408s Biochemical Society Transactions ( 1 993) 21
A low molecular weight (12-15kDa) protein fraction in rat
liver binds alpha-tocopherol
was eluted in three peaks; one in the 12-15 kDa molecular
weight fraction, another in the 30-40 kDa fraction and a further
DEREK J. LEISHMAN. FIONA M. CAMPBELL, MARGARET-].
GORDON,GARRY G.DUTHIE. and ASIM K. DUITA-ROY*
8000
Division of Biochemical Sciences, Rowett Research Institute,
Aberdeen, AB2 9SB, Scotland, U.K.
6000
D-a-tocopherol (vitamin E) is an essential dietary
constituent whose major biological role is to protect
polyunsaturated fatty acids and other components of cell
membranes from oxidation by free radicals (1). In addition,
vitamin E may have important roles in biological processes
which do not necessary relate to its function as a biological
antioxidant such as maintaining cell membrane integrity, antiinflammatory effects, DNA synthesis, and inhibiting smooth
muscle cell proliferation (2). Vitamin E is present in the
membranes of intracellular organelles where it plays an
important role in the suppression of lipid peroxidation.
Protection against the peroxidation of membrane lipids by
vitamin E is dependent on its incorporation into membranes
(3.4). and the extent of this protection is related to the quantity
of tocopherol present in the membranes (3). Regulation of atocopherol concentrations in membranes as well as in
intracellular organelles of a cell is therefore important in
mediating the free-radical induced events involved in the
pathogenesis of diseases in men and animals. Although it is
recognised that vitamin E plays an essential role in maintaining
optimum health, little is known about the mechanisms by which
it is absorbed and transported. Vitamin E is transported in
plasma primarily by lipoproteins (5). However, the intracellular
transport of vitamin E is not well understood. Recent studies
suggest that a 32kDa a-tocopherol-binding (TBP) in the liver
regulates plasma vitamin E concentrations by preferentially
incorporating the vitamin E homolog, RRR-a-tocopherol into
nascent VLDL (5,6). However, 32 kDa TBP is unique to the
hepatocyte whereas RRR-a-tocopherol is present in the cells of
all major tissues where it accumulates in organelles where
oxygen radical production is greatest such as heavy and light
mitochondria and endoplasmic reticulum (2). Moreover,
although 32 kDa TBP has been assumed to play a major role in
the discrimination and retention of RRR-a-tocopherol in the
plasma through preferential incorporation into VLDL in the
hepatocyte, no information is available on the distribution and
intracellular transport of RRR-a-tocopherol in liver. We have
recently purified a 14.2 kDa a-tocopherol-binding protein from
rabbit heart cytosol (7). Here we report the identification of a
12-15 kDa protein fraction in the rat liver which binds a tocopherol.
Hooded Lister rats (100-150 g) were killed by cervical
dislocation and their livers were perfused with ice-cold buffer
(20 mM Tris-HCI buffer, pH 7.4, containing 0.25 mM sucrose
and 1 mM EDTA) and then rapidly excised. Rat livers were
prepared after homogenisation of tissues (25g) in 4 volumes of
the same buffer, containing 0.25 mM sucrose, 1 mM EDTA, 1
mM PMSF, 0.145 mM KCI, and 1 mM DTT at 4OC. The
homogenate was then centrifuged at 26o00xg for 10 min. and
the supernatant was recentrifuged at 110,OOOxg for 1h 20 min..
In order to determine which molecular weight fractions bind atocopherol, the supernatant (-25 mg protein, 1 ml) was then
incubated with 100 nM [3H]a-tocopherol (specific activity 55
Ci/mmol) for 30 min. at 23OC and then applied to an FPLC
Sephacryl S-300 column (2.6x60cm) for gel permeation
chromatography. The column was equilibrated with 10 mM
Tris-HCI buffer, pH 7.4 containing 5 mM mercaptoethanol, 0.1
M KCI, 1 mM EDTA and 5% glycerol. The protein was eluted
with the same buffer with a flow rate of 0.75 d m i n at 4OC.
The elution was monitored for protein at 280 nm using UV-M
monitor (Pharmacia, LKB). Fractions of 5 ml were collected,
and protein and radioactivity were measured. [3H]a-tocopherol
'.O
I
h
E
0
4000
0.5
$
?d
e
2000
w
2
0
100
0
200
300
400
Elution h l u m e (ml)
Figure: Gel filtration of rat liver cytosol on Sephacryl S-300
with [3H]a-tocopherol (100 nM).
high molecular weight fraction in the void volume (Figure).
The purification of TBP from 12-15 kDa fraction was carried
out as described (7.8). The supernatant obtained after
centrifugation of rat liver cytosol at llOOOOxg was treated with
70% (NH4)2S04.After centrifugation at 30000xg for 30 min
the supematant was dialysed against 5 mM Tris-HCL buffer,
pH 7.4 at 4OC for 24 h. The dialysed fraction was then
concentrated by ultrafiltration using a filter with mol.wt. cut-off
3500. The concentrated fraction(-20 mg protein) was applied to
a Sephacryl S-300 column as described before. The fractions
which emerged in the elution volume of (250-400 m1)(12-15
kDa fractions) were pooled together, and concentrated.
Binding of [3H]a-tocopherol to the 12-15 kDa fraction was
examined using the Lipidex-lo00 column method (7.9). SDSpolyacrylarnide gel electrophoresis of the active fraction eluted
from the Sephacryl S-300 column showed the partial
purification of the low molecular weight TBP from rat liver
c ytosol.
In conclusion, we have identified and partially purified
the low mol. wt. TBP in the rat liver cytosol. Further
purification and characterisation are needed to understand the
roles of these two (32 kDa and low mol wt. 12-15kDa) protein
in vitamin E metabolism in liver.
We acknowledge the financial support from the Scottish Office
Agriculture and Fisheries Department.
* To whom all correspondence should be addressed
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