RESUMEN
The yeast exocyst complex (also called Sec6/8 complex in higher eukaryotes) is a multiprotein complex essential for targeting exocytic vesicles to specific docking sites on the plasma membrane. It is composed of eight proteins (Sec3, -5, -6, -8, -10, and -15, and Exo70 and -84), with molecular weights ranging from 70 to 144 kDa. Mammalian orthologues for seven of these proteins have been described and here we report the cloning and initial characterization of the remaining subunit, Sec3. Human Sec3 (hSec3) shares 17% sequence identity with yeast Sec3p, interacts in the two-hybrid system with other subunits of the complex (Sec5 and Sec8), and is expressed in almost all tissues tested. In yeast, Sec3p has been proposed to be a spatial landmark for polarized secretion (1), and its localization depends on its interaction with Rho1p (2). We demonstrate here that hSec3 lacks the potential Rho1-binding site and GFP-fusions of hSec3 are cytosolic. Green fluorescent protein (GFP)-fusions of nearly every subunit of the mammalian Sec6/8 complex were expressed in Madin-Darby canine kidney (MDCK) cells, but they failed to assemble into a complex with endogenous proteins and localized in the cytosol. Of the subunits tested, only GFP-Exo70 localized to lateral membrane sites of cell-cell contact when expressed in MDCK cells. Cells overexpressing GFP-Exo70 fail to form a tight monolayer, suggesting the Exo70 targeting interaction is critical for normal development of polarized epithelial cells.
Asunto(s)
Proteínas Portadoras/metabolismo , Transporte de Proteínas , Proteínas de Saccharomyces cerevisiae , Secuencia de Aminoácidos , Animales , Línea Celular , Polaridad Celular , Clonación Molecular , Perros , Exocitosis , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Perfilación de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Sustancias Macromoleculares , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Complejos Multiproteicos , Unión Proteica , Estructura Terciaria de Proteína , Subunidades de Proteína , ARN Mensajero/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Técnicas del Sistema de Dos Híbridos , Proteínas de Transporte VesicularRESUMEN
A novel microsomal beta-glucosidase was recently purified and characterized from human liver that catalyzes the hydrolysis of bile acid 3-O-glucosides as endogenous compounds. The primary structure of this bile acid beta-glucosidase was deduced by cDNA cloning on the basis of the amino acid sequences of peptides obtained from the purified enzyme by proteinase digestion. The isolated cDNA comprises 3639 base pairs containing 524 nucleotides of 5'-untranslated and 334 nucleotides of 3'-untranslated sequences including the poly(A) tail. The open reading frame predicts a 927-amino acid protein with a calculated M(r) of 104,648 containing one putative transmembrane domain. Data base searches revealed no homology with any known glycosyl hydrolase or other functionally identified protein. The cDNA sequence was found with significant identity in the human chromosome 9 clone RP11-112J3 of the human genome project. The recombinant enzyme was expressed in a tagged form in COS-7 cells where it displayed bile acid beta-glucosidase activity. Northern blot analysis of various human tissues revealed high levels of expression of the bile acid beta-glucosidase mRNA (3.6-kilobase message) in brain, heart, skeletal muscle, kidney, and placenta and lower levels of expression in the liver and other organs.
Asunto(s)
Ácidos y Sales Biliares/metabolismo , beta-Glucosidasa/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células COS , Clonación Molecular , ADN Complementario , Etiquetas de Secuencia Expresada , Humanos , Datos de Secuencia Molecular , beta-Glucosidasa/metabolismoRESUMEN
Now that whole genome sequences are available for many eukaryotic organisms from yeast to man, we can form broad hypotheses on the basis of the relative expansion of protein families. To investigate the molecular mechanisms responsible for the organization of membrane compartments, we identified members of the SNARE, coat complex, Rab and Sec1 protein families in four eukaryotic genomes. Of these families only the Rab family expanded from the unicellular yeast to the multicellular fly and worm. All families were expanded in humans, where we find 35 SNAREs, 60 Rabs and 53 coat complex subunits. In addition, we were able to resolve the SNARE class of proteins into four distinct subfamilies.
