Biosynthetis of phytoquinones. Biosynthetic origins of the nuclei and satellite methyl groups of plastoquinone, tocopherols and tocopherolquinones in maize shoots, bean shoots and ivy leaves

1. By using dl-[ring-(14)C]phenylalanine, dl-[beta-(14)C]phenylalanine, dl-[alpha-(14)C]-tyrosine and dl-[beta-(14)C]tyrosine it was shown that in maize shoots (Zea mays) the nucleus and one nuclear methyl group of each of the following compounds, plastoquinone, gamma-tocopherol (aromatic nucleus) and alpha-tocopherolquinone, are formed from the nuclear carbon atoms and beta-carbon atom respectively of either exogenous phenylalanine or exogenous tyrosine. With ubiquinone only the aromatic ring of the amino acid is used in the synthesis of the quinone nucleus. Chemical degradation of plastoquinone and gamma-tocopherol molecules labelled from l-[U-(14)C]tyrosine established that a C(6)-C(1) unit directly derived from the amino acid is involved in the synthesis of these compounds. Radioactivity from [beta-(14)C]cinnamic acid is not incorporated into plastoquinone, tocopherols or tocopherolquinones, demonstrating that the C(6)-C(1) unit is not formed from any of the C(6)-C(1) phenolic acids associated with the metabolism of this compound. 2. The incorporation of radioactivity from l-[U-(14)C]tyrosine, dl-[beta-(14)C]tyrosine and dl-[U-(14)C]phenylalanine into bean shoots (Phaseolus vulgaris) and dl-[beta-(14)C]tyrosine and l-[Me-(14)C]methionine into ivy leaves (Hedera helix) was also investigated. Similar results were obtained to those reported for maize, except that in beans phenylalanine is only used for ubiquinone biosynthesis. This is attributed to the absence of phenylalanine hydroxylase from these tissues. In ivy leaves it is found that the beta-carbon atom of tyrosine gives rise to the 8-methyl group of delta-tocopherol, and it is suggested that for all other compounds examined it will give rise to the nuclear methyl group meta to the polyprenyl unit. 3. Preliminary investigations with the alga Euglena gracilis showed that in this organism ring-opening of tyrosine occurs to such an extent that the incorporation data from radiochemical experiments are meaningless. 4. The above results, coupled with previous observations, are interpreted as showing that in higher plants the nucleus of ubiquinone can be formed from either phenylalanine or tyrosine by a pathway involving as intermediates p-coumaric acid and p-hydroxybenzoic acid. Plastoquinone, tocopherols and alpha-tocopherolquinone are formed from p-hydroxyphenylpyruvate by a pathway in which the aromatic ring and C-3 of the side chain give rise respectively to the nucleus and to one nuclear methyl group. 5. Dilution experiments provided evidence that in maize shoots p-hydroxyphenylpyruvic acid and homogentisic acid (produced from p-hydroxyphenylpyruvic acid) are involved in plastoquinone biosynthesis, and presumably the biosynthesis of related compounds: however, other possible intermediates in the conversion including toluquinol (the aglycone of the proposed key intermediate) showed no dilution effects. Further, radioactivity from [Me-(14)C]toluquinol is not incorporated into any of the compounds examined. 6. Dilution experiments with 3,4-dihydroxybenzaldehyde and radioactive-labelling experiments with 3,4-dihydroxy[U-(14)C]benzoic acid demonstrated that these compounds are not involved in the biosynthesis of either ubiquinone or phylloquinone in maize shoots. 7. Evidence is also presented to show that in maize shoots ring-opening of the aromatic amino acids takes place. The suggestion is offered that this may take place via homogentisic acid, as in animals and some micro-organisms.     

