RESUMEN
A cDNA for the human cytoplasmic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase (EC 4.1.3.5) was subcloned and expressed from a T7-based vector in Escherichia coli. The over-produced enzyme was purified using a three-step protocol that generated 20 to 30 mg protein/liter cell culture. The physical and catalytic properties of the recombinant synthase are similar to those reported for the nonrecombinant enzymes from chicken liver [Clinkenbeard et al. (1975a) J. Biol. Chem. 250, 3124-3135] and rat liver [Mehrabian et al. (1986) J. Biol. Chem. 261, 16249-16255]. Mutation of Cys129 to serine or alanine destroys HMG-CoA synthase activity by disrupting the first catalytic step in HMG-CoA synthesis, enzyme acetylation by acetyl coenzyme A. Furthermore, unlike the wild-type enzyme, neither mutant was capable of covalent modification by the beta-lactone inhibitor, L-659,699 [Greenspan et al. (1987) Proc. Natl. Acad. Sci. USA 84, 7488-7492]. Kinetic analysis of the inhibition by L-659,699 revealed that this compound is a potent inhibitor of the recombinant human synthase, with an inhibition constant of 53.7 nM and an inactivation rate constant of 1.06 min-1.
Asunto(s)
Cisteína/genética , Hidroximetilglutaril-CoA Sintasa/genética , Hidroximetilglutaril-CoA Sintasa/metabolismo , Mutación , Acetilcoenzima A/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Escherichia coli/genética , Ácidos Grasos Insaturados/química , Ácidos Grasos Insaturados/farmacología , Colorantes Fluorescentes/química , Colorantes Fluorescentes/farmacología , Biblioteca de Genes , Humanos , Hidroximetilglutaril-CoA Sintasa/antagonistas & inhibidores , Hidroximetilglutaril-CoA Sintasa/aislamiento & purificación , Lactonas/química , Lactonas/farmacología , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
Stromelysin-1 is a member of a tissue metalloproteinase family whose members are all capable of degrading extracellular matrix components. A truncated form of human fibroblast prostromelysin 1 lacking the C-terminal, hemopexin-like domain has been expressed in Escherichia coli and purified to homogeneity. Treatment of this short form of prostromelysin with (aminophenyl)mercuric acetate resulted in activation and loss of the propeptide in a manner identical with the wild-type, full-length protein. Kinetic comparisons using Nle11-substance P as a substrate showed that the wild-type stromelysin and the truncated form of the enzyme had similar kcat and Km values. Likewise, both enzymes displayed similar Ki values for a hydroxamate-containing peptide inhibitor. Taken together, these results indicate that the C-terminal portion of stromelysin is not required for proper folding of the catalytic domain, maintenance of the enzyme in a latent form, activation with an organomercurial, cleavage of a peptide substrate, or interaction with an inhibitor. Moreover, the active short form of stromelysin displayed a reduction in the C-terminal heterogeneity, a characteristic degradation of the full-length stromelysin, and thereby provides a more suitable protein for future structural studies.
