RESUMEN
We recently suggested that soluble beta-amyloid (betaA4) is a ligand of the low density lipoprotein receptor-related protein and heparan sulfate proteoglycan pathway. In the blood and in the cerebrospinal fluid, betaA4 is bound to apolipoprotein E containing lipoproteins. We examined how binding of betaA4 to beta-very low density lipoproteins (betaVLDL) alters their cellular metabolism. Compared with betaVLDL alone, complexes of betaVLDL and betaA4 were internalized, but not degraded at increased rates in fibroblasts and in rat hippocampal cells. The uptake of complexes of betaVLDL and betaA4 was not mediated by the low density lipoprotein receptor. BetaA4 not complexed to betaVLDL competed with the endocytosis of alpha2-macroglobulin and apolipoprotein E-enriched betaVLDL. The uptake of complexes of betaVLDL and betaA4 was inhibited by heparin, suramin, lactoferrin, the 39-kd receptor-associated protein, and alpha2-macroglobulin. Complexes of betaVLDL and betaA4 were taken up at reduced rates in Chinese hamster ovary cells partially (pgsB-650) or completely lacking (pgsA-745) proteoglycans. BetaA4 in which the positively charged amino acids between positions 13 and 17 (HHQKL) were replaced by glycine (GGQGL) failed to enhance the uptake of betaVLDL. Together, the data suggest that binding of betaA4 to betaVLDL produces particles that are endocytosed by low density lipoprotein receptor-related protein and HSPG. Complexes of betaVLDL and betaA4 had an intracellular half-life 4-fold that of native betaVLDL, did not undergo lysosomal degradation, and were resecreted into the culture medium. These findings represent the first identification of an endocytotic pathway for betaA4 and may be of relevance to the pathobiochemistry of neurodegenerative disorders.
Asunto(s)
Péptidos beta-Amiloides/química , Proteoglicanos de Heparán Sulfato/metabolismo , Lipoproteínas VLDL/metabolismo , Receptores de LDL/metabolismo , Alimentación Animal , Animales , Células CHO , Células Cultivadas , Colesterol en la Dieta/farmacología , Cricetinae , Fibroblastos/metabolismo , Hipocampo/citología , Hipocampo/metabolismo , Humanos , Lipoproteínas VLDL/química , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad , Conejos , Ratas , Ratas WistarRESUMEN
The genetic polymorphism of apolipoprotein E (apoE) is associated with the age of onset and relative risk of Alzheimer's disease (AD). In contrast to apoE3, the wild type allele, apoE4 confers an increased risk of late-onset AD. We demonstrate that the beta-amyloid peptide isoforms Abeta (1-28), Abeta (1-40), and Abeta (1-43) compete for the cellular metabolism of apoE3 and apoE4 containing beta-very low density lipoproteins. An antibody raised against Abeta (1-28) cross-reacted with recombinant apoE. Epitope mapping revealed positive amino acid clusters as common epitopes of Abeta (13 through 17; HHQKL) and apoE (residues 144 through 148; LRKRL), both regions known to be heparin binding domains. Abeta in which amino acids 13 through 17 (HHQKL) were replaced by glycine (GGQGL) failed to compete with the cellular uptake of apoE enriched betaVLDL. These observations indicate that Abeta and apoE are taken up into cells by a common pathway involving heparan sulfate proteoglycans.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Apolipoproteínas E/genética , Endocitosis/fisiología , Fragmentos de Péptidos/metabolismo , Receptores de Superficie Celular/metabolismo , Secuencia de Aminoácidos , Péptidos beta-Amiloides/inmunología , Anticuerpos/metabolismo , Apolipoproteína E3 , Apolipoproteínas E/metabolismo , Unión Competitiva , Reacciones Cruzadas/inmunología , Mapeo Epitopo , Fibroblastos , Heparitina Sulfato/metabolismo , Humanos , Lipoproteínas VLDL/metabolismo , Glicoproteínas de Membrana/metabolismo , Datos de Secuencia Molecular , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/farmacología , Polimorfismo Genético/genética , Factores de RiesgoRESUMEN
