Download Parathyroid hormone raises the Pi concentration in a cultured

BIOCHEMICAL SOCIETY TRANSACTIONS
10 I0
After feeding of rats with the raw soyabean diet, serum
polyamine concentrations (primarily spermidine) were
significantly increased at 6 and 12 h (Table I). N o similar
increases were apparent in the control animals. After 24 h,
the circulating levels of polyamines returned to control
values and subsequently the total polyamine content in the
blood was below normal. These later changes suggest that
clearance of polyamines from the circulation occurred at
higher rates in soyabean-fed rats than in controls.
In another experiment, feeding of rats ( 7 g/day per rat) for
7 days with a lactalbumin-based diet containing purified
trypsin inhibitors ( 2 8 g/kg diet) resulted in a considerable
increase in pancreatic dry weight ( + 82 f 14 mg) and polyamine content ( + 1345 f 350 nmol/pancreas) compared
with that obtained for controls. The lectin ( 7 g/kg diet) had a
similar effect upon pancreatic weight ( + 60 k 14 mg) and
polyamine content ( + 928 f 179 nmol/pancreas). The
control values for pancreatic dry weight were 158 f 14 mg
and for polyamine content 2209 f 179 nmol/pancreas.
Treatment of rats with 1200- 1300 mg/day per kg rat body
weight of a-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC [5],lowered pancreatic activity of
ODC in test and control groups from 3.0 k 0.5 to 1.Of 0.5
nmol of putrescinc/h per pancreas. However. it did not reduce
pancreatic polyamine accumulation or growth induced by
the trypsin inhibitors or the lectin. Therefore, it appears that
most of the additional polyamines found in the pancreata of
rats fed with soyabean. trypsin inhibitors o r lectin were not
synthesized de m v o .
Pancreatic activity o f spcrmidinc/spermine W-acetyltransferase (SAT) 161 was high (25 f 9 nmol o f N1-acetylspermidine/h per pancreas). Thus, much of the required
polyamines could have been generated in the pancreas via
the interconversion pathway if they were available from the
circulation. However, since SAT activity was not significantly
increased by feeding of rats with soyabean, trypsin inhibitors
or lectin, accumulation of polyamines could only occur if the
rate of uptake of polyamines from circulation into the
pancreas was higher in these rats than in controls. This
would be consistent with the elevated rates of polyamine
clearance from thc circulation found in soyabean-fed rats
(Table I ) and in accordance with the finding [ 71 that dietary
kidncy bean lectin could stimulate the uptake o f "C-labelled
polyamines by the pancreas.
Since luminally applied polyamines are rapidly taken up
systemically [ 8 ] and the raw soyabean protein diet contained
4 10 f 16 nmol of total polyamines (primarily spermidine)/g
of diet. it is possible that the high levels of circulating polyarnines at 6 and 12 h after exposure to soyabean and the
polyamines subsequently accumulated in the pancreata
(Table 1) could have originated indirectly from these
luminally supplied polyamines. However, the semi-synthetic
diets used in the studies with the purified trypsin inhibitors
and lectin contained < 0.5 nmol of total polyamines/g. The
required polyamines could not, therefore, have been
supplied through the diet and were probably derived from
either bacterial [Y, 101 or systemic sources. Thus, the high
serum polyarnine concentrations observed at 6 and 12 h in
soyabean-fed rats may have, in part, been due t o release of
stored polyamines in response to the higher requirements o f
the pancreas for polyamines.
We gratefully acknowledge the generous gift o f DFMO from the
Merrel I h w International Research Institute, Strasbourg Centre,
France.
I . Liener, 1. E. ( 1 98 1 ) J. Am. Oil C%c,m..So(.. 58. 306-4 I 5
2 Grant, G., Watt, W. B., Stewart, J. C. & Pusztai, A. ( 1987) hlecl.
P i . Kes. 15. 1197-1 198
3. Grant, G.. Bardocz. S.. Brown, D. S. & Pusztai, A. (1989)
Biochem. SOC. Trutis. 17, 527-528
4. Russell, D. & Snyder, S. H. ( 1 968) BI~i~hi~mI.s/ty
60,
1420- I427
5. Luk.G.D.&Yang,P.(I9X7)~~1t~28,95-101
6 . Seiler, N. ( 1987) C ' m . J . /%y.sio/. I'hnrmcrcd. 65, 2024-203s
7. Bardocz. S., Grant, G.. Brown. D. S.. Ewen, S. W. B. & Pusztai.
A. ( 1989) Med. Sci. Kes. 17. 309-3 I I
8. Bardocz, S., Brown, D. S., Grant, G. bz Pusztai, A. (1990)
Hioc,/linr.Riop/ry.v.Acrcr. in the press
9. Sarhan. S., Knodgen. U. & Sciler, N. ( 1989) Atiricwrcer Kes. 9.
2 15-224
1 0 . Osborne, D. L. & Seidel, E. R. (1989) Am. J. I%y.sio/. 256,
G 1049-G I057
1 1 . Seiler,N.& Knodgen, U.( 1980) J. C'/ir~inicr/ogr.
