An early effect of diphtheria toxin on the metabolism of mammalian cells growing in culture.

At concentrations of 0.1 Lf (0.25 microg.)/ml., or greater, diphtheria toxin produces an immediate decrease (40 +/- 5 per cent) in the steady-state level of incorporation of inorganic phosphate into ATP by cultures of normal human kidney cells growing at 37 degrees C. in the presence of glucose. The effect is readily reversible by specific antitoxin for a period of 30 minutes or more after adding toxin. Protein synthesis in these cells continues at a normal rate for 60 minutes after adding toxin and the toxin-treated cells are able to take up and concentrate potassium ions normally for at least 1 hour. Even high concentrations of toxin (5 Lf/ml. or more) fail to effect either protein synthesis or ATP formation by cultures of spleen cells from the mouse, an animal that is relatively resistant to the lethal action of the toxin. Of various inhibitors studied, antimycin A most closely resembles toxin in its action, both on protein synthesis and on ATP formation. Mouse cells are considerably more resistant to antimycin A than are human kidney cells. Human kidney cells treated with saturating doses of toxin continue to form RNA at a normal rate for about 1 hour, after which the rates of both P(i) (32) incorporation and of 6-C(14)-orotic acid incorporation into RNA sharply decline and continue at about 40 to 50 per cent their initial rate. At 37 degrees C., S(35)-methionine incorporation into cell protein ceases altogether 60 to 75 minutes after addition of a saturating dose of toxin. The effect of saturating doses of toxin on S(35)-methionine incorporation into human kidney cell protein at different temperatures, has been studied. It is concluded from the present studies that diphtheria toxin exerts a primary, reversible effect at the surface of susceptible cells where it inhibits cytochrome-linked phosphorylation concerned with transport of inorganic phosphate across the cell membrane.