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Characterization of ATP-dependent proton transport in medullary bone-derived microsomes
1Department of Anatomy, University of Oulu, Finland.
Abstract:
Proton transport in microsomal vesicles derived from medullary bone of laying hens was observed to be inhibited in a dose-dependent manner with fusidic acid, 7-chloro-4-nitrobenz-2-oxa-1,3-diatzole (NBD-Cl), duramycin and dicyclohexylcarbodiimide (DCCD). The IC50 values were 570 microM, 4.5 microM, 10 micrograms/ml and 32 microM for fusidic acid, NBD-Cl, duramycin and DCCD, respectively. 14C-DCCD labeled a single protein band of 15-17 kDa from bone-derived microsomes in SDS-electrophoresis. A protein of this size is a proton-conducting subunit of the vacuolar ATPases. Further, the proton transport was found to be electrogenic, thus it generates the membrane potential across the vesicle membrane. The generation of membrane potential was inhibited using 100 nM bafilomycin A1, which in low concentrations is a specific inhibitor of vacuolar ATPases. The presence of Cl- was essential for maximal proton transport activity. These results confirm the electrogenicity and extend the characterization of the osteoclastic H(+)-ATPase.
Insights
Researchers investigated proton transport in laying hen medullary bone, identifying vacuolar ATPases as key players. Inhibitor studies confirmed the electrogenic nature of this proton transport, crucial for bone function.
Area of Science:
- Biochemistry
- Cell Biology
- Bone Physiology
Background:
- Medullary bone in laying hens serves as a calcium reservoir.
- Osteoclasts are crucial for bone resorption, a process involving proton transport.
- The specific mechanisms of proton transport in avian bone are not fully elucidated.
Purpose of the Study:
- To characterize the proton transport system in medullary bone microsomes.
- To identify the molecular components involved in proton transport.
- To confirm the electrogenic nature and functional properties of the osteoclastic H(+)-ATPase.
Main Methods:
- Microsomal vesicles were isolated from laying hen medullary bone.
- Proton transport was measured using various inhibitors: fusidic acid, NBD-Cl, duramycin, and DCCD.
- Protein labeling with 14C-DCCD followed by SDS-electrophoresis was performed.
- Membrane potential generation was assessed and inhibited by bafilomycin A1.
- The role of chloride ions (Cl-) was investigated.
Main Results:
- Proton transport was inhibited dose-dependently by fusidic acid, NBD-Cl, duramycin, and DCCD, with distinct IC50 values.
- 14C-DCCD labeled a 15-17 kDa protein band, consistent with a vacuolar ATPase subunit.
- Proton transport was electrogenic, generating a membrane potential.
- Bafilomycin A1 specifically inhibited membrane potential generation.
- Chloride ions were essential for maximal proton transport activity.
Conclusions:
- The osteoclastic H(+)-ATPase is involved in proton transport in medullary bone.
- The proton transport system is electrogenic and requires chloride ions.
- These findings contribute to understanding the molecular mechanisms of bone resorption in avian species.