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Putrescine and Spermidine Changes in Heat-Synchronized Tetrahymena Populations

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Putrescine and Spermidine Changes in Heat-Synchronized Tetrahymena Populations 604 B. HOLM AND O. HEBY Putrescine and Spermidine Changes in Heat-synchronized Tetrahymena Populations BORIS HOLM and OLLE HEBY Institute of Zoophysiology, University of Lund, S-223 62 Lund, Sweden (Z. Naturforsdi. 26 b, 604—606 [1971] ; received March 3, 1971) Putrescine and spermidine were the dominant polyamines in Tetrahymena cells. Spermine was present only in very small amounts. Changes in the amount of putrescine during and after the synchronizing heat treatment followed a definite quantitative pattern. Changes in the amount of spermidine were similar, though not so pronounced. The changes may be due to a varied influx of polyamines into the cells. Moreover, the exchange of polyamines between the cells and the culture medium must be specifically regulated since there seems to be a relatively greater accumulation of spermidine than of putrescine in the cells, and since spermine was almost excluded from the cells. Putrescine, spermidine and spermine have been medium, which consisted of 2 per cent proteose pep- found in all cells so far examined. Spermidine and tone (Difco), 0.3 per cent yeast extract (Difco) and inorganic salts in the following amounts per 1: 10 g spermine are dominant in animal cells, while MgS04x7H,0, 0.5 g CuCl2 x 2 H20, 7.5 g CaCl2 x putrescine and spermidine are the most frequent of 6 H20, 0.125 g FeCl3x6H20, 0.05 g MnCl2x4H20, these amines in bacteriaIn most cases, these 6 mg ZnAc x 2 H20, 2.5 g Fe(NH4) (S04) 2 x 6 H20. amines are referred to as polyamines, although After inoculation of each flask with 5 ml of 2-days putrescine is a diamine. Their postulated role as old stock cultures, and subsequent exponentiil growth stabilizers of nucleic acids has attracted much atten- for 14 hours, the cultures were synchronized by 7 heat shocks at 34 °C, lasting 30 min each, during a total tion 2' 3. In vitro experiments have yielded evidence period of 7 hours, according to SCHERBAUM and that polyamines compete with metal ions at the ZEUTHEN 9. The cultures were mixed and distributed organization of ribosomes (for references see again during the interval at 28 °C between the fourth STEVENS *) as well as with histones at nucleopro- and fifth shocks. 200 ml samples were withdrawn, two tein formation 4. at a time, at appropriate intervals during the heat treat- ment and the subsequent period up to and including The presence of putrescine, spermidine and sper- the second synchronous division. The samples were mine in Tetrahymena cells has earlier been estab- centrifuged and the cells were rapidly washed in 0.4 lished by WELLER, RAINA and JOHNSTONE5, and per cent NaCl, frozen and stored at — 20 °C. Small ARLOCK, HEBY and HOLM 6. These observations are samples (2 xl ml) were removed from each 200 ml sample before centrifugation and the number of cells confirmed in the present paper. The high amounts per ml was counted in a Celloscope. The times and of polyamines in the cell suggest that they may the degree of synchronous divisions were established participate in ionic regulation. The polyamine con- by calculating the maximum values of the ratio between tent during and after heat treatment changes in- the number of cells in division and the total number of versely with the amount of bound Mg and in parallel cells. with the stability of DNA previously registered 7' 8. Preparation and analysis of polyamines: Frozen cell samples of 2.0 —2.5 ml volume were homogenized in ice-cold 0.9 per cent NaCl with a Teflon homogenizer. Material and Methods The proteins in the homogenates were precipitated by addition of ice-cold trichloroacetic acid and removed Experimental cultivation and sampling: The ciliate by filtration. The supernatants were extracted three Tetrahymena pyriformis, amicronucleate strain GL, was times with diethyl ether to remove the acid, and the cultured at 28 °C in Fernbach flasks containing 500 ml amines were then extracted into n-butanol according Reprints request to Dr. BORIS HOLM, Institute of Zoophy- 5 D. L. WELLER, A. RAINA, and D. B. JOHNSTONE, Biochim. siology University of Lund, S-223 62 Lund, Sweden. biophysica Acta [Amsterdam] 157, 558 [1968]. 1 L. STEVENS, Biol. Rev. 45,1 [1970]. 6 P. ARLOCK, O. HEBY, and B. HOLM, J. Protozool. 16 Suppl. 2 R. R. MACGREGOR and H. R. MAHLER, Arch. Biochem. 33 [1969], Biophysics 120, 136 [1967]. 7 B. HOLM, Exp. Cell Res. 53,18 [1968], 3 O. HEBY and I. AGRELL, Hoppe-Seyler's Z. physiol. Chem. 8 B. HOLM, J. Protozool. 17, 615 [1970], 352, 29 [1971]. 9 O. SCHERBAUM and E. ZEUTHEN, Exp. Cell Res., Suppl., 4 I. AGRELL and O. HEBY, Hoppe-Seyler's Z. physiol. Chem. 3,312 [1955]. 