Regulation of xanthine oxidase activity by substrates at active sites via cooperative interactions between catalytic subunits: implication to drug pharmacokinetics.

Three xanthine oxidase substrates (i.e., xanthine, adenine, and 2-amino-4-hydroxypterin) show a "substrate inhibition" pattern (i.e., slower turnover rates at higher substrate concentrations), whereas another two substrates (i.e., xanthopterin and lumazine) show a "substrate activation" pattern (i.e., higher turnover rates at higher substrate concentrations). Binding of a 6-formylpterin at one of the two xanthine oxidase active sites slows down the turnover rate of xanthine at the adjacent active site from 17.0 s(-1) to 10.5 s(-1), and converts the V-[S] plot from "substrate inhibition" pattern to a classical Michaelis-Menten hyperbolic saturation pattern. In contrast, binding of xanthine at an active site accelerates the turnover rate of 6-formylpterin at the neighboring active site. The experimental results demonstrate that a substrate can regulate the activity of xanthine oxidase via binding at the active sites; or a xanthine oxidase catalytic subunit can simultaneously serve as a regulatory unit. Theoretical simulation based on the velocity equation derived from the extended Michaelis-Menten model shows that the substrate inhibition and the substrate activation behavior in the V-[S] plots could be obtained by introducing cooperative interactions between two catalytic subunits in homodimeric enzymes. The current work confirms that there exist very strong cooperative interactions between the two catalytic subunits of xanthine oxidase.

Cytotoxicity of xanthopterin and isoxanthopterin in MCF-7 cells.

Human mammary carcinoma MCF-7 cell line responsiveness to the pteridines xanthopterin and isoxanthopterin was studied using the MTS assay for measurement of cell viability. The pteridines were tested at concentrations ranging from 7.8 to 500 microM singly and in 11 isoxanthopterin:xanthopterin ratios. IC50s of xanthopterin and isoxanthopterin were 109+/-13 microM (mean+/-SEM of y estimate) and 103+/-9 microM, respectively. The IC50 values for pteridine mixtures were similar although 3:1 and 4:1 isoxanthopterin:xanthopterin ratios seemed slightly more cytotoxic than other mixtures. However, ANOVA revealed no statistical differences in the cytotoxicity of mixtures.

Differential effect of xanthopterin and biopterin on cell growth.

1. Xanthopterin inhibited proliferation of primary renal proximal tubule cells (RPTC) and LLC-PK1 cells while in a growth phase but when incubated at confluence the cells were relatively insensitive. 2. The growth of malignant human prostate PC-3 cells was also inhibited by xanthopterin in a concentration and time dependent manner. 3. Dunning R3327 AT-3 rat prostate tumor cells which were exposed to xanthopterin in vitro before their in vivo inoculation resulted in smaller tumours while in vivo administration of xanthopterin following implantation also resulted in smaller tumors. 4. Xanthopterin exerts differential effects on cell growth dependent upon the cell origin and their state of proliferation.

Effect of pteridines on normal and neoplastic cell lines of Xiphophorus.

The effect of three pteridines namely biopterin, isoxanthopterin and xanthopterin on cell growth, viability and morphology of three embryo and one melanoma derived cell lines was studied. Pteridines were assayed in the range of 10(-6) M to 10(-4) M. Biopterin exerted no influence on all cell lines tested, whereas isoxanthopterin and xanthopterin caused a decrease in growth rate with increasing concentrations. The strongest decrease of cell growth was exerted by 10(-4) M xanthopterin. Furthermore at this concentration xanthopterin reduced the viability of normal cells and influenced the morphology of both normal and neoplastic cells. In general, normal cells were more sensitive to pteridines than neoplastic cells.

Inhibition of GTP cyclohydrolase I by pterins.

