Abstract
Translocation of apolipoprotein (apo) B across the endoplasmic reticulum membrane is a likely site for regulation of secretion of very low density lipoproteins from the liver. When primary rat hepatocytes are enriched with the phospholipid phosphatidylmonomethylethanolamine, the secretion of apoB, but not other proteins such as apoprotein A1 and albumin, is disrupted (Vance, J. E. (1991) J. Lipid Res. 32, 1971-1982). Moreover, less apoB enters the microsomal lumen and the intracellular degradation of apoB is increased (Rusiñol, A. E., Chan, E. Y. W., and Vance, J. E. (1993a) J. Biol. Chem. 268, 25168-25175). In the present study we have used McArdle 7777 rat hepatoma cells stably transfected with carboxyl-terminal-truncated variants of human apoB100 and have demonstrated that the reduction in apoB secretion induced by phosphatidylmonomethylethanolamine is not a function of assembly of the apoB into a buoyant lipoprotein particle. In addition, inhibition of the intracellular degradation of the apoproteins B does not restore apoB secretion, suggesting that the effect of phosphatidylmonomethylethanolamine enrichment on apoB degradation is secondary to the effect on translocation of the protein into the endoplasmic reticulum lumen. Furthermore, supplementation of the culture medium with oleic acid does not increase apoB secretion, reduce the intracellular degradation of apoB or reverse the effects of phosphatidylmonomethylethanolamine enrichment on these processes. Our data support the hypothesis that translocation of apoB protein across the endoplasmic reticulum membrane, regardless of the association of the apoB with neutral lipids, may be a key regulatory step in very low density lipoprotein secretion.
Highlights
The iron complex of a-azamesoporphyrin XIII was combined with apomyoglobin to investigate influence of the meso nitrogen on ligand binding properties in the reconstituted protein
The efficient and general synthesis is essential because it first allows us to prepare sufficient quantity of the monoazaporphyrin bearing a molecular architecture like native porphyrins
We employ in the present work o-azamesoporphyrin XIII, a new compound derived through the established synthetic route (Engel et al, 1978), as the prosthetic group of Mb
Summary
Dicarboxylic acid (Fuhrhop and Smith, 1975), was refluxed in 20 ml of methanol for 16 h in the presence of 500 mg of sodium azide (Engel et al, 1978). The resultant erude o-azamesoporphyrin XIII, after evaporation of the solvent, was purified with silica gel column using 2%. Slow evaporation of the concentrated solution from methanol-dichloromethane mixture (1:1, v/v) under low heating afforded the title azaporphyrin as deep violet microneedles (20 mg, 39%). FeClz·nHzO in dimethylformamide under nitrogen atmosphere (Adler et al, 1970), oxidized with air, and purified on a silica gel column with chloroform-methanol (85 mM-I em-I), 457 mixt (8.7), ure (9:1, v/v). The monoazahemin ester was hydrolyzed in a refluxing methanol-water mixture containing 1% KOH as described by Fuhrhop and Smith (1975). ApoMb (Teale, 1959), mixed with a 1.2-equivalent amount of the azahemin, was dialyzed against cold 10 mM bis-Tris buffer, pH 6.0
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