Nitrogenase , Sequência de Aminoácidos , Fragmentos de Peptídeos/análise , Tripsina
10.
J Biol Chem ; 252(20): 7089-92, 1977 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-561782

RESUMO

A total of 10 cyanogen bromide peptides were isolated from the S-beta-carboxymethyl iron protein of nitrogenase. Purification of these peptides was performed mainly by gel filtration on Sephadex G-50; by ascending paper chromatography using the solvent system of pyridine, isoamyl alcohol, 0.1 M ammonium hydroxide; and also, in some cases, with additional steps such as anion exchange column chromatography on Dowex 1-X2 or ascending paper chromatography in an acidic solvent system or by pyridine precipitation of the cyanogen bromide fragment. Sequenator analyses of three large cyanogen bromide peptides (53 to 72 residues) provided tryptic peptide overlap data for the inner portion of the protein. The cyanogen bromide peptides accounted for all of the 273 amino acid residues which were present in the tryptic peptides isolated from carboxymethyl-iron protein (Tanaka, M., Haniu, M., Yasunobu, K. T., and Mortenson, L. E. (1977) J. Biol. Chem. 252, 7081-7088).


Assuntos
Clostridium/enzimologia , Ferredoxinas , Nitrogenase , Sequência de Aminoácidos , Brometo de Cianogênio , Fragmentos de Peptídeos/análise
11.
Biochem Biophys Res Commun ; 116(1): 30-8, 1983 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-6639664

RESUMO

Reactions of the Pseudomonas putida cytochrome P-450-substrate complex or enzyme alone with 14C-labeled iodoacetic acid have been investigated at pH 7.0. After subsequent conversion of all of the cysteine residues to S-beta-carboxymethylcysteinyl residues, tryptic peptides of the derivative were separated by either high performance liquid chromatography or two dimensional electrophoresis, and their amino acid compositions and partial sequences were determined. All but cysteine residue-134 reacted to some extent. This result implicated residue-134 as the thiol group which is the axial heme ligand since the heme was intact in all of the derivatives made. Reaction of the enzyme-substrate complex with "cold" iodoacetic acid followed by substrate removal and reaction with 14C-labeled iodoacetic acid resulted in radiolabeling of mainly cysteine-240. This suggested cysteine-240 to be an active site cysteine residue.


Assuntos
Sistema Enzimático do Citocromo P-450 , Sítios de Ligação , Cisteína , Heme/metabolismo , Cinética , Pseudomonas/enzimologia , Relação Estrutura-Atividade
12.
J Biol Chem ; 259(22): 13703-11, 1984 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-6438080

RESUMO

Detergent-solubilized NADPH-cytochrome P-450 reductase was purified from porcine hepatic microsomes and compared to the rabbit enzyme isolated under identical conditions. The porcine enzyme had an equivalent specific activity toward cytochrome c compared to the rabbit enzyme. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the porcine enzyme exhibited a major band at Mr = 80,000 and two additional bands at Mr = 20,000 and 60,000. The 20-kDa fragment was shown to be the COOH-terminal portion of the protein which contains a hydrophobic sequence of 28 residues homologous to the pyrophosphate-binding portion of the FAD-binding protein p-hydroxybenzoate hydroxylase. The 60-kDa fragment corresponded to the NH2-terminal portion of the protein since this peptide and the intact protein have blocked NH2 terminal. The trypsin-solubilized porcine enzyme has an NH2-terminal sequence which is homologous to the equivalent trypsin-solubilized enzymes from rat and rabbit (80% sequence homology). Eight cysteine-containing peptides were isolated from a tryptic digest of the S-carboxymethylated pig enzyme. Significant sequence homology was not found between these peptides and other flavoproteins, except for one peptide (Glu-Val-Gly-Glu-Thr-Leu-Leu-Tyr-Tyr-Gly-Cys-Arg) which exhibited partial homology with the known NADPH-binding site of glutathione reductase. When the NADPH-protected enzyme was first S-alkylated with unlabeled iodoacetate, NADPH depleted, and further alkylated with 14C-labeled iodoacetate, the above radiolabeled peptide was isolated from a tryptic digest. The equivalent peptide was also isolated by a similar procedure from rabbit liver cytochrome P-450 reductase.


