J.H. Fellman

Assay, properties and tissue distribution of p-hydroxyphenylpyruvate hydroxylase

Abstract Two methods for the assay of p -hydroxyphenylpyruvate hydroxylase ( p -hydroxyphenylpyruvate, ascorbate: O 2 oxidoreductase (hydroxylating), EC 1.14.2.2) activity are described. (1) Chemical method: The homogentisate formed was condensed with cysteine to yield a 1,4-thiazine with an absorption maximum at 390 nm. (2) Radiochemical assay: The release of 14 CO 2 from p -hydroxyphenyl[ carboxy - 14 C]-pyruvate was used as a measure of enzyme activity. Enzyme activation by certain reducing agents, pH optimum, tissue distribution, and fetal and adult enzyme activities are reported. Phenylpyruvate and 3,4-dihydroxyphenylpyruvate were substrates of the enzyme and were mutually competitive to p -hydroxyphenylpyruvate oxidation. The substrates, p -hydroxyphenylpyruvate and oxygen, are inhibitory at high concentration. Hydrogen peroxide, generated by the substrate and oxygen, may account for the observed substrate inhibition. The enzyme is found only in liver and kidney. The significance of these findings to phenylketonuria and tyrosinemia is discussed.

Photosensitization and the effect of ultraviolet radiation on the production of unpaired eletrons in the presence of furocoumarins (psoralens)

The mechanism of biological photosensitization induced by furocoumarins (psoralens) has been investigated. Psoralen and other furocoumarins were found to exhibit fluorescence and to undergo electronic transitions involving singlet → triplet inter-combinations. The wavelengths emitted during phosphorescence were longer than those emitted during fluorescence. The biologically active and inactive compounds studied showed phosphorescence in the region of 460–470 and 500–525 mμ, respectively. Solutions of three psoralen derivatives (i.e., psoralen, 8-methoxypsoralen and 4,5′-dihydro-5-ethylcarbamyl-8-methoxypsoralen) in various solvents were irradiated with ultraviolet light (wavelengths greater than 310 mμ) at the temperature of liquid oxygen and examined with an electron spin-resonance spectrometer for free-radical formation. In the presence of either psoralen or 8-methoxypsoralen substantial electron-resonance signals were produced in solvent systems such as alcohol, alcohol plus water and glycerol plus water. Smaller signals were observed when water, glycerol and benzene were used alone as solvetns. The electron-resonance spectra of psoralen and 8-methoxypsoralen in alcohol solvents differed markedly from those of the same substances in solvents containing water. The biologically inactive derivative, 4′5′-dihydro-5-ethylcarbamyl-8-methoxypsoralen, produced weak free-radical signals in ethanol, but failed to produce signals in several other solvents. This study indicates that the reactive species in the biological photosensitization induced by psoralens is the triplet state of the sensitizing molecule. Exciation of the psoralen molecule to the triplet state may lead to free-radical formation. The mechanism of biological photosensitization appears to involve excitation of molecules to a triplet state and generation of free radicals which eventually evoke biological changes in the irradiated system.

The metabolism of taurine to isethionate

Abstract The biosynthesis of isethionate from taurine in mammalian tissue has been reexamined. In vivo metabolism of taurine to isethionate was demonstrated but it was shown that a number of acyltaurine metabolites also behave like isethionate on the conventional dual column ion-exchange chromatographic analytical system. Hydrolysis of these column effluates coupled with high-voltage electrophoresis resolves this ambiguity. In vivo formation of isethionate from taurine in mammals seems to occur from gut microorganism metabolism since: (a) germ-free mice could not convert taurine to isethionate, (b) gut anaerobes were able to metabolize taurine, (c) in vitro rat and mouse tissue failed to metabolize taurine to isethionate. These findings are in conflict with earlier reports.

Decarboxylation to tyramine is not a major route of tyrosine metabolism in mammals.

