Purpose. The aim of this study was to investigate the mechanism of permeation and cytotoxicity of vanadium compounds, [VO(acac)2], [VO(ma)2], and vanadate. Methods. Absorptive transport were carried out in Caco-2 monolayers grown on transwell inserts. Vanadium was quantified using inductively coupled plasma atomic emission spectrometry (ICP-AES). The change of Caco-2 cells in the microvilli morphology and F-actin structure was visualized by transmission electron microscopy and confocal laser scanning microscopy. Results. The three vanadium compounds were taken up by Caco-2 cells via simple passive diffusion. [VO(acac)2] were mainly transcellularly transported and exhibited the highest apparent permeabilty coefficients (8.2 × 10−6 cm-1). The cell accumulation of [VO(acac)2] was found to be greater than that of [VO(ma)2], and vanadate caused much less accumulation than the other two compounds. Vanadium compounds induced intracellular reactive oxygen species, reduced the transepithelial electric resistance, caused morphological change in microvilli, and led to different perturbation of F-actin structure. Conclusions. The three compounds exhibited different permeability due to different diffusion process and cellular uptake. The toxicity of vanadium complexes on Caco-2 monolayer involved F-actin-related change of tight junction and impairment of microvilli. The toxicity was also related to elevated intracellular reactive oxygen species (ROS) and their cellular accumulation.

Lanthanum ion (La3+) has been reported to affect proliferation or apoptosis of different cells. In the present study, La3+ was confirmed to promote both proliferation and apoptosis of NIH 3T3 cells at the same concentrations. La3+ was shown to promote proliferation by helping the cells to pass through the G1/S restriction point and enter S phase, however, the proliferating cells induced by incubation with La3+ eventually underwent apoptosis. The proliferation and apoptosis of NIH 3T3 cells induced by La3+ were well correlated with cell-cycle alterations. La3+ caused the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2; while inhibition of ERK phosphorylation by 2'-amino-3'-methoxyflavone (PD98059) suppressed both proliferation and apoptosis induced by La3+. Based on the above experimental results, we postulated that La3+-promoted proliferation of NIH 3T3 cells could be interconnected with the cell apoptosis, possibly through cell-cycle machinery. Our results thus support the recent hypothesis that proliferation and apoptosis of cell are intrinsically coordinated.

Binding of La3+ to calmodulin (CaM) and its effects on the complexes of CaM and CaMbinding peptide, polistes mastoparan (Mas), were investigated by nuclear magnetic resonance (NMR) spectroscopy, fluorescence and circular dichroism spectroscopy, and by the fluorescence stopped-flow method. The four binding sites of La3+ on CaM were identified as the same as the binding sites of Ca2+ on CaM through NMR titration of La3+ to uniformly 15N-labeled CaM. La3+ showed a slightly higher affinity to the binding sites on the N-terminal domain of CaM than that to the C-terminal. Large differences between the 1H-15N heteronuclear single quantum coherence (HSQC) spectra of Ca4CaM and La4CaM suggest conformational differences between the two complexes. Fluorescence and CD spectra also exhibited structural differences. In the presence of Ca2+ and La3+, a hybrid complex, Ca2La2CaM, was formed, and the binding of La3+ to the N-terminal domain of CaM seemed preferable over binding to the C-terminal domain. Through fluorescence titration, it was shown that La4CaM and Ca2La2CaM had similar affinities to Mas as Ca4CaM. Fluorescence stopped-flow experiments showed that the dissociation rate of La3+ from the C-terminal domain of CaM was higher than that from the N-terminal. However, in the presence of Mas, the dissociation rate of La3+ decreased and the dissociation processes from both global domains were indistinguishable. In addition, compared with the case of Ca4CaM-Mas, the slower dissociations of Mas from La4CaM-Mas and Ca2La2CaM-Mas complexes indicate that in the presence of La3+, the CaM-Mas complex became kinetically inert. A possible role of La3+ in the Ca2+-CaM-dependent pathway is discussed.

