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The 35 kDa DCCD-binding protein from pig heart mitochondria is the mitochondrial porin
Abstract:
The protein which can be labelled by low concentrations of dicyclohexylcarbodiimide in the Mr region of 30 000-35 000 has been purified from pig heart mitochondria with a high yield and as a single band of apparent Mr 35 000 in dodecyl sulphate-containing gels. The protein is not identical with the phosphate carrier as suggested before, since the two proteins behave differently during isolation. Incorporation of the isolated 35 kDa dicyclohexylcarbodiimide-binding protein into lipid bilayer membranes causes an increase of the membrane conductance in definite steps, due to the formation of pores. The specific pore-forming activity increases during the purification procedure. The single pore conductance is about 4.0 nS, suggesting a diameter of 1.7 nm of the open pore. The pore conductance is dependent on the voltage across the membrane. Anion permeability of the pore is higher than cation permeability. These properties are similar to those described for isolated mitochondrial and bacterial porins. It is concluded that the 35 kDa dicyclohexylcarbodiimide-binding protein from pig heart mitochondria is identical with porin from outer mitochondrial membrane.
Insights
Researchers purified a 35 kDa protein from pig heart mitochondria that forms pores in membranes. This dicyclohexylcarbodiimide-binding protein is identified as mitochondrial porin, crucial for outer membrane transport.
Area of Science:
- Mitochondrial biochemistry
- Membrane protein research
- Cellular transport mechanisms
Background:
- Mitochondria possess a unique outer membrane with specific transport proteins.
- Previous studies suggested a dicyclohexylcarbodiimide (DCC)-binding protein might be the phosphate carrier.
- Characterization of mitochondrial porins is essential for understanding metabolite transport.
Purpose of the Study:
- To purify and characterize a DCC-binding protein from pig heart mitochondria.
- To determine if this protein is identical to the phosphate carrier.
- To investigate the functional properties of the purified protein in lipid bilayers.
Main Methods:
- Purification of a 35 kDa protein from pig heart mitochondria using DCC labeling.
- Analysis of protein purity via SDS-PAGE.
- Reconstitution of the purified protein into lipid bilayer membranes.
- Electrophysiological measurements of membrane conductance and pore formation.
Main Results:
- A high-yield purification of a single 35 kDa DCC-binding protein was achieved.
- The purified protein is distinct from the mitochondrial phosphate carrier.
- Incorporation into lipid bilayers induced stepwise conductance increases, indicating pore formation.
- The pore exhibited a specific conductance of 4.0 nS, a diameter of 1.7 nm, voltage-dependence, and higher anion than cation permeability.
Conclusions:
- The 35 kDa DCC-binding protein from pig heart mitochondria is identified as porin.
- This porin is a component of the outer mitochondrial membrane.
- The pore-forming and transport properties align with known mitochondrial and bacterial porins.