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Purification of a bone-inducing substance (osteogenic factor) from a murine osteosarcoma is reported. The final preparation of the osteogenic factor appeared homogeneous in Sephacryl S-200 gel chromatography and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The factor is a glycoprotein with a molecular weight of 22,000. The yield of the factor was 1 mg from l0 g of osteosarcoma. Allo-implantation of the factor provoked ectopic bone formation in situ in three to four weeks.KEY WORDS osteogenic factor / osteosarcoma / ectopic bone formation It has been speculated that normal (7) and transformed osteoblasts (9) produce a boneinducing substance which provokes cytodifferentiation of immature mesenchymal cells into osteoblasts, but the substance has not been identified.In 1973, we reported the presence of boneinducing activity (osteogenic factor) in freezedried powder of a murine osteosarcoma (BF osteosarcoma). Iso-or allo-implantation of this insoluble powder into muscles of mice induced ectopic bone tissue in sin: in 3 to 4 weeks (1, 5). The osteogenic factor was concluded to be a protein because its bone-inducing activity was lost when it was digested with proteolytic enzymes (2). The main difficulty in purification of the bone-inducing substance was its solubilization, but recently we succeeded in solubilizing it with 4M guanidine-HCl (pH 5.5), and recovered the activity from the solution in the form of an insoluble precipitate by dialysis against deionized water (12). We now report further purification of this osteogenic factor and determination of its molecular weight. and biochemical characters. MATERIALS AND METHODS BFO OsteosarcomaThe BF osteosarcoma, originating from Thelma Dunn osteosarcoma (6), was maintained in CBA strain mice in Argonne National Laboratory (Ill., U.S.A.) by Dr M. P. Finkel and was given to our group for study in 1972. We transferred the sarcoma to a cell culture system and established a clonal cell line (3) (BF-Osaka cells: BFO cells). The biological properties of the cells have been reported (4). An inoculum of 1><10'*' BFO cells was injected subcutaneously into a young male CBA mouse. A tumor (BFO osteosarcoma) was formed in sim in 4 weeks and was serially transplanted into male CBA mice (4~8 weeks old). The tumors harvested were stocked in a deep freezer at --70°C a.nd thawed before use. About 100 g wet weight of tumor tissue was used in the following series of experiments. Seplracryl S-200 Gel ChromatographyTwo Sephacryl S-200 gel columns (4 >< 150 cm and 2.6 >< 150cm) were prepared and equilibrated BONE-INDUCING SUBSTANCE with 4M guanidine-HCl (pH 5.5). Samples dissolved in 4M guanidine-HCl (pH 5.5) were applied to the columns and eluted with the same solution at a. fiow rate of 15 ml/hr. Fractions of 7 ml were collected automatically with a fraction collector. DialysisDialyses were done against 10 mM phosphate buffer solution (pH 7.0) at 4°C unless otherwise stated. Dialysis tubing (Nakarai Chemical Industries, Kyoto, Japan) with a molecular cut-off si...

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SummaryMale mice of three strains, C57BL, DBA and C3H/He, were fed on commercial food with 10% (v/v) ethanol solution as drinking liquid ad libitum for eighty days, and the changes in the activities of enzymes in the metabolic pathway of ethanol in the liver were examined. C57BL and C3H/He mice showed a preference for drinking the 10% (v/v) ethanol solution, while DBA mice did not. The ethanol intake g/g of body weight of C3H/He mice showed the highest value among all three strains and that of C57BL mice tended to show higher value than that of DBA mice. The liver weights of C57BL and C3H/He mice increased significantly following chronic ethanol administration, but that of DBA did not. The cytosolic enzyme alcohol dehydrogenase (ADH) showed no changes in any of the strains following chronic ethanol administration. The microsomal ethanol-oxidizing system (MEOS) of C57BL mice ex hibited approximately 2-fold higher activity compared to that of DBA and C3H/He mice but did not increase in any strain following chronic ethanol administration. However, the microsomal aniline hydroxylase activity in the liver increased significantly in C57BL and C3H/He mice following chronic administration of ethanol. The microsomal cytochrome P-450 content also tended to slightly increase in the same strains of mice. It seemed that cytochrome P-450IIEl was induced in the liver microsomes of these strains. Total aldehyde dehydrogenase (AlDH) activities togeth er with high-Km AlDH activity increased markedly in the microsomes of C57BL mice and tended to increase in C3H/He mice, while it did not change in DBA mice following chronic ethanol administration. In the mitochondria of C57BL, total AlDH activities increased slightly and high-Km AlDH activities tended to increase. These mitochondrial AlDH