Purification of a fibroblast growth factor from bovine pituitary.

D Gospodarowicz

doi:10.1016/s0021-9258(19)41631-1

Abstract

The purification from bovine pituitary gland of a growth factor responsible for the control of animal cell division in tissue culture is reported. This growth factor is a polypeptide of 13,300 molecular weight and is homogeneous when analyzed by polyacrylamide gel electrophoresis, carboxymethyl-Sephadex gradient elution chromatography, and Sephadex G-50 chromatography. The yield of growth factor is 5 mg per kg of pituitary. It is active in stimulating DNA synthesis in 3T3 cells at concentrations as low as 2 times 10-13 M with saturation at 1 times 10-10 M.

Highlights

MethodsMaterials-Bovine pituitaries were obtained from both the Endocrine Research Supply and the Talone Meat Company.Carboxymethyl-SephadexC-50 (CM-Sephadex), Sephadex G-50, and Senhadex G-75 were obtained from Pharmacia
We have recently reported the presence in bovine pituitary extracts of an agent which stimulates the division of fibroblasts in tissue culture [1]
In this communication we describe the isolation of this agent and present evidence which allows us to conclude that it is distinct from all previously described pituitary hormones

Summary

Methods

Materials-Bovine pituitaries were obtained from both the Endocrine Research Supply and the Talone Meat Company.Carboxymethyl-SephadexC-50 (CM-Sephadex), Sephadex G-50, and Senhadex G-75 were obtained from Pharmacia. Materials-Bovine pituitaries were obtained from both the Endocrine Research Supply and the Talone Meat Company. Bovine NIH-LH-B7, which contains the growth factor as a contaminant, was taken as a reference standard. Eight hours later the medium was removed and replaced with 5 ml of DME’ containing 0.4% calf serum. Twenty-four hours later, an identical medium change was made. Within a day of this final medium change, the cells entered a resting stage, at which time, various concentrations of samples dissolved in DME with 0.5yc crystalline bovine albumin (to minimize nonspecific adsorption) were added to the cultures. Sixteen hours after sample addition, 50 ~1 of DME containing 5 PCi of [methyl-sH]thymidine (New England Nuclear) and 6 rg of cold thymidine were added to each dish. DNA was precipitated by adding trichloroacetic acid to a final concentration of 30%. The precipitates were collected on Whatman GF/A glass fiber filters, washed twice with 15% trichloroacetic acid, and counted (scintillation counter with toluene-2,5-diphenyloxazole (PPO) - 1,4 - bis[2 - (5 -phenyloxazolyl)]benzene (POPOP)

Results

Discussion

Conclusion