1994 Volume 57 Issue 3 Pages 198-216
We attempted to isolate hemagglutinins from cells of non-fimbriated Prevotella intermedia strain E18 and to characterize the active fractions.
Sucrose density ultracentrifugation at 35,000 rpm for 20 h revealed hemagglutination activities in three different fractions (designated A, B and C). Negative staining revealed amorphous structures in fraction A, while vesicle-like structures were a major consti-tuent of fractions B and C. In the API ZYM system, all fractions, as well as whole cells of strain E18, produced alkaline phosphatase, acid phosphatase, phosphoamidase and α-glucosidase. Hemagglutination activity of fraction A was eliminated by heating at 60℃ for l0min, while 70℃ was required for fractions B and C. All fractions were sensitive to trypsin, D-glucosamine, D-galactosamine and N-acetylneuraminic acid. In addition, chymotrypsin, protease type IV and L-arginine caused hemagglutination inhibition in fraction A. The SDS-PAGE pattern of fraction B was similar to that of C, but different from A. However, a protein of approximately 70kDa was common to all Fractions. Scanning and transmission electron microscopy revealed small particles in fraction A, and vesicle-like structures surrounding fractions B and C. Addition of anti-fraction B or C serum caused hemagglutination inhibition in fractions B and C, as well as A. Moreover, the Ouchterlony method revealed common antigens against anti-fractions B and C serum in all fractions. The precipitating lines between fractions A and anti-fraction B and C were located in the center of wells, although the lines between fraction B and anti-fraction B and between fraction C and anti-fraction C, were located on the side of the antigens. Negative staining revealed amorphous structures surrounding vesicle-like structures.
These results indicate that when amorphous particles, which are glycoprotein in nature, surround vesicle-like structures, they may mediate hemagglutination.