Asunto(s)
Membrana Celular/fisiología , Proteína Coat de Complejo I/fisiología , Proteínas de la Membrana/fisiología , Proteínas del Tejido Nervioso/fisiología , Proteínas de Transporte Vesicular , Proteínas de Unión al GTP rab/fisiología , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiología , Compartimento Celular , Membrana Celular/genética , Proteína Coat de Complejo I/genética , Drosophila melanogaster/genética , Drosophila melanogaster/fisiología , Genoma , Genoma Humano , Humanos , Proteínas de la Membrana/genética , Proteínas Munc18 , Proteínas del Tejido Nervioso/genética , Proteínas SNARE , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Proteínas de Saccharomyces cerevisiae , Vesículas Transportadoras/genética , Vesículas Transportadoras/fisiología , Proteínas de Unión al GTP rab/genéticaRESUMEN
Through two-hybrid interactions, protein affinity and localization studies, we previously identified Yip1p, an integral yeast Golgi membrane protein able to bind the Ras-like GTPases Ypt1p and Ypt31p in their GDP-bound conformation. In a further two-hybrid screen, we identified Yif1p as an interacting factor of Yip1p. We show that Yif1p is an evolutionarily conserved, essential 35.5 kDa transmembrane protein that forms a tight complex with Yip1p on Golgi membranes. The hydrophilic N-terminal half of Yif1p faces the cytosol, and according to two-hybrid analyses can interact with the transport GTPases Ypt1p, Ypt31p and Sec4p, but in contrast to Yip1p, this interaction is dispensable for Yif1 protein function. Loss of Yif1p function in conditional-lethal mutants results in a block of endoplasmic reticulum (ER)-to-Golgi protein transport and in an accumulation of ER membranes and 40-50 nm vesicles. Genetic analyses suggest that Yif1p acts downstream of Yip1p. It is inferred that Ypt GTPase binding to the Yip1p-Yif1p complex is essential for and precedes vesicle docking and fusion.
Asunto(s)
GTP Fosfohidrolasas/metabolismo , Aparato de Golgi/metabolismo , Proteínas de la Membrana/fisiología , Proteínas de Saccharomyces cerevisiae , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Unión Proteica , Homología de Secuencia de Aminoácido , Proteínas de Transporte VesicularRESUMEN
The regulation of vesicular transport in eukaryotic cells involves Ras-like GTPases of the Ypt/Rab family. Studies in yeast and mammalian cells indicate that individual family members act in vesicle docking/fusion to specific target membranes. Using the two-hybrid system, we have now identified a 248 amino acid, integral membrane protein, termed Yip1, that specifically binds to the transport GTPases Ypt1p and Ypt31p. Evidence for physical interaction of these GTPases with Yip1p was also demonstrated by affinity chromatography and/or co-immunoprecipitation. Like the two GTPases, Yip1p is essential for yeast cell viability and, according to subcellular fractionation and indirect immunofluorescence, is located to Golgi membranes at steady state. Mutant cells depleted of Yip1p and conditionally lethal yip1 mutants at the non-permissive temperature massively accumulate endoplasmic reticulum membranes and display aberrations in protein secretion and glycosylation of secreted invertase. The results suggests for a role for Yip1p in recruiting the two GTPases to Golgi target membranes in preparation for fusion.
Asunto(s)
GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/metabolismo , Aparato de Golgi/metabolismo , Proteínas de Saccharomyces cerevisiae , Proteínas de Unión al GTP rab , Secuencia de Aminoácidos , Transporte Biológico , Compartimento Celular , GTP Fosfohidrolasas/genética , Proteínas de Unión al GTP/genética , Glicosilación , Datos de Secuencia Molecular , Unión Proteica , Procesamiento Proteico-Postraduccional , Proteínas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fracciones SubcelularesRESUMEN
A human liver microsomal beta-glucosidase has been purified to apparent homogeneity in sodium dodecyl sulfate-polyacrylamide gel electrophoresis where a single protein band of Mr 100,000 was obtained under reducing conditions. The enzyme was enriched about 73, 000-fold over starting microsomal membranes by polyethylene glycol fractionation, anion exchange chromatographies on DEAE-Trisacryl, and Mono Q followed by affinity chromatography on N-(9-carboxynonyl)-1-deoxynojirimycin-AH-Sepharose 4B. The purified enzyme had a pH optimum between 5.0 and 6.4, was activated by divalent metal ions, and required phospholipids for exhibition of activity. The enzyme catalyzed the hydrolysis of 3beta-D-glucosido-lithocholic and 3beta-D-glucosido-chenodeoxycholic acids with high affinity (Km, 1.7 and 6.2 microM, respectively) and of the beta-D-glucoside (Km, 210 microM) and the beta-D-galactoside of 4-methylumbelliferone. The ratio of relative reaction rates for these substrates was about 6:3:11:1. No activity was detectable toward 6beta-D-glucosido-hyodeoxycholic acid, glucocerebroside, and the following glycosides of 4-methylumbelliferone: alpha-D-glucoside, alpha-L-arabinoside, beta-D-fucoside or beta-D-xyloside. Immunoinhibition and immunoprecipitation studies using antibodies prepared against lysosomal glucocerebrosidase showed no cross-reactivity with microsomal beta-glucosidase suggesting that these two enzymes are antigenically unrelated.
