In a 96-well microtiter plate, 500 g of partially purified BTD from plasma and 12 L of 10 mM putative BTD inhibitors (120 nmoles/well; 1.0 mM final concentration) were mixed with 88 L of 54 mM sodium phosphate buffer (pH 6.0), containing 1.08 mM disodium EDTA and 4.3 mM cysteamine hydrochloride (prepared fresh); samples were preincubated at 37C for 60 min. chemically synthesized and tested for their ability to inhibit human BTD. Seven of these compounds inhibited BTD by 26% to 80%. Biotinyl-methyl 4-(amidomethyl) benzoate had the largest effect on BTD, causing an 80% inhibition at 1 mM concentration. Enzyme kinetics studies were conducted to determine Vmax, Km, and Ki for the seven inhibitors; kinetics were consistent with the hypothesis that biotinyl-methyl 4-(amidomethyl) benzoate and the other compounds acted by competitive inhibition of BTD. Finally, biotinyl-methyl 4-(amidomethyl) benzoate did not affect biotin transport in human cells, suggesting specificity in regard to biotin-related processes. [16,17]. Biotinylation of histones is mediated by both HCS [1,16] and BTD [8], but evidence has been provided that HCS is the dominant histone-biotinyl ligase [16]. Biotinylation of histones is a reversible modification. Ballard et al. suggested that debiotinylation of histones might be mediated by BTD [18]. The regulation of BTD to favor debiotinylation of histones over biotinylation of histones by the same enzyme is unknown. A number of variables may regulate the catalytic activity of BTD. First, the availability of substrate might favor either biotinylation or debiotinylation of histones. For example, locally high concentrations of biocytin might shift the reaction equilibrium towards biotinylation of histones [8,19]. Second, proteins Kenpaullone may interact with BTD at the chromatin level, favoring either biotinylation or debiotinylation of histones. Third, three alternatively spliced variants of BTD have been identified [20]. Theoretically, these variants may have unique functions with regard to histone biotinylation. Fourth, BTD possesses six glycosylation sites [21,22]; glycosylation of BTD might affect its cellular location [23]. Our long-term goal is to identify the roles of BTD in biotinylation and debiotinylation of histones. As a first step towards this goal, we generated a first generation of synthetic inhibitors of BTD, and we developed a 96-well plate assay for high-throughput screening of putative BTD inhibitors. Previous studies have proposed using biotin, di-isopropylfluorophosphate, and thiol reagents such as < 0.05 compared with inhibitor-free control). 2.3. BTD assay BTD activity was measured as the rate of hydrolysis of N-biotinyl-4-aminobenzoic acid to release 4-aminobenzoic acid (PABA). Kenpaullone The latter was quantified using N-1-naphthylethylenediamine dihydrochloride as described by Knappe et al. [26] and Backman-Gullers et al. [27], and modified by Nilsson & Ronge [28]. These protocols were adapted for microtiter plates as follows. In a 96-well microtiter plate, 500 g of partially purified BTD from plasma and 12 L of 10 mM putative BTD inhibitors (120 nmoles/well; 1.0 mM final concentration) were mixed with 88 L of 54 mM sodium phosphate buffer (pH 6.0), containing 1.08 mM disodium EDTA and 4.3 mM cysteamine hydrochloride (prepared fresh); samples were preincubated at 37C for 60 min. Then, 10 L of 6mM for 10 min) and the supernatant was transferred to a new plate and Kenpaullone the absorbance was measured at 546 nm. Previous Kenpaullone studies suggested that BTD activity is maximal at 37C and pH 6.0 [24] and, thus, all tests were run under these conditions. One unit of BTD FZD10 activity is defined as the amount of protein required to release 1 nanomole of PABA 120 min?1 under the conditions of the assay. 2.4. Enzyme kinetics Km, Vmax, and Ki [29] were determined as follows. The concentration of inhibitors was kept constant (0.5 mM) in enzyme assays as described above, while the concentration of the substrate N-(+)-biotinyl-PABA was varied from 0.05 mM to 1 1 mM. The enzyme kinetics module of Sigmaplot 10.0 was used for calculations [30]. 2.5. Biotin transport Theoretically, the biotin analogs tested here might affect both biotin transport into human cells and BTD activity. Here, biotin transport was quantified using a physiological concentration of [3H]biotin (475 pM) in the presence or absence of putative BTD inhibitors (0.5 mM) as described [31]; the Km of biotin transporters is in the low micromolar range [32]. Human Jurkat cells were used for biotin transport studies [33]. 2.6. Statistical analysis Heterogeneous variances were identified by using Bartletts test, and data were log transformed where applicable [34]. Significance of differences was tested by one-way ANOVA. Fishers Protected Least Significant Difference procedure was used for posthoc testing..