Observations on the biosynthesis of phytoterpenoid quinone and chromanol nuclei

1. p-Hydroxy[U-(14)C]benzoic acid, except for loss of the carboxyl group, is effectively incorporated into the nucleus of ubiquinone and an unidentified prenylphenol by maize roots, maize shoots, french-bean leaves, french-bean cotyledons and Ochromonas danica. Plastoquinone, alpha-tocopherol, gamma-tocopherol and alpha-tocopherolquinone are all unlabelled from this substrate. The high radioactivity of the prenylphenol and its behaviour in a pulse-labelling experiment with maize shoots suggested that it may be a ubiquinone precursor. 2. Members of the 2-polyprenylphenol and 6-methoxy-2-polyprenylphenol series, compounds that are known ubiquinone precursors in Rhodospirillum rubrum, could not be detected in maize tissues, but possibly they may occur as their glycosides. 3. [G-(14)C]Shikimic acid is incorporated into the nuclei of phylloquinone, plastoquinone, alpha-tocopherolquinone, gamma-tocopherol, alpha-tocopherol and ubiquinone in maize shoots, showing that in plant tissues the nuclei of these compounds arise via the shikimic acid pathway of aromatic biosynthesis. 4. l-[U-(14)C]Phenylalanine and l-[U-(14)C]tyrosine are incorporated into plastoquinone, gamma-tocopherol, alpha-tocopherolquinone and ubiquinone. alpha-Tocopherol, which is absent from shoots incubated with l-[U-(14)C]tyrosine, is also labelled from l-[U-(14)C]phenylalanine. Degradation studies showed that there is little (14)C radioactivity in the terpenoid portions of the molecules and from this it is concluded that the aromatic portions of these amino acids are giving rise to the quinone and chromanol nuclei. 5. It is proposed that in maize the nucleus of ubiquinone can be formed from either phenylalanine or tyrosine by a pathway involving p-coumaric acid and p-hydroxybenzoic acid. Plastoquinone, tocopherols and tocopherolquinones are formed from tyrosine by some pathway in which the aromatic ring and C-3 of the side chain of this amino acid gives rise to the nucleus and one methyl substituent respectively of these compounds.     

Mechanism of irreversible inactivation of phenylalanine-4- and tryptophan-5-hydroxylases by [4-36Cl, 2-14C]p-chlorophenylalanine: A revision

Intraperitoneal injection of [4-³⁶Cl, 2-¹⁴C]p-chlorophenylalanine (pCPA) (300 mg/kg) in rats revealed absence of chlorine in pure hepatic phenylalanine hydroxyase, while the carbon label appeared as 1–4 moles/mole of [¹⁴C]tyrosine in the inactivated phenylalanine and cerebral tryptophan-5-hydroxylase. Crystalline muscle aldolase and tyrosine hydroxylase also revealed the presence of [2-¹⁴C]tyrosine from [2-¹⁴C]pCPA without inactivating these enzymes. Injection of L-[(U)-¹⁴C] tyrosine led to its incorporation into the above enzymes, but to a different degree without altering the enzyme activity. Repeated injections ofp-chlorophenylacetic acid had no effect on phenylalanine or tryptophan-hydroxylase. Administration of pCPA did not change the levels of cerebral biopterins. Reexamination of the effect of cycloheximide on reversing enzymic inactivation by pCPA failed to confirm our earlier observation.     

The contribution of phenylalanine to tyrosine metabolism in vivo. Studies in the post-absorptive and phenylalanine-loaded rat.

