Volume 1, Issue 4

An enzyme which reduces 6-pyruvoyl-tetrahydropterin has been purified to apparent homogeneity from human liver. It consists of a single polypeptide chain with a molecular weight of 35 kDa, has an isoelectric point of 5.9 ± 0.1 and contains no glycosyl residues. The pure enzyme has a specific activity of 450 mU/mg protein at pH 7.0 in 10 mM potassium phosphate buffer. It converts 6-pyruvoyl-tetrahydropterin to 6-lactoyltetrahydropterin by transfer of the pro 4R-hydrogen of NADPH to form the side chain -OH at position C(2') of the substrate. Km values are 1.8 J..lM for 6-pyruvoyl-tetrahydropterin and 5.5 J..lM for NADPH. Polyclonal antibodies raised against the purified enzyme recognize 6-pyruvoyl-tetrahydropterin reductase in Western blot and ELISA but do not cross-react with human sepiapterin reductase. The enzyme appears to be identical with aldose reductase.

A series of new amino acid-tetrahydropterin-conjugates has been synthesized starting from N 2-isobutyryl-6,7- dimethyl-5,6,7,8-tetrahydropterin (7). The amino acids have been attached in form of their esters (10 - 16) via a succinyl spacer to the N-5 position (17 - 23) or directly via the carboxyl function applying CBZprotected activated esters (35, 39). Acylation of 7 with N-benzyloxycarbonylglutamic acid anhydride led to the y-glutamyl derivative 41. Selective deblocking experiments were achieved by catalytic hydrogenolysis to cleave the benzyl esters (24-28, 36, 42) and by DBU treatment to eliminate the p-nitrophenylethyl group (24, 25) respectively. Further reaction with ammonia hydrolysed the isobutyryl group to give the free tetrahydropterin-amino acid-conjugates 29 - 32, 37 and 43. Also a carbamoyl-type conjugate (45) was obtained from 7 and benzyl phenylalanine N-carbonylimidazolide (44). The newly synthesized compounds have been characterized by elemental analysis, UV and IH-NMR spectra.

An enzyme-linked immunosorbent assay (ELISA) was calibrated and used for quantitation of the folate binding protein in human cerebrospinal fluid. The precision of the assay in terms of the coefficient of variation was 6.3% intra-assay (10 replicate single determinations) and 4.8% inter-assay (8 duplicate determinations). The concentration of folate binding protein in cerebrospinal fluid specimens from 20 patients with no complaints or diseases of neurological type was 0.14-0.38, 0.24 nmol FBP/I (range and median value). The gelfiltration profiles of radioligand bound and immunoreactive folate binding protein were identical, both consisting of one major peak (M, '" 25000) and one minor peak (M, '" 1 00000).

The effects of a diet of 85% casein on the activities of the phenylalanine hydroxylases of rat liver and kidney have been compared. Whereas only the tetrahydrobiopterin-dependent activity of rat hepatic phenylalanine hydroxylase is significantly stimulated, both the tetrahydrobiopterin-dependent and the dimethyltetrahydropterin- dependent activities of the renal enzyme are significantly decreased, after five days of feeding a casein diet. The animals fed a high protein diet for seven days have an increased rate of phenylalanine catabolism in vivo, which is also reflected in increased flux of label from phenylalanine into glucose. The regulation of phenylalanine metabolism, under these conditions, is discussed.