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, terminal amino acids) (B) of TpeL (AB262081) TcdB (CAJ67492)
, 1) according to the UP- GMA method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (100 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site, TcsL6018 (X82638) and TcnA
, Figure 2. TpeL N-terminal part glucosylates Ras but not Rho, Rac, and Cdc42. (A) SDS- PAGE of recombinant N-terminal part of TpeL stained with Blue Coomassie R-250
, ?g recombinant protein) by TpeL N-terminal part (10 -7 M) in the presence of UDP, p.14
, Glucose or UDP-[ 14 C]-N-acetylglucosamine for 1 h at 37°C. The proteins were run on a SDS-PAGE
, Figure 3 TpeL uses preferentially UDP-N-acetylglucosamine as cosubstrate. A kinetics of Ras glucosylation by rN-TpeL. Ras (1 ?g) was incubated with rN-TpeL (10 -7 M) for 1, 5, 10, 15 or 30 min at 37°C in glucosylation buffer containing 7 ?M UDP-[ 14 C]-Nacetylglucosamine or UDP, The samples were electrophoresed on a SDS- (14%)PAGE, autoradiographed, and band intensity was quantified by ImageJ
, Kinetics of Ras glucosylation by rN-TpeL with UDP-[ 14 C]-glucose as in A but with a different scale of Y axis. Note that Ras glucosylation with UDP-[ 14 C]-glucose is about 100 fold less than with UDP-[ 14 C]-N-acetylglucosamine. (C) Competition assay of Ras glucosylation with various hexose derivatives. Ras (1 ?g) was incubated in glucosylation buffer with rN-Tpel (10 -7 M), Experiments were done in triplicate, p.14
, acetylglucosamine was added and the preparations were incubated at 37°C for additional 30
, min and processed as described above. (D) Quantification of the competition assays