568th MEETING, ABERDEEN K. R. H. thanks the Science Research Council for a Research Studentship.

Meijer, A. J. & Van Dam, K. (1974) Biochim. Biophys. Acta 346,213-244 Mowbray, J. & Ottaway, J. H. (1973) Eur. J. Biochem. 36, 362-379 Ottaway, J. H. & Mowbray, J. (1974) Abstr. FEBS Meet. 9th 111 Stanley, K. K. & Tubbs, P. K. (1974) FEBS Lett. 39,325-328 Whereat, A. F.,Orishimo, M. W., Nelson, J. &Phillips,S. J. (1969)J. Biol. Chem. 244,6498-6506 Williamson, J. R., Safer, B., LaNoue, K. F., Smith, C. M. & Walajtys, E. (1973) Symp. SOC. Exp. Biol. 27,241-281

Release of Polyamines from Growing and Serum-Deprived BHI(-21/C13 Cells MAUREEN A. L. MELVIN and HAMISH M. KEIR

Department of Biochemistry, University of Aberdeen, Marischal College, Aberdeen AB9 IAS, Scotland, U.K. Intracellular concentrations of the polyamines putrescine, spermidine and spermine tend to be correlated positivelywith the rate of growth of cell systems (Cohen, 1971;Tabor & Tabor, 1972; Bachrach, 1973). Enhanced accumulation of polyamines is characteristic of rapidly growing cell systems, whereas slowly growing cell systems contain decreased intracellular concentrations of polyamines. Much is known about the mechanisms of biosynthesis of polyamines and how their production may be regulated (Williams-Ashman et al., 1973). However, the means by which a cell may decrease its intracellular content of polyamines are less well understood. A major degradative pathway for polyamines may be via their oxidative deamination by amine oxidases, resulting in the formation of the corresponding aldehydes as suggested by Bachrach (1973). Another possibility is that polyamines produced in excess of the cell's requirements are released from the cell; indeed it has been shown by Pohjanpelto & Raina (1972) that putrescine normally leaks out of some mammalian cells growing in culture. The present study was undertaken to examine the fate of intracellular polyamines when the growth of BHK (baby hamster kidney)-21/C13 cells in culture is arrested by depriving the cells of serum (Howard et al., 1974). The intracellular concentration of polyamines decreases markedly within 24h after transfer of the cells from medium containing 10%(v/v) serum to medium containing 1 % (v/v) serum (Table 1). Intracellular polyamines were radioactively labelled by growing the cells in medium containing [3H]putrescinedihydrochloride, followed by incubation in unlabelled medium for several hours before transfer of the cells to medium containing either 10 or 1% (v/v) horse serum. There was an efflux of polyamines into both types of medium within the first few hours after transfer of the cells (Table 2). Thereafter there was no increase in the 3H content of the medium containing 10% serum, for at least 24h; indeed some uptake of polyamines by the cells from the medium usually occurred. In contrast, the 3H content of medium containing only 1 % serum increased continuously for a h , by which time most of the label originally associated with the cells was found free in the medium. This

Table 1. Concentration ofpolyarnines(putrescine+spermidine+spermine)in BHK-21IC13 cells after transfer to fresh medium containing either 10% (v/v) or 1% (v/u) horse serum

Initial cell culture in 10% serum Culture after 24h in 1% serum

Vol. 5

No. of cells/ culture dish 1.3~10~ 1.6~10~

Total polyamines (nmOl/lO6 cells) 5.58





Table 2. Release of 3H-lahelledpolyamines from BHK-211C13 cells into medium afier transfer of cells tofresh medium containingeither 10 % (vlv)or 1% (v/v) horse serum

3H in medium Time after transfer 3H radioactivity [c.p.m./ to fresh medium (h) dish (in medium)] (% of total) Initial cell culture Cells in 10 % serum

Cells in 1 % serum



6 24 6 24

12893 10 762 11227 32 628


30 25 30.3 71

release of polyamines from cells in ‘low-serum’ medium is not a consequence of cell death, as has been demonstrated by several criteria. These results illustrate that decrease of the cell content of polyamines after ‘step-down’ of growing cells into serum-depleted medium is accompanied by release of significant amounts of polyamines from the cells into the culture medium. This work was supported by grant no. G975/38/C from the Medical Research Council. Bachrach, U. (1973) Funcfionof Naturally Occurring Polyamines, pp. 74-81,96107, Academic Press,London and New York Cohen, S. S. (1971) Itttroduciion to the Polyamines, pp. 2847, PrenticeHall, Englewood Cliffs Howard, D. K., Hay, J., Melvin, W. T. & Durham, J. P. (1974) Exp. CeNRes. 86, 31-42 Pohjanpelto, P. & Raina, A. (1972) Nafure(London) 235, 247-249 Tabor. H. & Tabor, C. W. (11972) Adv. Enzymol. Relat. Areas Mol. Biol. 36,203-269 Williams-Ashman, H. G., Coppoc, G. L., Schenone, A. & Weber, G. (1973)in Polyamines in NormalandNeoplasticGrowfh(Russell, D. H., ed.), pp. 181-197, Raven Press, New York

A New Method for Immunochemical Quantification CHRISTINA GLAD and ANDERS 0. GRUBB Department of Clinical Chemistry, University of Lund, Malmo General Hospital, S-214 01 Malmo, Sweden

A large number of methods for immunochemical quantification have been developed during the last 50 years. To bring about the necessary interaction between antigen and antibody these methods use either diffusion, as in single radial immunodiffusion (Mancini et al., 1965), an electrophoretic process, as in electroimmunoassay (Laurell, 1966), or mixing as in radioimmunoassay (Ekins, 1960; Yalow & Berson, 1960) and 1973). This report demonstrates that the capillary force in a nephelometry (Ritchie et d., porous material can also be used to bring about an antigen-antibody interaction and that this can be used for quantitative purposes. Antibodies were attached to porous insoluble supports, such as cellulose acetate sheets (Sartorius, Gottingen, W. Germany), filter paper Munktell 00 (Glycksbo Pappersbruk, Sweden) and polyvinyl chloride sheets containing silica gel as a filler (Amerace Erna Corporation, New York, NY, U.S.A.). Methods used for attaching antibodies were either covalent linkage with CNBr (Nishikawa & Bailon, 1975) or cross-linkingwith glutardialdehyde for cellulose acetate and fiter paper, and adsorption from 0.1 M-Tris/HCI buffer, pH8.0, containing 0.5% (wlv) goat or rabbit antibodies for polyvinyl chloride sheets. The antibody-containingporous sheets were then used in tw6 different ways. In the fkst procedure, 1-5p1 of sample solutionswas applied on a line 1cm from the edge of a sheet, and the sheet was placed in a chromatographic chamber with 5 m m of it inserted in O.l~-Tris/HCl,pH8.0, and the capillary migration of the buffer was allowed to proceed upwards. When the buffer had migrated 6cm, the sheet 1977

C13 cells [proceedings].

711 568th MEETING, ABERDEEN K. R. H. thanks the Science Research Council for a Research Studentship. Meijer, A. J. & Van Dam, K. (1974) Biochim. Bio...
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