424
PURIFICATION AND SYNTHESIS OF HORMONES
[36]
yield of the required peptide is considerably reduced, although it is still comparable with that obtained with the hydrogen fluoride method. We have found that the extent of fragmentation at proline varies considerably from analogue to analogue and in some instances is virtually absent.
Biologic Results The measurements of L H - R H activities by the in vivo and in vitro stimulation of L H and F S H release are described in detail elsewhere in this volume. Serum L H levels of the two synthetic L H - R H samples are compared with those obtained after the administration of saline and natural L H - R H . Measurements are made at two dose levels. The two synthetic peptides possessed maximal potencies. The activities of several analogues we have synthesized are also given in Table I.
[36] P r e p a r a t i o n
of Epidermal
Growth
Factor
B y STANLEY COHEN and C. RICHARD SAVAGE, JR. I. Introduction . . . . . . . . . . . . . . . . . . II. Assay . . . . . . . . . . . . . . . . . . . . III. Isolation Procedure . . . . . . . . . . . . . . . . A. Preparation of Salivary Gland Extracts . . . . . . . . . B. Bio-Gel P-10 Chromatography . . . . . . . . . . . . C. Diethylaminoethyl-Cellulose Ion-Exchange Chromatography D. Isolation of EGF Lacking the COOH-Terminal Leu-Arg Residues (EGF-2) . . . . . . . . . . . . . . . . . . IV. Yield of EGF . . . . . . . . . . . . . . . . . . V. Properties of EGF . . . . . . . . . . . . . . . .
424 425 427 427 427 428 428 429 430
I. I n t r o d u c t i o n Epidermal growth factor (EGF) is a polypeptide (MW 6045) isolated from the submaxillary glands of adult male mice, 1 which stimulates the proliferation of a number of epithelial tissues both in vivo and in vitro. ~,3 Among the metabolic events in epidermal tissue affected by E G F are the s~imulation of protein and R N A synthesis, the conversion of pre1S. Cohen, J. Biol. Chem. 237, 1555 (1962). 2S. Cohen and J. M. Taylor, in "Epidermal Wound Healing" (H. I. Maibach and D. T. Rovee, eds.), p. 203. Yearbook Publ., Chicago, Illinois, 1972. s C. R. Savage, Jr., and S. Cohen, Exp. Eye Res. 15, 361 (1973).
[36]
PREPARATION OF EPIDERMAL GROWTH FACTOR
425
viously existing ribosomal monomers into polysomes, and the induction of ornithine decarboxylase with the concomitant accumulation of intracellular polyamines. These studies have been reviewed recently. 2 Although methods for the isolation of E G F have been described, 1,~ the yields are low and the multistep procedures are time-consuming. We are describing our present procedure for the isolation of E G F 2 The method is based on the observation that, at low pH, columns of polyacrylamide (Bio-Gel) are capable of selectively adsorbing E G F from crude homogenates of the male mouse submaxillary gland. II. Assay The biologic assay for E G F is based on precocious eyelid opening in the newborn mouse. 1 Daily subcutaneous injections of the preparation are nlade into newborn mice with a volume of 0.01 ml/g body weight. The injections are started within 12 hours after birth. Since the volume of daily injection is varied in direct proportion to the body weight, the number of micrograms of material injected per gram body weight is constant throughout the experimental period. The animals are examined and weighed at 24-hour intervals; the days on which the eyes open are recorded. Three or four animals receive injections at each dosage level tested (a series of twofold dilutions). The material may be injected with either distilled water or dilute salt solution as diluent. This assay is useful within the dosage range of 0.3-5 t*g of pure E G F per gram body weight per day, and is sensitive to twofold changes in concentration of the active material. A representative assay is shown ill Table I. One may define a biologic unit of activity as that quantity of material injected per gram body weight per day to cause eyelid opening in 9 days. Therefore, within the limits of this assay procedure, pure E G F contains approximately 1000-2000 units/rag. Rabbit antibodies to E G F may be prepared with the aid of Freund's adjuvant as described previously. 1 Antiserum to E G F has proved to be very useful for both semiquantitative and quantitative estimation of the amounts of E G F in solution. The semiquantitative ring test described by Taylor e t al. ~ may be used for the rapid determination of approximate amounts of E G F in unknown solutions (e.g., column eluates, see Fig. 1). Presumably, a more precise estimation of the E G F concentration could be obtained by quan~J. M. Taylor, S. Cohen, and W. M. Mitchell, Proc. Nat. Acad. Sci. U.S. 67, 164 (1970). C. R. Savage, Jr. and S. Cohen, J. Biol. Chem. 247, 7609 (1972).
426
PURIFICATION
EFFECT
OF I N J E C T I O N
AND
SYNTHESIS
TABLE I EGF
OF P U R E
OF
[35]
HORMONES
INTO NEWBORN
Dosage (#g/g body weight/day)
Eyelids open (day)
4.8 2.4 1.2 0.6 0.3 0
6,6,7 7,7,8 8,8,8 9,9,10 10,10,10 13,13,14
MICE a
Newborn mice were treated with daily injections of 0.010 ml/g of body weight. Three animals were used for each dosage.