1. Rates of appearance and oxidation of plasma L-leucine, L-phenylalanine and L-tyrosine, as well as conversion of plasma phenylalanine into plasma tyrosine, were determined in 90-120 g rats after overnight starvation and while receiving 115-120 mumol of L-phenylalanine/h. 2. In the post-absorptive state, plasma tyrosine and phenylalanine appearances were similar, despite the fact that 22% of plasma tyrosine appearance could be attributed to the hydroxylation of phenylalanine. 3. A constant infusion of 115-120 mumol of L-phenylalanine/h did not significantly alter plasma leucine kinetics, but increased appearance of plasma phenylalanine and tyrosine. The percentage of phenylalanine and tyrosine appearance that was oxidized increased from 12.1% and 24.4% to 37.3% and 48.0% respectively. In phenylalanine-loaded rats, 72% of plasma tyrosine appearance could be attributed to the conversion of phenylalanine. 4. Whole-body tyrosine oxidation measured from a continuous infusion of either L-[14C]tyrosine or L-[14C]phenylalanine differed by 165%. 5. It can be concluded that, in the post-absorptive state, phenylalanine hydroxylation makes a substantial contribution to the plasma appearance of tyrosine and is significantly increased when phenylalanine is administered. The disposal of excess infused phenylalanine is a result of a greater percentage of plasma phenylalanine being converted into tyrosine and a greater proportion of tyrosine being further oxidized. However, apparent tyrosine oxidation rates estimated from plasma tyrosine specific radioactivities and appearance of expired 14CO2 during administration of [14C]tyrosine are underestimates of true rates, in part because tyrosine generated from phenylalanine hydroxylation is catabolized without freely equilibrating with the plasma compartment.     

Biosynthesis of 3,4-dihydroxyphenylalanine in Vicia faba

Radioactive shikimic acid and l-tyrosine were shown to be efficient precursors of 3,4-dihydroxyphenylalanine (DOPA) in Vicia faba. [1-¹⁴C]Acetate and l[U-¹⁴C]phenylalanine were not incorporated into tyrosine or DOPA. Thus the synthesis of DOPA occurs via the shikimic acid pathway and tyrosine or a very closely related metabolise. Phenolase was present in etiolated plants in much larger quantities after a brief light exposure whereas DOPA concentration was relatively constant during all stages of plant growth. Partially purified phenolase did not catalyze the conversion of tyrosine to DOPA and does not appear to have a role in DOPA synthesis.     

Biosynthesis of an unusual amino acyl moiety contained in bestatin

The biosynthetic pathway of an unusual amino acyl [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl (AHP)] moiety which is contained in bestatin has been studied by testing the incorporation of potential precursors. L-[U-14C]-Phenylalanine, L-[U-14C]leucine, and [U-14C]acetic acid were efficiently incorporated into bestatin, but the radioactivity of L-[1-14C]phenylalanine, [1-14C]glyoxylic acid, and [14C]oxalic acid were not incorporated. Incorporation of acetic acid into 1- and 2-carbon of the AHP moiety was confirmed by incorporation of [13C]acetic acid. Thus, the AHP moiety was shown to be biosynthesized from L-phenylalanine and two carbon atoms of acetic acid, accompanied by decarboxylation of the phenylalanine.     

Biosynthesis of piperlongumine

The incorporation of l-[U-¹⁴C]lysine and l-[U-¹⁴C]phenylalanine into piperlongumine has been demonstrated in Piper longum. The subsequent stepwise degradation to methyl-(3,4,5-trimethoxyphenyl)-propanoate and δ-aminovaleric acid revealed that the C₆-C₃ moiety of the alkamide arises from phenylalanine; the heterocyclic ring is biosynthesised from lysine. It has also been shown that dl-[2-¹⁴C]tyrosine and [2-¹⁴C]sodium acetate are poor precursors of piperlongumine.     

The pathways of oxalate formation from phenylalanine,tyrosine, tryptophan and ascorbic acid in the rat