Assuntos
Microssomos Hepáticos/enzimologia , NADPH-Ferri-Hemoproteína Redutase/análise , Fragmentos de Peptídeos/análise , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Cromatografia Líquida de Alta Pressão , Cisteína/análise , Eletroforese em Gel de Poliacrilamida , Glutationa Redutase/metabolismo , Peso Molecular , NADP/metabolismo , Coelhos , Ratos , Suínos , Tripsina/metabolismo
13.
Biochemistry ; 25(24): 7906-11, 1986 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-3099837

RESUMO

The complete amino acid sequence of porcine hepatic microsomal NADPH-cytochrome P-450 reductase has been determined by microsequence analysis on several sets of proteolytic fragments. Sequence studies were performed initially on a 20-kilodalton (kDa) fragment and then on 80-kDa fragment. The amino-terminal end of the mature protein was blocked with an acetyl group, followed by 676 amino acid residues. It has been revealed that the COOH-terminal 20-kDa fragment has been derived from original enzyme by cleavage at the Asn-Gly (residues 502-503) linkage by an unknown mechanism. An NADPH-protected cysteine residue is located at residue 565, near a region exhibiting high sequence homology with ferredoxin-NADP+ reductase. The FMN and FAD binding regions are possibly located in the amino-terminal region and the middle part of the protein molecule, respectively, as suggested by Porter and Kasper [Porter, T. D., & Kasper, C. B. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 973-977]. When this sequence is compared with that of rat enzyme, 60 amino acid residues are substituted, probably due to species differences. However, total sequence homology between these enzymes is 90%. Hydropathy plot analysis reveals that two regions from residues 27-43 and from residues 523-544 exhibit a high degree of hydrophobicity, suggesting membrane binding or interaction with cytochrome P-450.


Assuntos
Microssomos Hepáticos/enzimologia , NADPH-Ferri-Hemoproteína Redutase , Sequência de Aminoácidos , Animais , NADPH-Ferri-Hemoproteína Redutase/isolamento & purificação , Fragmentos de Peptídeos/análise , Conformação Proteica , Suínos
14.
Biochemistry ; 28(21): 8639-45, 1989 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-2513880

RESUMO

The complete amino acid sequence of human liver NADPH-cytochrome P-450 reductase has been determined by microsequence analysis and mass spectrometry. The total sequence consists of 676 amino acids initiated by an amino-terminal acetyl group. There is no evidence for posttranslational modifications, including Asn-linked glycosylation. The human enzyme exhibits sequence homology in the range of 92-95% with other mammalian enzymes. Sequence differences were mainly confined to several hydrophilic regions in the NH2-terminal and COOH-terminal domains. Since the human enzyme is immunochemically distinct from the rabbit enzyme despite similar enzymatic properties, it is likely that these variable hydrophilic regions are potential antigenic determinants. The NADPH-depleted enzyme is inactivated by either fluorescein isothiocyanate, a lysine-specific reagent, or 5-(iodoacetamido)fluorescein, a cysteine-specific reagent. In both cases, protection by NADP(H) prevents enzyme inactivation by the reagents. Isolation of fluorescent peptide from 5-(iodoacetamido)fluorescein-inactivated enzyme identified Cys 565 as the specifically NADPH-protected residue.