The yeast PRD1 gene, encoding proteinase yscD, was cloned by complementation of the prd1-6 point mutation. Sequencing of the gene revealed an open reading frame of 2.136 kb, encoding a protein of 712 amino acids with a calculated molecular mass of 81.8 kDa. The sequence HEGLG beginning at residue 501 represents the HEXXH motif, unique for the zinc metallo-peptidases. Sequence comparison revealed complete identity of the proteinase yscD gene with a recently published open reading frame of yeast chromosome III. We found 34.8% identity between proteinase yscD and rat metalloendopeptidase (thimet oligopeptidase, EP 24.15). Proteinase yscD hydrolyzes several chromogenic and fluorogenic peptides that are substrates of thimet oligopeptidase. N-[1-(RS)-carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoic acid, a compound designed as specific inhibitor of EP 24.15, is also a strong inhibitor of the yeast enzyme. Proteinase yscD is a nonvacuolar enzyme. 3-5% of the total enzyme activity can be detected in the intermembrane space of mitochondria. In a mutant carrying a deletion of the PRD1 gene no proteinase yscD activity is detectable in the cytoplasm and in mitochondria of these cells. They do not show any grossly altered phenotype but exhibit a decrease in the intracellular degradation of peptides. This suggests a function of proteinase yscD in the late stages of protein degradation.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Genes Fúngicos , Metaloendopeptidasas/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Animales , Bacillus/enzimología , Bacillus/genética , Secuencia de Bases , Cromosomas Fúngicos , Clonación Molecular , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Escherichia coli , Eliminación de Gen , Prueba de Complementación Genética , Genotipo , Hígado/enzimología , Mamíferos , Metaloendopeptidasas/química , Metaloendopeptidasas/genética , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Mutación Puntual , Ratas , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
Aminopeptidase yscXVI was purified from the yeast Saccharomyces cerevisiae. By SDS-PAGE the enzyme has a molecular weight of 45,000 Da, and in chromatofocusing, elution was observed at pH 6.2. The synthetic substrate cystinyl-4-nitroanilide (Km 22.5 microM, Vmax 12.9 mU/mg) is cleaved most efficiently in the pH range 7-8. Besides cleaving this standard substrate, aminopeptidase yscXVI acts on several other 4-nitroanilide substrates with unsubstituted N-terminal L-amino acids. Highest hydrolysis rate was measured with Lys-4-nitroanilide and Leu-4-nitroanilide. The activity of aminopeptidase yscXVI is abolished by chelating agents and restored by Zn2+, Mn2+ and Co2+ ions. Bestatin and amastatin are both strong inhibitors of the enzyme, with Ki values of 0.53 microM and 0.93 microM, respectively. Aminopeptidase yscXVI is detectable in the logarithmic growth phase, stationary phase, and in starved cultures of yeast.
Asunto(s)
Aminopeptidasas/aislamiento & purificación , Saccharomyces cerevisiae/enzimología , Secuencia de Aminoácidos , Aminopeptidasas/antagonistas & inhibidores , Aminopeptidasas/metabolismo , Cinética , Metales/farmacología , Datos de Secuencia Molecular , Especificidad por Sustrato , Vacuolas/enzimologíaRESUMEN
Thimet oligopeptidase (EC 3.4.24.15) is a thiol-dependent metallo-endopeptidase also known as Pz-peptidase, collagenase-like peptidase, endooligopeptidase A, soluble metallo-endopeptidase and endopeptidase 24.15. The enzyme is closely related to the yeast proteinase yscD. Thimet oligopeptidase (M(r) 74000) is widely distributed in animals and plants. In rat liver it exists in a cytoplasmic and mitochondrial form; a membrane-bound form of the enzyme was discovered in rat brain. Thimet oligopeptidase hydrolyses small peptides but does not act on proteins. In rat brain thimet oligopeptidase is involved in the generation of enkephalins and inactivation of bioactive peptides and experiments with yeast provided good evidence that the enzyme is involved in the late stages of cytoplasmatic protein degradation.