Asunto(s)
Ácidos y Sales Biliares/metabolismo , Glucósidos/metabolismo , Microsomas Hepáticos/enzimología , beta-Glucosidasa/aislamiento & purificación , Ácido Quenodesoxicólico/análogos & derivados , Ácido Quenodesoxicólico/metabolismo , Cromatografía de Afinidad , Reacciones Cruzadas , Detergentes/farmacología , Inhibidores Enzimáticos/farmacología , Glucosilceramidasa/inmunología , Humanos , Ácido Litocólico/análogos & derivados , Ácido Litocólico/metabolismo , Fosfolípidos/farmacología , Especificidad por Sustrato , beta-Glucosidasa/antagonistas & inhibidores , beta-Glucosidasa/inmunología , beta-Glucosidasa/metabolismoRESUMEN
A gene (brnQ), encoding a carrier for branched-chain amino acids in Lactobacillus delbrückii subsp. lactis DSM7290 was cloned in the low-copy-number vector pLG339 by complementation of a transport-deficient Escherichia coli strain. The plasmid carrying the cloned gene restored growth of an E. coli strain mutated in 4 different branched-chain amino acid transport genes at low concentrations of isoleucine, and increased its sensitivity to valine. Transport assays showed that leucine, isoleucine and valine are transported by this carrier and that transport is driven by the proton motive force. Nucleotide sequence analysis revealed an open reading frame of 1338 bp encoding a hydrophobic protein of 446 amino acids with a calculated molecular mass of 47864 Daltons. The start site of brnQ transcription was determined by primer extension analysis using mRNA from Lactobacillus delbrückii subsp. lactis DSM7290. The hydropathy profile suggests the existence of at least 12 hydrophobic domains that probably form membrane-associated alpha-helices. Comparisons of the nucleotide sequence of brnQ from Lactobacillus delbrückii subsp. lactis DSM7290, the amino acid sequence of its product and the topology of the hydrophobic domains with those of the respective carrier genes and proteins of Salmonella typhimurium and Pseudomonas aeruginosa revealed extensive homology.
Asunto(s)
Aminoácidos de Cadena Ramificada/metabolismo , Proteínas Bacterianas/genética , Proteínas Portadoras/genética , Genes Bacterianos , Lactobacillus/genética , Secuencia de Aminoácidos , Secuencia de Bases , Transporte Biológico , Clonación Molecular , Escherichia coli/genética , Eliminación de Gen , Prueba de Complementación Genética , Datos de Secuencia Molecular , Conformación Proteica , Análisis de Secuencia de ADN , Homología de Secuencia , Especificidad de la EspecieRESUMEN
From a genomic library of Lactobacillus delbrueckii subsp. lactis (DSM7290) DNA, in the low-copy-number vector pLG339, a recombinant clone was selected, which complemented a mutation in the prolidase gene (pepQ) of Escherichia coli UK173. Nucleotide sequence analysis revealed an open reading frame of 1104 nucleotides corresponding to a protein of 368 amino acids with a calculated pI of 4.64 and a molecular mass of 41,087 Da. The start site of pepQ transcription was determined by primer extension analysis with mRNA prepared from L. delbrueckii. Based on homology of the gene product to various peptidases and on the substrate specificity determined, the peptidase was designated PepQ. The influence of various protease inhibitors and cations on peptidase activity indicated that PepQ is a metalloprotease. The absence of a membrane-spanning domain and a signal peptide sequence argues for a cytoplasmic localization of the enzyme.
