Separation, identification and mechanism of action of the granulocytic chalone

Abstract

The preparation of highly purified granulocytic chalone from bone marrow conditioned medium is described. A sequence of gel-chromatographic steps on Sephadex G-25, G-15 and Biogel P-6 is applied. The end product has a sigmoid dose-response-curve with a plateau at 40-50% inhibition of thymidine incorporation. The inhibition is only observed with bone marrow cells as target cells whereas lymphoid cells are not affected. When bone marrow cells are treated for 1 min with 0.005% trypsin and subsequently used for assaying chalone activity in the presence of cycloheximide, no inhibition is found. These results may indicate that chalone specific receptor sites (of protein nature) are present on the surface of the target cells, which are destroyed by the trypsin treatment. Granulocytic chalone is soluble in aqueous solvents and in polar organic solvent mixtures. Its activity is destroyed by trypsin but only after prolonged treatment. The elution behaviour of chalone is discussed in detail. Chromatography on Sephadex G-75 and Ultrafiltration point to a molecular weight of several thousands, whereas chromatography on G-25 or G-15 or on Biogel P-6 would indicate a rather low molecular weight.