Cellular processing of a ricin-antibody conjugate. A kinetic analysis of the rate-limiting step.
Upon exposure to holoricin-antibody conjugates, target cells display a period of no measurable intoxication followed by a concentration-dependent exponential decline in protein synthesis. The rate of this decline is increased by conjugate structural variables such as addition of a second ricin to the antibody or introduction of a peptide spacer between the toxin and antibody. Additionally, the rate is enhanced by the addition of ammonia or monensin. The relationship between immunotoxin concentration and the rate of protein synthesis inhibition is shown to obey typical Michaelis-Menten kinetics. By displacing bound immunotoxin with native antibody, it was demonstrated that the rate-limiting saturable process is not due to antibody-surface antigen interaction. Although addition of a second ricin as well as addition of ammonia elevated the measured rate of protein synthesis inhibition, curve-fitting techniques indicated that they did not elevate the maximal rate; however, introduction of a peptide spacer into the conjugate or addition of monensin increased the maximal rate of this saturable process. Additionally, the kinetic examination of the potentiation effects of ammonia and monensin indicates that they operate on different cellular processes involved with the intracellular routing toward intoxication. An equational model is developed which helps interpret the known features of the conjugate intoxication process.
Marsh
J W
JW
Laboratory of Molecular Biology, National Institute of Mental Health, Bethesda, Maryland 20892.
D000641:Ammonia / Q000494:pharmacology; D002460:Cell Line; D000922:Immunotoxins / Q000494:pharmacology*; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D014176:Protein Biosynthesis; D011500:Protein Synthesis Inhibitors; D012276:Ricin / Q000494:pharmacology*
D000922:Immunotoxins; D011500:Protein Synthesis Inhibitors; D000641:Ammonia; D012276:Ricin; D007930:Leucine