Asunto(s)
Dipeptidasas/genética , Genes Bacterianos , Lactobacillus/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Dipeptidasas/metabolismo , Metaloendopeptidasas/genética , Datos de Secuencia Molecular , Filogenia , Regiones Promotoras Genéticas , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
A genomic library of Lactobacillus delbrueckii subsp. lactis DSM7290 DNA fragments from a Sau3A partial digestion in the low-copy-number vector pLG339, was used to screen Escherichia coli for the presence of peptidases. Using the chromogenic substrate leucine-beta-naphthylamide (Leu-NH-Nap) and E. coli strain CM89 lacking the corresponding enzyme activity in an enzymic plate assay, allowed the isolation of two peptidase genes; the newly described pepL and the recently cloned and sequenced pepN. Clones could be distinguished not only by the restriction pattern of isolated plasmids but also by the rate and intensity of their colour reaction with Leu-NH-Nap. Three out of five clones were identified to express the Lactobacillus pepN gene; the others were shown to express a second aminopeptidase gene, designated pepL. This gene, together with 200 bp upstream of the proposed AUG initiation codon, was further subcloned and sequenced. The corresponding open reading frame of 897 nucleotides is predicted to encode a protein of 299 amino acids (34,541 Da). Searching the EMBL database revealed similarity to the prolinase of Lactobacillus helveticus (45.8% identity), to the iminopeptidases of Lb. delbrueckii subsp. lactis and Lb. delbrueckii subsp. bulgaricus (25.5%), and to the Bacillus coagulans prolinase (21.5%). Minor similarities were detected for hydrolytic enzymes with serine active sites. The product encoded by the pepL gene was functional but could not be visualized on Coomassie-blue-stained polyacrylamide gels. High level expression of peptidase L in E. coli was achieved by placing the gene under the control of the T7 promoter.
Asunto(s)
Lactobacillus/genética , Leucil Aminopeptidasa/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Bacteriano , Hidrólisis , Lactobacillus/enzimología , Leucil Aminopeptidasa/metabolismo , Datos de Secuencia Molecular , ARN Mensajero/genética , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
Formation of bile acid glucosides occurs in rat liver homogenate with a specific enzyme activity of 0.014 +/- 0.001 nmol per min per mg protein. Subcellular fractionation of rat liver by differential centrifugation revealed an enrichment of bile acid glucosyltransferase activity both in the mitochondrial-lysosomal fraction and in microsomes with a recovery of 38.8 +/- 4.6% and 37.7 +/- 1.7%, respectively, of enzyme activity in the homogenate. Subfractionation of the mitochondrial-lysosomal fraction after treatment of rats with Triton WR 1339 showed an almost exclusive association of bile acid glucosyltransferase activity with purified lysosomes ("tritosomes"). After subfractionation of microsomes by analytical gradients, bile acid glucosyltransferase was bimodally distributed with peaks at modal densities of 1.09 g/cm3 and 1.16 g/cm3, respectively. If microsomes were pretreated with pyrophosphate, a membrane perturbant known to strip ribosomes, only the peak of bile acid glucosyltransferase at higher density (1.16 g/cm3) and UDP-glucuronosyltransferase (marker of endoplasmic reticulum) shifted to a similar lower equilibrium density. Both enzymes were unaffected in their distribution by pretreatment of microsomes with digitonin. In contrast, markers of plasma membranes (5'-nucleotidase) and the Golgi-complex (galactosyltransferase) shifted to higher equilibrium densities after digitonin treatment, but were unaltered in their distribution after pyrophosphate. Bile acid glucosyltransferase activity in the lower density range with a peak at 1.09 g/cm3 did not show any association with the density distributions of known marker enzymes. In purified microsomal fractions obtained by preparative gradients, bile acid glucosyltransferase activity was enriched in enzyme activity by 1.4-fold in rough and by 2.3-fold in smooth endoplasmic reticulum, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Ácidos y Sales Biliares/metabolismo , Glucosiltransferasas/metabolismo , Hígado/enzimología , Fracciones Subcelulares/metabolismo , Animales , Centrifugación , Retículo Endoplásmico/enzimología , Femenino , Aparato de Golgi/enzimología , Lisosomas/enzimología , Microsomas/enzimología , Ratas , Ratas Sprague-Dawley , Distribución TisularRESUMEN
Cell extracts of Lactobacillus delbrueckii subsp. lactis DSM 7290 were found to exhibit unique peptolytic ability against unusual beta-alanyl-dipeptides. In order to clone the gene encoding this activity, designated pepV, a gene library of strain DSM 7290 genomic DNA, prepared in the low-copy-number plasmid pLG339, was screened for heterologous expression in Escherichia coli. Recombinant clones harbouring pepV were identified by their ability to allow the utilization of carnosine (beta-alanyl-histidine) as a source of histidine by the E. coli mutant strain UK197 (pepD, hisG). Complementation was observed in a colony harbouring a recombinant plasmid (pKV101), carrying pepV. A 2.4 kb fragment containing pepV was subcloned and its nucleotide sequence revealed an open reading frame (ORF) of 1413 nucleotides, corresponding to a protein with predicted molecular mass of 51998 Da. A single transcription initiation site 71 bp upstream of the ATG translational start codon was identified by primer extension. No significant homology was detected between pepV or its deduced amino acid sequence with any entry in the databases. The only similarity was found in a region conserved in the ArgE/DapE/CPG2/YscS family of proteins. This observation, and protease inhibitor studies, indicated that pepV is of the metalloprotease type. A second ORF present in the sequenced fragment showed extensive homology to a variety of amino acid permeases from E. coli and Saccharomyces cerevisiae.
