As part of a broader analysis of the mechanism(s) by which the most sensitive (type III) paroxysmal nocturnal hemoglobinuria (PNH) erythrocytes are excessively sensitive to reactive lysis by isolated C5b6, C7, C8, and C9, we have compared type III PNH (PNH-III) and normal human E in respect to both total specific binding of 125I-C9 and the proportion of cell-bound C9 appearing in high molecular weight (HMW) complexes. In a previous report, we found that after exposure to purified C5b6 and 125I-C7, specific C7 binding and, by implication, EC5b-7 formation were equal for PNH-III E and normal E. In the present study, C8-dependent binding of 125I-C9 to PNH-III EC5b-7 and normal EC5b-7 was also similar, although lysis of the PNH-III E was up to five times greater; that is, PNH-III E required fewer bound C9 molecules to produce an effective lytic site than did normal E. To quantify radioactivity in monomeric and HMW forms of membrane-bound C9, lysed and unlysed E were subjected to low ionic strength buffers to convert all E to ghosts. These ghosts were solubilized in 0.1 or 2% SDS (without reduction) and electrophoresed on 2.4-11% polyacrylamide gradient gels followed by autoradiography and densitometric scanning. With 0.1% SDS, broad, heterodisperse zones of HMW C9 were recovered from both PNH and normal ghosts; the amounts of C9 incorporated into the HMW complexes were similar for PNH-III E and normal E. In selected experiments, 125I-C7 could be shown in these same HMW bands. When membranes were solubilized in 2% SDS, the overall proportion of HMW C9 complexes compared with dimer and monomer C9 was reduced on both PNH and normal membranes. In many, but not all experiments, more of the highest mol wt C9 complexes were detected from PNH-III E membranes solubilized in 2% SDS than from normal or PNH-II E membranes similarly treated. When antibody-sensitized E were lysed by purified C1-C9, PNH-III EA bound far more C9 than did normal EA, and both lysis and C9 incorporation into HMW complexes were markedly and proportionately increased over normal; however, lytic efficiency of 125I-C9 bound to PNH EA was equal to or less than that bound to normal EA.(ABSTRACT TRUNCATED AT 250 WORDS)
Production and characterisation of a marine Halomonas surface-active exopolymer
