RESUMO
The lipolysis-stimulated receptor (LSR) is a lipoprotein receptor primarily expressed in the liver and activated by free fatty acids. Antibodies inhibiting LSR functions showed that the receptor is a heterotrimer or tetramer consisting of 68-kDa (alpha) and 56-kDa (beta) subunits associated through disulfide bridges. Screening of expression libraries with these antibodies led to identification of mRNAs derived by alternate splicing from a single gene and coding for proteins with molecular masses matching that of LSR alpha and beta. Antibodies directed against a synthetic peptide of LSR alpha and beta putative ligand binding domains inhibited LSR activity. Western blotting identified two liver proteins with the same apparent molecular mass as that of LSR alpha and beta. Transient transfections of LSR alpha alone in Chinese hamster ovary cells increased oleate-induced binding and uptake of lipoproteins, while cotransfection of both LSR alpha and beta increased oleate-induced proteolytic degradation of the particles. The ligand specificity of LSR expressed in cotransfected Chinese hamster ovary cells closely matched that previously described using fibroblasts from subjects lacking the low density lipoprotein receptor. LSR affinity is highest for the triglyceride-rich lipoproteins, chylomicrons, and very low density lipoprotein. We speculate that LSR is a rate-limiting step for the clearance of dietary triglycerides and plays a role in determining their partitioning between the liver and peripheral tissues.
Assuntos
Fígado/metabolismo , Receptores de LDL/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Clonagem Molecular , Cricetinae , DNA Complementar , Cinética , Lipólise , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Receptores de LDL/metabolismo , TransfecçãoRESUMO
A simple and reliable automated method was developed for the detection of amplified products after PCR, which provides an alternative to the time-consuming Southern blotting and hybridization procedure. For the determination and quantification of hepatitis C viremia, the digoxigenin labelling process was applied during the PCR of the amplicons, followed by an inverse hybridization assay performed with biotinylated probes. The detection of the PCR amplified products could be processed as simply as an ELISA, or by means of an automated analyser.