Asunto(s)
Dipeptidasas/genética , Lactobacillus/genética , Secuencia de Aminoácidos , Secuencia de Bases , Sondas de ADN , Dipeptidasas/aislamiento & purificación , Activación Enzimática/genética , Escherichia coli/genética , Lactobacillus/enzimología , Datos de Secuencia Molecular , Plásmidos , ARN Bacteriano/genética , Saccharomyces cerevisiae/genética , Alineación de SecuenciaRESUMEN
In order to establish the position of conjugation of bile acids with glucose or N-acetylglucosamine, glucosides of chenodeoxycholic and hyodeoxycholic acids and of 13C-labeled cholic, lithocholic, chenodeoxycholic, hyodeoxycholic, and ursodeoxycholic acids, and N-acetylglucosaminides of ursodeoxycholic, isoursodeoxycholic, 3-dehydro-ursodeoxycholic, and ursodeoxycholylglycine were synthesized in vitro. The conjugates were purified by anion-exchange chromatography and reversed-phase HLPC and were analyzed by gas chromatography-mass spectrometry. The glucosides of chenodeoxycholic and hyodeoxycholic acids were also analyzed after periodate and chronic acid oxidation. All conjugates were analyzed by fast atom bombardment mass spectrometry with collision-induced dissociation. Glucose conjugation was shown to occur at C-3 in all bile acid glucosides studied. In contrast, the selective N-acetylglucosaminidation of 7 beta-hydroxy bile acids was shown to occur at the 7 beta-position.
Asunto(s)
Ácidos y Sales Biliares/química , Glicoconjugados/química , Acetilglucosamina/química , Ácidos y Sales Biliares/síntesis química , Ácidos y Sales Biliares/metabolismo , Ácido Quenodesoxicólico/análogos & derivados , Ácido Quenodesoxicólico/química , Ácido Quenodesoxicólico/metabolismo , Ácido Desoxicólico/síntesis química , Ácido Desoxicólico/química , Ácido Desoxicólico/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Glucosa/química , Glicoconjugados/síntesis química , Glicoconjugados/metabolismo , Humanos , Técnicas In Vitro , Estructura Molecular , Espectrometría de Masa Bombardeada por Átomos VelocesRESUMEN
The transcriptional start points of ten Lactobacillus delbrückii ssp. lactis DSM7290 genes were determined by primer extension. The upstream located promoter regions, including potential -35 and -10 regions and the spacing between them were compared to the well-known Escherichia coli and Bacillus subtilis promoters. The Lb. delbrückii -35 consensus sequence (TTGACA) seems to be less conserved then the E. coli sequence. The nucleotides TGC were often found upstream of the -10 region (TATAAT). The most frequently observed spacing between the two core promoter regions was 17 nt and the main distance between the -10 region and the transcriptional start point was mostly determined to be 6 nt in contrast to 7 nt, as described for E. coli promoters. The preferred initiation nucleotides in Lb. delbrückii were shown to be definitely purines (A or G). The ribosome binding sites located downstream of the promoters revealed the consensus sequence 3'-UCCUCCU-5', being the predicted 3'-OH end of the Lactobacillus 16S rRNA with a high degree of homology to known 16S rRNAs.