Assuntos
Fígado/enzimologia , NADPH-Ferri-Hemoproteína Redutase , Adulto , Sequência de Aminoácidos , Animais , Sítios de Ligação , Fluoresceína-5-Isotiocianato , Fluoresceínas/farmacologia , Humanos , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Dados de Sequência Molecular , NADPH-Ferri-Hemoproteína Redutase/antagonistas & inibidores , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Coelhos , Ratos , Homologia de Sequência do Ácido Nucleico , Suínos , Tiocianatos/farmacologia
15.
Arch Biochem Biophys ; 254(1): 380-4, 1987 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-3495238

RESUMO

Adrenal microsomal 21-hydroxylase is essential for biosynthesis or metabolism of various steroid hormones. The complete amino acid sequence of this cytochrome P-450 from pig adrenal was determined by sequence analysis on several sets of proteolytic fragments. The mature protein consists of 492 amino acid residues, corresponding to a molecular weight of 55,484. Seven out of nine total cysteine residues are localized in the amino terminal half of the molecule. The carboxyl terminal half contains only two cysteines, one of which is located at the highly conserved heme-binding region proposed in all cytochromes P-450. A structural comparison between 21-hydroxylase and 17 alpha-hydroxylase reveals that there is a preponderance of sequence homology at the carboxyl terminal region. These studies indicate that a single gene product is expressed for steroid 21-hydroxylase in porcine adrenal glands.


Assuntos
Glândulas Suprarrenais/enzimologia , Esteroide 21-Hidroxilase , Esteroide Hidroxilases , Sequência de Aminoácidos , Animais , Sítios de Ligação , Microssomos/enzimologia , Esteroide 17-alfa-Hidroxilase , Esteroide 21-Hidroxilase/genética , Esteroide 21-Hidroxilase/metabolismo , Esteroide Hidroxilases/genética , Esteroide Hidroxilases/metabolismo , Suínos
16.
Biochemistry ; 26(2): 657-62, 1987 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-3493807

RESUMO

The affinity alkylating progesterone analogue 17-(bromoacetoxy)progesterone has been used to label the active site of a microsomal cytochrome P-450 enzyme from neonatal pig testis. The enzyme causes removal of the C20 and C21 side chains from the substrates progesterone and pregnenolone by catalyzing both 17-hydroxylase and C17,20-lyase reactions, which produce the corresponding C19 steroidal precursors of testosterone. The progesterone analogue causes simultaneous inactivation of the two catalytic activities of the enzyme by a first-order kinetic process that obeys saturation kinetics. Progesterone and 17-hydroxyprogesterone each protect the enzyme against inactivation. The progesterone and analogue is a competitive inhibitor of the enzyme with Ki values of 8.4 microM and 7.8 microM for progesterone and 17-hydroxyprogesterone, respectively. The enzyme inactivation and kinetic data are consistent with a theory proposing that the analogue and the two substrates compete for the same active site. The radioactive analogue 17-[( 14C]bromoacetoxy)progesterone causes inactivation of the enzyme with incorporation of 1.5-2.2 mol of the analogue per mole of inactivated enzyme. When this experiment is carried out in the presence of a substrate, then 0.9-1.2 mol of radioactive analogue is incorporated per mole of inactivated enzyme. The data suggest that the analogue can bind to two different sites, one of which is related to the catalytic site. Radiolabeled enzyme samples, from reactions of the 14C-labeled analogue with the enzyme alone or with enzyme in the presence of a substrate, were subjected to amino acid analysis and also to tryptic digestion and peptide mapping.(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
Marcadores de Afinidade/metabolismo , Cisteína , Sistema Enzimático do Citocromo P-450/metabolismo , Hidroxiprogesteronas/metabolismo , Esteroide 21-Hidroxilase/metabolismo , Esteroide Hidroxilases/metabolismo , Testículo/metabolismo , Alquilação , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Sítios de Ligação , Cinética , Masculino , Microssomos/metabolismo , Suínos
17.
Arch Biochem Biophys ; 244(1): 323-37, 1986 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-3947063