The metabolic pathway by which L-[¹⁴C₁]phenylalanine, L-[¹⁴C₁]tyrosine, L-[¹⁴C₁]tryptophan, and L-[¹⁴C₁]ascorbic acid are converted to [¹⁴C]oxalate have been investigated in the male rate. Only [¹⁴C]oxalate was detected in the urine of rats injected with L-[¹⁴C₁]ascorbic acid, but [¹⁴C]-labeled oxalate, glycolate, glyoxylate, glycolaldehyde, glycine, and serine were recovered from the [¹⁴C₁]-labeled aromatic amino acids. DL-Phenyllactate, an inhibitor of glycolic acid oxidase and glycolic acid dehydrogenase, reduced the amount of [¹⁴C]oxalate recovered in the urine of rats given the [¹⁴C₁]-labeled aromatic amino acids, but increased the amount of [¹⁴C]glycolate formed from L-[¹⁴C₁]-phenylalanine and L-[¹⁴C₁]tyrosine and the amount of [¹⁴C]glycolate produced from [¹⁴C₁]tryptophan. Based on the [¹⁴C]labeled intermediates identified and the relative distribution of the radioactivity, it is postulated that phenylalanine and tyrosine are converted to oxalate via glycolate which is oxidized directly to oxalate by glycolic acid dehydrogenase. Tryptophan is metabolized via glyxylate which is oxidized directly to oxalate by glycolic acid oxidase. Neither glycolate, glyoxylate, glycolic acid oxidase or glycolic acid dehydrogenase are involved in the formation of oxalate from ascorbic acid.     

In vivo regulation of phenylalanine hydroxylation to tyrosine, studied using enrichment in apoB-100

Phenylalanine hydroxylation is necessary for the conversion of phenylalanine to tyrosine and disposal of excess phenylalanine. Studies of in vivo regulation of phenylalanine hydroxylation suffer from the lack of a method to determine intrahepatocyte enrichment of phenylalanine and tyrosine. apoB-100, a hepatic export protein, is synthesized from intrahepatocyte amino acids. We designed an in vivo multi-isotope study, [(15)N]phenylalanine and [2H2]tyrosine to determine rates of phenylalanine hydroxylation from plasma enrichments in free amino acids and apoB-100. For independent verification of apoB-100 as a reflection of enrichment in the intrahepatocyte pool, [1-(13)C]lysine was used as an indicator amino acid (IAA) to measure in vivo changes in protein synthesis in response to tyrosine supplementation. Adult men (n = 6) were fed an amino acid-based diet with low phenylalanine (9 mg.kg(-1).day(-1), 4.54 mumol.kg(-1).,h(-1)) and seven graded intakes of tyrosine from 2.5 (deficient) to 12.5 (excess) mg.kg(-1).day(-1). Gas chromatography-quadrupole mass spectrometry did not detect any tracer in apoB-100 tyrosine. A new and more sensitive method to measure label enrichment in proteins using isotope ratio mass spectrometry demonstrated that phenylalanine hydroxylation measured in apoB-100 decreased linearly in response to increasing tyrosine intake and reached a break point at 6.8 mg.kg(-1).day(-1). IAA oxidation decreased with increased tyrosine intake and reached a break point at 6.0 mg.kg(-1).day(-1). We conclude: apoB-100 is an accurate and useful measure of changes in phenylalanine hydroxylation; the synthesis of tyrosine via phenylalanine hydroxylation is regulated to meet the needs for protein synthesis; and that plasma phenylalanine does not reflect changes in protein synthesis.     

INCORPORATION OF PHENYLALANINE AS A SINGLE UNIT INTO RAT BRAIN PROTEIN: RECIPROCAL INHIBITION BY PHENYLALANINE AND TYROSINE OF THEIR RESPECTIVE INCORPORATIONS