Asunto(s)
Genes Bacterianos/genética , Lactobacillus/genética , Regiones Promotoras Genéticas/genética , Secuencia de Bases , Secuencia Conservada , Datos de Secuencia Molecular , Transcripción GenéticaRESUMEN
A rapid and sensitive radioassay for measuring UDP-glucuronosyltransferase activities (EC 2.4.1.17) toward the major endogenous substrates hyodeoxycholic and hyocholic acids, bilirubin, estriol, androsterone, and testosterone has been developed. In this assay, 14C-labeled glucuronides are formed from the enzyme-catalyzed reaction of 14C-labeled UDP-glucuronic acid with the unlabeled aglycones. Following incubation, the 14C-labeled glucuronides are separated under acidic conditions from the unreacted 14C-labeled UDP-glucuronic acid by a single extraction with ethyl acetate. The recovery of glucuronides into ethyl acetate was greater than 90%, whereas the carryover of unreacted UDP-glucuronic acid into the organic phase was approximately 0.2%. The reaction products extracted into ethyl acetate were characterized by their mobilities in thin-layer chromatography and identified as glucuronides by their sensitivity to hydrolysis with beta-glucuronidase and inhibition of hydrolysis by the specific beta-glucuronidase inhibitor D-saccharic acid-1,4-lactone. The optimal conditions of enzyme reactions with the individual aglycones have been defined with human liver microsomes as enzyme source. For all aglycones investigated, 10-30 micrograms of microsomal protein are sufficient for enzyme estimation. The assay is applicable to biochemical studies of UDP-glucuronosyltransferases, as well as to measurement of these enzyme activities from small amounts of clinical liver specimens.
Asunto(s)
Autorradiografía/métodos , Glucuronosiltransferasa/análisis , Uridina Difosfato Ácido Glucurónico/metabolismo , Radioisótopos de Carbono , Glucuronosiltransferasa/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Sensibilidad y Especificidad , Solventes , Especificidad por SustratoRESUMEN
Uridine 5'-diphosphate-glucose dolichyl-phosphate glucosyltransferase and bile acid glucosyltransferase were quantitatively determined in subcellular fractions obtained by differential centrifugation of human liver homogenate. Both enzymes were exclusively enriched in the microsomal fraction with a recovery of total enzyme activity of 65.9 +/- 9.9% and 69.1 +/- 13.8%, respectively. Microsomal preparations were further subfractionated by isopycnic centrifugation on a continuous sucrose density gradient. Both glucosyltransferases closely followed marker constituents of endoplasmic reticulum, as shown by similar distribution profiles in the gradient, but differed in their quantitative distribution among the endoplasmic reticulum membranes. The bile acid glucosyltransferase showed an almost identical distribution with NADPH-cytochrome c reductase as marker of smooth endoplasmic reticulum with a modal density of 1.16 g/cm3. The uridine 5'-diphosphate-glucose dolichyl-phosphate glucosyltransferase equilibrated at a higher density with a peak at a model density of 1.174 g/cm3. Its marked overlap with the distribution of NADPH-cytochrome c reductase suggests that the major activity of uridine 5'-diphosphate-glucose dolichyl-phosphate glucosyltransferase is also associated with smooth endoplasmic reticulum membranes, whereas minor proportions of enzyme activity are present in the rough endoplasmic reticulum. Association of both glucosyltransferases with membranes derived from Golgi-complex or plasma membranes could be excluded by treatment of microsomes with membrane reagents prior to isopycnic centrifugation. Digitonin did not alter the equilibrium densities of the glucosyltransferases and endoplasmic reticulum markers in contrast to markers of plasma membranes and the Golgi-complex shifting to higher densities. The reversed effect was observed in case of pretreatment of microsomes with pyrophosphate known to detach ribosomes.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Ácidos y Sales Biliares/metabolismo , Retículo Endoplásmico/enzimología , Glucosiltransferasas/análisis , Microsomas Hepáticos/enzimología , Anciano , Centrifugación por Gradiente de Densidad , Humanos , Microscopía Electrónica , Persona de Mediana EdadRESUMEN
Human liver contains hydrolytic activity toward 3 beta-glucosido-chenodeoxycholic acid. This beta-glucosidase activity, localized predominantly in the microsomal fraction, was optimally active in the presence of divalent metal ions close to pH 5.0 and was inhibited by EDTA. Kinetic parameters and other catalytic properties of hydrolytic activity towards 3 beta-glucosido-chenodeoxycholic acid from human liver microsomes are described.
