RESUMEN
Apolipoprotein (apo) A-I is the major protein component of high-density lipoproteins (HDLs), which are responsible for reverse cholesterol transport from peripheral tissues to the liver. A low level of plasma HDL is correlated with susceptibility to atherosclerosis and coronary heart disease. Mammalian apo A-I synthesis has been attributed mainly to liver and intestine. Recently, apo A-I expression has been shown in porcine brain capillaries, suggesting an independent lipid metabolism within the brain. In this study, protein synthesis and secretion were investigated in primary cultures of porcine brain microvascular endothelial cells and compared with those in large vessel endothelium. Active protein synthesis in vitro was demonstrated by metabolic labeling. Cerebral endothelial cells were shown to secrete apo A-I into the culture supernatant, whereas aortic endothelial cells were negative for apo A-I expression. Further studies of transcriptional regulation showed that cerebral endothelium was responsive to apo A-I-inducing agents, such as cholesterol, insulin, and retinoic acid, as previously shown in human hepatoma HepG2 cells. Thus, cultures of porcine cerebral endothelial cells may represent a suitable model for physiological studies of apo A-I-regulation with regard to brain lipid metabolism and blood-brain barrier function. To investigate the interspecies conservation of regulatory elements, 178 bp of the 5' flanking region of the porcine apo A-I gene was cloned using PCR techniques. Alignments of the cDNA, of the deduced apo A-I protein sequence, and of the 5' promoter region with the corresponding genomic sequences of different species show a high degree of similarity between the porcine and the primate apo A-I genes, thus indicating a similar function and possibly common regulatory mechanisms in those species. In contrast, the rodent and avian apolipoprotein A-I promoter sequences differed significantly.
Asunto(s)
Apolipoproteína A-I/metabolismo , Circulación Cerebrovascular , Endotelio Vascular/metabolismo , Secuencia de Aminoácidos , Animales , Apolipoproteína A-I/genética , Secuencia de Bases , Células Cultivadas , ADN Complementario/genética , Endotelio Vascular/citología , Exones , Técnica del Anticuerpo Fluorescente , Humanos , Intrones , Datos de Secuencia Molecular , Sondas de Oligonucleótidos/genética , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Porcinos , Transcripción Genética , Células Tumorales CultivadasRESUMEN
The translocation of the mRNA in relation to the ribosome during peptide synthesis represents an example for a mechanochemical reaction in which the chemical bond energy of GTP is transformed into coordinated motion. We demonstrate here that translocation can be explained simply by binding equilibria between the tRNA, the mRNA, and their binding sites on the ribosome. The presence of two cognate tRNAs shifts the association constant for the 70 S ribosome . AUGU3 complex from 6.8 x 10(5) to 2.2 x 10(8) M-1. The elongation factor G and GTP or guanosine-5'-(beta,gamma-methylene)triphosphate GMP-PCP) displace the methionine tRNAs which can be formylated (tRNAfMet) from the quaternary complex 70 S . AUGU3 . tRNAfMet . tRNAPhe. Only the ternary complex Phe-tRNAPhe . elongation factor Tu . GMP-PCP shows an absolute preference for the aminoacyl-tRNA binding site (A site) (K a = 6.6 x 10(6) M-1). AcPhe-tRNAPhe, (N alpha-acetylphenylalanyl-tRNA) an analogue of a peptidyl-tRNA exhibits a 20-fold higher affinity to the peptidyl-tRNA binding site (P site) (K a = 3.5 x 10(6) M-1) as against the A site (K a = 1.8 x 10(6) M-1) at 8 mM Mg2+. Compared to aminoacyl-tRNA and tRNA, peptidyl-tRNA shows a 3- to 15-fold higher affinity toward complementary oligonucleotides both in the binary complex and in the presence of 70 S ribosomes (UUCA . AcPhe-tRNAPhe: K a = 1.9 x 10(5) M-1), UUCA . tRNAPhe:K a = 3.2 x 10(4) M-1). This indicates a stabilization of the peptidyl-tRNA . mRNA complex during translocation. Our data support a concept of mRNA translocation in which the removal of the deacylated tRNA from the P site requires GTP energy and a peptidyl-tRNA . mRNA complex diffuses from its low affinity site (A) to its high affinity binding site (P).