Vanillic acid, a metabolite of 4-hydroxy-3-methoxyphenylglycol and 4-hydroxy-3-methoxymandelic acid in man

Abstract: 4-Hydroxy-3-methoxyphenylglycol (HMPG) labelled with ¹⁴C was used to study the metabolic fate of HMPG in six healthy volunteers. Besides conjugation and oxidation to 4-hydroxy-3-methoxymandelic acid (HMMA, VMA) a minor portion, 8.4 ± 1.1% (mean ± SEM) was excreted as ¹⁴C-labelled vantllic acid (VA). To study if VA was formed from HMPG or HMMA (VMA), deuterium-labelled HMPG ([²H₃]HMPG) and HMMA ([²H₆]HMMA) were simultaneously injected intravenously to seven healthy volunteers. The recovery of [²H₃]VA from [²H₃]HMPG was 8.3 ± 2.1% and the recovery of [²H₆]VA from [²H₆]HMMA was 9.0 ± 2.1%. The ²H-labelled VAs were probably formed by a decar boxylation reaction, in the case of HMPG after previous oxidation to HMMA.     

Effects of Osmotic Pressure on the Oxidative Metabolism of Leishmania major Promastigotes

Leishmania major promastigotes were washed and resuspended in an iso-osmotic buffer. The rate of oxidation of ¹⁴C-labeled substrates was then measured as a function of osmolality. An acute decrease in osmolality (achieved by adding H₂O to the cell suspension) caused an increase in the rates of ¹⁴CO₂ production from [6-¹⁴C]glucose and, to a lesser extent, from [1, (3)-¹⁴C]glycerol. An acute increase in osmolality (achieved by adding NaCl, KCl, or mannitol) strongly inhibited the rates of ¹⁴CO₂ production from [1-: ¹⁴C]alanine, [1-¹⁴C]glutamate, and [1, (3)-¹⁴C]glycerol. The rates of ¹⁴CO₂ formation from [1-¹⁴C]laurate, [1-¹⁴C]acetate, and [2-¹⁴C]glucose (all of which form [1-¹⁴C]acetyl CoA prior to oxidation) were also inhibited, but less strongly, by increasing osmolality. These data suggest that with increasing osmolality there is an inhibition of mitochondrial oxidative capacity, which could facilitate the increase in alanine pool size that occurs in response to hyper-osmotic stress. Similarly, an increase in oxidative capacity would help prevent a rebuild up of the alanine pool after its rapid loss to the medium in response to hypo-osmotic stress.     

Oligodendroglial Glycerophospholipid Synthesis: Incorporation of Radioactive Precursors into Ethanolamine Glycerophospholipids by Calf Oligodendroglia Prepared by a Percoll Procedure and Maintained in Suspension Culture

Abstract: Oligodendroglia prepared from minced calf cerebral white matter by trypsinization at pH 7.4, screening, and isosmotic Percoll (polyvinylpyr-rolidone-coated silica gel) density gradient centrifugation survived in culture on polylysine-coated glass, extending processes and maintaining phenotypic characteristics of oligodendroglia. In the present study, ethanolamine glycerophospholipid (EGP) metabolism of the freshly isolated cells was examined during short-term suspension culture by dual label time course and substrate concentration dependence experiments with [2-³H]glycerol and either [1,2-¹⁴C]ethanolamine or L-[U-¹⁴C]serine. Rates of incorporation of ³H from the glycerol and of ¹⁴C from the ethanolamine into EGP were constant for 14 h. In medium containing 3 mM-[1,2-¹⁴C]ethanolamine and 4.8 mM-[2-³H]glycerol, rates of incorporation of ¹⁴C and ³H into diacyl glycerophosphoethanolamine (diacyl GPE) were similar. Under the same conditions, ³H specific activities of alkylacyl GPE and alkenylacyl GPE were much lower than ¹⁴C specific activities, likely as a result of the loss of tritium during synthesis of these forms of EGP via dihydroxyacetone phosphate. L-[U-¹⁴C]serine was incorporated into serine glycerophospholipid (SGP) by base exchange rather than de novo synthesis. ¹⁴C from L-[U-¹⁴C]serine also appeared in EGP after an initial lag period of several hours. Methylation of oligodendroglial EGP to choline glycerophospholipid (CGP) was not detected.     

CHOLINE ACETYLTRANSFERASE–EVIDENCE FOR ACETYL TRANSFER BY A HISTIDINE RESIDUE

Abstract— Choline acetyltransferase from bovine brain has been extensively purified to a specific activity of 2.5 μmol ACh/min mg protein. Attempts to isolate an acetyl enzyme intermediate after incubation of the enzyme with [1-¹⁴C]acetyl-CoA were unsuccessful. Such an intermediate could only be isolated using a 30-fold less purified enzyme preparation. The protein, binding ¹⁴C in this preparation, did not correspond to choline acetyltransferase as shown by disc-electrophoresis. The highly purified enzyme could, however, be labelled when choline acetyltransferase was immobilized on a mercuribenzoate sepharose gel and incubated with [1-¹⁴C]acetyl-CoA. Subsequently, the immobilized labelled enzyme or the labelled enzyme which had been released by cysteine from the gel. formed ACh after incubation with choline. The labelling and the following formation of [¹⁴C]ACh was pH dependent.
Masking htstidine residues of the enzyme with diethylpyrocarbonate almost abolished the labelling of the immobilized enzyme and completely abolished the formation of [¹⁴C]ACh. Enzyme inhibited with 5.5'-dithiobis(2-nitrobenzoate) was partially reactivated when the thionitrobenzoatederivative was cleaved by KCN treatment to a thiocyanatederivalive. A reaction mechanism for ChAT is proposed based on the present data.     

