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Purpose. The purpose of this study was to isolate Caco-2 subclones that express high levels of multidrug resistance protein (MDR1) and to characterize their kinetics and affinity parameters for MDR1 substrate/inhibitors. Methods. The subclones were selected by a dilution cloning technique. The polarized efflux of [³H]-vinblastine across subclone cell monolayers was quantified by measuring the apparent permeability coefficients (P[sub app]) of [³H]-vinblastine in the basolateral (BL)-toapical (AP) direction and in the AP-to-BL direction (P[sub app BL-to-AP]/ P[sub app AP-to-BL]) across the cell monolayers. The expression of MDR1 in the Caco-2 subclones compared with the parental Caco-2 cells was confirmed by Western blotting analysis. The kinetics parameters (K[sub m], V[sub max]) of [³H]-vinblastine and the inhibitory constants (K[sub I]) of several known MDR1 substrates/inhibitors on the transport of [³H]-digoxin determined in the parental Caco-2 cells and Caco-2 subclones were also compared. Results. Three subclones (#1, #20, #21) were selected based on their polarized efflux of [³H]-vinblastine. The P[sub app BL-to-AP]/P[sub app AP-to-BL] ratios for #1, #20, and #21 were 110, 140, and 112, respectively, and were about 6-fold higher than the ratio observed for the parental Caco-2 cells. In the presence of GF-120918 (2 µM), a known MDR1specific inhibitor, the P[sub app BL-to-AP]/P[sub app AP-to-BL] ratios were significantly decreased, suggesting that these cells were overexpressing MDRI. The K[sub m] values observed for vinblastine in the Caco-2 subclones were nearly identical to the value observed in the parental Caco-2 cells. In contrast, the V[sub max] values observed in the subclones were approximate 26-69% higher. The K[sub I] values observed for various known MDR1 substrates/inhibitors on [³H]-digoxin transport were nearly identical to those in the parental Caco-2 cells and Caco-2 subclones. The high functional efflux... [ABSTRACT FROM AUTHOR]

Human placental S-adenosylhomocysteine (AdoHcy) hydrolase was subjected to limited papain digestion. The multiple cleavage sites in the enzyme were identified to be Lys94-Ala95, Tyr100-Ala101, Glu243-Ile244, Met367-Ala368, Gln369-Ile370, and Gly382-Val383. Despite multiple cleavage sites in the backbone of the protein, the digested enzyme was able to maintain its quaternary structure and retain its full catalytic activity. The enzyme activity of the partially digested AdoHcy hydrolase was essentially identical to that of the native enzyme at several pH values. The thermal stabilities of the native and partially digested enzymes were only slightly different at all temperatures tested. The stability of both native and partially digested enzymes were examined in guanidine hydrochloride and equilibrium unfolding transitions were monitored by CD spectroscopy and tryptophan fluorescence spectroscopy. The results of these experiments can be summarized as follows: (1) CD spectroscopic analysis showed that the overall secondary and tertiary structures of the partially digested enzyme are essentially identical with those of the native enzyme; and (2) tryptophan fluorescence spectroscopic analysis indicated that there are small differences in the environments of surface-exposed tryptophan residues between the partially digested enzyme and the native enzyme under unfolding conditions. The differences in the free energy of unfolding, Δ(Δ Gu) [Δ Gu (native)-Δ Gu(digested)], is approximately 1.3 kcal/mol. When NAD+ was removed from the partially digested enzyme, the secondary and tertiary structures of the apo form of the digested AdoHcy hydrolase were completely lost and the enzymatic activity could not be recovered by incubation with excess NAD+. These results suggest that AdoHcy hydrolase exists as a very compact enzyme with extensive intramolecular bonding, which contributes significantly to the overall global protein stabilization. Identification of the surface-exposed peptide bonds, which are susceptible to papain digestion, has provided some constraints on the spatial orientations of subunits of the enzyme. This information, in turn, has provided supplemental data for X-ray crystallographic studies currently ongoing in our laboratories. [ABSTRACT FROM AUTHOR]

Conjugated diene 5–7 and enyne 8 analogs derived from adenosine and uridine were synthesized employing Pd-catalyzed cross-coupling reactions. [ABSTRACT FROM AUTHOR]