Pterins inhibit rat liver GTP cyclohydrolase I activity noncompetitively. Reduced pterins, such as 7,8-dihydro-D-neopterin, (6R,S)-5,6,7,8-tetrahydro-D-neopterin, 7,8-dihydro-L-biopterin, (6R)-5,6,7,8-tetrahydro-L-biopterin, L-sepiapterin, and DL-6-methyl-5,6,7,8-tetrahydropterin are approximately 12-times more potent as inhibitors than are oxidized pterins, such as D-neopterin, L-biopterin, and isoxanthopterin. They are also 12-times more potent than folates, such as folic acid, dihydrofolic acid, (+/-)-L-tetrahydrofolic acid, and aminopterin. The Ki values for 7,8-dihydro-D-neopterin, 7,8-dihydro-L-biopterin, and (6R)-5,6,7,8-tetrahydro-L-biopterin are 12.7 microM, 14.4 microM, and 15.7 microM, respectively. These results suggest that mammalian GTP cyclohydrolase I may be regulated by its metabolic end products.

Chemotaxis towards pteridines during development of Dictyostelium.

Following a previous study indicating a sensitivity to folate during the developmental phase of Dictyostelium discoideum, a series of pteridines were investigated for their ability to induce amoebal chemotaxis during development of this organism. Several compounds were found to resemble folate in their ability to induce chemotaxis of both vegetative amoebae and amoebae developing during the first few hours of starvation. One compound, L-monapterin, was distinct in showing chemotactic activity only during the developmental phase after approximately 12 h of starvation. Tests using the polymerization of cytoskeletal actin as an assay for a cellular response correlated with chemotaxis showed that 10 nM-L-monapterin was a potent inducer of this response and that responsiveness appeared only after 12 h of development. The timing of these events may be correlated with the formation of tips containing stalk cells that occurs in multicellular aggregates at approximately 12 h, and suggests a role for L-monapterin (or a naturally occurring, closely related pteridine) in cell sorting. The evolutionary significance of the roles of pteridines is discussed.

Pterins and the regulation of lymphocyte activation on the mode of xanthopterin action.

Some intermediates of pterin anabolism amplify the lectin-induced lymphocyte stimulation while the catabolites xanthopterin and isoxanthopterin terminate their proliferation (Ziegler, I. et al., Cancer Res. 43, 5356 (1983). In the present investigation, we analysed the effect of xanthopterin on total RNA synthesis and on DNA synthesis in both concanavalin A-stimulated lymphocytes and in the lymphoblastoid cell line L 1210. The time courses at various inhibitor concentrations indicated that xanthopterin inhibits RNA synthesis prior to DNA synthesis. Further analysis of the RNA species was performed by double-labeling and subsequent polyacrylamide-gel electrophoresis. Pulse and pulse-chase experiments revealed that an inhibition of 45 S pre-RNA is closer to the target of xanthopterin inhibition than is DNA synthesis.

The influence of some pterins on the circadian rhythmicity of hydroxyindole-O-methyl transferase in the pineal gland of 42-day old male Wistar rats.

The influence of three pterin derivatives on the diurnal fluctuations of HIOMT (hydroxyindole-O-methyl transferase) activity was studied in the isolated pineal glands of 42-day old male Wistar rats during the month of October. The method used permitted the separate determination of four HIOMT activities. --Reduced neopterin stimulated the methylation of the substances, 5-HTP, 5-HT and 5-HIAA (see abbreviations in Material and methods), during the night. HIOMT action on the combinations N-Ac-5-HT/5-HTL was shifted to a later moment in the dark period. --Pterin-6-aldehyde stimulated HIOMT action on 5-HT during the daytime. HIOMT action on the substrates 5-HTP, 5-HIAA and N-Ac-5-HT/5-HTL was shifted towards an earlier period. --Isoxanthopterin did not exert any influence on diurnal variation in the four HIOMT activities. It may be concluded that reduced neopterin and pterin-6-aldehyde influenced the activity and the circadian rhythmicity of 5-methoxyindole synthesis. Those alterations might be important in the regulation of reproduction.