Partial purification and characterization of human megakaryocyte colony-stimulating factor (Meg-CSF).
Megakaryocyte colony-stimulating factor (Meg-CSF) in urinary extracts from patients with aplastic anemia was partially characterized and purified. Using Meg-CSF-enriched fractions, we established that the moiety has the following characteristics: 1) portions of the molecules having Meg-CSF activity have sialic acid, probably with a biantennary structure, and beta-galactose residues as the terminal and penultimate sugars; 2) disulfide residues are an essential chemical group of the molecule and are located on its surface; and 3) Meg-CSF activity is stable in n-propanol, but not in acetonitrile with trifluoroacetic acid. Partial purification of Meg-CSF by a four-step procedure of ethanol precipitation, CM Affi-Gel Blue chromatography, wheat germ agglutinin-sepharose chromatography, and high-resolution hydroxyapatite chromatography, yielded a concentrate with a 430- to 630-fold increase in specific activity. The partially purified Meg-CSF fractions stimulated both human and murine megakaryocytopoiesis in vitro (CFU-meg). When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreduced conditions, Meg-CSF activity was recovered in the 29-34 kDa molecular weight fractions. We have also shown that Meg-CSF, purified from the urine of aplastic anemia patients, stimulated murine megakaryocytopoiesis and platelet production in vivo. Final purification of human urinary Meg-CSF is currently in progress.
Ogata
K
K
Hipple Cancer Research Center, Dayton, Ohio 45439-2092.
Zhang
Z G
ZG
Abe
K
K
Murphy
M J
MJ
D000741:Anemia, Aplastic / Q000652:urine; D000818:Animals; D001798:Blood Proteins / Q000652:urine*; D002846:Chromatography, Affinity; D003115:Colony-Stimulating Factors / Q000652:urine*; D058851:GPI-Linked Proteins; D006801:Humans; D008533:Megakaryocytes / Q000032:analysis*; D008562:Membrane Glycoproteins; D000090204:Mesothelin; D009439:Neuraminidase; D011506:Proteins / Q000302:isolation & purification*; D051381:Rats; D012997:Solvents; D013439:Sulfhydryl Reagents
D001798:Blood Proteins; D003115:Colony-Stimulating Factors; D058851:GPI-Linked Proteins; D008562:Membrane Glycoproteins; D011506:Proteins; D012997:Solvents; D013439:Sulfhydryl Reagents; D009439:Neuraminidase; D000090204:Mesothelin