Purification and Characterization of Small Molecular Weight Myeloperoxidase from Human Promyelocytic Leukemia HL-60 Cells (1)
The human leukemia line HL-60 cells are promyelocytic cells and can be induced to differentiate into granulocytes or macrophages by dimethyl sulfoxide, 12-O-tetradecanoyl phorbor-13-acetate, or tunicamycin.
Myeloperoxidase (MPO) from HL-60 cells consisted of a small size MPO, Mw 79 000, in addition to large size MPO, Mw 153000. The small MPO differed in immunological properties from the large MPO, and the large MPO appeared heterogeneous on CM-Sepharose and Con A-Sepharose column chromatography.
HL-60 cells were transplanted subcutaneously into the back of male athymic nude mice (nu/nu) with a BALB/C genetic background and solid tumors were obtained from the animals 1.5 months later. Human MPO was extracted with 1% CETAB from the particulate fraction precipitated at 20000g from an HL-60 cell homogenate. In CM-Sepharose CL-6B preparation, MPO activity was separated into three fractions, 1, II, and III, which were recovered in tubes 30-33, 35-38, and 39-44, respectively. In Sephacryl S-200 preparation, MPO I was separated into two distinct activities, named IA (Mw 150,000) and IB, on the column. On gel filtration, all the activity of MPO II and III was eluted in exactly the same position as MPO IA, and none in fractions corresponding to MPO IB.
Myeloperoxidase IB activity amounted to 8% of the total MPO activity at the final stage of purification. The yield of total activity of MPO was 62% from the extract.
The absorption spectra of native and reduced MPO IB were nearly identical with those of the large MPO IA, II and III. The absorption bands of native and reduced absorption MPO IA, IB, II, and III are shown.
In sucrose density gradient centrifugation, the molecular weight of MPO IB was calculated to be 79000 from the S20,w value of 5.2 S and the molecular weights of MPO IA, II, and III were calculated to be 153000 from their average S20,w of 8.07 S. In SDS-PAGE analysis, two bands of proteins at positions corresponding to molecular weights of 59350 and 10150 were obtained with all four MPO. The sum of the molecular weights of the two bands is 69500.
In Con A-Sepharose column chromatography, MPO IA was eluted at 24 mM methyl a-D-mannoside, and MPO II and III were eluted at 47 and 44 mM methyl a-D-mannoside, respectively. The small MPO IB was eluted at 20 mM methyl a-D-mannoside under the same conditions.
All four MPO reacted with antiserum against MPO III in the immunological reactions on agarose gel.
The amounts of complement fixed by the antiserum against MPO III with the large MPO IA, II, and III were not significantly different, but the amount fixed by reaction of various amounts of small myeloperoxidase IB was much less. The largest amounts of complement fixed by reaction of the antiserum with the small MPO IB and with the large myeloperoxidase III were distinctly different.
MPO were calculated to constitute 2.5% of the total protein in HL-60 cells. Large and small MPO when treated with 2-mercaptoethanol and sodium dodecyl sulfate gave rise to two protein components of M, 10150 and 59 300, respectively.