RESUMO
Ligand affinity chromatography was used to purify a cell surface alpha 2-macroglobulin (alpha 2M) receptor. Detergent extracts of human placenta were applied to an affinity matrix consisting of alpha 2M, previously reacted with methylamine, coupled to Sepharose. Elution with EDTA specifically released polypeptides with apparent molecular masses of 420 and 39 kD. In some preparations, small amounts of a 90-kD polypeptide were observed. The 420- and 39-kD polypeptides appear specific for the forms of alpha 2M activated by reaction with proteinases or methylamine and do not bind to an affinity matrix consisting of native alpha 2M coupled to Sepharose. Separation of these two polypeptides was accomplished by anion exchange chromatography, and binding activity was exclusively associated with the 420-kD polypeptide. The purified 420-kD protein binds to the conformationally altered forms of alpha 2M that are known to specifically interact with alpha 2M receptors and does not bind to native alpha 2M. Binding of the 420-kD polypeptide to immobilized wheat germ agglutinin indicates that this polypeptide is a glycoprotein. The cell surface localization of the 420-kD glycoprotein was confirmed by affinity chromatography of extracts from surface radioiodinated fibroblasts. These properties suggest that the 420-kD polypeptide is a cell surface receptor for the activated forms of alpha 2M.
Assuntos
Glicoproteínas de Membrana/metabolismo , Receptores Imunológicos/metabolismo , alfa-Macroglobulinas/metabolismo , Animais , Linhagem Celular , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Cinética , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Glicoproteínas de Membrana/isolamento & purificação , Peso Molecular , Placenta/metabolismo , Gravidez , Conformação Proteica , Receptores Imunológicos/isolamento & purificação , alfa-Macroglobulinas/ultraestruturaRESUMO
Half-of-the-sites reactivity of the catalytic site thiol groups of UDPglucose dehydrogenase (UDPglucose:NAD+ 6-oxidoreductase, EC 1.1.1.22) can be ascribed either to the induction of conformational asymmetry following derivatization of one half of the subunits or to intrinsic conformational differences in the subunits of the native enzyme. If the half-sites reactivity behavior is due to induction effects, the magnitude of the induction could be expected to depend on the nature of the covalent modification. On the other hand, if the half-sites reactivity behavior is due to pre-existing asymmetry and there is no communication between catalytic centers, the properties of unmodified sub-units should be independent of the nature of the covalent derivative introduced on the modified subunits. According to the induced asymmetry hypothesis, the catalytic activity of half-sites modified enzyme might be different for different covalent modifications, whereas for the rigid pre-existing asymmetry hypothesis the catalytic activity of half-sites modified enzyme should be the same regardless of the modifying group. During the course of catalytic site thiol group modification by a number of thiol specific reagents, the loss of enzyme activity was equivalent to the degree of modification for most of the reagents employed. However, with iodoacetate and 5-(iodoacetamidoethyl)aminonaphthalene-1-sulfonic acid, half-sites modification of UDPglucose dehydrogenase reduced catalytic activity by 58 and 78%, respectively, of the initial activity. These observations are consistent with a model in which there is communication between catalytic sites. Electron microscopy shows that the six subunits of UDPglucose dehydrogenase are arranged as a hexagonal planar ensemble.
Assuntos
Desidrogenases de Carboidrato/antagonistas & inibidores , Fígado/enzimologia , Uridina Difosfato Glucose Desidrogenase/antagonistas & inibidores , Animais , Sítios de Ligação/efeitos dos fármacos , Bovinos , Iodoacetamida/análogos & derivados , Iodoacetamida/farmacologia , Iodoacetatos/farmacologia , Microscopia Eletrônica , Naftalenossulfonatos/farmacologia , Conformação Proteica/efeitos dos fármacosRESUMO
The activities of 3 lysosomal proteases in the nematode Caenorhabditis elegans are markedly lower in older animals. The aspartyl protease cathepsin D declines about 10-fold from day 3 (early adulthood) to day 11 (near the mean lifespan); this reflects a net decline in the amount of cathepsin D antigen. The specific activity of the thiol protease cathepsin Ce1 declines about 2.5-fold over the same period, and the specific activity of the thiol protease cathepsin Ce2 declines about 8-fold. The activity of a new non-lysosomal protease, designated cathepsin CeX, is invariant with age. The data are consistent with the hypothesis that reduced protease activity in older animals may cause a decline in the rate of protein turnover with age, but do not prove this hypothesis.
