Platelet-interactive products of Streptococcus sanguis protoplasts.
To isolate a more native, platelet-interactive macromolecule (class II antigen) of Streptococcus sanguis, cultured protoplasts were used as a source. Protoplasts were optimally prepared from fresh washed cells by digestion with 80 U of mutanolysin per ml for 75 min at 37 degrees C while osmotically stabilized in 26% (wt/vol) raffinose. Osmotically stabilized forms were surrounded by a 9-nm bilaminar membrane, as shown by transmission electron microscopy. Protoplasts were cultured in chemically defined synthetic medium and osmotically stabilized by ammonium chloride. Spent culture media were harvested daily for 7 days. Each day, soluble proteins were isolated from media, preincubated with platelet-rich plasma, and tested for inhibition of platelet aggregation induced by S. sanguis cells. Products released from S. sanguis protoplasts and reactive with an anti-class II antigen immunoaffinity matrix were able to inhibit S. sanguis-induced platelet aggregation. As resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, anti-class II-reactive protoplast products included silver-stained bands of 67, 79, 115, 216, and 248 kDa. The 115-kDa protein fraction was isolated by gel filtration and ion-exchange chromatography. This form of the class II antigen contained N-formylmethionine at its amino terminus. Rhamnose constituted 18.2% of the total residual dry weight and nearly half of its carbohydrate content. Diester phosphorus constituted 1% of this fraction. After trypsinization of the protoplast products from either preparation, a 65-kDa protein fragment was recovered. This protoplast protein fragment and the S. sanguis cell-derived 65-kDa class II antigen, previously implicated in the induction of platelet aggregation, were shown to be functionally and immunologically identical.
Herzberg
M C
MC
Department of Preventive Sciences, School of Dentistry, University of Minnesota, Minneapolis 55455.
Erickson
P R
PR
Kane
P K
PK
Clawson
D J
DJ
Clawson
C C
CC
Hoff
F A
FA
D000596:Amino Acids / Q000032:analysis; D000949:Histocompatibility Antigens Class II / Q000032:analysis / Q000302:isolation & purification*; D006801:Humans; D008854:Microscopy, Electron; D008970:Molecular Weight; D010974:Platelet Aggregation; D011523:Protoplasts / Q000276:immunology / Q000648:ultrastructure; D012210:Rhamnose / Q000032:analysis; D013298:Streptococcus sanguis / Q000276:immunology* / Q000648:ultrastructure
D000596:Amino Acids; D000949:Histocompatibility Antigens Class II; D012210:Rhamnose