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Biochemical characteristics of alveolar macrophage-specific peroxidase activities in the rat
I de Mendez1, K R Young, J Bignon
1University of Alabama, Birmingham.
Abstract:
The biochemical characteristics of endogenous macrophage peroxidases (Po), and their relationship to myeloperoxidase (MPO), have heretofore been poorly understood and were examined in the current study. Rat alveolar macrophages (AM) were homogenized and fractionated by differential centrifugation into lysosomal and microsomal fractions. The Po activities in both fractions were separated using HPLC gel-filtration and two main activities were detected. One, in the lysosomal fraction, had a relative molecular mass (Mr) of 58,000, while the other, associated with the microsomal fraction corresponded to Mr 74,000. By comparison, MPO from rat polymorphonuclear neutrophils (PMN) had Mr 140,000. The 58- and 74-kDa Po activities also differed from MPO with respect to their apparent Km for H2O2 and optimum pH of activity. Using o-dianisidine as a substrate, the Km for H2O2 of the 58- and 74-kDa Po species was 0.4 and 0.19 mM, respectively, compared to 0.011 mM for MPO. Using monochlorodimedon, the corresponding values were 0.22 and 0.195 mM for the 58- and 74-kDa activities and 0.026 mM for MPO. With either substrate, MPO exhibited optimum activity at pH 5.4, compared to 5.2 for the 58-kDa activity and 4.8 for the 74-kDa species. Thus, rat AM contain two endogenous Po activities with biochemical characteristics distinct from those of MPO. Our findings suggest that these activities represent novel peroxidases that may play an important role in the oxidative metabolism of AM.
Insights
Rat alveolar macrophages contain two distinct endogenous peroxidases (Po) with biochemical properties different from myeloperoxidase (MPO). These novel peroxidases may be crucial for macrophage oxidative metabolism.
Area of Science:
- Biochemistry
- Cell Biology
- Immunology
Background:
- Endogenous macrophage peroxidases (Po) and their relation to myeloperoxidase (MPO) are not well understood.
- Macrophage oxidative metabolism plays a critical role in immune responses.
Purpose of the Study:
- To biochemically characterize endogenous peroxidases in rat alveolar macrophages (AM).
- To compare the properties of AM peroxidases with myeloperoxidase (MPO).
Main Methods:
- Rat AM were homogenized and fractionated into lysosomal and microsomal components.
- High-performance liquid chromatography (HPLC) gel-filtration was used to separate Po activities.
- Kinetic parameters (Km for H2O2) and optimal pH were determined for identified Po species and MPO.
Main Results:
- Two distinct Po activities were identified in rat AM: a 58-kDa species in lysosomes and a 74-kDa species in microsomes.
- These AM Po activities exhibited different molecular masses, Km values for H2O2, and optimal pH compared to MPO (140 kDa).
- The 58-kDa and 74-kDa Po species had significantly higher Km for H2O2 and lower optimal pH than MPO.
Conclusions:
- Rat alveolar macrophages possess two endogenous peroxidase activities biochemically distinct from MPO.
- These findings suggest the presence of novel peroxidases in AM that likely contribute to their oxidative metabolism.
- Further research is warranted to elucidate the specific roles of these novel AM peroxidases in cellular functions.