Co-isolation of proteolipids and calcium-phospholipid-phosphate complexes

Summary

This study demonstrates that calciumphospholipid-phosphate complexes (CPLX) and calcifiable proteolipid are associatedin vivo by establishing that they can be co-isolated from calcified bacteria. Both of these membrane constituents, which support apatite formationin vitro, have been isolated independently fromBacterionema matruchotii. However, isolation of proteolipid was preceded by demineralization in 2N formic acid, thereby dissociating bound Ca, whereas isolation of CPLX included sonication of calcified bacteria in 2:1:1.5 chloroform:methanol:Tris buffer, thereby dissociating any protein. Co-isolation is possible by demineralizing the calcified bacteria with 50 mM phthalic acid, pH 5.5, followed by extraction with 2:1 chloroform:methanol, and precipitation of crude phospholipid with acetone. CPLX and proteolipid are present in all Sephadex LH-20 chromatographic fractions of the crude phospholipid and of diethyl ether precipitates of the crude phospholipid. CPLXs contain protein:phospholipid:Ca:P_i but differ in relative composition from each other and from independently isolated CPLX. The Ca:phospholipid:P_i molar ratio of diethyl ether precipitable proteolipid-CPLX is most similar to previously published values for CPLX. The protein content of CPLX accounts for all of the proteolipid apoprotein in each Sephadex LH-20 fraction.