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Some preliminary observations on the biochemical and biological properties of an epithelial growth factor
Biochemistry of epithelial growth factor 2. Ficq, A, Monographie No. 9, Institut Interuniversitaire des Sciences Nucleaires. Bruxelles (1961). 3. Freeman, S B, Biol bull 135 (1968) 501. 4. Ghiara, G, Arch zoo1 Ital 45 (1960) 9. 5. Humphries, A A, Jr, Biol bull ‘120 (1961) 29. 6. - Devel biol 13 (1966) 214. 7. Humphries, A A, Jr & Hughes, W N, Biol bull 116 (1959) 446. 8. Humphries, A A, Jr &Workman, W M, Am zoo1 6 (1966) 611. 9. Humphries, A A, Jr, Freeman, S B & Workman, W M, Biol bull 134 (1968) 266. 10. Kambara, S, Annot zoo1 Japon 29 (1956) 146. 11. - Ibid 30 (1957) 143. 12. Katagiri, S, Zoo1 mag 72 (1963) 23. 13. Kelley, J W, Protoplasma 43 (1954) 329. 14. Lee, P A, J exptl zoo1 166 (1967) 99. 15. Machlin, L J, Pearson, P B & Denton, C A, J biol them 212 (1955) 469. 16. McLaughlin, E W, Doctoral dissertation, Emory University (1967). 17. Minganti, A, Exptl cell res, Suppl 3 (1955) 248. 18. Minganti, A & D’Anna, T, Ricerca sci 28 (1958) 2090. 19. Monroy, A, Chemistry and physiology of fertilization. Holt, Rinehart & Winston, New York (1965). 20. Nate, G W, Suyama, T & Smith, N, Symposium on germ cells and development, p. 564. Inst Intern Embryo Fondazione A, Baselli (1960). 21. Richards, 0 W, J exptl zoo1 83 (1940) 401. 22. Rogers, A W, Techniques of autoradiography, p. 267. Elsevier, Amsterdam (1967). 23. Shivers, C A. Personal communication. 24. Subtelny, S & Bradt, C, Devel biol 3 (1961) 96. Received September 15, 1969 Revised version received October 28, 1969
SOME PRELIMINARY OBSERVATIONS ON THE BIOCHEMICAL AND BIOLOGICAL PROPERTIES OF AN EPITHELIAL GROWTH FACTOR R. 0. JONES and M. J. ASHWOOD-SMITH, M. R. C. Radiobiology Unit, Harwell, Didcot, Berks, UK
Summary Preliminary observations have shown that epithelial growth factor is separable from skin growth factor, and has proteolytic activity and a mol wt of 22,500.
It has been reported [3] that a growth factor extractable from male mouse submandibular salivary gland induces extensive proliferation of epithelia in organs cultured in an otherwise chemically defined medium. The epithelial
161
growth effect was originally detected utilising a relatively crude proteinaceous extract. The present work describes some of the properties of a partially purified epithelial growth factor, and distinguishes it from an epidermal growth factor [2], also detected in the crude exctract. Material and Methods The crude extract was prepared as described earlier [3], the distilled water was removed by freeze-drying and the protein dissolved in 0.1 M Tris (hydroxymethyl) aminomethane buffer pH 8.3, containing 0.1 M NaCI, at a concentration of 250 O.D. units/ml. (One Optical Density unit of protein is defined as giving an absorbance reading of 1.0 at 2,800 A for a path length of 10 mm; this is approximately equivalent to 1 mg/mI of albumin). An aliquot (2 ml) of this solution was placed on the top of a column of DEAE Sephadex A.50 (90 cm), equilibrated in 0.1 M Tris buffer pH 8.3 containing 0.1 M NaCl. After the extract had passed below the surface of the Sephadex, a gradient of increasing salinity (0.1 M-O.3 M NaCl) was passed through the column, and approx. 5.0 ml samples were collected every 20 min (fig. 1). A good fractionation of the crude extract was obtained and the contents of the tubes from each peak were pooled, dialysed against distilled water and freeze-dried. Each fraction was dissolved in a known volume of distilled water, and after measurement of the protein concentration was tested against rat prostate [3], and 16 day rat embryo back skin in vitro. Epithelial growth factor was confined to neak 2, with some slight activity detectable in peak 1. The skin or epidermal growth factor was confined to the last fractions in peaks 5 and 6 (fig. 1). Activity was assessed on an arbitrary --/scale on histological preparations of the cultures, taking the response observed with the crude extract [3] as the maximum (4 +). The skin growth response was assessed on the height of keratinized epidermis apparent after 24 h in vitro in the presence of growth factor, as compared to controls of uncultured skin from the same embryo, and skin maintained on chemically defined medium for the same period with or without IO % horse serum.
