Restoration of the fusion capacity of human erythrocyte ghosts by SH blocking reagents

The fusion capacity of the intact human erythrocyte membrane is lost upon hemolysis. Erythrocyte ghosts, unlike intact human erythrocytes, cannot be fused when incubated either with Sendai virus or with the non-viral fusogenic agent glycerylmonooleate. Various SH blocking reagents were found to restore the fusion capacity of human erythrocyte ghosts (HEG). Mercurial compounds, such as p-chloromercuribenzoate (PCMB), p-chloromer-curibenzene sulfonate (PCMBS) and chlormerodrin were found to have the most potent effect and were able to restore the fusion potentiality of HEG at concentrations as low as 0.05 mM. N-Ethyl maleimide (NEM) and 5.5 dithio-bis(2-nitrobenzoate) (DTNB) were less effective and as high as 2 mM were required for achieving fusible ghosts. Iodoacetate (IAA) and iodoacetamide (IAM) (0.1-20 mM) were without effect. Fusion of HEG whose SH groups were blocked could be promoted either by Sendai virus or by glycerylmonooleate. Addition of SH blocking reagents at 37 °C after the ghosts have been agglutinated by the virus also induced their fusion. The virus agglutinated both treated and untreated HEG. No differences were detected in the ability of the virus to induce leakage of [¹²⁵I]BSA (as a measure of viral hemolytic activity) or to hydrolyse membrane sialic acid when incubated with control or with HEG treated with SH blocking reagents. Thus it was concluded that the ability of erythrocyte membrane to react with the virus was not disturbed during hemolysis. Membrane ATPases were found to be inactive in fusible SH-treated erythrocyte ghosts. The present work clearly shows that intact viral particles or non-viral fusogenic agents are not necessarily sufficient for inducing fusion of biological membranes. The interpretation of the data suggests that in order to undergo fusion the erythrocyte membrane has to possess a certain property which is lost during hemolysis and can be restored by SH blocking reagents.