Resolution of ion translocating proteolipid subclasses active in bacterial calcification

Area of Science:

• Biochemistry
• Microbiology
• Biomineralization

Background:

• Calcium hydroxyapatite formation involves membrane-surface interactions with calcium, phosphate, phospholipids, and proteolipids.
• Proteolipids from Bacterionema matruchotti function as ionophores when reconstituted into proteoliposomes.
• Ionophoric activity of these proteolipids is inhibited by [14C]dicyclohexylcarbodiimide ([14C]DCCD), which binds to a Mr 8500 protein.

Purpose of the Study:

• To investigate whether proteins other than the [14C]DCCD-binding protein are involved in proteolipid ionophoric activity.
• To determine the roles of different proteolipid species extracted using solvents of varying polarity.

Main Methods:

• Proteolipids were extracted from Bacterionema matruchotti using chloroform:methanol (2:1) and chloroform:methanol:HCl (200:100:1) solvents.
• Proteolipid species were isolated using Sephadex LH-20 chromatography and analyzed by SDS-PAGE.
• Ionophoric activity was assessed by measuring proton translocation in reconstituted bacteriorhodopsin-proteoliposomes.

Main Results:

• SDS-PAGE revealed a Mr 10,000 proteolipid in the chloroform:methanol extract, absent in the acidified extract.
• The acidified extract contained a Mr 8500 proteolipid that co-migrated with the [14C]DCCD-binding protein.
• Combining proteolipids from both extracts significantly increased the rate and extent of proton translocation.

Conclusions:

• At least two distinct proteolipid proteins are required for ionophoric activity.
• A Mr 10,000 protein (extracted by non-acidified solvent) and the Mr 8500 [14C]DCCD-binding protein (extracted by acidified solvent) are both necessary.
• These findings elucidate the cooperative function of multiple proteolipids in bacterial ion transport and calcification.