We have examined cytokine regulation of nitric oxide synthase (NOS) in human umbilical vein endothelial cells (HUVEC). 24-h treatment with IFN-'y (200 U/ml) plus TNF (200 U/ml) or IL-1,8 (5 U /ml) increased NOS activity in HUVEC lysates, measured as conversion of I '4C IL-arginine to Essentially, all NOS activity in these cells was calcium dependent and membrane associated. Histamine-induced nitric oxide release, measured by chemiluminescence, was greater in cytokine-treated cells than in control cells. Paradoxically, steadystate mRNA levels of endothelial NOS fell by 94±2.0% after cytokine treatment. Supplementation of HUVEC lysates with exogenous tetrahydrobiopterin (3 ,M) greatly increased total NOS activity, and under these assay conditions, cytokine treatment decreased maximal NOS activity. IFN-y plus TNF or IL-1,8 increased endogenous tetrahydrobiopterin levels and GTP cyclohydrolase I activity, the rate-limiting enzyme oftetrahydrobiopterin synthesis. Intracellular tetrahydrobiopterin levels were higher in freshly isolated HUVEC than in cultured cells, but were still limiting. We conclude that inflammatory cytokines increase NOS activity in cultured human endothelial cells by increasing tetrahydrobiopterin levels in the face of falling total enzyme; similar regulation appears possible in vivo.

The onset of neurologic symptoms in a child who had markedly elevated blood phenylalanine levels during the first two weeks of life and who was promptly treated with a low phenylalanine diet, with excellent control of serum phenylalanine levels, suggested that this child had an unusual form of phenylketonuria. In assays of the components of the phenylalanine hydroxylating system (open liver biopsy at 14 months), the activity of phenylalanine hydroxylase was 20 per cent of the average normal adult value. By contrast, no dihydropteridine reductase activity was detected in the patient's liver, brain or cultured skin fibroblasts. Since dihydropteridine reductase is also essential for the biosynthesis of dopamine, norepinephrine, and serotonin, disturbed neurotransmitter function may be responsible for the patient's neurologic deterioration. On the basis of these results, assay of reductase in cultured skin fibroblasts may be advisable in the initial diagnosis of phenylketonuria.

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We have examined the interaction of phenylalanine hydroxylase with phenylalanine, tetrahydropterin cofactors, and an activating phospholipid, lysophosphatidylcholine. Incubation of native phenylalanine hydroxylase with phenylalanine or lysophosphatidylcholine results in an increase in the fluorescence emission of the enzyme at 360 nm, which closely parallels the increase in tetrahydrobiopterin-dependent activity observed under these conditions. The presence of tetrahydrobiopterin in the absence of phenylalanine results in quenching of the enzyme fluorescence emission; this quenching exhibits a sharp end point at about 1 mol of tetrahydrobiopterin bound/mol of enzyme subunit. The binding of tetrahydrobiopterin under these conditions is unexpectedly tight, with an estimated KD of 10-20 nM, while in the presence of lysophosphatidylcholine, the KD is increased to about 25 microM. Quenching experiments with sodium iodide indicate greater exposure of tryptophan residues in the phenylalanine-activated enzyme. The ultraviolet difference spectrum of phenylalanine hydroxylase in the presence of phenylalanine exhibits a peak at 238 nm, which correlates with the fluorescence increase and activation, as well as additional changes in the aromatic region, which do not correlate well with activation. Phenylalanine does not alter the far-ultraviolet circular dichroism spectrum of phenylalanine hydroxylase. In contrast, lysophosphatidylcholine appears to induce a dramatic change in enzyme secondary structure upon activation. These results suggest that activation of phenylalanine hydroxylase results in a conformation change and the exposure of buried tryptophan(s) and possibly a cysteine residue.

Mechanical stretch of embryonic chicken skeletal myotubes developed in vitro leads to many of the biochemical changes seen in skeletal muscle hypertrophy. These include increased amino acid accumulation, increased incorporation of amino acids into general cellular proteins and myosin heavy chains, and increased accumulation of total protein and myosin heavy chains. This model system should aid in understanding how the growth rate of skeletal muscle is regulated by its activity.

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The product of the aerobic oxidation of tetrahydrobiopterin, quinonoid dihydrobiopterin, is unstable and rapidly rearranges to form a 7,s-dihydropteridine. Kaufman [Kaufman, S. (1967) J. Biol. Chem. 242,3934-39431 identified the stable product produced in 0.1 M phosphate pH 6.8, as 7,s-dihydrobiopterin. However, Armarego et al. [Armarego, W. L. F., Randles, D. and Taguchi, H. (1983) Eur. J. Biochem. 135 393-4031 questioned this assignment because they found that the dihydroxypropyl group on C-6 was eliminated and 7,s-dihydropterin was the predominant product when the aerobic oxidation was performed in 0.1 M Tris pH 7.6. In the present study we demonstrate that the rearrangement of the unstable quinonoid dihydrobiopterin results in a mixture of these two 7,8-dihydropteridines at neutral pH, 25 "C. Furthermore, we find that the loss or retention of the alkyl sidechain is not solely dependent on the pH of the reaction mixture, as was previously assumed by Armarego et al., but rather is strongly influenced by the temperature and the type of buffer. In addition, we describe a new method for quantifying the relative amounts of these two 7,8-dihydropteridines in mixtures of unknown concentrations. This method relies on multicomponent analysis of second derivative spectra and results in values which agree with the concentrations determined directly by HPLC.A reduced pteridine cofactor is necessary for phenylalanine hydroxylase to catalyze the conversion of phenylalanine to tyrosine [l]. The natural cofactor for this enzyme (as well as for tyrosine hydroxylase [2-51 and tryptophan hydroxylase [6, 71 is tetrahydrobiopterin, (BH,) [S]. During the hydroxylation reaction at neutral pH, there is a stoichiometric oxidation of BH4 to quinonoid dihydrobiopterin (qBH2) [9, 101. This latter species can then be reduced back to BH4 by the NADH-dependent enzyme, dihydropteridine reductase [ l l -151. qBH2 is unstable and, in the absence of the reductase, rapidly rearranges to a 7,s-dihydropteridine [9]. It was concluded that 7,s-dihydrobiopterin (BH,) was the product of the tautomerization of qBH2 [16,17] because when the auto-oxidation of BH4 was performed in 0.1 M phosphate pH 6.8 at ambient temperature, the ultraviolet spectrum of the product was identical to that of authentic BH2 [16]. This conclusion was also strongly supported by the finding that one of the products of the aerobic oxidation of BH4 in phosphate buffer was a substrate for sepiapterin reductase, an enzyme that catalyzes the NADP-dependent oxidation of the 2'-hydroxyl group on the side-chain of BH2 [16].However, it has become apparent that the unstable qBH2 does not always isomerize to BH,. Whereas the oxidation of Correspondence to