PREFERENTIAL PROCESSING OF THE S1 SUBUNIT OF PERTUSSIS TOXIN THAT IS BOUND TO EUKARYOTIC CELLS

Labelled [I-125]-pertussis toxin was prepared and used to measure the association of pertussis toxin (PT) to eukaryotic cells. PT was radioi odinated by the lactoperoxidase method which preferentially radioiodin ated the S1 subunit. PT was radioiodinated at a high specific activity and possessed the same cytotoxicity as native PT as demonstrated by t he ability to cluster Chinese hamster ovary (CHO) cells. Cell associat ion of [I-125]PT was not inhibited by excess non-radiolabelled PT, whi ch indicated that the initial interaction between PT and CHO cells inv olved a large number of low-affinity receptors. At 37 degrees C, the S 1 within cell-associated PT was preferentially processed to an S1 with a lower apparent molecular weight (termed S1p). This processing was i nhibited by the addition of unlabelled PT, indicating that the process ing event was saturable and specific. S1 processing occurred in CHO, M adin-Darby canine kidney (MDCK) cells, and pig kidney (LLC-PK1) cells. A pulse-chase experiment showed that, at 37 degrees C but not at 22 d egrees C, essentially all of the cell-associated S1 was processed with in 3h of a chase. Reagents that were previously shown to inhibit the a bility of PT to ADP-ribosylate Gi proteins in intact CHO cells also in hibited the preferential processing of S1 within cell-associated PT, i n the order of efficiency: 22 degrees C > chloroquine > nocodazole > b refeldin A. This indicates that S1 processing requires an early endoso mal function.