Abstract
We have investigated the steps by which retinol, released from plasma retinol-binding protein (RBP), enters the cells and is accumulated for the most part as a retinyl-ester, only a small fraction of it being present as a complex with cytoplasmic retinol-binding protein (CRBP). For this purpose, we have developed a cell-free system composed of plasma membrane-enriched fractions from bovine retinal pigment epithelium which selectively incorporates exogenous vitamin A when presented as a retinol-RBP complex. Upon incubation in the presence of [3H]retinol-RBP, isolated plasma membrane fractions take up and esterify retinol. A 4-fold reduction of total vitamin A incorporation is observed in conditions which specifically inhibit retinyl-ester formation, thus indicating that the two processes of retinol uptake and esterification are functionally coupled. Evidence is presented that retinol bound to a plasma membrane receptor sharing functional and structural similarities with CRBP is the actual substrate for esterification. Vitamin A accumulation seems to require retinol esterification to allow the recycling of a limited number of free, plasma membrane-associated, retinol receptors. Mobilization of retinol stored as a membrane-bound retinyl-ester is mediated by a membrane-associated hydrolase activity selectively controlled by the level of apo-CRBP which acts as a carrier for the released retinol. Up to 90% of membrane-bound vitamin A is released upon incubation in the presence of apo-CRBP (11 microM) with concomitant formation of retinol-CRBP. The overall process, in which retinol never needs to leave its binding proteins, allows the accumulation of vitamin A in the form of a membrane-bound retinyl-ester and its regulated mobilization as a retinol-CRBP complex.
Highlights
RETINOL TRANSFER FROM PLASMA RETINOL-BINDING PROTEIN TO CYTOPLASMIC RETINOL-BINDING PROTEIN WITH RETINYL-ESTER FORMATION AS THE INTERMEDIATE STEP*
As a complex with cytoplasmic retinol-binding protein Dietary vitamin A, which is absorbed as a retinyl-ester from (CRBP)
We have developed a cell- the intestine, is transported to the liver in association with free system composed of plasma membrane-enriched the chylomicrons and is released into the circulatory system fractionsfrombovineretinapl igmenet pithelium after having beencomplexedwitha protein called retinolwhich selectively incorporates exogenous vitamin A binding protein(RBP‘; mass = 21 kDa)
Summary
This result was interpreted as evidencefor the + retinyl-esters) present as retinyl-ester was determined by existence of a specificplasma membrane receptor for retinol. TLC analysis using retinyl palmitate as standard. To gain a mordeetailed understandingof the uptakeprocess of the membranes prior to the uptake experiment did not and of its possible relation with theformation of retinyl- affect this result (TableI), showing that retinol uptake esters, we havedeveloped a call-free system, composed of and esterification are authentic membrane processes which plasmamembrane-enrichedfractions frombovine RPE, do notdepend on theavailability of soluble components. Which carries out the uptake and esterificatioonf retinol and Since membrane fraction P 3 is not pure, we cannot rigorthe hydrolysis of membrane bound retinyl-esters. We describe the propertiesof this system and demon- of retinol esterification.
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