AMethodforAssayingDeubiquitinatingEnzymes2

To validate further the assay method, ¹²⁵I-labeled Ub-PESTc was incubated with increasing amounts of YUH1 for 30 min at 37 °C. The samples were then subjected to polyacrylamide gel electrophoresis in duplicate under denaturing conditions. After electrophoresis, one of the gels was stained with Coomassie R-250. Fig. 2A shows that the intensity of a protein band corresponding to the size of Ub increases upon incubation with increasing amounts of YUH1.

In this gel, however, we could not find the band corresponding to the PESTc peptide, which might have been diffused out during the staining and destaining process. Therefore, the other gel was covered with a Saran wrap and directly exposed on an X-ray film. Upon the autoradiography, we were able to detect the band of the PESTc peptide, whose intensity also increased upon incubation with increasing amount of YUH1. In addition, this increase in the band intensity was approximately proportional to the increase in the release of acid-soluble radioactivity (data not shown). Furthermore, the amino acid sequence of the acid-soluble product determined by Edman degradation after separation from undigested ¹²⁵I-labeled Ub-PESTc by gel filtration was shown to be identical with the N-terminal sequence of PESTc (see Fig. 8 of ref. 13). Thus, it is clear that the acid-soluble radioactivity represents PESTc released from ¹²⁵I-labeled PESTc by the action of YUH1.

Surprisingly, however, the band intensity of Ub upon the autoradiography was far lower than the others, despite the fact that Ub itself has a single tyrosine residue at the 59th position that can also be labeled by ¹²⁵I. Typically, chloramine T is used for radioiodination of Ub molecules. In our studies, we used IODO-BEADS, in which chloramine T is immobilized on non-porous polystyrene beads. Perhaps, the tyrosine residue in Ub is not accessible to electrophilic idodine species (i.e., I⁺), which are produced by the immobilized chloramine T, due to structural barrier, unlike that in PESTc, that is fused to the flexible C-terminal region of Ub. In any event, we could obtain ¹²⁵I-labeled Ub-PESTc, in which PESTc was almost exclusively radioiodinated. This fortuitous finding allowed us to determine rapidly the activity of YUH1 as well as of other DUBs and to quantify precisely cleavage products by simple measurement of the radioactivity released into the acid-soluble products.

Using the assay method, we have previously purified several novel DUBs from chick skeletal muscle and yeast, including cUBP41 (15), cUCH-1 (16), cUCH-6 (13), cUCH-8 (17), and yUBP-6 (18), using conventional chromatographic procedures. To compare the activities of these DUBs against ¹²⁵I-Ub-PESTc, the same amount of each enzyme was incubated with the substrate for various periods. Fig. 3 shows that the hydrolytic rates differ markedly from each other. Among the DUBs, cUCH-6 hydrolyzed Ub-PESTc most rapidly. The K_m values for the enzymes were also different, ranging from 5 to 65 μM (e.g., 5.1 μM for cUCH-6 and 64.5 μM for yUBP-6.)

Ub ends with the amino acid sequence of -RLRGG⁷⁶. Based on the C-terminal sequence, Stein and coworkers (19) have synthesized a variety of fluorogenic peptides by conjugating AMC to the C-termini of the peptides and used them as substrates for determination of the specificity of isopeptidase T, which exists in all mammalian cells and hydrolyzes the isopeptide linkages of poly-Ub chains (27). Of these, Cbz-LRGG-AMC was used in the present studies to compare its sensitivity to the purified DUBs to that of ¹²⁵I-Ub-PESTc. Table 2 shows that all of the DUBs have at least three order higher specific activities against ¹²⁵I-Ub-PESTc than those against Cbz-LRGG-AMC, indicating that the assay method using ¹²⁵I-Ub-PESTc is much more sensitive for determining the activities of DUBs than that using the fluorogenic peptide substrate. In addition, we have recently found that the extracts of E. coli by themselves are capable of releasing AMC from the peptide substrate (data not shown), implying that E. coli contains a protease(s), which specifically cleaves off AMC from the peptide substrate. On the other hand, the same extracts could not hydrolyze ¹²⁵I-Ub-PESTc at all. Thus, the protease(s) is likely to interfere with the assay for overproduced eukaryotic DUBs in E. coli cells, when Cbz-LRGG-AMC was used as a substrate. Furthermore, extracts prepared from most of eukaryotic cells also contain a protease(s) that rapidly cleaves Cbz-LRGG-AMC but not ¹²⁵I-Ub-PESTc. Thus, the assay method using ¹²⁵I-Ub-PESTc should be appropriate for identification of DUBs in eukaryotic cells and their clones expressed in E. coli. In fact, we were able to identify and partially purify at least 10 different DUBs from the extract of chick skeletal muscle (13) and to purify several novel chick DUBs cloned and expressed in E. coli (15,28).

Recently, Stein and coworkers (29) have developed a new assay method for DUBs based on the substrate Ub C-terminal AMC (Ub-AMC). They showed that the rate constants (kc/K_m ) for the hydrolysis of Ub-AMC are 10⁴- and 10⁷-fold over those for the cleavage of Cbz-LRGG-AMC for isopeptidase T andUCH-L3 respectively. UCH--L3 is a 26 kDa Ub C-terminal hydrolase (30) isolated from rabbit reticulocytes. However, it has not yet been tested whether Ub-AMC is susceptible to any protease in bacterial or eukaryotic cells other than DUBs.

We are grateful to Dr. Martin Rechsteiner (University of Utah) for providing E. coli strain AR13 carrying pNMHUB-PESTc, for which Ub-PESTc was purified. This work was supported by grants from Korea Science and Engineering foundation through Research Center for Cell Differentiation, Lotte Foundation, and Korea Ministry of Education (BSRI-97-4415).