Asunto(s)
Metaloendopeptidasas/metabolismo , Secuencia de Aminoácidos , Animales , Humanos , Datos de Secuencia MolecularAsunto(s)
Proteínas de la Matriz Extracelular , Metaloendopeptidasas , Metaloendopeptidasas/clasificación , Secuencia de Aminoácidos , Animales , Pollos/metabolismo , Hígado/enzimología , Metaloendopeptidasas/metabolismo , Datos de Secuencia Molecular , Inhibidores de Proteasas/farmacología , Especificidad por SustratoRESUMEN
Thimet peptidase has been purified from rat liver mitochondria and found to share the characteristics of a thiol-dependent metallo-endopeptidase previously described for an enzyme in the cytosolic fraction from rat testis: inhibition by EDTA, reactivation by Zn2+, requirement of dithiothreitol for maximal and stable activity, and inhibition by N-ethylmaleimide. The Ki for inhibition by N-[1-(RS)-carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoic acid is 2.6 microM, 100-fold higher than the value for the cytosolic form. The mitochondrial form is not inhibited by antisera against the cytosolic form, and the two forms of the enzyme show quantitative differences in substrate specificity. The name thimet peptidase II is suggested for the enzyme from rat mitochondria.
Asunto(s)
Isoenzimas/aislamiento & purificación , Metaloendopeptidasas/aislamiento & purificación , Mitocondrias Hepáticas/enzimología , Secuencia de Aminoácidos , Animales , Cromatografía DEAE-Celulosa , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Citosol/enzimología , Isoenzimas/metabolismo , Cinética , Hígado/enzimología , Metaloendopeptidasas/metabolismo , Datos de Secuencia Molecular , Inhibidores de Proteasas/farmacología , Ratas , Especificidad por SustratoRESUMEN
Pz-peptidase was purified from rat testis and rabbit muscle. Zinc was detectable in the rat enzyme. The activity of the enzyme from both species was slowly but completely abolished by EDTA and restored by Zn2+. Free thiol groups were also important for the catalytic activity of rat Pz-peptidase, as previously reported for the rabbit enzyme. We conclude that in both species Pz-peptidase has the characteristics of a thiol-dependent metallo-endopeptidase.
Asunto(s)
Ditiotreitol/farmacología , Endopeptidasas/metabolismo , Metaloendopeptidasas/metabolismo , Secuencia de Aminoácidos , Animales , Cromatografía DEAE-Celulosa , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Endopeptidasas/aislamiento & purificación , Etilmaleimida/farmacología , Cinética , Masculino , Datos de Secuencia Molecular , Peso Molecular , Músculos/enzimología , Conejos , Ratas , Especificidad por SustratoRESUMEN
7-Methoxycoumarin-3-carboxylyl-Pro-Leu-Gly-Pro-D-Lys(2,4-dinitr oph enyl) is introduced as a new quenched fluorescence substrate for assaying Pz-peptidase (also known as soluble metallo-endopeptidase and endo-oligopeptidase). The value of Km for partially purified Pz-peptidase from rat muscle was 8.6 microM. High protein concentrations did not interfere with the assay, so that for the first time continuous assays of Pz-peptidase in crude tissue extracts became possible.
Asunto(s)
Endopeptidasas/análisis , Metaloendopeptidasas , Oligopéptidos/metabolismo , Secuencia de Aminoácidos , Fenómenos Químicos , Química , Fluorescencia , Hidrólisis , Cinética , Datos de Secuencia Molecular , Especificidad por SustratoRESUMEN
Pz-peptidase was purified from rabbit muscle by acid precipitation of tissue homogenate followed by cation- and anion-exchange chromatography, gel chromatography, and immunoadsorption. In analytical gel chromatography, one single peak of protein with corresponding Pz-peptidase activity was obtained. The enzyme had an apparent Mr of 74,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was eluted at pH 4.8 in chromatofocusing. No metals were detectable in the protein by neutron activation analysis. Purified Pz-peptidase hydrolyzed Dnp-Pro-Leu-Gly-Pro-Trp-D-Lys (Km 7.2 microM) most effectively in the presence of 5 mM 2-mercaptoethanol and 10 mM CaCl2. No inhibition was observed with inhibitors of serine proteinases, aspartic proteinases, or metalloproteinases, apart from some nonspecific reversible inhibition by 1,10-phenanthroline. The activation by Ca2+ was reversed by EDTA. The enzyme was not inhibited by E-64, cystatin, or leupeptin, but was irreversibly inactivated by iodoacetate, iodoacetamide, and N-ethylmaleimide. It was therefore concluded that rabbit muscle Pz-peptidase is not a typical member of any of the four recognized catalytic classes of proteinases, but may be an atypical cysteine endopeptidase. The peptidase was not bound by alpha 2-macroglobulin. No hydrolysis of gelatin or fibronectin by the enzyme was detected, nor was there any activation of latent collagenase.