Assuntos
Envelhecimento/metabolismo , Caenorhabditis/enzimologia , Lisossomos/enzimologia , Peptídeo Hidrolases/metabolismo , Animais , Catepsina D/metabolismo , Centrifugação Isopícnica , Immunoblotting , Focalização IsoelétricaRESUMO
Women smokers concerned about weight gain (N = 219) were randomly assigned to 1 of 3 adjunct treatments accompanying group smoking cessation counseling: (a) behavioral weight control to prevent weight gain (weight control); (b) cognitive-behavioral therapy (CBT) to directly reduce weight concern, in which dieting was discouraged; and (c) standard counseling alone (standard), in which weight gain was not explicitly addressed. Ten sessions were conducted over 7 weeks, and no medication was provided. Continuous abstinence was significantly higher at posttreatment and at 6 and 12 months of follow-up for CBT (56%, 28%, and 21%, respectively), but not for weight control (44%, 18%, and 13%, respectively), relative to standard (31%, 12%, and 9%, respectively). However, weight control, and to a lesser extent CBT, was associated with attenuation of negative mood after quitting. Prequit body mass index, but not change in weight or in weight concerns postquit, predicted cessation outcome at 1 year. In sum, CBT to reduce weight concerns, but not behavioral weight control counseling to prevent weight gain, improves smoking cessation outcome in weight-concerned women.
Assuntos
Terapia Cognitivo-Comportamental , Abandono do Hábito de Fumar , Aumento de Peso , Adolescente , Adulto , Feminino , Humanos , Pessoa de Meia-IdadeRESUMO
Sensitivity in responses to one drug may relate to sensitivity to other drugs, suggesting broad individual differences in characteristic responsivity across drugs. Data from two separate studies of smokers were reanalyzed to examine associations between acute subjective and cardiovascular effects of nicotine vs. caffeine and between nicotine vs. alcohol. Typical intakes of cigarettes, alcohol, and caffeine were included as covariates when they were correlated with the responses of interest. Significant associations between nicotine and caffeine were seen for most of the subjective measures and for blood pressure responses. Fewer significant associations were observed between nicotine and alcohol. Responses associated between nicotine and both of the other drugs tended to reflect psychomotor stimulation. These results suggest that smokers who are more responsive to some of nicotine's subjective and blood pressure effects are also more sensitive to the same effects of caffeine and, to a lesser extent, of alcohol.
Assuntos
Cafeína/farmacologia , Depressores do Sistema Nervoso Central/farmacologia , Estimulantes do Sistema Nervoso Central/farmacologia , Etanol/farmacologia , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Fumar/psicologia , Administração Intranasal , Adulto , Afeto/efeitos dos fármacos , Feminino , Hemodinâmica/efeitos dos fármacos , Humanos , Masculino , Nicotina/administração & dosagem , Agonistas Nicotínicos/administração & dosagem , PsicometriaRESUMO
Self-quenched fluorogenic substrates for proteolytic enzymes have been prepared by alkylation of thiol groups in reduced bovine serum albumin with iodoacetamidofluorescein or iodoacetamidoeosin. Substrates immobilized by adsorption onto nitrocellulose membranes or by incorporation into agarose gel slabs are suitable for fluorescence zymography after electrophoretic separation of catalytically active proteases, including cathepsin D.
Assuntos
Catepsina D/análise , Peptídeo Hidrolases/análise , Quimotripsina/análise , Fluoresceínas , Focalização Isoelétrica , Soroalbumina Bovina/análise , Espectrometria de Fluorescência/métodos , Tripsina/análiseRESUMO
The venoms of predatory marine cone snails, Conus species, contain numerous peptides and proteins with remarkably diverse pharmacological properties. One group of peptides are the alpha-conotoxins, which consist of 13-19 amino acids constrained by two disulphide bonds. A biologically active fluorescein derivative of Conus geographus alpha-conotoxin GI (FGI) was used in novel solution-phase-binding assays with purified Torpedo californica nicotinic acetylcholine receptor (nAchR) and monoclonal antibodies developed against the toxin. The binding of FGI to nAchR or antibody had apparent dissociation constants of 10-100 nM. Structure-function studies with alpha-conotoxin GI analogues composed of a single disulphide loop revealed that different conformational restraints are necessary for effective toxin interactions with nAchR or antibodies.