Further differences were observed between the epithelial growth factor and the skin growth factor. On heating in a water bath at 100°C for 30 min the epithelial growth factor lost its activity to induce proliferation of epithelial cell layers, whereas the skin growth factor was heat-stable, and maintained its ability to induce epidermal proliferation even at a concentration of 0.0037, O.D. units/ml. Furthermore, in dilutions of Exptl Cell Res 59
162 R. 0. Jones & 44. J. Ashwood-Smith
(4 (b) Abscissa: fraction no.; ordinate: E (2800 A). Fig. I. Separation of EGF on DEAE 50. Scale (a): Molar&y of NaCl in 0.1 M tris, pH 8.3; Scale(b): Units of protease activity, EGF, epithelial growth factor; SGF, skin growth factor.
both the unheated crude extract and epithelial growth factor from the DEAE fractionation (peak 2), epithelial growth activity disappeared at concentrations between 0.09 and 0.009, O.D. units/ml. Skin proliferative activity was still detectable at 0.0018, O.D. units/ ml. This suggests that epithelial growth factor is either less stable or less active than the skin growth factor. After dialysis against distilled water, the epithelial growth factor of peak 2 of the DEAE fractionation was freeze-dried, dissolved in 0.01 M phosphate buffer pH 7.4, and passed through a column of Sephadex GlOO (90 cm) equilibrated in the same buffer. Only one peak was obtained and it was assumed that the epithelial growth factor was now relatively homogeneous. After passage through the Sephadex GlOO it was estimated that purification had been increased lZfold, assessed on yield obtained from an initial sample of 250 O.D. units/ml of crude extract. Molecular weight estimations were carried out according to the method of Andrews [l]. A series of proteins of known molecular weight were individually passed through a stabilised column of Sephadex GlOO (90 cm), Exptl Cell Res 59
equilibrated with 0.01 M phosphate buffer pH 7.4. The effluent volume of each was accurately measured, and from this a curve was constructed plotting effluent volume against known molecular weight (fig. 2). Two samples of purified epithelial growth factor were separately passed through the column, the effluent volumes measured and the molecular weight estimated by comparison with the standard curve. The molecular weight of epithelial growth factor is approx. 22,500. However, electrophoresis on cellulose acetate in 0.01 M Verona1 buffer pH 8.4 showed two bands migrating towards the anode (3 h at 20 V/cm width and stained with nigrosine). This implies that the epithelial growth factor consists of at least two further components, both of which may be involved in the induction of epithelial proliferation. This has not yet been resolved. In a separate series of experiments it had been observed that cultures of male mouse submandibular salivary gland autolysed in the presence of other cultured organs. These organ cultures simultaneously showed epithelial proliferation. It was consequently considered possible that protease activity may be
EOF ____..*
Abscissa: vE (cm”); ordinate: mol. wt. Fig. 2. Estimation of molecular weight of EGF by gel filtration on GlOO (Andrews’ method). 1, Glucagon; 2, cytochrome c; 3, RNAase (a); 4, trypsin inhibitor (S.B.); 5, chymotrypsinogen A (II); 6, serum albumin (v); 7, globulin (Bov. II).