4.0 E m 0 3.0 P,i I-~ 2.0 z m riO 1.0
>-
COLUMN 4+
PURIFICATION AND SYNTHESIS OF HORMONES
[36]
yield of the required peptide is considerably reduced, although it is still comparable with that obtained with the hydrogen fluoride method. We have found that the extent of fragmentation at proline varies considerably from analogue to analogue and in some instances is virtually absent.
Biologic Results The measurements of L H - R H activities by the in vivo and in vitro stimulation of L H and F S H release are described in detail elsewhere in this volume. Serum L H levels of the two synthetic L H - R H samples are compared with those obtained after the administration of saline and natural L H - R H . Measurements are made at two dose levels. The two synthetic peptides possessed maximal potencies. The activities of several analogues we have synthesized are also given in Table I.
[36] P r e p a r a t i o n
of Epidermal
Growth
Factor
B y STANLEY COHEN and C. RICHARD SAVAGE, JR. I. Introduction . . . . . . . . . . . . . . . . . . II. Assay . . . . . . . . . . . . . . . . . . . . III. Isolation Procedure . . . . . . . . . . . . . . . . A. Preparation of Salivary Gland Extracts . . . . . . . . . B. Bio-Gel P-10 Chromatography . . . . . . . . . . . . C. Diethylaminoethyl-Cellulose Ion-Exchange Chromatography D. Isolation of EGF Lacking the COOH-Terminal Leu-Arg Residues (EGF-2) . . . . . . . . . . . . . . . . . . IV. Yield of EGF . . . . . . . . . . . . . . . . . . V. Properties of EGF . . . . . . . . . . . . . . . .
424 425 427 427 427 428 428 429 430
I. I n t r o d u c t i o n Epidermal growth factor (EGF) is a polypeptide (MW 6045) isolated from the submaxillary glands of adult male mice, 1 which stimulates the proliferation of a number of epithelial tissues both in vivo and in vitro. ~,3 Among the metabolic events in epidermal tissue affected by E G F are the s~imulation of protein and R N A synthesis, the conversion of pre1S. Cohen, J. Biol. Chem. 237, 1555 (1962). 2S. Cohen and J. M. Taylor, in "Epidermal Wound Healing" (H. I. Maibach and D. T. Rovee, eds.), p. 203. Yearbook Publ., Chicago, Illinois, 1972. s C. R. Savage, Jr., and S. Cohen, Exp. Eye Res. 15, 361 (1973).
[36]
PREPARATION OF EPIDERMAL GROWTH FACTOR
425
viously existing ribosomal monomers into polysomes, and the induction of ornithine decarboxylase with the concomitant accumulation of intracellular polyamines. These studies have been reviewed recently. 2 Although methods for the isolation of E G F have been described, 1,~ the yields are low and the multistep procedures are time-consuming. We are describing our present procedure for the isolation of E G F 2 The method is based on the observation that, at low pH, columns of polyacrylamide (Bio-Gel) are capable of selectively adsorbing E G F from crude homogenates of the male mouse submaxillary gland. II. Assay The biologic assay for E G F is based on precocious eyelid opening in the newborn mouse. 1 Daily subcutaneous injections of the preparation are nlade into newborn mice with a volume of 0.01 ml/g body weight. The injections are started within 12 hours after birth. Since the volume of daily injection is varied in direct proportion to the body weight, the number of micrograms of material injected per gram body weight is constant throughout the experimental period. The animals are examined and weighed at 24-hour intervals; the days on which the eyes open are recorded. Three or four animals receive injections at each dosage level tested (a series of twofold dilutions). The material may be injected with either distilled water or dilute salt solution as diluent. This assay is useful within the dosage range of 0.3-5 t*g of pure E G F per gram body weight per day, and is sensitive to twofold changes in concentration of the active material. A representative assay is shown ill Table I. One may define a biologic unit of activity as that quantity of material injected per gram body weight per day to cause eyelid opening in 9 days. Therefore, within the limits of this assay procedure, pure E G F contains approximately 1000-2000 units/rag. Rabbit antibodies to E G F may be prepared with the aid of Freund's adjuvant as described previously. 1 Antiserum to E G F has proved to be very useful for both semiquantitative and quantitative estimation of the amounts of E G F in solution. The semiquantitative ring test described by Taylor e t al. ~ may be used for the rapid determination of approximate amounts of E G F in unknown solutions (e.g., column eluates, see Fig. 1). Presumably, a more precise estimation of the E G F concentration could be obtained by quan~J. M. Taylor, S. Cohen, and W. M. Mitchell, Proc. Nat. Acad. Sci. U.S. 67, 164 (1970). C. R. Savage, Jr. and S. Cohen, J. Biol. Chem. 247, 7609 (1972).
426
PURIFICATION
EFFECT
OF I N J E C T I O N
AND
SYNTHESIS
TABLE I EGF
OF P U R E
OF
[35]
HORMONES
INTO NEWBORN
Dosage (#g/g body weight/day)
Eyelids open (day)
4.8 2.4 1.2 0.6 0.3 0
6,6,7 7,7,8 8,8,8 9,9,10 10,10,10 13,13,14
MICE a
Newborn mice were treated with daily injections of 0.010 ml/g of body weight. Three animals were used for each dosage.
4.0 E m 0 3.0 P,i I-~ 2.0 z m riO 1.0
>-
COLUMN 4+
