Human (h) DNA topoisomerase I has been identified as a major SUMO1 target in camptothecin-treated cells. In response to TOP1-mediated DNA damage induced by camptothecin, multiple SUMO1 molecules are conjugated to the N-terminal domain of a single TOP1 molecule. To investigate the molecular mechanism of SUMO1 conjugation to TOP1, an in vitro system using purified SAE1/2, Ubc9, SUMO1, and TOP1 peptides was developed. Consistent with results from in vivo studies, multiple SUMO1 molecules were found to be conjugated to the N-terminal domain of a single TOP1 molecule. Systematic analysis has identified a single major SUMO1 conjugation site located between amino acid residues 110 and 125 that contains a single lysine residue at 117 (Lys-117). Using a short peptide spanning this region, we showed that a poly-SUMO1 chain was assembled in this peptide at Lys-117. Interestingly, a Ubc9-poly-SUMO1 intermediate had accumulated to a high level when the sumoylation assay was performed in the absence of hTOP1 substrate, suggesting a possibility that the poly-SUMO1 chain is formed on Ubc9 first and then transferred en bloc onto hTOP1. This is the first definitive demonstration of the assembly of a poly-SUMO1 chain on protein substrate. These results offer new insight into hTOP1 polysumoylation in response to TOP1-mediated DNA damage and may have general implications in protein polysumoylation.Human SUMO1 (small ubiquitin-related modifier) (also named UBL1, PIC1, GMP1, SMT3C, and sentrin in the literature) is a ubiquitin-like protein (1-3). It shares about 18% identity to ubiquitin (4). Human SUMO1 and its yeast homolog, Smt3p, also share a similar activation and conjugation pathway with ubiquitin but employ distinct sets of E1 2 and E2 enzymes (5-9). The E1 enzymes for activating human SUMO1 and yeast Smt3p are the heterodimeric proteins SAE1/SAE2 and Aos1p/Uba2p, respectively (6, 9). Ubc9 is the only E2 identified for SUMO1/Smt3p, although a dozen of E2 enzymes have been identified for ubiquitin in yeast (10 -14). Recently, proteases that specifically activate SUMO1/Smt3p precursors and cleave SUMO1/Smt3p from their protein conjugates have been identified in yeast and mammalian cells (15)(16)(17)(18). Many proteins such as RanGAP1 (19), promyelocytic leukemia protein (20), IB␣ (21), RAD51, RAD52 (22, 23), p53 (24), and centromere proteins (25, 26), which have diverse functions, have been shown to interact with Ubc9/SUMO1 or be covalently modified by SUMO1. SUMO1 appears to primarily target nuclear proteins (23). Although the function of SUMO1 is still elusive, there are a few interesting observations that may shed light on the function of SUMO1. First, Pods (promyelocytic leukemia protein oncogenic domains, also called nuclear bodies or ND10) (27) and the nuclear envelope appear to be the major site of localization of SUMO1 conjugates in the nucleus (28). Second, SUMO1 and ubiquitin appear to share the same conjugation site(s) on some target proteins (e.g. IB␣ and MDM2) (21). Third, stress-caused protein unfolding (e.g. heat s...

A disposable copper nanoparticle-plated screen-printed carbon electrode (designated as Cun-SPE(100-nm)) provides a new material for the determination of native amino acids. All 20 underivatized amino acids can be sensitively determined at 0.0 V vs. Ag/AgCl in pH 8 phosphate buffer solution. The precisely controlled copper nanoparticles can boost up the CuIIO/CuI2O redox signal on the working surface without any prior pretreatment procedure. The formation of a reversible 1:1 CuIIO-amino acid complex on the Cun-SPE(100-nm) was proposed to play a key role in the reaction mechanism. Stable detection responses were obtained for all amino acids by flow injection analysis with detection limits (S/N = 3) that lie in the range of 24 nM-2.7 microM. Selected amino acids from six representative chemical natures were separated by HPLC and detected at the Cun-SPE(100-nm) with promising results.

Peak overlap in voltammetry poses challenges for the quantitative analysis of electroactive species. Dopamine and uric acid are typically challenging to determine voltammetrically because of their very similar oxidation peak potentials. We report preliminary results of the use of a screen-printed carbon electrode for the determination of dopamine and uric acid in an electrolyte solution maintained above ambient temperatures. Higher temperatures resulted in dramatic shifting of the dopamine oxidation peak toward lower potentials, while the uric acid peak was essentially stationary. Ascorbic acid, an interference in voltammetric uric acid determinations, is effectively suppressed at higher temperatures. This resulted in a greater peak separation of dopamine from uric acid at higher temperatures, which is desirable for better peak integration. In addition, greater current responses for both species were recorded at higher temperatures. The cause for such an increase in peak current is unraveled using ac impedance measurements. Presented are preliminary results for determining dopamine and uric acid at temperatures higher than ambient. Much improved voltammetric peak separation and sensitivity is obtained at these higher temperatures compared to ambient.

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