Assuntos
Anticorpos Monoclonais/metabolismo , Conotoxinas , Venenos de Moluscos/metabolismo , Peptídeos Cíclicos/metabolismo , Receptores Nicotínicos/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/farmacologia , Sítios de Ligação de Anticorpos , Centrifugação , Cromatografia em Gel , Fluoresceínas/metabolismo , Dose Letal Mediana , Camundongos , Dados de Sequência Molecular , Venenos de Moluscos/imunologia , Peptídeos Cíclicos/imunologia , Ligação Proteica , Soluções , Espectrometria de Fluorescência , TorpedoRESUMO
6,6-Dithiodinicotinate shows half-of-the-sites reactivity towards the six catalytic-site thiol groups of bovine liver UDP-glucose dehydrogenase. The reagent introduces three intrasubunit disulphide linkages between catalytic-site thiol groups and non-catalytic-site thiol groups and abrogates 60% of the catalytic activity of the hexameric enzyme; excess 2-mercaptoethanol rapidly restores full catalytic activity. These results show the half-of-the-sites behaviour of the enzyme with the reagent and the presence of a non-catalytic-site thiol group capable of forming a disulphide linkage with a catalytic-site thiol group on the same subunit without irreversible denaturation.
Assuntos
Desidrogenases de Carboidrato/metabolismo , Ácidos Nicotínicos/metabolismo , Uridina Difosfato Glucose Desidrogenase/metabolismo , Sítios de Ligação , OxirreduçãoRESUMO
The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR/LRP) consists of two polypeptides, 515 and 85 kDa, that are noncovalently associated. A 39-kDa polypeptide, termed the receptor-associated protein (RAP), interacts with the 515-kDa subunit after biosynthesis of these molecules and remains associated on the cell surface. This molecule regulates ligand binding of alpha 2MR/LRP (Herz, J., Goldstein, J. L., Strickland, D. K., Ho, Y. K., and Brown, M. S. (1991) J. Biol. Chem. 266, 21232-21238). Titration and binding studies indicate that RAP binds to two equivalent binding sites on alpha 2MR/LRP, with a KD of 14 nM. Heterologous ligand displacement experiments demonstrated that RAP completely inhibits the binding of 125I-activated alpha 2M to human fibroblasts and to the purified alpha 2MR/LRP, with a Ki of 23 and 26 nM, respectively. A direct correlation between the degree of binding of RAP to the receptor and the degree of ligand inhibition was observed, indicating that as the RAP binding sites are saturated, alpha 2MR/LRP loses its ability to bind ligands. Thus, the amount of RAP bound to alpha 2MR/LRP dictates the level of receptor activity. A model is proposed in which alpha 2MR/LRP contains multiple ligand binding sites, each regulated by a separate RAP site.
Assuntos
Receptores Imunológicos/metabolismo , alfa-Macroglobulinas/metabolismo , Sequência de Bases , Sítios de Ligação , Cálcio/metabolismo , Cátions Bivalentes , DNA/genética , Humanos , Concentração de Íons de Hidrogênio , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Dados de Sequência Molecular , Receptores de LDL/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
The catalytic-site thiol groups of UDP-glucose dehydrogenase from bovine liver were carboxymethylated with iodo[2-14C]acetate or with iodoacetamidofluorescein. After the residual thiol groups were carboxymethylated with iodoacetate, the proteins were digested with trypsin. The 14C-labelled peptide from the carboxymethylated enzyme was purified to homogeneity by successive thick-layer chromatography on silica gel, paper electrophoresis and chromatography, and column chromatography on Bio-Gel P-6. Homogeneous fluoresceincarboxamidomethylated peptide was prepared from a tryptic digest of fluoresceincarboxamidomethylated enzyme by specific adsorption--desorption from Sephadex G-25. The sequences of either peptide determined by the manual Edman dansyl procedure is: Ala-Ser-Val-Gly-Phe-Gly-Gly-Ser-Cys-Phe-Glx-Glx-Gly-Lys.
