The use of bee venom melittin to assess the topography of membrane vesicles derived from Paracoccus denitrificans

Publisher Website
Google Scholar
Abstract
There exists considerable controversy regarding membrane topography in vesicles derived by osmotic lysis of spheroplasts of gram-negative bacteria. It was reported by others that bee venom can be used to quantitate the portion of a heterogeneous vesicle population, with an inside-out orientation by determining the degree of loss of crypticity of NADH dehydrogenase activity. A major component of bee venom, melittin, causes an increase in the activity of several different respiratory enzymes in isolated membrane vesicles of P. denitrificans. The degree of stimulation produced by melittin is dependent upon the nature of the respiratory substrates, pH, presence of Mg2+, melittin:membrane protein ratio, and the growth history of the cells from which the membrane vesicles were derived. Melittin-induced enhancement of TMPD[N,N,N''N''-tetramethyl-p-phenylenediamine]:ascorbate and cytochrome c oxidase activities cannot be accounted for by increased accessibility of nonpermeant substrate to the interior of the vesicle. The stimulatory effect of melittin may rely in part on its ability to alter the proton permeability of the membrane, thereby abolishing respiratory control. Collectively, these observations call into question the usefulness of bee venom melittin in quantitative analyses of membrane topography. These results are consistent with the postulated existence of a homogeneous vesicle population in which the topography of the NADH dehydrogenase is different from that of the intact cell.