Asunto(s)
Endopeptidasas/aislamiento & purificación , Metaloendopeptidasas , Músculos/enzimología , Secuencia de Aminoácidos , Animales , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Endopeptidasas/metabolismo , Cinética , Peso Molecular , Oligopéptidos , Inhibidores de Proteasas/farmacología , Conejos , Especificidad por SustratoRESUMEN
It was found that Pz-peptidase (assayed with 2,4-dinitrophenyl-Pro-Leu-Gly-Pro-Trp-D-Lys) and endopeptidase 24.15 (assayed with benzoyl-Gly-Ala-Ala-Phe-p-aminobenzoate) were co-purified from rat skeletal muscle, were co-eluted in high-resolution gel chromatography and co-existed in a homogeneous preparation of rat testis endopeptidase 24.15. The action of partially purified Pz-peptidase from rat testis on 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg was blocked by an inhibitor of endopeptidase 24.15, and also by a substrate of this enzyme. The partially purified enzyme hydrolysed two substrates of endopeptidase 24.15 with Km values similar to those published previously, and its action on 2,4-dinitrophenyl-Pro-Leu-Gly-Pro-Trp-D-Lys was inhibited by compounds that are considered specific for endopeptidase 24.15. We conclude that the activities previously attributed to two distinct enzymes are due to only one, and that the merging of the two literatures may lead to new lines of research.
Asunto(s)
Endopeptidasas/metabolismo , Metaloendopeptidasas/metabolismo , Animales , Endopeptidasas/aislamiento & purificación , Metaloendopeptidasas/aislamiento & purificación , Inhibidores de Proteasas , RatasRESUMEN
During purification of endo-oligopeptidase from rabbit heart, activities cleaving bradykinin, a substrate of endo-oligopeptidase, and Mcc-Pro-Leu-Gly-Pro-D-Lys(Dnp), a substrate of Pz-peptidase, were found in the same fractions. The hydrolysis of both substrates was inhibited by antisera against endo-oligo-peptidase and Pz-peptidase, and reversibly inhibited by 1, 10-phenanthroline. The purified enzyme hydrolysed Dnp-Pro-Leu-Gly-Pro-Trp-D-Lys, another substrate of Pz-peptidase. Purified Pz-peptidase from rabbit muscle degraded bradykinin and was inhibited by an antiserum against endo-oligopeptidase.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Endopeptidasas/metabolismo , Metaloendopeptidasas , Animales , Cromatografía , Inhibidores de Cisteína Proteinasa , Técnicas Inmunológicas , Fenantrolinas/farmacología , Inhibidores de Proteasas , ConejosRESUMEN
The peptide derivative N alpha-(2,4-dinitrophenyl)-L-prolyl-L-leucyl-glycyl-L-prolyl-L-tryptophanyl-D- lysine (Dnp-Pro-Leu-Gly-Pro-Trp-D-Lys) has been found to be a convenient substrate for the assay of clostridial collagenase and Pz-peptidase. The substrate shows a 25-fold enhancement of fluorescence (gamma ex. 283 nm, lambda em. 350 nm) following hydrolysis of the Leu2-Gly3 peptide bond. The value of Km for clostridial collagenase was 17 microM. The substrate for the first time makes possible continuous fluorimetric assays for Pz-peptidase and clostridial collagenase.
Asunto(s)
Clostridium/enzimología , Endopeptidasas/análisis , Metaloendopeptidasas , Colagenasa Microbiana/análisis , FluorometríaRESUMEN
The plasma clearance rate of 131I-insulin was studied in diabetic and obese patients and healthy persons. The aim of the investigation was to find out whether an accelerated degradation of insulin-probably due to the metabolism of insulin by adipocytes-causes slight forms of the diabetic syndrome. For the evaluation of the data a three-compartment model was used whereby the third compartment reflects the degradation of the intravenously administered 131I-insulin in the tissue. In this compartment 131I-insulin has a half life of 38.7 +/- 7.0 min for control persons (n = 19), 47.4 +/- 27.4 min for patients with a latent diabetes mellitus (n = 7), 34.2 +/- 8.5 min for the group with the subclinical diabetes (n = 5) and 47.6 +/- 16.2 min for the patients with an onset diabetes mellitus (n = 5). No significant difference was observed in the plasma clearance rate between healthy and diabetic persons. There is also no detectable correlation between the grade of obesity and the insulin clearance rate.
