The mechanism of uptake of retinol by plasma-membrane vesicles.
The mechanism of retinol uptake by human placental brush-border membrane vesicles was investigated using initial-velocity studies of [3H]retinol uptake from the [3H]retinol-RBP (retinol-binding protein) complex. The process was rapid and time- and temperature-dependent. The uptake was specifically reversed by the addition of native or apo-RBP, but not by serum albumin. By contrast, uptake of free [3H]retinol was temperature-independent, partially reversible and showed no requirement for a specific protein for reversibility. Treatment of membrane vesicles with p-chloromercuribenzenesulphonate (PCMBS), which inhibited 125I-RBP binding, also inhibited the uptake of retinol from RBP, but the uptake of free retinol was unaffected. Addition of PCMBS after the attainment of steady-state uptake equilibrium abolished the binding of RBP, but did not affect the retinol already taken up from RBP. The results suggest that binding of RBP to its specific receptor is obligatory for the subsequent delivery of retinol to the membrane. Since the studies were carried out on isolated membrane vesicles, endocytosis of RBP is most unlikely to be involved in the placental transport of retinol. A double-reciprocal plot of initial velocity versus [3H]retinol-RBP concentration gave an apparent Km of 116 +/- 13 nM. Transthyretin decreased the rate of uptake of [3H]retinol from RBP without substantially altering the steady-state uptake levels, suggesting that membranes take up retinol from uncomplexed RBP. High-pressure gel-filtration chromatography showed that [3H]retinol is largely transferred to a membrane component with an apparent molecular mass of 125 kDa.
Sivaprasadarao
A
A
Department of Biochemistry, University of Leeds, U.K.
Findlay
J B
JB
D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D002850:Chromatography, Gel; D005260:Female; D006801:Humans; D007700:Kinetics; D008871:Microvilli / Q000187:drug effects / Q000378:metabolism*; D010920:Placenta / Q000187:drug effects / Q000378:metabolism; D011228:Prealbumin / Q000494:pharmacology; D011247:Pregnancy; D012177:Retinol-Binding Proteins / Q000378:metabolism; D054839:Retinol-Binding Proteins, Plasma; D013696:Temperature; D014357:Trypsin / Q000494:pharmacology; D014801:Vitamin A / Q000493:pharmacokinetics*
D011228:Prealbumin; D012177:Retinol-Binding Proteins; D054839:Retinol-Binding Proteins, Plasma; D014801:Vitamin A; D002731:4-Chloromercuribenzenesulfonate; D014357:Trypsin