PURIFICATION AND CHARACTERIZATION OF PROTEASOMES FROM TRYPANOSOMA-BRUCEI

Proteasomes are multisubunit proteases that exist universally among eu karyotes. They have multiple proteolytic activities, and are believed to have important roles in regulating cell cycle, selective intracellu lar proteolysis, and antigen presentation. To determine the possible r ole that proteasomes may play in controlling the life cycle of African trypanosomes, we have isolated proteasomes from the bloodstream and t he insect (procyclic) forms of Trypanosoma brucei by DEAE-cellulose ch romatography and glycerol gradient fractionation in the presence of AT P. No 26 S proteasome homologs was identified in T. brucei under these experimental conditions. The proteasomes isolated from these two form s of T. brucei are very similar to the rat blood cell 20 S proteasome in their general appearance under the electron microscope. The profile of trypanosome proteasome subunits in sodium dodecyl sulfate polyacry lamide gel electrophoresis (SDS-PAGE) has eight visible protein bands with molecular weights ranging from 23 to 34 kDa, and cross-reacted ve ry poorly with the anti-human 20 S proteasome antibodies on immunoblot s. Two-dimensional gel electrophoresis of the parasite proteasomes sho ws a similar number of major subunits with pi's ranging from 4.5 to 7. Using a variety of fluorogenic peptides as substrates, the trypanosom e proteasomes exhibited unusually high trypsin-like, but somewhat lowe r chymotrypsin-like activities, as compared to the rat 20 S proteasome . These proteolytic activities were, however, insensitive to phenylmet hylsulfonyl fluoride (PMSF), tosyl-phenylalanine chloromethylketone (T PCK), tosyl-lysine chloromethylketone (TLCK) and trans-epoxy succinyl- L-leucylamido-(4 guanidino) butane (E-64), but the trypsin-like activi ty of trypanosome proteasomes was inhibited by leupeptin, an aldehyde known to inhibit the trypsin-like activity of mammalian proteasomes, t hus ruling out possible contamination by other serine or cysteine prot eases. Some quantitative differences in the substrate specificities be tween the proteasomes from bloodstream and procyclic forms were indica ted, which may play a role in determining the differential protein tur novers at two different stages of development of T. brucei.