221, 227-235
Received h March I990
Parathyroid hormone raises the Pi concentration in a cultured osteoblast model
ABDULLAH AHMADO,* ALAN BEVINGTON,t
R. GRAHAM G. RUSSELL* and
DIANE F. GUILLAND-CUMMlNG*
*I)epurrtrietirof tlirmuri Meruholism urid c'litiicul
Riochemisrty, Medicul School, Beech Hill Road, Shefield
S I O ZRX, U.K . utid t Department of Nephrology, Leicesrer
Generul tIospiruI9 Gweridoleti Koud, Leicesrer LES 4P W,
U.K.
In a recent detailed study [ I ] , it was demonstrated that parathyroid hormone (PTH) stimulates active (Na -dependent)
uptake o f P, in the osteoblast-like cell line UMR 106. It was
proposed that this effect might be o f importance in vivo in
the regulation o f P, transport from the systemic extracellular
fluid to bone extracellular fluid, where it may influence the
rate o f bone mineral deposition. As this might involve transcellular movement o f P, across the bone-lining cells, and as
we havc already demonstrated effects o f calcitonin [ 21 and
+
Abbreviations used: PTH. parathyroid hormone; PTHrp. hypercalcaemic PI'H-related peptidc.
1,25-dihydroxyvitamin D, [ 31 on the PI concentration in
UMR 106, we have now examined whether changes in intracellular P, metabolism accompany stimulation of these cells
with PTH.
Monolayer cultures of rat osteosarcoma UMR 106 (subclone -06) were incubated with rat 1-34 PTH (Sigma
P-4282) or hypercalcaemic PTH-related peptidc (PTHrp)14)
for 80 min in Eagle's minimum essential medium without
serum, supplemented with globulin-free bovinc serum
albumin ( 1 mg/ml). 3-O-Methyl-~-[l-3H]glucose was also
present during this incubation, for determination of thc ccll
water volume 151. Twenty minutes before the end of the
incubation, j2P,was added t o the cultures t o assess P, influx.
At the end of the incubation, the cells were rapidly rinsed
with Hepes/Ringer balanced salt solution (pH 7.4 at 37°C)
and immediately deproteinized with ice-cold perchloric acid.
P, was measured in the deproteinized neutralized cxtracts
using a selective colorimetric assay 161. '?P-labcllcd PI and
organic phosphates were separated by addition of acidified
molybdate to the neutralized cell extracts, t o convert the P, t o
a phosphomolybdic acid complex which was extracted into a
I01I
634th MEETING, BATH
mixture of 2-methylpropan- 1-01 and petroleum spirit 17 I. “P,
uptakc is expressed as the distribution ratio: (c.p.m./l of cell
water)/(c.p.m./l of extracellular water). All results are
expressed as means f S.E.M.
In agreement with the observations of Selz et ul. [ 11, PTH
stimulated ,>P,uptake in UMR 106-06 cells in nine experiments. over a dose range o f 10- I I - 10- M (from a Fricdman
two-way analysis o f variance, Fr = 23, I’< 0.00 1 ). In a
separate series of four experiments over the same dose
range. this was accompanied by a comparable fractional
increase in the cellular PI concentration (i;,= IS. I’< 0.0 1 ).
For example, at a dose of lo-’ M, the cellular P, conccntration was 7.5 f 1.0 mmol/l of cell water, compared with
4.8 f 0.6 in control cells; whereas, the total j2P distribution
ratio was 9.6 f 1.3 with PTH, compared with 6.5 f 0.9 in
control cells. This implied that PTH brings about a net
movement of P, from the extracellular medium into the cell.
However. o n measuring the incorporation of extracellular
‘?P, into cellular PI and organic phosphates, it was unexpectedly found that the fraction of cellular ,‘P in the form
o f P, did not increase in response to PTH ( i t . stimulation o f
total cellular jZP labelling by incubation with PTH was
accompanied by a similar fractional increase in the labelling
of both cellular PI and organic phosphates). In control cells,
even after only 20 min in the presence o f extracellular 32P,,
the majority (70 f 5%, ti = 5)of the cellular 3?Pincorporated
into the cells was in the form of organic phosphates (in agreement with our earlier report [XI), and this was virtually
unaltered (65 f 2%. t i = S), even in the presence of lo-’ MPTH. Assuming that the specific radioactivity of the cellular
P, pool which exchanges with the organic phosphates 181 is
unaltered by PTH, a possible explanation is that PTH stimulates the metabolic flux from P, into organic phosphates, in
addition t o its effects on the Na’-linked PI transporter at the
plasma membrane.
Finally, stimulation of P, uptake in UMR 106-06 was not
confined t o PTH. Over the dose range 1 0 - 1 1 - 1 0 - 7M, both
PTH ( n = 4 ) and PTHrp ( r i = S ) gave a comparable percentage stimulation of total ‘?P, uptake. (At 10+ M. PTH
stimulated total ’?P, uptake by SS f 12% relative t o control
cells. whereas, with PTHrp, the stimulation was 56 f I3”X.)