An inhibitable thymidine uptake system associated with the epithelial growth factor, and tests for proteolytic activity were carried out according to Rick [4]. It was observed that most of the fractions separated on DEAE Sephadex showed proteolytic activity (fig. l), the highest activity being displayed by the epithelial growth factor fraction. (A protease unit was defined as an increase in optical density at 2800 A of trichloroacetic acid soluble material, released from a standard solution of denatured casein, 1 % w/v, when treated for 1 h at 37°C in 0.1 M phosphate buffer pH 7.4, with a solution of protease which itself has an absorbance of 2.0 at 2800 A). Proteolytic activity was similarly present in the purified epithelial growth factor after passage through Sephadex GlOO. After storage at - 15°C for several weeks, a gradual reduction in proteolytic activity as well as epithelial growth activity was observed. This does not occur with the crude extract and it is therefore assumed that the purified epithelial growth factor is unstable. It is possible that the relative instability of the epithelial growth factor with increasing purifications may account for the low yield finally obtained. It remains to be shown, however, that the proteolytic activity is indeed a property of the pure epithelial growth factor, and not that of a proteinaceous moiety which separates out with it. It is possible also that the active component of the epithelial growth factor exists as a part of a larger molecule, some of the other properties of which might be proteolytic. In any event the provisional assessment of the molecular weight will almost certainly have to be recalculated after further purification. After storage at - 15°C for several weeks, a gradual reduction in proteolytic activity as well as epithelial growth activity was observed. This does not occur with the crude extract and it is therefore assumed that the
163
purified epithelial growth factor is unstable. It is possible that the relative instability of the epithelial growth factor with increasing purification, may account for the low yield finally obtained. REFERENCES 1. 2. 3. 4.
Andrews, P, Biochem j 91 (1964) 222. Cohen, S, J biol them 237 (1962) 1555. Jones, R 0, Exptl cell res 43 (1966) 645. Rick, W, Methods of enzymatic analysis (ed H U Bergmeyer) p. 811. Academic Press, New York (1963).
Received September 15, 1969
SOME CHARACTERISTICS INHIBITABLE THYMIDINE SYSTEM IN MAMMALIAN
OF AN UPTAKE CELLS
G. S. SCHUSTER and J. D. HARE, Departments of Microbiology and Dental Research, University of Rochester Medical Center, Rochester, N. Y. 14620, USA
Summary Some characteristics of the thymidine uptake process in mammalian cells are described. The uptake is differentially inhibited by organic and inorganic mercurials. It is shown to be different in nature and/or location from the phenylalanine uptake process, which shows a pattern of inhibition by the same compounds which is distinct from that of thymidine. Speculation on the nature of the uptake system is based on possible modes of action of the inhibiting agents.
It has been shown that pyrimidine nucleosides and their analogues may be taken into mammalian cells by an uptake process other than simple diffusion. This is most readily seen when they are at very low concentrations [l]. Hare [2] examined the relationship between extracellular concentration of thymidine (TdR) and the 5-minute uptake into an acidsoluble fraction and found evidence of a nonlinear portion of the uptake curve at low substrate concentrations. At higher concentrations a linear portion was seen. This could be interpreted as indicating that at low concentrations a mediated process predominated, Exptl Cell Res 59
SOME PRELIMINARY OBSERVATIONS ON THE BIOCHEMICAL AND BIOLOGICAL PROPERTIES OF AN EPITHELIAL GROWTH FACTOR R. 0. JONES and M. J. ASHWOOD-SMITH, M. R. C. Radiobiology Unit, Harwell, Didcot, Berks, UK
Summary Preliminary observations have shown that epithelial growth factor is separable from skin growth factor, and has proteolytic activity and a mol wt of 22,500.
