Interaction between tetanolysin and Mycoplasma cell membrane.

1. A partially purified tetanolysin preparation lysed the sterol-requiring Mycoplasma capricolum cells but had no effect on M. capricolum cells adapted to grow with no or very little cholesterol. The sterol-non-requiring Acholeplasma laidlawii cells grown either in a cholesterol-rich or a cholesterol-poor medium were unaffected by the tetanolysin preparation. 2. The lysis of M. capricolum cells by the tetanolysin preparation was temperature dependent, inhibited by cholesterol, sublytic concentrations of lucensomycin, and Mg2+. The sensitivity to lysis was greatly affected by the age of the culture, being highest in cells from the early logarithmic phase of growth and declining sharply thereafter. 3. Isolated M. capricolum membranes were capable of binding large amounts of the tetanolysin activity (up to 30 hemolytic units per mug membrane protein), 20 times as much as membranes of the adapted strain. The binding of tetanolysin activity to membranes was almost the same at 4,22, or 37 degrees C, and was very little affected by the age of the culture. The binding capacity of the membranes was not affected by the removal of 60-70% of membrane proteins by pronase digestion but markedly decreased with the removal of membrane lipids. 4. Of the five polypeptide bands detected in electrophorograms of the partially purified tetanolysin preparation, two bands (mol. wt. 44 000 and 42 000) were found to bind to the cholesterol-containing mycoplasma membrane preparation. EPR spectrometry revealed that the freedom of motion of fatty acid spin labels in the tetanolysin-treated membranes was markedly higher than that in untreated membranes. 5. The concept that tetanolysin interacts specifically with membrane cholesterol resulting in the shielding of cholesterol from its interaction with membrane phospholipids is discussed.