RESUMO

The complete amino acid sequence of rat liver microsomal cytochrome P-450d (induced by isosafrole) was deduced by microsequence analysis of the tryptic peptides after separation by reverse-phase HPLC and alignment by comparison to the cDNA sequence reported by K. Kawajiri, O. Gotoh, K. Sogawa, Y. Tagashira, M. Muramatsu, and Y. Fujii-Kuriyama (1984, Proc. Natl. Acad. Sci. USA 81, 1649-1653). Results from the two approaches are in complete agreement with the exception of two residues, Lys-30 (Arg in cDNA) and Phe-261 (Ser in cDNA). As previously reported by us (L. H. Botelho, D. E. Ryan, P.-M. Yuan, R. Kutney, J. E. Shively, and W. Levin (1982, Biochemistry 21, 1152-1155) the NH2-terminal sequence of the mature protein lacks the NH2-terminal Met residue. Comparison of the rat cytochrome P-450d sequence with the mouse cytochrome P3-450 cDNA sequence reported by S. Kimura, F.J. Gonzalez, and D.W. Nebert (1984, Nucl. Acids Res. 12, 2917-2928) reveals a high sequence homology with a total of 32 amino acid differences including six conferring charge changes. Prediction of the secondary structure of cytochrome P-450d yields a maximum of 17 helices, two of which may be poly(Pro)-like helices adjacent to potential membrane-spanning alpha-helices. Four of the alpha-helices are sufficiently hydrophobic to traverse the endoplasmic reticulum. The remaining helices are largely amphiphilic. Analysis of the helices in reference to predicted membrane topology suggests that cytochrome P-450d either has one large and one small globular domain separated by a transmembrane domain and anchored by NH2-terminal and COOH-terminal transmembrane domains, or has one large globular domain anchored at both ends by transmembrane domains.


Assuntos
Sistema Enzimático do Citocromo P-450/isolamento & purificação , Microssomos Hepáticos/enzimologia , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Dicroísmo Circular , Clonagem Molecular , Masculino , Microquímica , Conformação Proteica , Ratos , Tripsina
18.
J Biol Chem ; 261(8): 3556-62, 1986 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-3485096

RESUMO

In order to establish the nature of the aldosterone synthetase activity in the adrenal cortex, we have used porcine adrenal, bovine adrenal cortex, highly purified bovine and porcine 11 beta-/18-hydroxylase, and antibodies raised against the latter enzyme. Mitochondria from two zones (glomerulosa and fasciculata) of the bovine cortex synthesize aldosterone, but those from glomerulosa are much more active than those from fasciculata. Partially purified (cholate-extracted plus ammonium sulfate-precipitated) extracts of mitochondria from the two zones are equally active in catalyzing all three steps in the conversion of 11-deoxycorticosterone to aldosterone. 18-Hydroxylase and aldehyde synthetase activities (18-hydroxycorticosterone----aldosterone) were completely precipitated from cholate extracts of mitochondria from bovine adrenal by antibodies to the pure porcine enzyme. No activity corresponding to any of the three steps in the conversion of 11-deoxycorticosterone to aldosterone was found in extramitochondrial fractions of the bovine cortex. Synthesis of aldosterone by the pure porcine enzyme was inhibited by antibodies to this enzyme and by metyrapone (an inhibitor of 11 beta-/18-hydroxylase). When fractions of porcine adrenal, resulting from purification of the enzyme from mitochondria, were exhaustively tested for any of the enzyme activities required for the synthesis of aldosterone, activity was found only in those fractions containing the 11 beta-/18-hydroxylase, i.e. no additional enzyme was discarded during the purification procedure. It is concluded that the only adrenocortical enzyme capable of synthesizing aldosterone in bovine and porcine adrenal is the well known 11 beta-hydroxylase, that the conversion of 18-hydroxycorticosterone to aldosterone is catalyzed by this cytochrome P-450, and that this step (aldehyde synthetase) requires the heme of the P-450 as demonstrated by the photochemical action spectrum.