Abstract— Of seven amino acids studied, glutamic acid and phenylalanine were incorporated in highest amounts into the hot-TCA-insoluble material of the 100,000 g supernatant fraction of rat brain homogenate. The system for incorporation of phenylalanine was RNase-insensitive and required ATP (apparent K_m = 0.64 m m ), KC1 (apparent K_m = 14 m m ) and MgCl₂ (optimal concentration range 4-15 m m ). The apparent K_m for phenylalanine was 2.9 m m . [¹⁴C]Phenylalanine did not undergo modification before incorporation. Tyrosine and phenylalanine inhibited the incorporation, respectively, of [¹⁴C]phenylalanine and [¹⁴C]tyrosine when incubated simultaneously or successively. The K_m and K_t (3.3 m m ) values for phenylalanine in the incorporation reaction and as inhibitor of the incorporation of [¹⁴C]tyrosine were similar. We suggest that both the enzyme and the acceptor for the incorporation of these two amino acids are the same. [¹⁴C]Phenylalanine and [¹⁴C]tyrosine entered into COOH-terminal positions in the reactions described. Brain exhibited a 25- to 100-fold higher capacity to incorporate phenylalanine than that of liver, kidney or thyroid. The acceptor capacity in rat brain rapidly decreased from day 5 to day 15 of postnatal age and then slowly until age 150 days.     

Tyrosine and Phenylalanine Ammonia Lyase Activities during Shoot Initiation in Tobacco Callus Cultures

Both phenylalanine ammonia lyase and tyrosine ammonia lyase were detected in tobacco (Nicotiana tabacum L. Wisconsin 38) callus. The enzymes were separated from each other by Sephadex G-200 column chromatography. Increased activity of tyrosine ammonia lyase was observed during culture of tobacco callus under shoot-forming conditions, while activity of phenylalanine ammonia lyase increased during culture under non-organ-forming conditions. Confirmation of these findings was obtained by examining the incorporation of [¹⁴C]tyrosine and [¹⁴C]phenylalanine into p-coumarate and trans-cinnamate, respectively.     

Cerebral lipid metabolism in experimental hyperphenylalaninaemia: incorporation of 14C-labelled glucose into total lipids

—1. Effects of the administration of phenylalanine to rats on incorporation in vivo or in vitro of [U-¹⁴C]glucose into cerebral lipids were studied during the first 5–10 days of postnatal development. In addition, the effects of added phenylalanine and its deaminated metabolites on incorporation of [U-¹⁴C]glucose by homogenates into lipids of developing rat brain were investigated. Hyperphenylalaninaemia reduced incorporation both in vivo and in vitro of [U-¹⁴C]glucose into cerebral lipids. 2. Phenylalanine or tyrosine added in vitro at concentrations equivalent to those in the brain of the hyperphenylalaninaemic rat (0-1 μmole/ml incubation medium) did not inhibit incorporation of [U-¹⁴C)glucose into lipids, although at much higher concentrations of phenylalanine (36 μumoles/ml incubation medium) slight inhibition (10 per cent) of incorporation of [U-¹⁴C]glucose into lipids was observed. 3. In contrast, the deaminated metabolites in general exerted greater inhibitory effects at lower concentrations. Phenyllactic acid, in comparison to phenylpyruvic and phenyl-acetic acid, was the most potent inhibitor of the incorporation in vitro of [U-¹⁴C]glucose into cerebral lipids. These results indicated that these metabolites of phenylalanine were the more potent inhibitors of cerebral lipid metabolism in immature animals.     

The effects of individual amino acids on the incorporation of labelled amino acids into proteins by brain homogenates

Abstract— The effects of phenylalanine and other amino acids on incorporation of several different ¹⁴C-labelled amino acids into cerebral protein were studied in brain homogenates. Excess of some amino acids had a varied effect with different ¹⁴C-labelled amino acids. Of the unlabelled-labelled amino acid combinations tested the maximal inhibition was obtained with the following: (1) phenylalanine, which inhibited the incorporation of [¹⁴C]tyrosine, and (2) leucine, which inhibited incorporation of [¹⁴C]isoleucine. In both cases the inhibition occurred principally in proteins that were recovered in the 800 g and 13,000 g sediments. Only a small degree of inhibition occurred in proteins that sedimented at 100,000 g, and no inhibition occurred in proteins of the 100,000 g supernatant.     

Metabolism of L-(U-14C)valine, L-(U-14C)leucine, L-(U-14C)histidine and L-(U-14C) phenylalanine by the isolated perfused lactating guinea-pig mammary gland.

