RESUMO
We report the cloning, overexpression, kinetic analysis, and modeling of the tertiary structure of an unusual plant cysteine proteinase. Ananain (EC 3.4.22.31), from Ananas comosus (pineapple) is distinguished from all other cysteine proteinases in the papain superfamily by having a unique combination of acidic amino acids. As well as lacking the acidic residue immediately preceding the active site histidine (position 158 in papain), it also lacks the extensive surface network of acidic residues that were postulated to compensate for the loss of charge at position 158 in mammalian cathepsins. Ananain has the fewest acidic residues, so far reported, of any plant cysteine proteinase, but two of the carboxyl residues (E50 and E35) postulated to have an enabling role in catalysis, the so-called "electrostatic switch", remain conserved. Comparisons of the kinetics of recombinant wild-type ananain with E50A and E35A mutants proves that these charged groups are not essential for catalysis. Hence this research does not confirm the presence of an electrostatic switch in this cysteine proteinase, and the role of acidic residues in the enhancement of catalytic competence in these enzymes is discussed in light of this new evidence.
Assuntos
Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Mutagênese Sítio-Dirigida , Proteínas de Plantas/química , Proteínas de Plantas/genética , Alanina/genética , Alanina/metabolismo , Sequência de Aminoácidos , Asparagina/genética , Asparagina/metabolismo , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Sítios de Ligação/genética , Clonagem Molecular , Cisteína Endopeptidases/biossíntese , Cisteína Endopeptidases/metabolismo , DNA Complementar/isolamento & purificação , Escherichia coli/enzimologia , Escherichia coli/genética , Ácido Glutâmico/genética , Ácido Glutâmico/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Magnoliopsida , Modelos Moleculares , Dados de Sequência Molecular , Proteínas de Plantas/metabolismo , Precursores de Proteínas/biossíntese , Precursores de Proteínas/genética , Precursores de Proteínas/isolamento & purificação , Precursores de Proteínas/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
(+)-trans-delta 8-Tetrahydrocannabinol (THC) was synthesized by condensation of (1R)-cis-verbenol and olivetol, and its in vitro and in vivo hepatic metabolism was studied in male mice. Metabolites were isolated by solvent extraction, purified, in the case of the in vivo study, by chromatography on Sephadex LH-20, and identified by combined gas chromatography/mass spectrometry. Twenty-three metabolites were identified. These were similar to those from the (-)-isomer in that the major metabolic route was hydroxylation at C-11 followed, in vivo, by oxidation to the corresponding acid. Further hydroxylation of these compounds at C-7 and in the side chain led to a series of disubstituted metabolites. The major sites of side chain hydroxylation were 1'-, 3'-, and 4'-. Less 2'-hydroxylation was observed than with the (-)-isomer, but the major difference between the two isomers was the production of 1'-hydroxy metabolites only from the (+)-isomer. Much less hydroxylation occurred at C-7 than with the (-)-isomer so that the 7,11-disubstituted compounds, which were major metabolites of (-)-trans-delta 8-THC, were formed in insignificant concentrations. Another difference between the metabolism of the two isomers, in vitro, was the presence of substantial concentrations of monohydroxy metabolites containing hydroxylation in the side chain; these compounds are absent in metabolic profiles from (-)-trans-delta 8-THC. An epoxide and its derived glycol were detected in vitro. Another major in vitro metabolic route was the formation of an acetate derivative of 11-hydroxy-(+)-trans-delta 8-THC.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Dronabinol/metabolismo , Animais , Biotransformação , Cromatografia Gasosa-Espectrometria de Massas , Hidroxilação , Técnicas In Vitro , Espectroscopia de Ressonância Magnética , Masculino , CamundongosRESUMO
The metabolism of antipyrene and delta-9-tetrahydrocannabinol (delta-9-THC) was studied in vitro with mouse liver supernatant and 105,000 g microsomes at 450 atm pressure. No significant change in rate of antipyrene metabolism was detected, whereas a slight (5.6%) but nonsignificant reduction in the rate of metabolism of delta-9-THC was found. Pressure did, however, produce a significant (23%) reduction in the formation of the 11-hydroxy metabolite from delta-9-THC, but at the same time had no effect on the production of the other major metabolite, 8-alpha-hydroxy-delta-9-THC. This is possibly due to a differential effect on cytochrome P-450 isozymes. Mechanisms for the possible cause of the inhibition of metabolism are discussed.
Assuntos
Antipirina/metabolismo , Dronabinol/metabolismo , Microssomos Hepáticos/metabolismo , Pressão , Animais , Antipirina/farmacologia , Sistema Enzimático do Citocromo P-450/fisiologia , Cromatografia Gasosa-Espectrometria de Massas , Masculino , Camundongos , Camundongos Endogâmicos , OxirreduçãoRESUMO
Constituent cytochrome P-450 isozymes from mouse hepatic microsomes were fractionated by chromatofocusing on Polybuffer Exchanger 94 over the pH range 5.3-8.3 and characterized by polyacrylamide gel electrophoresis. Eight isozymes were detected in fractions from unpretreated mice and seven from phenobarbitone-pretreated animals. Isozyme-containing fractions were reconstituted and shown to produce specific monohydroxy metabolites from both testosterone and delta 9-tetrahydrocannabinol.