Biochemical Society Transactions
Difference in patterns of proteins isolated from polysomes in free, cytoskeletonbound and membrane-bound fractions in MPC-I I cells incubated with insulin
Rigmor Moss, Ian F. Pryme and Anni Vedeler
Department of Biochemistry, University of Bergen, Arstadveien 19, N-5009 Bergen, Norway
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W e have earlier described the isolation and characterization of free (FP), cytoskeleton-bound (CRP)
and membrane-bound (MBP) polysomes in Krebs
I1 ascites and 3T3 cells [ 13. Using Northern hybridization the three polysome fractions were shown to
contain different amounts of individual mRNA
species (actin, c-myc, /3-2-microglobulin) [2, 31.
It is known that insulin causes an increase in
protein synthesis during short-term incubation in a
Abbreviations used: FI', free polysomes; CRP, cytoskeleton-bound polysomes; MHI', membrane-bound
polysomes.
variety of cell types [4].The increase is a result of
regulation of the rate of peptide-chain initiation.
Both insulin and step-up conditions were shown to
cause changes in the distribution of ribosomes
within the three polysome populations during
short-term incubation [S]. It is thus apparent that
ribosomes can be diverted from one population of
polysomes to another, presumably depending on
the requirements for protein synthesis in the individual subcellular compartments. The possibility
that proteins may play a role in the diversion of
ribosomes was suggested by results of Olsen et al.
[6], where it was shown that the profile of proteins
Fig. I
Gel scans of proteins present in high-salt extracts from FP, CBP and MBP
isolated from MPC-I I cells
Fractions containing FP, CBP and MBP were isolated from MPC- I I cells incubated in the
absence or presence of 0 I rnlU insultn/ml of culture for I h Proteins in high-salt extracts
of polysornes were separated by PAGE and visualized using silver staining (see text) (0
and b ) , FP, (c and d ) , CBP. and (e and fl, MBP Arrows indicate the positions of protein
peaks (kDa) calculated from the migration of standard markers
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Cytoskeleton: i t s Role in Cellular Function
in the high-salt wash of total polysomes isolated
from 1,-929 cells varied during the cell cycle in synchronized cell populations. Since the respective
amounts of FP and MBP have been shown to vary
during the cell cycle in MPC-11 cells [7], it was of
interest to investigate whether or not changes occur
in the proteins associated with FP, CBP and MBP in
this cell line during short-term incubation with
insulin.
MPC-11 cells were grown in roller suspension culture as previously described [8]. FP, CBP
and MBP were prepared from MPC-11 cells using
the differential detergendsalt extraction technique
of Vedeler et al. [l]. Using this cell line 0.1%
Nonidet-P40 (NP-40), 0.05% Triton X-100 and
0.05% deoxycholate (DOC) were used. Polysomes
were pelleted through a cushion of 20% (w/v)
sucrose, resuspended in buffer containing 1 M-KCI
and re-pelleted. Proteins in the high-salt wash were
separated by SDS/PAGE and bands visualized
using silver staining.
The results indicate that there are differences
in the protein species present in high-salt wash
extracts prepared from the three polysome populations (Fig. 1). Proteins of 34, 47, 56 and 120 kDa
were unique in the FP high-salt wash, while a protein of 26 kDa seemed to be unique in the comparative preparation from MBP. A protein of 45 kDa
was present in all fractions. In all three fractions
bands were evident at 99 and 110 kDa, though they
were only weak in the CRP and MRP high-saltwash extracts. Proteins of 37 and 42 k n a were
present in FP and CHP but not in the MBP high-salt
wash.
Following insulin stimulation, proteins of 3 1,
32.5 and 50 kDa increased significantly in amount
in the FP high-salt wash. A 99 kDa protein also
increased but not to the same extent as the above
proteins. In the MBP high-salt wash there were also
increases in the amounts of proteins of 26 and 50
kDa. There was, however, a decrease in the amount
of a 29.5 kDa protein in FP, while increases were
observed in the other two high-salt-wash fractions.
A 64 kDa protein in the FP high-salt wash
increased in amount after insulin stimulation. This
protein, though not evident in the CBP fraction in
unstimulated cells, appeared in a small amount after
insulin stimulation. it was not observed in the MBP
salt wash.
The results further substantiate earlier observations [l-3, 51 demonstrating that there was a
variety of differences among the fractions containing FP, CBP and MRP. It is evident that insulin
alters the pattern of proteins associated with ribosomes/mFWA in the three polysome preparations,
although the identity and exact localization of these
proteins is not yet known.
1. Vedeler, A,, Pryme, I. F. & Hesketh, J. E. (1991) Mol.
Cell Hiochem. 100, 183-193
2. Hesketh, J. E. & Pryme, I. F.(1991) Biochem. J. 277,
1-10
3. Hesketh. J. E., Campbell. G. P. & Whitelaw, 1’. F.
(1991) Hiochem. J. 274,607-609
4. Almis, B., Pryme, 1. F., Vedeler, A. & Hesketh, J. E.
(1901) Int. J. Hiochem. in the press
5. Vedeler, A,, Pryme, I. F. & Hesketh, J. E. (1990) Cell
Biol. Int. Resp. 14, 21 1-218
6. Olsen, L. C., Pryme, I. F. & Hassse, C.-F. (1988) HioFactors 1, 161-165
7. Abraham, K. A,, Pryme, I. F., Abro, A. & Dowben,
K.M. (1973) Exp. Cell K ~ s 82.9.5-102
.
8. Pryme. I. F. (1986) Hiochem. Int. 13,287-293
Received 28 June 199 1
1991
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