1. The incorporation of L-[U-14C]leucine, L[U-14C]histidine and L-[U-14C]phenylalanine into casein secreted during perfusion of isolated guinea-pig mammary glands was demonstrated. 2. The extent of incorporation of label into casein residues was consistent with their being derived from free amino acids of the perfusate plasma. 3. The mean transit time of the amino acids from perfusate into secreted casein was approx. 100 min. 4. Whereas radioactive histidine and phenylalanine were incorporated solely into milk protein, radioactivity from [U-14C]valine was also transferred to CO2 and to an unidentified plasma component, and from [U-14C]leucine to plasma glutamic acid. 5. Evidence from experiments with [U-14C]phenylalanine suggests that, as in rats, but in contrast with ruminant species, guinea-pig mammary tissue does not possess phenyl alanine hydroxylase activity. 6. The results are discussed in relation to the possible role of essential amino acid catabolism in the control of milk-protein synthesis.     

Some properties of rat-liver glucose–adenosine triphosphate phosphotransferases

1. Bacilysin, a peptide which yields l-alanine and l-tyrosine on acid hydrolysis, was produced by a strain of Bacillus subtilis (A 14) in a chemically defined medium containing glucose, ammonium acetate or ammonium chloride, potassium phosphate and other inorganic salts, and ferric citrate. 2. Under the conditions used growth was diphasic. Bacilysin was formed during the second phase of slower growth, and there was little production during the stationary phase. Nevertheless, bacilysin production occurred when protein synthesis was inhibited by chloramphenicol. It thus appears that there is no obligatory coupling of protein synthesis and bacilysin synthesis. 3. When dl-[1-(14)C]alanine was added to a growing culture of B. subtilis, (14)C was incorporated into bacilysin, which contains an N-terminal alanine residue. 4. Under similar conditions virtually no (14)C was incorporated into bacilysin from dl-[2-(14)C]tyrosine, l-[U-(14)C]tyrosine or [1-(14)C]acetate, although these compounds were used by the cell for the biosynthesis of other substances. These results indicate that neither tyrosine nor acetate is a precursor of the fragment of bacilysin which yields tyrosine on hydrolysis with hot 6n-hydrochloric acid. 5. The tyrosine-yielding fragment of bacilysin was labelled with (14)C from [1,6-ring-(14)C(2)]shikimic acid. The biosynthesis of bacilysin thus appears to involve a diversion from the pathway leading to aromatic amino acids at the shikimic acid stage, or a subsequent one.     

Phenol biosynthesis in higher plants. Gallic acid

The biosynthesis of gallic acid in a number of higher plants was investigated by using l-[U-(14)C]phenylalanine, (-)-[G-(14)C]shikimic acid, d-[1-(14)C]glucose and d-[6-(14)C]glucose as tracers. The results are compared with those obtained similarly for caffeic acid and are interpreted in terms of the dehydrogenation of 5-dehydroshikimic acid as a normal route of metabolism for gallic acid.     

Preparation, Characterization, and Microbial Degradation of Specifically Radiolabeled [14C]Lignocelluloses from Marine and Freshwater Macrophytes

