Involvement of membrane-associated sialic acid in the resistance of sheep erythrocytes to lysis by mouse complement

Role of C4-Binding Protein in the Defective Lysis of Sensitized Sheep Erythrocytes by Mouse Complement

“…1, table I), the affinity fractionation procedure caused a considerable increase in C activity as mea sured with both normal and desialylated ShEA in VSB2+. In earlier communications [20,23] these C as says were shown to be specific for mouse classical C pathway activity. The enhanced classical C pathway activity upon C4-bp depletion and the inhibitory ac tivity of the eluate suggest that C-regulation by C4-bp (table I) and sialic acid [20] are independent pheno mena.…”

“…The mechanisms of the relatively inefficient lysis of ShEA by mouse C was studied in further detail [20], Reason for focusing on C4-bp was that this molecule is a known regulatory protein of the classical C path way [8,14], that it forms stable complexes with C4 in mouse serum but not in plasma [8], and that mouse serum rapidly loses C4 activity during storage [3,11]. Partial C4-bp depletion of mouse plasma was achieved by passing it over an affinity column pre pared with a crude anti-C4-bp serum absorbed with ShEAMoC lq Mo to eliminate contaminating antibod ies.…”

“…In brief, 200 pi of mouse plasma preparations, serially diluted in VSB:*, was mixed with 100 pi of 1.5 x 10s RaEA, ShE A or desialylated ShEA per millilitre. The mix ture was incubated for 60 min at 30°C where RaEA were concerned [23] and at 37°C with the two sorts of ShEA [20], The lytic reaction was stopped by addition of 2.2 ml of ice-cold VSB:*, the cells were b Mean ± standard error of the mean out of 3 independent experiments. c n.t.…”

“…This treatment resulted in a loss of membrane sialic acid of approximately 80% [20,24]. Upon desialylation the cells were washed 3 times using VSB These cells, normal ShE and RaE were photometrically adjusted to numbers of 3 x lOVml in VSB: *.…”

“…The assay for measuring mouse classical C pathway activity was as described elsewhere [20,23]. In brief, 200 pi of mouse plasma preparations, serially diluted in VSB:*, was mixed with 100 pi of 1.5 x 10s RaEA, ShE A or desialylated ShEA per millilitre.…”