Characterization of deglycosylated human urinary colony-stimulating factor (CSF-1).
1. Isolation of CSF-1 from human urine was performed through five purification steps. These include concentration by dialysis, silica gel absorption, hydrophobic chromatography and phenyl-Sepharose CL-6B, Fast Protein Liquid Chromatography (FPLC) and finally preparative electrophoresis on polyacrylamide gels. These methods have been reported in a previous paper (Tao et al., 1987). 2. The isolated CSF-1 which exhibits a one band pattern on SDS-PAGE under non-reducing conditions after Coomassie Blue and silver stainings. CSF-1 was purified 100,000-fold and has a specific activity of 2.16 x 10(7) units/mg protein. Its apparent Mr is 57,000 with an isoelectric point pI = 5.8-6.0 CSF-1 is a glycoprotein with 40% of carbohydrate (w/w). 3. An almost complete removal of the carbohydrate moiety from CSF-1 was obtained after treatment with trifluoromethanesulfonic (TFMS) acid followed by gel filtration on Sephadex G-25 (Fine). The deglycosylated (DG) CSF-1 possesses an apparent Mr of 38,000 and an isoelectric point, pI: 6.2 as compared to native-CSF-1 (N-CSF-1), Mr = 57,000 and pI = 5.8 respectively. 4. The TFMS treatment did not alter the activities of CSF-1 as shown by biological assay and receptor binding assay. The thermostability experiment revealed that DG-CSF-1 was less stable than N-CSF-1. The circular dichroism spectra (CD) of N-CSF-1 and DG-CSF-1 were different. 5. The features of interaction of iodinated-N-CSF-1 and iodinated-DG-CSF-1 with single cell suspensions from human peritoneal macrophage were studied. The binding activity of peritoneal macrophage was the highest among all cells examined.(ABSTRACT TRUNCATED AT 250 WORDS)
Gao
G
G
Department of Biochemistry, University of Nanjing, People's Republic of China.
Tao
X
X
Zhu
D X
DX
Lamblin
G
G
Shing
Y W
YW
Han
K K
KK
D003115:Colony-Stimulating Factors / Q000652:urine*; D006031:Glycosylation; D006801:Humans; D007526:Isoelectric Point; D007700:Kinetics; D008698:Mesylates; D008970:Molecular Weight
D003115:Colony-Stimulating Factors; D008698:Mesylates; C012077:trifluoromethanesulfonic acid