Journal of Lipid Research, Vol 16, 200-210, Copyright © 1975 by Lipid Research, Inc.
Vitamin A transport in chicken plasma: isolation and characterization of retinol-binding protein (RBP), prealbumin (PA), and RBP--PA complex
T Abe, Y Muto and N Hosoya
Vitamin A-transporting protein in chicken plasma was purified by column
chromatography on DEAE-Sephadex and Sephadex G-100; the protein formed a
complex of retinol-binding protein (RBP) with prealbumin (PA). The
molecular weight of the 1:1 molar complex was estimated to be 76,000 by gel
filtration, and the sedimentation coefficient (S20,W) was found to be 5.2
S. RBP and PA were dissociated from the purified complex by means of
CM-Sephadex column chromatography. Purified RBP contained 1 mole of vitamin
A bound per mole of RBP. The molecular weight of RBP was determined to be
20,000 by gel filtration on Sephadex G-75, 19,000 by SDS-disc gel
electrophoresis, and 20,500 by sedimentation equilibrium analysis. The
S20,W was calculated to be 2.0 S. The molecular weight of PA was determined
to be 56,000 by gel filtration, 52,000 by sedimentation equilibrium
analysis, and 13,000 by SDS-disc gel electrophoresis. The S20,W was
calculated to be 3.9 S. From these findings it was concluded that PA
consists of four subunits, each with a molecular weight of approximately
13,000. Peptide mapping experiments suggested that the subunits were
identical. No carbohydrates were detected in either RBP or PA. Chicken RBP
and PA were immunologically distinct from those of human and rat. It is
well established that vitamin A is transported bound to a specific plasma
protein, retinol- binding protein (RBP), in both man (1,2) and rat (3).
Purified human and rat plasma RBP have a single binding site for one
molecule of retinol, alpha mobility on disc gel electrophoresis, and a
molecular weight of approximately 20,000. In both species, RBP forms a
tight complex with plasma prealbumin (PA) and normally circulates as a 1:1
molar protein-protein complex with PA (1-5). Despite these similarities, no
immunological cross-reactivity between human and rat RBP has been observed
(3,6). The present study was undertaken to explore whether or not a similar
transport system for vitamin A exists in the chicken, a nonmammalian
vertebrate. During the course of this study, Mokady and Tal (7) reported
the isolation of RBP from chicken plasma and some physicochemical
properties, e.g., a molecular weight of about 19,000. On the other hand,
Muto, Smith, and Goodman (6) had already observed that the molecular weight
of vitamin A-containing protein in fresh chicken plasma is approximately
60,000-80,000, as determined by gel filtration. However, no convincing
information is available regarding an entire system of vitamin A transport
in chicken plasma. We now describe procedures for the isolation of the
RBA-PA complex of chicken plasma and the dissociation into the component
proteins, RBP and PA. We also describe in detail the physicochemical
properties of the individual proteins. It is also clearly demonstrated that
chicken RBP and PA are immunologically distinct and different from the
respective proteins in man and rat. Moreover, purified chicken PA appears
to be a tetramer of four identical subunits and is thus similar to human
and rat PA.
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