It has been reported [3] that a growth factor extractable from male mouse submandibular salivary gland induces extensive proliferation of epithelia in organs cultured in an otherwise chemically defined medium. The epithelial
161
growth effect was originally detected utilising a relatively crude proteinaceous extract. The present work describes some of the properties of a partially purified epithelial growth factor, and distinguishes it from an epidermal growth factor [2], also detected in the crude exctract. Material and Methods The crude extract was prepared as described earlier [3], the distilled water was removed by freeze-drying and the protein dissolved in 0.1 M Tris (hydroxymethyl) aminomethane buffer pH 8.3, containing 0.1 M NaCI, at a concentration of 250 O.D. units/ml. (One Optical Density unit of protein is defined as giving an absorbance reading of 1.0 at 2,800 A for a path length of 10 mm; this is approximately equivalent to 1 mg/mI of albumin). An aliquot (2 ml) of this solution was placed on the top of a column of DEAE Sephadex A.50 (90 cm), equilibrated in 0.1 M Tris buffer pH 8.3 containing 0.1 M NaCl. After the extract had passed below the surface of the Sephadex, a gradient of increasing salinity (0.1 M-O.3 M NaCl) was passed through the column, and approx. 5.0 ml samples were collected every 20 min (fig. 1). A good fractionation of the crude extract was obtained and the contents of the tubes from each peak were pooled, dialysed against distilled water and freeze-dried. Each fraction was dissolved in a known volume of distilled water, and after measurement of the protein concentration was tested against rat prostate [3], and 16 day rat embryo back skin in vitro. Epithelial growth factor was confined to neak 2, with some slight activity detectable in peak 1. The skin or epidermal growth factor was confined to the last fractions in peaks 5 and 6 (fig. 1). Activity was assessed on an arbitrary --/scale on histological preparations of the cultures, taking the response observed with the crude extract [3] as the maximum (4 +). The skin growth response was assessed on the height of keratinized epidermis apparent after 24 h in vitro in the presence of growth factor, as compared to controls of uncultured skin from the same embryo, and skin maintained on chemically defined medium for the same period with or without IO % horse serum.
Further differences were observed between the epithelial growth factor and the skin growth factor. On heating in a water bath at 100°C for 30 min the epithelial growth factor lost its activity to induce proliferation of epithelial cell layers, whereas the skin growth factor was heat-stable, and maintained its ability to induce epidermal proliferation even at a concentration of 0.0037, O.D. units/ml. Furthermore, in dilutions of Exptl Cell Res 59
162 R. 0. Jones & 44. J. Ashwood-Smith
(4 (b) Abscissa: fraction no.; ordinate: E (2800 A). Fig. I. Separation of EGF on DEAE 50. Scale (a): Molar&y of NaCl in 0.1 M tris, pH 8.3; Scale(b): Units of protease activity, EGF, epithelial growth factor; SGF, skin growth factor.
both the unheated crude extract and epithelial growth factor from the DEAE fractionation (peak 2), epithelial growth activity disappeared at concentrations between 0.09 and 0.009, O.D. units/ml. Skin proliferative activity was still detectable at 0.0018, O.D. units/ ml. This suggests that epithelial growth factor is either less stable or less active than the skin growth factor. After dialysis against distilled water, the epithelial growth factor of peak 2 of the DEAE fractionation was freeze-dried, dissolved in 0.01 M phosphate buffer pH 7.4, and passed through a column of Sephadex GlOO (90 cm) equilibrated in the same buffer. Only one peak was obtained and it was assumed that the epithelial growth factor was now relatively homogeneous. After passage through the Sephadex GlOO it was estimated that purification had been increased lZfold, assessed on yield obtained from an initial sample of 250 O.D. units/ml of crude extract. Molecular weight estimations were carried out according to the method of Andrews [l]. A series of proteins of known molecular weight were individually passed through a stabilised column of Sephadex GlOO (90 cm), Exptl Cell Res 59
equilibrated with 0.01 M phosphate buffer pH 7.4. The effluent volume of each was accurately measured, and from this a curve was constructed plotting effluent volume against known molecular weight (fig. 2). Two samples of purified epithelial growth factor were separately passed through the column, the effluent volumes measured and the molecular weight estimated by comparison with the standard curve. The molecular weight of epithelial growth factor is approx. 22,500. However, electrophoresis on cellulose acetate in 0.01 M Verona1 buffer pH 8.4 showed two bands migrating towards the anode (3 h at 20 V/cm width and stained with nigrosine). This implies that the epithelial growth factor consists of at least two further components, both of which may be involved in the induction of epithelial proliferation. This has not yet been resolved. In a separate series of experiments it had been observed that cultures of male mouse submandibular salivary gland autolysed in the presence of other cultured organs. These organ cultures simultaneously showed epithelial proliferation. It was consequently considered possible that protease activity may be
EOF ____..*
Abscissa: vE (cm”); ordinate: mol. wt. Fig. 2. Estimation of molecular weight of EGF by gel filtration on GlOO (Andrews’ method). 1, Glucagon; 2, cytochrome c; 3, RNAase (a); 4, trypsin inhibitor (S.B.); 5, chymotrypsinogen A (II); 6, serum albumin (v); 7, globulin (Bov. II).
