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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.