What question did this study set out to answer?

This research aims to elucidate the structure and expression of myeloperoxidase during myeloid differentiation.

February 1, 1985Blood183 citationsOpen Access

Myeloperoxidase: its structure and expression during myeloid differentiation

Key Points

This research aims to elucidate the structure and expression of myeloperoxidase during myeloid differentiation.
Used HL-60 cell line to study differentiation into granulocytes and macrophagelike cells.
Isolated mRNA from different HL-60 cell types and normal human granulocytes.
Conducted SDS-PAGE and pulse-chase experiments to analyze MPO synthesis and expression.
HL-60 blasts exhibited a major radioactivity band at 77,000 daltons corresponding to MPO synthesis.
MPO synthesis was almost negligible in differentiated HL-60 granulocytes and macrophages.
MPO remained present in granulocytes and macrophages despite no new synthesis after differentiation.

Abstract

Myeloperoxidase (MPO) is a major protein present in myeloid cells and is used by these cells to help kill microbes. The human promyelocytic HL-60 line can be induced to differentiate to granulocytes or macrophagelike cells. Poly (A) containing RNA was isolated from HL-60 granulocytes, HL-60 macrophages, HL-60 blasts, and normal human granulocytes. The mRNA was translated in a reticulocyte lysate system in the presence of 35S-methionine. The MPO was precipitated from the lysate with rabbit IgG antiserum to human MPO. The resulting precipitate from HL-60 blasts gave a major band of radioactivity of approximately 77,000 daltons and another band at approximately 46,000 daltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The MPO identity of the labeled bands was confirmed by cold competition. The relative mRNA activity expressed as a percentage of radioactivity incorporated into MPO (77,000-dalton band) as compared with total trichloracetic acid (TCA) precipitable radioactivity was 0.2%. Negligible mRNA activity for MPO was present in HL-60 granulocytes, HL-60 macrophages, and normal human granulocytes. Pulse-chase experiments showed that MPO was an approximate 75,000-dalton major band and 77,000-dalton minor band of radioactivity after HL-60 blasts were labeled for 1/2 hour with 35S-methionine and the cell lysate immunoprecipitated and subjected to SDS-PAGE. The chase experiments (one to 24 hours) showed that the 77,000- and 75,000-dalton bands of radioactivity were replaced with two major bands (55,000 and 15,000 daltons) and one minor band (approximately 39,000 daltons) of radioactivity. Six-hour 35S-methionine labeling experiments showed that the relative rate of MPO synthesis compared with total TCA precipitable radioactivity was 0.5% in HL-60 blasts and almost negligible in HL-60 macrophages and granulocytes, normal human granulocytes, and B-lymphocytes. The KG-1 myeloblasts and KG-1a early myeloblasts synthesized a small amount of the 75,000-dalton MPO protein. Although HL-60 cells no longer synthesized MPO after differentiation, HL-60 granulocytes and HL-60 macrophages continued to contain MPO as measured by enzyme activity.(ABSTRACT TRUNCATED AT 400 WORDS)

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