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Purpose: Because of the evolutionary loss of the uricolytic pathway, humans accumulate poorly soluble urate as the final product of purine catabolism. Restoration of uricolysis through enzyme therapy is a promising treatment for severe hyperuricemia caused by deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT). To this end, we studied the effect of PEG conjugation on the activity and stability of the enzymatic complement required for conversion of urate into the more soluble ( S)-allantoin. Methods: We produced in recombinant form three zebrafish enzymes required in the uricolytic pathway. We carried out a systematic study of the effect of PEGylation on the function and stability of the three enzymes by varying PEG length, chemistry and degree of conjugation. We assayed in vitro the uricolytic activity of the PEGylated enzymatic triad. Results: We defined conditions that allow PEGylated enzymes to retain native-like enzymatic activity even after lyophilization or prolonged storage. A combination of the three enzymes in an appropriate ratio allowed efficient conversion of urate to ( S)-allantoin with no accumulation of intermediate metabolites. Conclusions: Pharmaceutical restoration of the uricolytic pathway is a viable approach for the treatment of severe hyperuricemia. [ABSTRACT FROM AUTHOR]

Contents of purine alkaloids in different parts of tea (Camellia sinensis L. cv. Yabukita) seedlings, seeds and tissue cultures were determined with high‐performance liquid chromatography. More than 99% of the caffeine detected was in the leaves of the 4‐month‐old seedlings. The amount expressed per g fresh weight was higher in older leaves. Theobromine, a precursor of caffeine biosynthesis, was found only in younger leaves. Zero or only trace amounts of theophylline, a degradation product of caffeine, were found in the seedlings. Almost all the caffeine in tea seeds was found in the seed coats. Theobromine and theophilline could not be detected in any part of the seeds. Tracer experiments using [8‐14C]‐adenine indicate that (i) caffeine biosynthesis from [8‐14C]‐adenine occurs only in younger leaves,(ii) “salvage” of [8‐14C]‐adenine for nucleic acid synthesis takes place in all parts of the seedlings, (iii) considerable degradation of [8‐14C]‐adenine by conventional purine degradation pathway via uric acid takes place in roots and lower parts of stem tissue. The results strongly suggest that caffeine is synthesized in younger leaves and accumulated within the leaves. Both caffeine contents and its synthetic activity from adenine were extremely low in tissue culture of tea. [ABSTRACT FROM AUTHOR]