Purification of an endopeptide to homogenity and the verification of its selective inhibitory action on myeloid cell proliferation.
The purification of an endogenous regulatory peptide to homogeneity which selectively inhibits the proliferation of normal and leukaemic myeloid cells is described. Leucocytes isolated from calf spleen or horse blood were homogenized, extracted by acetone, chloroform and distilled water, ultrafiltrated by Amicon Diaflo XM 50 and PM 10 membranes. The lyophilized filtrate was chromatographed on Sephadex G-15 and G-10 columns. Fractions were tested for the selective inhibition of myeloid proliferation by 3H-TdR incorporation and capillary colony formation. The active fractions were submitted to paper electrophoresis at pH 6.5 and 1.9, and the peptides were re-tested. Finally a ninhydrin negative and chlor-tolidine positive oligopeptide was identified as granulocyte-specific inhibitor, effective at 0.2 to 3.0 pmol/ml MED value in vitro. The peptide is negatively charged at pH 6.5, its electrophoretic mobility as compared to aspartic acid is -0.60. The peptide has no charge at pH 1.9, the mobility related to epsilon-DNP lysine is 0.26. Details of the structure analysis of the inhibitory endopeptide is described in our next paper.
D000818:Animals; D001854:Bone Marrow Cells; D002417:Cattle; D002455:Cell Division / Q000187:drug effects; D003864:Depression, Chemical; D006098:Granulocytes / Q000166:cytology / Q000187:drug effects*; D006131:Growth Inhibitors / Q000302:isolation & purification*; D007942:Leukemia, Experimental / Q000473:pathology; D008297:Male; D051379:Mice; D010455:Peptides / Q000302:isolation & purification* / Q000494:pharmacology; D051381:Rats; D013154:Spleen / Q000032:analysis*; D013950:Thymus Gland / Q000166:cytology
D006131:Growth Inhibitors; D010455:Peptides