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Ca2+-dependent ATPase activity of alveolar macrophage plasma membrane
Abstract:
A plasma membrane fraction was isolated from lysates of Bacillus Calmette-Guérin-induced alveolar macrophages of rabbit. On the basis of morphological and biochemical criteria this fraction appeared to be minimally contaminated by other subcellular organelles. Concentrations of Ca2+, but not of Mg2+, from 6.10(-8) to 1.10(-5) M markedly stimulated the basal ATPase (EC 3.6.1.3) activity of the plasma membrane, with an apparent Km (Ca2+) of 1.10(-6) M. The specific activity of the Ca2+-ATPase assayed at pCa = 5.5 was enriched about 8-fold in the plasma membrane fraction over the macrophage lysate. In contrast, the specific activity of the K+, EDTA-activated ATPase, associated to macrophage myosin, increased only 1.3-fold. Oligomycin and -SH group reagents exerted no influence on the Ca2+-ATPase activity, which was on the contrary inhibited by detergents such as Triton X-100 and deoxycholate. The activity of the Ca2+-ATPase was maximal at pH 7, and was decreased by 50 mM Na+ and 5 mM K+. On the contrary, the activity of Mg2+-ATPase, also present in the plasma membrane fraction, had a peak at about pH 7.8, and was stimulated by Na+ plus K+. On account of its properties, it is suggested that the Ca2+-ATPase is a component of the plasma membrane of the alveolar macrophage, and that its function may be that of participating in the maintenance of low free Ca2+ concentrations in the macrophage cytosol.
Insights
This study identified a calcium-dependent ATPase (Ca2+-ATPase) in rabbit alveolar macrophage plasma membranes. This enzyme is crucial for maintaining low intracellular calcium levels in these immune cells.
Area of Science:
- Cell Biology
- Immunology
- Biochemistry
Background:
- Alveolar macrophages play a key role in lung immunity.
- Understanding macrophage plasma membrane function is vital for immune response research.
- Calcium ion homeostasis is critical for cellular processes.
Purpose of the Study:
- To isolate and characterize the Ca2+-ATPase in rabbit alveolar macrophage plasma membranes.
- To investigate the enzymatic properties and potential function of this Ca2+-ATPase.
- To differentiate Ca2+-ATPase activity from other ATPases in the plasma membrane fraction.
Main Methods:
- Isolation of a plasma membrane-rich fraction from Bacillus Calmette-Guérin-induced rabbit alveolar macrophages.
- Biochemical assays to measure ATPase activity, including Ca2+-ATPase and Mg2+-ATPase.
- Enzyme kinetics studies to determine substrate affinity (Km) and optimal conditions (pH, ion concentrations).
- Assessment of inhibitor effects (detergents, -SH reagents, oligomycin).
Main Results:
- A distinct Ca2+-ATPase activity was enriched approximately 8-fold in the plasma membrane fraction.
- The Ca2+-ATPase exhibited high affinity for Ca2+ (apparent Km = 1.10(-6) M) and was stimulated by micromolar Ca2+ concentrations.
- Enzyme activity was optimal at pH 7 and inhibited by Na+ and K+ ions.
- Detergents inhibited Ca2+-ATPase activity, while oligomycin and -SH reagents had no effect.
Conclusions:
- The characterized Ca2+-ATPase is a component of the alveolar macrophage plasma membrane.
- Its properties suggest a role in actively transporting calcium ions out of the cytosol.
- This function likely contributes to maintaining low intracellular free Ca2+ concentrations essential for macrophage physiology.