9- -hypoxan thine

Xanthine oxidase (XO) was the first enzyme studied from the family of enzymes now known as the molybdenum hydroxylases (HiUe 1999). XO, which catalyzes the hydroxylation of xanthine to uric acid is abundant in cow s milk and contains several cofactors, including FAD, two Fe-S centers, and a molybdenum cofactor, all of which are required for activity (Massey and Harris 1997). Purified XO has been shown to use xanthine, hypoxan-thine, and several aldehydes as substrates in the reduction of methylene blue (Booth 1938), used as an electron acceptor. Early studies also noted that cyanide was inhibitory but could only inactivate XO during preincubation, not during the reaction with xanthine (Dixon 1927). The target of cyanide inactivation was identified to be a labile sulfur atom, termed the cyanolyzable sulfur (Wahl and Rajagopalan 1982), which is also required for enzyme activity. 

In addition to the molybdenum hydroxylases mentioned above, a new selenium-dependent hydroxylase with specificity for purine and hypoxan-thine as substrates, termed purine hydroxylase, was uncovered during purification of XDH from C. purinolyticum (Self and Stadtman 2000). Purified PH was labeled with Se and was not reduced in the presence of xanthine as a substrate. As with other selenium-dependent molybdenum hydroxylases, selenium was removed by treatment with cyanide with parallel loss in catalytic activity. Selenium was also efficiently removed in the presence of low ionic strength buffer during final dialysis of PH, indicating that ionic strength affects the stability of the labile selenium cofactor in this enzyme. 

Successful fusion (2) is a rare event, but the frequency can be improved by adding polyethylene glycol (PEG). To obtain only successfully fused cells, incubation is required for an extended period in a primary culture with HAT medium (3), which contains hypoxan-thine, aminopterin, and thymidine. Amino-pterin, an analogue of dihydrofolic acid, competitively inhibits dihydrofolate reductase and thus inhibits the synthesis of dTMP (see p. 402). As dTMP is essential for DNA synthesis, myeloma cells cannot survive in the presence of aminopterin. Although spleen cells are able to circumvent the inhibitory effect of aminopterin by using hypoxanthine and thymidine, they have a limited lifespan and die. Only hybridomas survive culture in HAT medium, because they possess both the immortality of the myeloma cells and the spleen cells metabolic side pathway. 

Phosphorylases remove the ribose sugars to yield the bases guanine or hypoxan-thine (from adenine or inosine nucleosides). 

Hypouricemia. Deficiency of uric acid in the blood, along with xanthinuria, resulting from deficiency of xanthineoxidase, the enzyme required for conversion of hypoxan-thine to xanthine and of xanthine to uric acid. 

The bases of nucleic acids can also be considered prebiotic compounds. A possible prebiotic route to adenine has been described (Oro, 1960 Ord and Kimball, 1961 1962 see also Shapiro, 1995), as shown in Figure 3.4. For details see also Miller s review (Miller, 1998). Guanine, and the additional purines such as hypoxan-thine, xanthine, and diaminopurine could also have been synthesized by variations of the above synthesis (Sanchez et al, 1968). 

Orotic acid, uric acid, uridine, allantoin, cytidine, hypoxan-thine, xanthine, guanine, pseudopurines, pyrimidines Purine bases... 

Figure 8.8 Retention times for adenosine (22.45 pmol), inosine (22.37 pmol), and hypoxan-thine (44.08 pmol) (A) standard prepared in mobile phase (B) standard prepared in incubation fluid or cell suspension medium. Conditions mobile phase, 125 mM potassium dihydrogen phosphate, 1.5% (v/v) acetonitrile, 20 mM triethylamine, and 1.0 mM tetrabutylammonium hydrogen sulfate (TBAHS) (pH 6.5) flow rate, 0.5 ml/min. (Reprinted from Ref. 18 with permission.)...

The drug allopurinol, which is an inhibitor of xanthine oxidase, effectively treats gout. Allopurinol is structurally similar to hypoxan-thine, except that the 5-membered ring has the positions of the carbon and nitrogens reversed. 

The application of the HPLC assay method to studies on reaction mechanisms has been limited, and the reader is referred to the work of Sloan (1984). Sloan and his colleagues studied the formation of IMP or GMP (and pyrophosphate) from the substrates phosphoribosylpyrophosphate (PRPP) and either hypoxanthine or guanine. These reactions, catalyzed by hypoxan-thine/guanine phosphoribosyltransferase (GHPRTase), were studied by HPLC after a method was developed to separate all the reactants and products simultaneously. 

The determination of the purine-nucleotide metabolites xanthine, hypox-an thine, and inosine by biosensors is of special interest for the estimation of meat or fish freshness. After the death of a fish, adenosine triphosphate (ATP) in the fish tissue is quickly degraded to inosine monophosphate (IMP). Further enzymatic decomposition of IMP leads to the accumulation of hypoxan thine (Hx), which is used as an indicator of fish freshness. To quantify these compounds with biosensors, it is possible to perform amperometric measurements of the generated hydrogen peroxide or the consumed oxygen according to the following enzymatic reactions ... 

Examples of amination of oxopurines to aminopurines via silyl derivatives include conversion of inosine into adenosine with catalysis by mercury ions (76LA745), and hypoxan-thines have been converted directly into adenines with phosphoramides at 225-230 °C (69IZV655). 

Treatment of (205) with triethyl orthoformate gave an 8,9-cyclic derivative of hypoxan-thine (206) (67MI41000, 67MIP41000). 

Bromination has been widely studied and occurs readily with adenine, guanine, hypoxan-thine, xanthine, and methylated xanthines giving the 8-bromo analogs. 

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