Assuntos
Desidrogenases de Carboidrato/metabolismo , Fígado/enzimologia , Fragmentos de Peptídeos/isolamento & purificação , Uridina Difosfato Glucose Desidrogenase/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Catálise , Bovinos , Fluoresceínas , Corantes Fluorescentes , Compostos de Sulfidrila/metabolismo , Tripsina/metabolismoRESUMO
Crude homogenates of the soil nematode Caenorhabditis elegans exhibit strong proteolytic activity at acid pH. Several kinds of enzyme account for much of this activity: cathepsin D, a carboxyl protease which is inhibited by pepstatin and optimally active toward hemoglobin at pH 3; at least two isoelectrically distinct thiol proteases (cathepsins Ce1 and Ce2) which are inhibited by leupeptin and optimally active toward Z-Phe-Arg-7-amino-4-methylcoumarin amide at pH 5; and a thiol-independent leupeptin-insensitive protease (cathepsin Ce3) with optimal activity toward casein at pH 5.5. Cathepsin D is quantitatively most significant for digestion of macromolecular substrates in vitro, since proteolysis is inhibited greater than 95% by pepstatin. Cathepsin D and the leupeptin-sensitive proteases act synergistically, but the relative contribution of the leupeptin-sensitive proteases depends upon the protein substrate.
Assuntos
Caenorhabditis/enzimologia , Peptídeo Hidrolases/metabolismo , Animais , Catepsinas/antagonistas & inibidores , Cinética , Peptídeo Hidrolases/isolamento & purificação , Inibidores de Proteases/farmacologia , Especificidade por SubstratoRESUMO
Ten peptides, derived from human alpha 2-macroglobulin (alpha 2M) receptor by chemical or proteolytic digestion, were sequenced. Comparative analysis revealed that all of the resulting sequences were present within the cDNA-deduced structure of low density lipoprotein receptor-related protein (LRP) (Herz, J., Hamann, U., Rogne, S., Myklebost, O., Gausepohl, H., and Stanley, K. K. (1988) EMBO J. 7, 4119-4127). The findings provide evidence that the alpha 2M receptor and LRP are the same molecule. Further evidence comes from immunoprecipitation experiments using a monoclonal antibody specific for the alpha 2M receptor that show this molecule, like LRP, to contain two polypeptides of approximately 420 and 85 kDa that are noncovalently associated. An additional component of this receptor system is a 39-kDa polypeptide that co-purifies with the alpha 2M receptor during affinity chromatography. Solid phase binding studies reveal that the 39-kDa polypeptide binds with high affinity (Kd = 18 nM) to the 420-kDa component of the alpha 2M receptor. The apparent identity of LRP and the alpha 2M receptor suggests that this molecule is a multifunctional receptor with the capacity to bind diverse biological ligands and highlights a possible relationship between two previously unrelated biological processes, lipid metabolism and proteinase regulation.
Assuntos
Receptores Imunológicos/genética , Receptores de LDL/genética , Sequência de Aminoácidos , Feminino , Fibroblastos/metabolismo , Humanos , Immunoblotting , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Dados de Sequência Molecular , Placenta/metabolismo , Gravidez , Receptores Imunológicos/isolamento & purificação , Receptores Imunológicos/metabolismo , Homologia de Sequência do Ácido Nucleico , alfa-Macroglobulinas/metabolismoRESUMO
The alpha 2-macroglobulin (alpha 2M) receptor complex as purified by affinity chromatography contains three polypeptides: a 515-kDa heavy chain, an 85-kDa light chain, and a 39-kDa associated protein. Previous studies have established that the 515/85-kDa components are derived from a 600-kDa precursor whose complete sequence has been determined by cDNA cloning (Herz, J., Hamann, U., Rogne, S., Myklebost, O., Gassepohl, H., and Stanley, K. (1988) EMBO J. 7,4119-4127). We have now determined the primary structure of the human 39-kDa polypeptide, termed alpha 2M receptor-associated protein, by cDNA cloning. The deduced amino acid sequence contains a putative signal sequence that precedes the 323-residue mature protein. Comparative sequence analysis revealed that alpha 2M receptor-associated protein has 73% identity with a rat protein reported to be a pathogenic domain of Heymann nephritis antigen gp 330 and 77% identity to a mouse heparin-binding protein termed HBP-44. The high overall identity suggests that these molecules are interspecies homologues and indicates that the pathogenic domain, previously thought to be a portion of gp 330, is in fact a distinct protein. Further, the 120-residue carboxyl-terminal region of alpha 2M receptor-associated protein has 26% identity with a region of apolipoprotein E containing the low density lipoprotein receptor binding domain. Pulse-chase experiments revealed that the newly formed alpha 2M receptor-associated protein remains cell-associated, while surface labeling experiments followed by immunoprecipitation suggest that this protein is present on the cell surface forming a complex with the alpha 2M receptor heavy and light chains.