J. Ennever

Nucleation of Microbiologic Calcification by Proteolipid

Summary The component of crude phospholipid responsible for B. matruchotii calcification was isolated. Crude phospholipid, extracted from the microorganism, was separated into five fractions by column chromatography. A single, protein-containing fraction catalyzed apatite formation in a metastable calcium phosphate solution. The nucleating fraction was identified as a proteolipid. This work was supported by a grant from The Procter & Gamble Company. Amino acid analysis was done by Dr. E. P. Lazzari. Able technical assistance was provided by Mary Anita Stull.

Synthetic Medium for Calcification of Bacterionema matruchotii

A synthetic medium was developed for further study of intracellular calcification by Bacterionema matruchotii. The medium contained metastable calcium phosphate, six other salts, three purines, two pyrimidines, nine vitamins, pimelic and thioctic acids, vitamin-free casein hydrolysate, and glucose. The vehicle was 0.1 M N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid buffer at pH 7.4.

Calcium Hydroxyapatite Nucleation by Lipid Extract of Bacterionema matruchotii

Several investigators have established that Bacterionema matruchotii, a large, filamentous oral microorganism, will acquire intracellular calcium hydroxyapatite.1-3 Takazoe4 has shown that an early event in this calcification process is the concentrating of calcium within the cell by organic binding. Ennever, Vogel, and Takazoe5 have shown that the factor responsible for calcium binding by the microorganism could be removed from the cell by a lipid extraction. The initial purpose of this study was to determine whether the lipid extract of B matruchotii could induce apatite crystallinity in a metastable calcium phosphate solution. This was extended to include an examination of the crude phospholipid and nonphospholopid fractions.

Proteolipid and Bone Matrix Calcification In Vitro

Proteolipid was demonstrated to contain the nucleator of bone matrix calcification, in vitro. Crude Phospholipid extracted from bone matrix was fractionated by gel filtration. A single, protein-containing fraction induced apatite crystallization in a metastable calcium phosphate solution. The fraction was identified as proteolipid. The result supports the validity of a microbiologic analogue for vertebrate calcification.

Characterization of Bacterionema matruchotii Calcification Nucleator

The nucleator of Bacterionema matruchotii calcification was characterized. Parameters examined were: proteolipid purity and singularity, amino acid composition and relative polarity, phospholipid composition, apoprotein homogeneity, essentiality of the complex for nucleation, and ordered structure. The data fulfill a requirement for comparisons among apatite-nucleating proteolipids.

Lipid and Calculus Matrix Calcification In Vitro

Lipid is necessary for calcification of a calculus matrix. Matrix was prepared by decalcification of dental calculus. The matrix calcified when it was exposed to a metastable calcium phosphate solution. After extraction with chloroform-methanol, the matrix lost the capacity to calcify. The lipid extract was calcifiable.

Lipid and Bone Matrix Calcification in Vitro

Summary A calcifiable matrix was prepared by decalcification of marmoset femurs. Lipid extraction rendered the matrix noncalcifiable. The crude phospholipid fraction of the lipid extract induced apatite crystallinity in a metastable calcium phosphate solution. The acetone-soluble fraction did not. The results show lipid is involved in calcification of a bone matrix, in vitro, and that the nucleator is in the crude phospholipid fraction.

Calcification by proteolipid from atherosclerotic aorta

Calcified atherosclerotic aorta was examined for proteolipid capable of nucleating apatite, the crystal species of aortic calcification. Appropriate tissue pieces were decalcified with dilute formic acid and extracted with chloroform-methanol. Lipid fractionation yielded proteolipid which, upon incubation in metastable calcium phosphate solution, induced apatite crystallization. The proteolipid was partially characterized as a hydrophobic protein, acidic phospholipid complex. It resembles the nucleator previously demonstrated for bone matrix calcification.

Survey of Microorganisms for Calcification in a Synthetic Medium

Fourteen microorganisms, oral isolates from man or marmoset, were examined for calcification in a synthetic medium. The medium was essentially identical to that in which Bacterionema matruchotii acquires intracellular apatite. Four of the microorganisms, Escherichia coli, Alcaligenes marshalli, Aerobacter cloacae, and Proteus mirabilis, developed intracellular calcifications.

Phospholipids of a Bone Matrix Calcification Nucleator

Phospholipids of a bone matrix calcification nucleator wre identified as mono-and diphosphoinositides and phosphatidyl serine. The nucleator, a protein-phospholipid complex, was dissociated by acidified-solvent porous-glass column chromatography. Analysis was by gas-liquid chromatography.