Specifically radiolabeled [¹⁴C-lignin]lignocelluloses were prepared from the aquatic macrophytes Spartina alterniflora, Juncus roemerianus, Rhizophora mangle, and Carex walteriana by using [¹⁴C]phenylalanine, [¹⁴C]tyrosine, and [¹⁴C]cinnamic acid as precursors. Specifically radiolabeled [¹⁴C-polysaccharide]lignocelluloses were prepared by using [¹⁴C]glucose as precursor. The rates of microbial degradation varied among [¹⁴C-lignin]lignocelluloses labeled with different lignin precursors within the same plant species. To determine the causes of these differential rates, [¹⁴C-lignin]lignocelluloses were thoroughly characterized for the distribution of radioactivity in nonlignin contaminants and within the lignin macromolecule. In herbaceous plants, significant amounts (8 to 24%) of radioactivity from [¹⁴C]phenylalanine and [¹⁴C]tyrosine were found associated with protein, although very little (3%) radioactivity from [¹⁴C]cinnamic acid was associated with protein. Microbial degradation of radiolabeled protein resulted in overestimation of lignin degradation rates in lignocelluloses derived from herbaceous aquatic plants. Other differences in degradation rates among [¹⁴C-lignin]lignocelluloses from the same plant species were attributable to differences in the amount of label being associated with ester-linked subunits of peripheral lignin. After acid hydrolysis of [¹⁴C-polysaccharide]lignocelluloses, radioactivity was detected in several sugars, although most of the radioactivity was distributed between glucose and xylose. After 576 h of incubation with salt marsh sediments, 38% of the polysaccharide component and between 6 and 16% of the lignin component (depending on the precursor) of J. roemerianus lignocellulose was mineralized to ¹⁴CO₂; during the same incubation period, 30% of the polysaccharide component and between 12 and 18% of the lignin component of S. alterniflora lignocellulose was mineralized.     

Mammalian melanocytes do not use phenylalanine for melanin synthesis

Hamster melanoma cells (RPMI 3460) were examined for their ability to utilize phenylalanine for melanin biosynthesis. There was a small but significant incorporation of L-[1-1414C] phenylalanine into hot acid-insoluble cellular material in the presence of cycloheximide. However, this radioactivity was removable from the acid-insoluble fraction by pronase digestion. A similar percentage of L-[U-14C] leucine incorporation was likewise resistant to cycloheximide inhibition. Residual protein synthesis is apparently responsible for the incorporation of both amino acids. Cycloheximide did not inhibit melanin synthesis. These results suggest that mammalian melanocytes do not use phenylalanine for melanin synthesis. Phenylalanine is not incorporated directly into melanin, nor do the cells appear to convert it to tyrosine via a phenylalanine hydroxylase.     

Cell-free biosynthesis of arphamenine A

Arphamenine A was synthesized in a cell-free system obtained from the arphamenine-producing strain, Chromobacterium violaceum BMG361-CF4. L-[14C]-phenylalanine was converted to beta-phenylpyruvic acid by phenylalanine amino-transferase obtained from the 10,000 x g supernatant (S10 fraction). [14C]-Benzylmalic acid was synthesized from beta-phenylpyruvic acid with [14C]-acetyl-CoA in the S10 fraction. [14C]-Benzylsuccinic acid was formed from beta-phenylpyruvic acid with [14C]-acetyl-CoA and ATP in this fraction, as was [14C]-arphamenine A from benzylsuccinic acid and L-[14C]-arginine. Thus, the pathway of arphamenine A biosynthesis was confirmed by the cell-free biosynthesis of this antibiotic.     

Phenylalanine hydroxylase in melanoma cells.

A pigmented subclone of Cloudman S91 melanoma cells, PS1-wild type, can grow in medium lacking tyrosine. This ability is conferred by phenylalanine hydroxylase activity, and not by tryptophan hydroxylase, tyrosine hydroxylase or tyrosinase activities, although the latter activity is also present in these cells. Conversion of phenylalanine to tyrosine was measured in living cells by chromatographic identification of the metabolites of [14C]phenylalanine and in cell extracts using a sensitive assay for phenylalanine hydroxylase. Phenylalanine hydroxylase activity in melanoma cell extracts was identified by its inhibition with p-chlorophenylalanine and not with 6-fluorotryptophan, 3-iodotyrosine, phenylthiourea, tyrosine or tryptophan; and by adsorption with antiserum prepared against purified rat liver phenylalanine hydroxylase, and migration of immunoprecipitable activity with authentic phenylalanine hydroxylase subunits in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.