Asunto(s)
Metaloendopeptidasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Precursores Enzimáticos/aislamiento & purificación , Precursores Enzimáticos/metabolismo , Escherichia coli/genética , Fibroblastos/enzimología , Humanos , Ácidos Hidroxámicos/farmacología , Cinética , Sustancias Macromoleculares , Metaloproteinasa 3 de la Matriz , Metaloendopeptidasas/aislamiento & purificación , Metaloendopeptidasas/metabolismo , Colagenasa Microbiana/genética , Modelos Estructurales , Datos de Secuencia Molecular , Peso Molecular , Sondas de Oligonucleótidos , Plásmidos , Reacción en Cadena de la Polimerasa/métodos , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Homología de Secuencia de Ácido NucleicoRESUMEN
Granzyme B has been purified to homogeneity from the granules of a human cytolytic lymphocyte line, Q31, in an enzymatically active form by a three-step procedure. Q31 granzyme B hydrolyzed Na-t-butyloxycarbonyl-L-alanyl-L-alanyl-L-aspartyl (Boc-Ala-Ala-Asp) thiobenzyl ester with a kcat of 11 +/- 5 mol/s/mol enzyme and catalytic efficiency kcat/Km of 76,000 +/- 44,000 M-1 s-1. The hydrolysis of Boc-Ala-Ala-Asp thiobenzyl ester by crude Q31 Percoll fractions paralleled the tryptase activity for granule-containing fractions, which showed that granzyme B was associated with granules. When chromatographed on Sephacryl S-300, Q31 granzyme B eluted in two broad bands corresponding to dimer and monomer, both of which electrophoresed at 35 kDa in reducing NaDodSo4 polyacrylamide, and both of which showed a lag phase in assays. The lag phase in assays could be extended with 0.03 mM pepstatin. Upon elution from ion-exchange chromatography Q31 granzyme B electrophoresed at 32 kDa in reducing NaDodSO4 polyacrylamide and did not have a lag phase in assays. The amino-terminal sequence of the 32-kDa Q31 granzyme B was identical to four other human cytotoxic T-lymphocyte granzymes B in 18 of 18 positions sequenced. Purified Q31 granzyme B had a preference for substrates with Glu or Asp as the residue amino-terminal to the scissile bond; little or no activity was noted with oligopeptide substrates for trypsin-like, chymotrypsin-like, and elastase-like proteases. Human plasma alpha 1-protease inhibitor, human plasma alpha 2-protease macroglobulin, soybean and lima-bean trypsin inhibitors, bovine aprotinin, phosphoramidon, and chymostatin inhibited Q31 granzyme B. The inhibition by alpha 1-protease inhibitor was rapid enough to be of physiological significance.
Asunto(s)
Gránulos Citoplasmáticos/enzimología , Serina Endopeptidasas/aislamiento & purificación , Linfocitos T Citotóxicos/enzimología , Secuencia de Aminoácidos , Línea Celular , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Granzimas , Humanos , Cinética , Datos de Secuencia Molecular , Inhibidores de Proteasas/farmacología , Serina Endopeptidasas/metabolismo , Especificidad por SustratoRESUMEN
The leukotrienes, metabolites of arachidonic acid produced through the action of the enzyme 5-lipoxygenase, are important mediators of immediate hypersensitivity and inflammation. Among the variety of diseases in which the leukotrienes may play a symptomatic or causative role is the dermatological condition psoriasis, a chronic proliferative disease of the skin. This study reports the synthesis and comparative biological activities of various ortho-substituted phenols including 4-methoxyphenols, 6-hydroxy-1,2,3,4-tetrahydrobenzopyrans, 2,3-dihydro-5-benzofuranols, and 5-benzofuranols. The phenols prepared in this study were evaluated for their ability to inhibit the production of leukotriene B4(LTB4) in isolated human polymorphonuclear leukocytes (PMNs) and to inhibit a topical inflammatory response in the topical mouse ear (TME) model. In the former case, when the log IC50 was plotted versus the log of the octanol/water partition coefficient (log P), to eliminate the effect of lipophilicity, the 2,3-dihydro-5-benzofuranol ring system was shown to be more potent than the other ring systems examined throughout the range of partition coefficients studied. The ability to inhibit leukotriene production in vitro in human PMNs can be rationalized on the basis of a model that suggests that the observed inhibition is dependent on the kinetic ability of the inhibitor to reduce a radical species and on the fraction of inhibitor that is partitioned into the cell membrane. While the in vivo antiinflammatory activity as measured by the TME did not correlate with the in vitro data, it was felt that the TME represented a valuable measure of the ability of a compound to penetrate the skin to the site of an ongoing inflammatory response. Of the compounds synthesized in this study, 6-[1-[2-(hydroxymethyl)phenyl]-1-propen-3-yl]-2,3-dihydro-5-benzof uranol (1, L-651896) was chosen for further development.