An inhibitable thymidine uptake system associated with the epithelial growth factor, and tests for proteolytic activity were carried out according to Rick [4]. It was observed that most of the fractions separated on DEAE Sephadex showed proteolytic activity (fig. l), the highest activity being displayed by the epithelial growth factor fraction. (A protease unit was defined as an increase in optical density at 2800 A of trichloroacetic acid soluble material, released from a standard solution of denatured casein, 1 % w/v, when treated for 1 h at 37°C in 0.1 M phosphate buffer pH 7.4, with a solution of protease which itself has an absorbance of 2.0 at 2800 A). Proteolytic activity was similarly present in the purified epithelial growth factor after passage through Sephadex GlOO. After storage at - 15°C for several weeks, a gradual reduction in proteolytic activity as well as epithelial growth activity was observed. This does not occur with the crude extract and it is therefore assumed that the purified epithelial growth factor is unstable. It is possible that the relative instability of the epithelial growth factor with increasing purifications may account for the low yield finally obtained. It remains to be shown, however, that the proteolytic activity is indeed a property of the pure epithelial growth factor, and not that of a proteinaceous moiety which separates out with it. It is possible also that the active component of the epithelial growth factor exists as a part of a larger molecule, some of the other properties of which might be proteolytic. In any event the provisional assessment of the molecular weight will almost certainly have to be recalculated after further purification. After storage at - 15°C for several weeks, a gradual reduction in proteolytic activity as well as epithelial growth activity was observed. This does not occur with the crude extract and it is therefore assumed that the
163
purified epithelial growth factor is unstable. It is possible that the relative instability of the epithelial growth factor with increasing purification, may account for the low yield finally obtained. REFERENCES 1. 2. 3. 4.
Andrews, P, Biochem j 91 (1964) 222. Cohen, S, J biol them 237 (1962) 1555. Jones, R 0, Exptl cell res 43 (1966) 645. Rick, W, Methods of enzymatic analysis (ed H U Bergmeyer) p. 811. Academic Press, New York (1963).
Received September 15, 1969
SOME CHARACTERISTICS INHIBITABLE THYMIDINE SYSTEM IN MAMMALIAN
OF AN UPTAKE CELLS
G. S. SCHUSTER and J. D. HARE, Departments of Microbiology and Dental Research, University of Rochester Medical Center, Rochester, N. Y. 14620, USA
Summary Some characteristics of the thymidine uptake process in mammalian cells are described. The uptake is differentially inhibited by organic and inorganic mercurials. It is shown to be different in nature and/or location from the phenylalanine uptake process, which shows a pattern of inhibition by the same compounds which is distinct from that of thymidine. Speculation on the nature of the uptake system is based on possible modes of action of the inhibiting agents.
It has been shown that pyrimidine nucleosides and their analogues may be taken into mammalian cells by an uptake process other than simple diffusion. This is most readily seen when they are at very low concentrations [l]. Hare [2] examined the relationship between extracellular concentration of thymidine (TdR) and the 5-minute uptake into an acidsoluble fraction and found evidence of a nonlinear portion of the uptake curve at low substrate concentrations. At higher concentrations a linear portion was seen. This could be interpreted as indicating that at low concentrations a mediated process predominated, Exptl Cell Res 59