Asunto(s)
Hígado/enzimología , beta-Glucosidasa/metabolismo , Cationes Bivalentes/farmacología , Compartimento Celular , Ácido Quenodesoxicólico/análogos & derivados , Ácido Edético/farmacología , Humanos , Concentración de Iones de Hidrógeno , Microsomas/enzimología , Fracciones Subcelulares/enzimología , beta-Glucosidasa/antagonistas & inhibidoresRESUMEN
The aim of this study was to define whether N-acetylglucosaminidation is a selective conjugation pathway of structurally related bile acids in humans. The following bile acids released enzymatically from N-acetylglucosaminides were identified: 3 alpha,7 beta-dihydroxy-5 beta-cholanoic (ursodeoxycholic), 3 beta, 7 beta-dihydroxy-5 beta-cholanoic (isoursodeoxycholic), 3 beta,7 beta-dihydroxy-5 alpha-cholanoic (alloisoursodeoxycholic), 3 beta,7 beta-dihydroxy-5-cholenoic, 3 alpha,7 beta,12 alpha-trihydroxy-5 beta-cholanoic, and 3 alpha,6 alpha,7 beta-trihydroxy-5 beta-cholanoic acids. The selectivity of conjugation was studied by administration of 0.5 g ursodeoxycholic (UDCA) or hyodeoxycholic (HDCA) acids, labeled with 13C, to patients with extrahepatic cholestasis, and of 0.5 g of 13C-labeled chenodeoxycholic acid (CDCA) to patients with extra- or intrahepatic cholestasis. After administration of [24-13C]-CDCA, labeled glucosides, and the glucuronide of CDCA were excreted in similar amounts. Labeled N-acetylglucosaminides of UDCA and isoUDCA were also formed. When [24-13C]-UDCA was given, 13C-label was detected in the N-acetylglucosaminide, the glucosides, and the glucuronide of UDCA, and in the N-acetylglucosaminide of isoUDCA. In the patient studied, 32% of the total UDCA excreted in urine was conjugated with N-acetylglucosamine. In contrast, 96% of the excreted amount of [24-13C]HDCA was glucuronidated, and 13C-labeled glucosides but no N-acetylglucosaminide were detected. The selectivity of N-acetylglucosaminidation towards bile acids containing a 7 beta-hydroxyl group was confirmed in vitro using human liver and kidney microsomes and uridine diphosphate glucose (UDP)-N-acetylglucosamine. These studies show that N-acetylglucosaminidation is a selective conjugation pathway for 7 beta-hydroxylated bile acids.
Asunto(s)
Acetilglucosamina/metabolismo , Ácidos y Sales Biliares/metabolismo , Administración Oral , Ácidos y Sales Biliares/administración & dosificación , Ácido Quenodesoxicólico/metabolismo , Colestasis/metabolismo , Ácido Desoxicólico/metabolismo , Glicósidos/orina , Humanos , Hidroxilación , Hepatopatías/metabolismo , Espectrometría de Masas , Ácido Ursodesoxicólico/metabolismoRESUMEN
In order to study the glycosidic conjugation of chenodeoxycholic, hyodeoxycholic, and ursodeoxycholic acids in patients with cholestasis after oral administration of pharmacological amounts of the respective bile acids avoiding the application of radioactive tracers we synthesized [24-13C]chenodeoxycholic, [24-13C]hyodeoxycholic, and [24-13C]ursodeoxycholic acids. The reaction intermediates of the bile acid syntheses were characterized by infrared spectroscopy. Purity was confirmed using thin-layer chromatography as well as gas chromatography-mass spectrometry. The 13C atom excess of approximately 90% of the synthesized bile acids was the same as the 13C atom excess of the sodium [13C]cyanide used for the labeling reaction confirming the successful synthesis. After oral administration of 0.5 g of [24-13C]ursodeoxycholic acid to a healthy volunteer, 13C label was detected in the nonamidated and glycine- or taurine conjugated glucosides and the N-acetylglucosaminide of ursodeoxycholic acid in urine. This establishes ursodeoxycholic acid as the first bile acid so far known to undergo both of the recently described glycosidic conjugation reactions in humans.
