Separation of the pore-forming and cytotoxic activities from natural killer cell cytotoxic factor.

The influence of Ca2+ on the cytotoxic activity of natural killer cell cytotoxic factor (NKCF) was analyzed. When the natural killer susceptible cell line K562 was exposed to NKCF in the presence of 5 mM Ca2+, two peaks of cell damage were found. The first peak was observed after 30-40 min of incubation as a result of pore formation on the surface of target cells. The second was a peak of cytolytic activity which appeared after 24 h of incubation. Upon dilution of the NKCF preparation, only the first peak was observed. Therefore, NKCF produced by large granular lymphocytes in response to K562 consists of different proteins and represents pore-forming and cytolytic activities.

[Detection of a perforin-like protein from the soluble cytotoxic factor of natural killer cells]. / Obnaruzhenie perforinopodobnogo belka v sostave rastvorimogo tsitotoksicheskogo faktora natural'nykh killerov.

The protein composition of the cytolytic factor (NKCF) secreted by natural killer cells was analyzed. Natural killer cells were isolated from spleens of nude rats and purified in the Percole gradient. SDS-PAAG analysis of NKCF revealed the presence of major proteins with an approximate molecular weight of 62-67 kDa. Biochemical properties of these proteins such as fractionation on a TSK-3000 SW gel filtration column, electrophoretical mobility in reduced conditions, enhanced cytolytical activity in the presence of Ca2+ demonstrate the similarity with the pore-forming protein--perforin. Apart from the major component, proteins with m. w. 42 kDa, 31-28 kDa, 17 kDa and 12-14 kDa were also found. It is possible that the Ca2+-independent cytolytical activity is bound to one of these minor protein components of NKCF.

[The effect of catalytic subunit of cAMP-dependent protein kinase on the activity of cytotoxic factor from natural killer cells]. / Vliianie kataliticheskoi sub''edinitsy cAMP-zavisimoi proteinkinazy na aktivnost' tsitotoksicheskogo faktora natural'nykh killerov.

The catalytic subunit of cAMP-dependent protein kinase modifies proteins of NKCF. Several proteins of the treated NKCF can be labelled with 32P. Treatment of NKCF with protein kinase enhance the cytotoxicity of NKCF.

Effect of antibodies to granular perforin and calmodulin on the activity of natural killer cytotoxic factor.

It was previously established that the cell death induced by natural killer cytotoxic factor (NKCF) is a complex calcium-dependent two-stage process. In the initial stage, during the first 30 min of incubation of NKCF with target cells, pore formation on the surface of the target is observed, together with temporary membrane damage. The second stage cytolytic activity occurs after a 3 h latent period, reaches maximum at 24 h, and leads to cell death. A more detailed analysis of the NKCF-dependent lysis of cells was carried out in a study of the action of antibodies to granular perforin and calmodulin on this process. It was established that pore formation in the membrane after 30 min and cell lysis over the next 24 h are interdependent stages of a single process. The interaction of antibodies to perforin with the perforin-like protein of NKCF led to blockage of pore formation and to subsequent inhibition of target cell lysis. Treatment of NKCF with antibodies to calmodulin gave rise to the appearance of pore-forming activity in the first 30 min of incubation with target cells and to the subsequent acceleration of cell lysis over the next 24 h.