Assuntos
Córtex Suprarrenal/metabolismo , Aldeídos/metabolismo , Aldosterona/biossíntese , Esteroide 11-beta-Hidroxilase/análise , Esteroide Hidroxilases/análise , Córtex Suprarrenal/citologia , Córtex Suprarrenal/enzimologia , Animais , Bovinos , Precipitação Química , Corticosterona/metabolismo , Citocromo P-450 CYP11B2 , Hidroxilação , Técnicas In Vitro , Metirapona/farmacologia , Mitocôndrias/metabolismo , Fotoquímica , Suínos
19.
Am J Pathol ; 140(2): 337-43, 1992 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-1739127

RESUMO

The expression of aromatase was evaluated in 38 breast carcinomas by an immunohistochemical method (ABC) using an specific polyclonal antibody against human placental aromatase. Fifteen tumors (40%) showed significant immunoreactivity, as defined by cytoplasmic positivity of moderate intensity present in at least 15% of the cells. The results were correlated with the estrogen and progesterone hormone receptor status and several clinicopathologic parameters such as age, tumor size, lymph node status, and stage of the disease. There was a significant, but inverse, correlation between the aromatase activity and the estrogen receptor status (P = 0.04), indicating the likelihood of negative estrogen if substantial aromatase activity was present. No statistically significant correlation was found between the presence of intratumoral aromatase and the rest of the parameters studied (P greater than 0.7). Nor was there a correlation between the aromatase content of the tumors and the menopausal status. The degree of intratumoral heterogeneity of the aromatase content was minimal in six cases where multiple samples from each tumor were analyzed. This is the first study reporting the detection of aromatase in archival material from breast carcinomas using immunohistochemical techniques. The lack of biologic significance of its presence in breast cancer reported here and by others using biochemical assays should be validated in larger series with longer follow-up. The method described can be readily used for that objective.


Assuntos
Aromatase/análise , Neoplasias da Mama/enzimologia , Aromatase/imunologia , Sequência de Bases , Neoplasias da Mama/química , Neoplasias da Mama/patologia , Feminino , Humanos , Soros Imunes , Técnicas Imunoenzimáticas , Dados de Sequência Molecular , Placenta/enzimologia , Reação em Cadeia da Polimerase , RNA Mensageiro/isolamento & purificação , Receptores de Estrogênio/análise , Receptores de Progesterona/análise
20.
J Protein Chem ; 15(4): 351-8, 1996 May.
Artigo em Inglês | MEDLINE | ID: mdl-8819011

RESUMO

Human neurotrophin-3 (NT-3) is a member of the nerve growth factor (NGF) family of neurotrophic factors, and the recombinant protein is being developed as a therapeutic for neurodegenerative diseases. The final product purity and lot-to-lot variation are monitored routinely by peptide mapping. However, only the N-terminal region of NT-3 was susceptible to proteolysis under native conditions. Complete digestion required that the protein be chemically modified by reduction and S-alkylation prior to proteolysis. Complete proteolytic degradation of the protein was achieved simply by an initial denaturation of NT-3 in 6 M guanidinium chloride (pH6) for 2 hr at 37 degrees C, followed by a tenfold dilution with the digestion buffer (0.1 M Tris-HCl, 1 mM CaCl2 at pH 7.0) and immediate addition of chymotrypsin at 1% by weight. Direct comparison of the peptide map with an identical aliquot that had been reduced and alkylated also allowed the establishment of the cystine linkages present in NT-3: Cys14 to Cys79, Cys57 to Cys108, and Cys67 to Cys110. This disulfide structure is homologous to the NGF family of neurotrophic factors.


Assuntos
Dissulfetos/química , Fatores de Crescimento Neural/química , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Quimotripsina/metabolismo , Cisteína/química , Ditiotreitol/farmacologia , Escherichia coli/genética , Guanidina , Guanidinas/farmacologia , Humanos , Dados de Sequência Molecular , Fatores de Crescimento Neural/genética , Neurotrofina 3 , Fragmentos de Peptídeos/química , Mapeamento de Peptídeos , Desnaturação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Termolisina/metabolismo
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