Asunto(s)
Diabetes Mellitus/metabolismo , Insulina , Radioisótopos de Yodo , Obesidad , Femenino , Semivida , Humanos , Insulina/farmacocinética , Masculino , Tasa de Depuración MetabólicaRESUMEN
A method for the determination of proteolytic activity is described which uses fluorescein-labeled gelatin coupled to Sepharose 4B as substrate. The assay is simple and sensitive allowing detection of one nanogram of trypsin and is found suitable for the measurement of gelatinolytic activity in tissue samples.
Asunto(s)
Péptido Hidrolasas/análisis , Animales , Endometrio/enzimología , Femenino , Fluoresceína-5-Isotiocianato , Fluoresceínas , Gelatina , Embarazo , Conejos , Sefarosa , Espectrometría de Fluorescencia , Especificidad por Sustrato , Tiocianatos , Tripsina/análisisRESUMEN
Previous investigations have proved that proteinases are involved in implantation of the rabbit embryo into the uterine tissues. This study describes a trypsin-like enzyme found in the blastocyst fluid and uterine flushings at the time of implantation. The proteinase isolated from uterine flushings has a molecular mass of about 50 kDa and exists in two differently charged forms of pI 4.0 and 4.5. Tests with low molecular mass 4-nitroanilide substrates proved a marked cleavage selectivity of the enzyme for arginyl bonds. The catalytic activity is not affected by Ca2+ and EDTA but inhibited by aprotinin and a high concentration (10(-6)M) of lima bean trypsin inhibitor.
Asunto(s)
Desarrollo Embrionario , Péptido Hidrolasas/aislamiento & purificación , Útero/enzimología , Animales , Líquidos Corporales/enzimología , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Femenino , Concentración de Iones de Hidrógeno , Hidrólisis , Focalización Isoeléctrica , Péptido Hidrolasas/análisis , Embarazo , ConejosRESUMEN
L-3-iodo-alpha-methyltyrosine, labeled with either I-131 or I-123, has a high pancreatic specificity in mice. A pancreas-to-liver ratio of 8.6 +/- 2.7 is observed during the first hour after i.v. injection. Accumulation is also prominent in the kidneys, but excretion of the radioagent is rapid, 50% of the activity being eliminated during 90 min. Compared with L-[75Se]selenomethionine, the compound currently used for pancreatic imaging, L-3-[123I]or[131I]iodo-alpha-methyltyrosine has a higher pancreas-to-liver ratio, a shorter physical half-life and biological half-time, and better decay characteristics.
Asunto(s)
Radioisótopos de Yodo , Metiltirosinas , Páncreas/diagnóstico por imagen , Animales , Femenino , Semivida , Ratones , Cintigrafía , Distribución TisularRESUMEN
Seven patients suffering from maturity on-set diabetes mellitus were given orally 100 mg of 14C-labelled butylbiguanide, specific activity 1.40 or 1.23 muCi/mg, resp. Three days before oral administration, two of the patients had received an i.v. injection of 50 mg butylbiguanide labelled with 120 muCi 14C. The radioactivity in the blood of the patients was followed up during the first 12-h period after administration of the drug. For determination of the radioactivity in the urine aliquots of three 24-h portions were measured. Furthermore, the radioactivity was checked of each individual sample of faeces for the first 72 h after administration. The radioactivity in the exhaled air was also measured. By comparison of the excretion after i.v. and oral application an absorption efficiency of 90% to 92% was calculated. Butylbiguanide is almost exclusively and fast excreted via the kidney. 86.5% of the i.v. administered material was eliminated within 24 h and 88.1% within 3 d in the urine of a person without kidney disease. Elimination through faeces was negligible, 0.2% in a person without kidney disease and 0.7% in a patient with renal insufficiency. The data obtained from the exhaled air show that there is only a negligible break-down of butylbiguanide, if any, to CO2 in man.