Further application of a two-step heparin affinity chromatography method using divalent cations as eluents: purification and identification of membrane-bound heparin binding proteins from the mitochondrial fraction of HL-60 cells.
Membrane proteins were obtained from the mitochondrial fraction of HL-60 cells by solubilization with octyl glucoside and bound to heparin-gels. Bound proteins were successively eluted with solutions containing increasing concentrations of Mg(2+) in the first and increasing concentrations of Ca(2+) in the second chromatography. After SDS-PAGE and subsequent N-terminal amino acid analysis of proteins on each band, 13 proteins were identified. Fifteen out of the 37 proteins analysed were modified at their N-termini. These results show that this two-step affinity chromatography method using divalent cations as eluents can be applied to a variety of membranes for the isolation of specific proteins.
Iida
Tsukimi
T
Department of Science of Human Life, City College of Mie, 157, Ishinnden-nakano, Tsu, Mie 514-0112, Japan. iida@tsu-cc.ac.jp
Kamo
Masaharu
M
Uozumi
Nobuyuki
N
Inui
Takashi
T
Imai
Katsuyuki
K
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000032:analysis* / Q000378:metabolism; D002413:Cations, Divalent; D002458:Cell Fractionation; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D018922:HL-60 Cells; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis* / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D020539:Sequence Analysis, Protein
D002352:Carrier Proteins; D002413:Cations, Divalent; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D006493:Heparin