352, 39 [1971]. PUTRESCINE AND SPERMIDINE CHANGES 605 10 r 160 to RAINA . The rc-butanol layer was acidified and lyo- spermidine philized. The residues were dissolved in 0.1 M HCl and stored in small test-tubes until analysis. The polyamines were separated by thin-layer chro- -80 matography according to HAMMOND and HERBST U. 5 [A samples were applied on thin-layer plates coated with Whatman Cellulose CC41. The solvent system used was ethylene glycol monomethyl ether : propionic g L0 acid : water (70 : 15 : 15) saturated with NaCl. The solvent was removed by heating and the plates immer- sed in a 1 per cent solution of ninhydrin in absolute putrescine ethanol containing 1 per cent 2,4,6-trimethylpyridine. -160 The plates were developed at 60 °C for 15 minutes and the coloured spots were scraped off and aspirated quantitatively3 into 70 per cent ethanol. When the colour had been eluted, the cellulose powder was sedimented by centrifugation. The absorbance of poly- amines at 575 m/u was used for the quantitative deter- minations. L Standard solutions of spermine tetrahydrochloride, 0 spermidine trihydrochloride and putrescine dihydro- chloride (Calbiochem) were included in duplicate on EH 60 120 180 each thin-layer plate. min. after EH- Fig. 1. Changes in the amounts of putrescine and sper- Results midine during and after the synchronizing heat treatment of Tetrahymena cells. Standard errors are given for the mean Of the polyamines, putrescine and spermidine ap- values of samples. The vertical broken lines indicate the mean times for the first and second synchronous divisions. The ab- peared in the highest amounts in the Tetrahymena scissa shows the last four heat hocks, EH (end of heat treat- cells. Spermine was present, but only in very small ment) and the following 210 minutes. The values after the amounts. Cadaverine, which had earlier been ob- syndironous divisions increase in proportion to the actual number of cells, so that all values correspond to the number tained 6, could not be detected by the present of original cells obtained at EH. method. The changes in the amounts of intracellular putrescine and 6.8 ± 0.4 nmoles spermidine per mil- putrescine between the fourth shock and the second lion cells. The pure medium was found to contain synchronous division are shown in Fig. 1. The peak 180 nmoles putrescine, 47 nmoles spermidine and during the last shock is statistically significant (/ = 40 nmoles spermine per ml respectively. These 10, Z = 3.38 and P<0.01) as is the increase from values were compared with the polyamine concentra- EH (EH = end of heat treatment) to 60 minutes tion in the Tetrahymena cells. By estimating the cell after EH (/= 10, « = 3.49 and P<0.01). The peak volume at EH to be 70 ju\ per million cells, ac- between the synchronous divisions is also significant cording to ZEUTHEN and SCHERBAUM 12, it was (/ = 5, « = 3.05 and P<0.05). The changes in sper- calculated that the approximate concentration of midine content are not so pronounced (Fig. 1). The putrescine and spermidine in the cells was 5 and 8 peak at 60 minutes after EH is, however, significant times higher, respectively, than in the medium. (/= 10, t = 2.13 and P<0.05). The intracellular amount of putrescine exceeded that of spermidine by Discussion more than a factor of 2. The amounts of putrescine and spermidine in the The variation in the amount of putrescine in heat- heat-synchronized cell cultures at EH were about synchronized Tetrahymena cultures closely parallels three times higher than in exponentially growing cul- the change in the uptake of 3H-putrescine from the tures. The latter contained 21.7+1.8 nmoles medium13. As there are polyamines in the sur- 10 A. RAINA, Acta physiol. scand. 60, Suppl. 218, 1 [1963]. 12 E. ZEUTHEN and O. SCHERBAUM, in: Recent Developments 11 J. E. HAMMOND and E .J. HERBST, Analyt. Biochem. [New in Cell Physiology, (J. A. KITCHING, ed.). Butterworths, York] 22, 474 [1968]. London, 141, 1954. 13 B. HOLM and H. EMANUELSSON, Z. Zellforsch. 115, 593 [1971]. 606 PUTRESCINE AND SPERMIDINE CHANGES 606 rounding medium, it seems reasonable to assume These simple relationships between some cations that part of the increase may be due to an in- may not be relevant under the more complicated creased influx into the cells. Results earlier obtained conditions in vivo. However, the magnitude of the suggest a very low or non-existent spermidine syn- changes in the ion content of heat-synchronized Tetra- thesis in Tetrahymena; no 3H-putrescine was found hymena suggests that the variations in polyamine to be transformed to 3H-spermidine13. An endo- content must be taken into account. During the seventh genous production of putrescine in Tetrahymena shock the amount of bound Mg increased by about cannot be confirmed until it has been shown that 25 nmoles per million cells while the putrescine possible precursors such as arginine and ornithine content decreased by about 40 nmoles per million are incorporated into putrescine.
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