Abstract We have examined the question of the contribution of decarboxylation of tyrosine to tyramine to the overall metabolic fate of tyrosine in mammalian organisms. Since this enzymatic step is independent of oxygen while the loss of the carboxyl group via the transaminase-hydroxylase major pathway is oxygen dependent, we studied the rate of 14 CO 2 release from tyrosine in the presence and absence of oxygen. These studies indicate that a trivial amount of tyrosine is processed by direct decarboxylation to tyramine, even at high substrate levels. Tyrosine metabolism precedes via the well-established pathway which is launched by transamination.

The conformation of acetylcholine

The infrared absorption of the carbonyl peak of acetylcholine exhibits a shift toward the higher energy. 2-Dimethylamino ethyl acetate, 3-dimethylaminopropyl acetate, 4-dimethylaminobutyl acetate show the expected absorption spectra for the carbonyl peak in that they are precisely like ethyl acetate. 3-Dimethylaminopropyl acetate methiodide and 4-dimethylaminobutyl acetate methiodide do not exhibit absorption for the carbonyl peak like acetylcholine, but rather behave like the unquaternized esters. dl, cis 2-Dimethylamino-cyclohexanol acetate methiodide shows a similar shift in the infra-red absorption for the carbonyl peak of acetylcholine, the trans isomer does not. The above evidence was advanced in support of a cyclic conformation of acetylcholine which can explain the observed kinetic data for alkaline hydrolysis and acid catalysis of this substance, as well as explain the rates of acylation of hydroxylamine observed.

Substrate specificity of p-hydroxyphenylpyruvate hydroxylase

Abstract Evidence is presented that a single enzyme is responsible for hydroxylation of three naturally occurring aromatic keto acids; p-hydroxyphenylpyruvate, phenylpyruvate and 3,4-dihydroxyphenylpyruvate.

Further studies on the conformation of acetylcholine.

Abstract A reexamination of the hypothesis that acetylcholine exists in a cyclic conformation was undertaken. The determination of infrared-absorption spectra and the acylation rates of a number of analogues of acetylcholine were carried out. The assembled evidence indicated that the shift toward the higher energy of the infrared absorption of the carbonyl peak of acetylcholine and its higher rate of acylation argues against a cyclic conformation and for an inductive influence of the quaternary nitrogen.

Concentration and hydroxylation of free phenylalanine in adrenal glands.

Abstract The adrenal glands of man, beef, spider monkey, rabbit, guinea pig, and sheep contain large amounts of free phenylalanine. Beef-adrenal medulla is capable of hydroxylating phenylalanine by a mechanism suggestive of the chemical system of Udenfriend . The significance of these findings to aromatic hydroxylation products found in normal urine and those isolated from the urine of individuals afflicted with phenylpyruvic oligophrenia is discussed.

The biosynthesis of DOPA in albino skin

Abstract 1. 1. Tyrosine-hydroxylating activity from pigmented and albino rat skin has been characterized in part. Both sources of enzyme convert tyrosine to DOPA, are stimulated by DOPA, are inhibited by diethyldithiocarbamate, but not by α , α ′- dipyridyl. 2. 2. In contrast to the enzyme obtained from pigmented skin scrapings, tyrosine- hydroxylation activity is found in the soluble fraction of albino skin homogenates.

Metabolism and properties of 3-methoxy-4-hydroxyphenylpyruvate; a metabolite of dihydroxyphenylalanine

The pathway of 3,4-dihydroxyphenylalanine undergoing metabolism via transamination and subsequent oxidative rearrangement to 2,4,5-trihydroxy-phenylacetate was investigated. 3-Methoxytyrosine does not pursue an identical course, since its corresponding keto acid is not subject to action of p-hydroxy-phenylpyruvate hydroxylase. The radiochemical synthesis of 3-methoxy-4-hydroxy[carboxy-14C] pyruvate was accomplished. This metabolite was used to demonstrate that this keto acid does not proceed through oxidative rearrangement both in vitro and in vivo. The keto acid was found to be a competitive inhibitor of the hydroxylase and can help account for some of the metabolites observed in the urine of patients treated with 3,4-dihydroxyphenylalanine.