title: Matrix vesicle annexins exhibit proteolipid-like properties. Selective partitioning into lipophilic solvents under acidic conditions.
abstract: Calcifiable proteolipids present in mineralizing tissues have been postulated to enhance apatite deposition by structuring membrane phosphatidylserine molecules into a conformation conducive to mineral formation. To examine whether proteolipid-like molecules are present in mineralizable matrix vesicles (MV), the vesicles were first extracted with chloroform/methanol (2:1, v/v), and then with chloroform/methanol/HCl (200:100:1, v/v) and the organic-soluble proteins subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Protein fractions were analyzed by Coomassie Blue staining and by immunoblot analysis of electrophoretically transferred MV protein with antisera to the 33- and 36-kDa annexins. We found that several MV proteins selectively partitioned into the lipophilic milieu under acidic conditions; however, very little protein did so at neutral pH. The principal organic-soluble MV proteins had molecular masses of 14, 33, and 36 kDa, with lesser bands at 28, 30, and 68 kDa. Immunological analyses revealed that the 33- and 36-kDa proteins were the MV annexins; the 14-kDa protein appeared to be hemoglobin, based on NH2-terminal sequencing. Our findings indicate that under acidic conditions the 33- and 36-kDa MV annexins undergo a conformational change which imparts a marked increase in the hydrophobicity of the proteins. While these observations reveal that the annexins possess proteolipid-like properties, radiolabeling and immunoprecipitation studies using [3H]myristic acid in chondrocyte cultures indicate that the MV annexins are not myristylated. Amino-terminal sequence analysis of the peptides generated by site-specific cleavage of the 33- and the 36-kDa MV annexins at tryptophan residues indicate that the 33 kDa is highly homologous to anchorin CII, a protein known to bind type II collagen, while the 36-kDa protein shares close homology with endonexin II, a tyrosine kinase substrate.
authors.affiliation: Department of Chemistry, University of South Carolina, Columbia 29208.
authors.forename: B R
authors.initials: BR
authors.lastname: Genge
authors.forename: L N
authors.initials: LN
authors.lastname: Wu
authors.forename: H D
authors.initials: HD
authors.lastname: Adkisson
authors.forename: R E
authors.initials: RE
authors.lastname: Wuthier
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D002356:Cartilage / Q000737:chemistry* / Q000166:cytology; D002478:Cells, Cultured; D002645:Chickens; D004591:Electrophoresis, Polyacrylamide Gel; D016326:Extracellular Matrix Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011487:Protein Conformation; D011510:Proteolipids / Q000737:chemistry*
chemical_list: D002135:Calcium-Binding Proteins; D016326:Extracellular Matrix Proteins; D011510:Proteolipids