Resolution of ion translocating proteolipid subclasses active in bacterial calcification.
Formation of calcium hydroxyapatite occurs on membrane surfaces via interaction of calcium, inorganic phosphate, phospholipids, calcifiable proteolipids, and ion flux to and from the nucleating site. Recently, this laboratory reported that proteolipids from the calcifying bacterium, Bacterionema matruchotti, act as an ionophore when reconstituted into bacteriorhodopsin-proteoliposomes. This ionophoric activity is blocked by [14C]dicyclohexylcarbodiimide ([14C]DCCD). SDS-PAGE shows that [14C]DCCD binds to a single band of Mr 8500. To determine whether proteins other than the [14C]DCCD-binding protein are involved, we examined the function of proteolipid species extracted by solvents of differing polarity. Proteolipids were isolated independently from chloroform:methanol (2:1) and chloroform:methanol:HCl (200:100:1) extracts of the bacteria by Sephadex LH-20 chromatography and were electrophoresed on 12.5% acrylamide gels. The chloroform:methanol extract contained a major hand at Mr 10,000 that was not present in gels of proteolipid isolated by acidified solvent. Proteolipids extracted in chloroform:methanol:HCl included a broad band at Mr 8500, which co-migrated with the [14C] DCCD-binding protein. The rate and extent of proton translocation were not altered when either proteolipid extract was added individually to bacteriorhodopsin proteoliposomes. However, when proteolipids isolated from the chloroform:methanol and chloroform:methanol:HCl extracts were combined, the rate and extent of translocation were increased. These data demonstrate that at least two proteolipid proteins are necessary for ionophoric activity, the Mr 10,000 protein isolated by chloroform:methanol 2:1 and the [14C]DCCD-binding protein requiring acidified solvent for extraction.
Swain
L D
LD
Department of Orthopaedics, University of Texas Health Science Center,San Antonio 78284-7774.
Renthal
R D
RD
Boyan
B D
BD
D000191:Actinomycetaceae / Q000378:metabolism*; D001436:Bacteriorhodopsins / Q000378:metabolism; D002234:Carbodiimides / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D004024:Dicyclohexylcarbodiimide / Q000378:metabolism*; D017886:Durapatite; D006882:Hydroxyapatites / Q000378:metabolism*; D007473:Ion Channels / Q000378:metabolism*; D011510:Proteolipids / Q000378:metabolism*; D011522:Protons
D002234:Carbodiimides; D002352:Carrier Proteins; D006882:Hydroxyapatites; D007473:Ion Channels; D011510:Proteolipids; D011522:Protons; C026077:dicyclohexylcarbodiimide-binding proteolipid; D001436:Bacteriorhodopsins; D004024:Dicyclohexylcarbodiimide; D017886:Durapatite