Asunto(s)
Ácido Quenodesoxicólico/síntesis química , Colestasis/metabolismo , Ácido Desoxicólico/síntesis química , Marcaje Isotópico , Ácido Ursodesoxicólico/síntesis química , Administración Oral , Isótopos de Carbono , Ácido Quenodesoxicólico/administración & dosificación , Ácido Quenodesoxicólico/metabolismo , Colestasis/tratamiento farmacológico , Cromatografía en Capa Delgada , Ácido Desoxicólico/administración & dosificación , Ácido Desoxicólico/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Humanos , Espectrofotometría Infrarroja , Ácido Ursodesoxicólico/administración & dosificación , Ácido Ursodesoxicólico/metabolismoRESUMEN
The enzyme hyodeoxycholic-acid: UDP-glucuronosyltransferase was purified about 230-fold from a solubilized human liver microsomal preparation utilizing anion-exchange chromatography, ampholyte-displacement chromatography and UDP-hexanolamine--Sepharose affinity chromatography. The homogeneity of the final enzyme preparation was judged by two criteria: the appearance of a single band of Mr 52000 in SDS/PAGE; the elution of a single peak in reversed-phase FPLC. The isolated enzyme catalyzed the glucuronidation of the 6 alpha-hydroxy bile acids hyodeoxycholic and hyocholic acids, and of the steroid hormone estriol, with a ratio of relative reaction rates of 13:1:2.7. UDP-glucuronosyltransferase activities toward the 3 alpha-hydroxy bile acid lithocholic acid, androsterone, testosterone, bilirubin and p-nitrophenol were not detectable in the pure enzyme preparation and were shown to be separated from enzyme activity toward hyodeoxycholic acid during ampholyte-displacement chromatography and/or UDP-hexanolamine--Sepharose affinity chromatography. Two-substrate kinetic analysis of hyodeoxycholic-acid-conjugating activity gave a sequential mechanism with apparent Km values of 12 microM and 4 microM for hyodeoxycholic acid and UDP-glucuronic acid, respectively. Phospholipids were required for reconstitution of maximal activity toward hyodeoxycholic acid. Phosphatidylcholine was the most effective activator of enzyme activity.
Asunto(s)
Ácido Desoxicólico/aislamiento & purificación , Glucuronosiltransferasa/aislamiento & purificación , Microsomas Hepáticos/enzimología , Ácidos y Sales Biliares/metabolismo , Cromatografía Liquida , Ácido Desoxicólico/metabolismo , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Glucuronosiltransferasa/metabolismo , Humanos , Cinética , Fosfolípidos/metabolismo , Especificidad por Sustrato , Testosterona/metabolismoRESUMEN
Recently the formation of bile acid glucosides has been described as a novel conjugation mechanism in vitro and in vivo. In 10 patients with extrahepatic cholestasis caused by carcinoma of the head of the pancreas we investigated excretion rates and profiles of urinary bile acid glucosides. Urinary bile acid glucosides and, for comparison, bile acid glucuronides were extracted and characterized according to established methods. In controls total urinary bile acid glucoside excretion was 0.22 +/- 0.03 mumol/24 hr (mean +/- S.E.M.)-in the range of bile acid glucuronide excretion (0.41 +/- 0.06 mumol/24 hr; mean +/- S.E.M.). A gas chromatography-mass spectrometry-characterized trihydroxy bile acid glucoside of still-unknown hydroxyl positions accounted for 65% of total urinary bile acid glucosides. In extrahepatic cholestasis total urinary bile acid glucoside excretion was 0.52 +/- 0.13 mumol/24 hr (mean +/- SEM), yet significantly lower than bile acid glucuronide excretion (1.53 +/- 0.13 mumol/24 hr; mean +/- SEM; p less than 0.001). In cholestasis the primary bile acid derivatives cholic and chenodeoxycholic acid glucosides amounted to 90%, whereas the trihydroxy bile acid glucoside had decreased to 5% of total bile acid glucoside excretion, indicating its alteration during enterohepatic circulation. The data establish the composition and quantity of urinary bile acid glucosides in healthy controls and cholestasis and constitute a quantitative comparison with another glycosidic conjugation reaction, bile acid glucuronidation.