PRACTICAL CONSIDERATIONS REGARDING THE USE OF STREPTOLYSIN-O AS A PERMEABILISING AGENT FOR CELLS IN THE INVESTIGATION OF EXOCYTOSIS

Streptolysin-O is widely used in cell biological investigations in ord er to make large (>12 nm) pores in the plasma membrane and so to rende r the cytosol directly accessible to experimental manipulation. We hav e compared the effect of streptolysin-O commercially formulated (Murex Diagnostics) as a diagnostic reagent in pathology with two pure reage nts (a conventional purified protein, and a recombinant protein genera ted in E.coli) on exocytotic secretion from mast cells. For mast cells permeabilised by streptolysin obtained from the commercial source, ex ocytosis (of beta-D-N-acetylglucosaminidase) is dependent on provision of both Ca2+ and a guanine nucleotide. In contrast, for cells permeab ilised by either of the two pure proteins, a substantial extent of Ca2 --independent exocytosis can be elicited. When the Murex material is s ubject to dialysis or ultrafiltration, some secretion can be induced i n the absence of Ca2+, indicating a modulatory function of the low mol wt additives of formulation, mainly phosphate and cysteine. However, Ca2+-independent exocytosis is still manifest when the pure proteins a re reconstituted with ultrafiltrates from the Murex material. These ob servations indicate that reagents used to permeabilise cells should be characterised thoroughly and used with great care. Confirmation that the cytolytic activity of the Murex material derives from a cholestero l directed factor was demonstrated by inhibition of exocytosis when re d blood cell derived (and hence cholesterol containing) sonicated lipo somes were provided.