’
These effects contrast with those that we have described in
UMR 106-06 in response t o 1,25-dihydroxyvitamin D,,
which caused only a small increase in total ”P, uptake. but a
marked stimulation of the cellular PI concentration which we
attributed t o net degradation o f cellular organic phosphates
in response to the hormone 131. However, in spite o f these
quantitative differences, with both PTH and 1,25-dihydroxyvitamin D, it seems necessary t o consider changes in intracellular organic phosphate metabolism, in addition t o
changes in P, transport at the plasma membrane, to explain
the observed effects. Once again, this emphasizes the
importance of making combined measurements of cellular
12P labelling and cellular PI concentration in studies o f this
type.
A.A. thanks the Government o f Saudi Arabia and D.F.G.-C.
thanks the National Fund for Research into Crippling Iliseases for
financial support. U M R 106-06 cells and hypercalcaemic P I H related peptide were kindly provided by Professor T. J. Martin
(Melbourne).
I . Selz, T., Caverzasio. J. & Bonjour. J.-P. ( I 989) Ant. J. I’by.sio/.
256, E93-E I00
2. Kemp. G. J.. Khouja, H. 1.. Hevington. A., Ahmado, A. & Russell,
R. G. G. ( 1989) l3ioclti.m. Soc. Trcins. 17. 522-523
3. Ahmado, A,, Bevington. A . & Russell, R. G. G. ( 1990) Riockvn.
.so<..7rrIrl.s. 18. 624-625
4. Suva. L. J.. Winslow. G. A,, Wettenhall. R. E. H., Hammonds.
R. G.. Moseley. J. M., Iliefenbach-Jagger. H.. Rodda, C. P.,
Kemp. 13. E.. Rodriguez. H.. Chen. E. Y., Hudson. P. J.. Martin.
T. J. & Wad, W. I. ( 19x7) .S(’irtlc’e237. 893-XO6
5. Kletzien, R. F., Pariza. M. W., Beckcr. J. E. & Potter, V. K.( 1 9 7 5 )
/ l t l ( l / . / 3 i 0 1 ~ / l 1 ~ l l68.
/.
537-544
6 . 13evington. A.. Angicr. C . M.. Kemp. G. J . & Russell, K. Ci. < i .
( I 9 , X U ) /lt/(I/. Hioc’l/ert/.181. 130-134
7. Kemp. G. J.. Bevington, A,, Khodja, D., Challa. A. & Russell.
R. G. G. ( 1 989) Niockcm. J . 264, 729-730
8. Kemp. G. J.. Bevington, A,. Khouja. H. I. & Russell. R. G. Ci.
(1989) Hioch<,m..Sot. 7rcrtt.v. 17. 521-522
Received 7 March I990
Overproduction and isolation of elongation factor Tu using Escherichia coli grown on protiated and
deuterated succinate
TINA D. HOWARD, DEBRA BLOOR.
KAREN KENNEDY and JILL BARBER
Depurttrietit of’l’hurrnucy, Uiiiversity of’ Mutichester,
Munchester M I 3 W L , U . K .
Elongation factor Tu (EF-Tu) is a protein with an important
role in the elongation cycle during protein biosynthesis in
prokaryotcs. In Escherichiu coli it has a molecular mass of
43 225 Da [ I], its amino acid sequence is known [ 11 and the
crystal structure of the 39 kDa tryptic digest fragment has
bcen determined [21. The protein has a limited stability in
solution (a half-life in the order of a few hours at pH 7 and
4°C). Stability is increased by the presence of either C U P o r
the antibiotic kirromycin [ 11.
Kirromycin inhibits protein biosynthesis by binding to EFTu. As part of a programme aimed at elucidating, in detail,
the molecular basis of kirromycin action using ‘ H n.m.r.
Ahhreviation used: EF-Tu. elongation factor Tu.
Vol. 18
techniques, we have attempted t o produce large quantititcs
of pure dcuterated EF-Tu. Economic production o f
deuterated protein requires that the bacterial strain grows on
a medium with succinate as the only carbon source.
E. coli MRE 600 (wild-type) produces about 1 X lo-’
mol o f EF-Tu/l of culture medium ( L broth), as measured
using a GDP-exchange assay 131. Two overproducers were
available t o us: LBE 1 101 pGP82 (‘LB clone’, a gift from Dr
E. Vijigenboom and Professor L. Bosch) [4] and MRE600
pC18S7 pCP40 (‘TDH clone’, the plasmids being gifts from
Professor A. Parmeggiani) [ 51. EF-Tu production in both
clones grown on L broth and succinate media was monitored
by the GDP-exchange assay. On L broth the LB clone
produced up t o 3 x I O Y ’ mol of EF-Tu/ (see Fig. 1 ). As
shown in the Figure, the concentration of EF-Tu falls sharply
in early stationary phase, but quickly recovers achieving the
maximum value 1-2 h later. When grown on succinate,
however, this clone produces no more EF-Tu than wild-type
MRE600. This strain is therefore not immediately suitable
for production of deuterated EF-Tu.