Isolation and characterization of an unprocessed extracellular myeloperoxidase in HL-60 cell cultures.
Extracellular myeloperoxidase of human myeloid leukemia HL-60 cells was purified to homogeneity from its culture supernatant by ammonium sulfate fractionation, CM-Sepharose column chromatography, and monoclonal antibody-Sepharose affinity column chromatography. The yield of enzyme activity was 38% that of the ammonium sulfate fraction. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified preparation gave a single band of approximately 84 kDa. Analysis of protein blot with antibodies specific for the light and heavy chains of myeloperoxidase indicated that the enzyme contained a light and a heavy chain in a single polypeptide. The amino-terminal amino acid sequence of the enzyme began at amino acid residue 155 of the 745-amino acid sequence predicted from myeloperoxidase cDNA, indicating that the enzyme consisted of 591 amino acids. Sucrose density gradient centrifugation of the enzyme showed that the enzyme was a monomeric form. In pulse-chase experiments on HL-60 cells with [35S]methionine, pulse-labeled myeloperoxidase precursors were shown to be processed to a light chain and a heavy chain of cellular enzyme. During a 3-day chase period, newly formed processed monomeric enzyme was converted to a dimeric form.
Hur
S J
SJ
Institute for Protein Research, Osaka University, Japan.
Toda
H K
HK
Yamada
M
M
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002460:Cell Line; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009195:Peroxidase / Q000302:isolation & purification* / Q000378:metabolism
D003470:Culture Media; D009195:Peroxidase