In an effort to design more specific and potent inhibitors of S-adenosylhomocysteine (AdoHcy) hydrolase, we investigated the mechanisms by which 5',5',6',6'-tetradehydro-6'-deoxy-6'-halohomoadenosines (X=C1, Br, I) inactivated this enzyme. The 6'-chloro (a) and 6'-bromo (b) acetylenic nucleoside analogues produced partial (50%) loss of enzyme activity with a concomitant (50%) reduction of E-NAD+ to E-NADH. In addition, Ade and halide ions were released from the inhibitors in amounts suggestive of a process involving enzyme catalysis. AdoHcy hydrolase, which was inactivated with compound a, was shown to contain 2 mol of the inhibitor covalently bound to Lys318 of two subunits of the homotetramer. These data suggest that the enzyme-mediated water addition at the 5'position of compound a or b produces an R-halomethyl ketone intermediate, which is then attacked by a proximal nucleophile (i.e., Lys318) to form the enzyme-inhibitor covalent adduct (lethal event); in a parallel pathway (nonlethal event), addition of water at the 6'position produces an acyl halide, which is released into solution and chemically degrades into Ade, halide ion, and sugar-derived products. In contrast, compound c completely inactivated AdoHcy hydrolase by converting 2 equiv of E-NAD+ to E-NADH and causing the release of 2 equiv of E-NAD+ into solution. Four moles of the inhibitor was shown to be tightly bound to the tetrameric enzyme. These data suggest that compound cinactivates AdoHcy hydrolase by a mechanism similar to the acetylenic analogue of Ado described previously by Parry et al. [(1991) Biochemistry 30, 9988-9997].

S-Adenosylhomocysteine hydrolase (AdoHcy hydrolase) crystallizes from solutions containing the intermediate analogue neplanocin A with the analogue bound in its 3'-keto form at the active sites of all of its four subunits and the four tightly bound cofactors in their reduced (NADH) state. The enzyme is in the closed conformation, which corresponds to the structure in which the catalytic chemistry occurs. Examination of the structure in the light of available, very detailed kinetic studies [Porter, D. J., Boyd, F. L. (1991) J. Biol. Chem. 266, 21616-21625. Porter, D. J., Boyd, F. L. (1992) J. Biol. Chem. 267, 3205-3213. Porter, D. J. (1998) J. Biol. Chem. 268, 66-73] suggests elements of the catalytic strategy of AdoHcy hydrolase for acceleration of the reversible conversion of AdoHcy to adenosine (Ado) and homocysteine (Hcy). The enzyme, each subunit of which possesses a substrate-binding domain that in the absence of substrate is in rapid motion relative to the tetrameric core of the enzyme, first binds substrate and ceases motion. Probably concurrently with oxidation of the substrate to its 3'-keto form, the closed active site is "sealed off" from the environment, as indicated by a large (108-9-fold) reduction in the rate of departure of ligands, a feature that prevents exposure of the labile 3'-keto intermediates to the aqueous environment. Elimination of the 5'-substituent (Hcy in the hydrolytic direction, water in the synthetic direction) generates the central intermediate 4',5'-didehydro-5'-deoxy-3'-ketoadenosine. Abortive 3'-reduction of the central intermediate is prevented by a temporary suspension of all or part of the redox catalytic power of the enzyme during the existence of the central intermediate. The abortive reduction is 104-fold slower than the productive reductions at the ends of the catalytic cycle and has a rate constant similar to those of nonenzymic intramolecular model reactions. The mechanism for suspending the redox catalytic power appears to be a conformationally induced increase in the distance across which hydride transfer must occur between cofactor and substrate, the responsible conformational change again being that which "seals" the active site. The crystal structure reveals a well-defined chain of three water molecules leading from the active site to the subunit surface, which may serve as a relay for proton exchange between solvent and active site in the closed form of the enzyme, permitting maintenance of active-ite functional groups in catalytically suitable protonation states.