THE RELEASE OF AMINO ACIDS FROM THE HEMISECTED SPINAL CORD DURING STIMULATION

Abstract— Isolated frog or toad hemicords were incubated for 40 min with either [¹⁴C]glycine, [³H]GABA, l -[¹⁴C]glutamate. l -[¹⁴C]aspartate, l -[¹⁴C]serine, l [¹⁴C]threonine or l -[³H]leucine, and the release of these compounds from the cord was measured under resting conditions and during electrical stimulation. Stimulation of spinal roots produced no significant change in the efflux of any of the compounds tested. Direct stimulation of the rostral cord however, produced a large increase in the efflux of [¹⁴C]glycine, [³H]GABA, l -[¹⁴C]glutamate and l -[¹⁴C]aspartate. These increased effluxes were calcium dependent, the effects of stimulation being reduced in a calcium-free, or magnesium-supplemented (10 mM) medium. Stimulation failed to produce an increase in the efflux of l -[¹⁴C]serine, l -[¹⁴C]threonine, l -[¹⁴H]leucine, [¹⁴C]mannitol or [¹⁴C]urea. These results are consistent with the suggestions that glycine, GABA, glutamate and aspartate may be synaptic transmitters in the spinal cord.     

ADENOSINE KINASE OF MAMMALIAN BRAIN: PARTIAL PURIFICATION AND ITS ROLE FOR THE UPTAKE OF ADENOSINE

Abstract— Adenosine metabolism in the homogenate of brain mainly undergoes deamination to inosine and hypoxanthine, while uniformly labelled [¹⁴C]adenosine injected into the carotid artery or [8-¹⁴C]adenosine incubated with brain slices was mostly phosphorylated to [¹⁴C]adenine nucleotides in brain cells. Adenosine kinase has now been partially purified from homogenates of guinea pig brain. The kinase preparation was free of adenosine deaminase, almost free of adenosine triphosphatase and had a K_m of the order of 2 × 10^-5M for adenosine.
Kinetic studies with brain slices showed that adenosine reached the cells by diffusion and that the diffusion was facilitated by subsequent phosphorylation to adenine nucleotides. From the following experimental results, it is concluded that the phosphorylation is catalysed by adenosine kinase quantitatively. (1) During the uptake and phosphorylation of adenosine by brain slices, the nucleoside did not split to adenine and ribose moieties. (2) The rate of formation of adenine nucleotides in the slices was a hyperbolic function of the concentration of adenosine in the medium, showing an apparent K_m foradenosine of the order of 2 × 10^-5 M. (3) Some analogues of adenosine inhibited both the facilitated diffusion of adenosine and the kinase activity, but ouabain (0.005 mM) did not inhibit either.     

Localization of newly synthesized indoIe-3-methylglucosinolate (= glucobrassicin) in vacuoles from horseradish (Armoracia rusticana)

The presence and concentration of indole-3-methylglucosinolate [= glucobrassicin; 0.49 μmol (g dry weight)-1] and its 1-methoxy derivative [0.38 umol (g dry weight)⁻¹] in Armoracia rusticana P. Gärtner, B. Meyer and Scherb, storage roots were measured. The storage tissue contains L-tryptophan [1.63 μmol (g dry weight)^-1], which is the common precursor amino acid of the indoleglucosinolates. Tissue cylinders convert [¹⁴C]- l -tryptophan efficiently to the indoleglucosinolates (25%) in vivo. The conversion of [¹⁴C]- l -tryptophan to indole-3-methylgluco-sinolate shows biphasic kinetics.
A fraction rich in vacuoles was prepared from tissue sections to which [¹⁴C]- l -tryp-tophan had been fed and allowed to be metabolized. The predominantly vacuolar location of both L-tryptophan and the indoleglucosinolates is demonstrated by correlation with the vacuolar marker acid phosphatase.
The significance for the regulation of the indoleglucosinolate biosynthesis and the role of indole-3-methylglucosinolate as a potential auxin precursor are discussed.     

CAPABILITY OF TUBULIN AND MICROTUBULES TO INCORPORATE AND TO RELEASE TYROSINE AND PHENYLALANINE AND THE EFFECT OF THE INCORPORATION OF THESE AMINO ACIDS ON TUBULIN ASSEMBLY

Abstract— Incorporation of [¹⁴C]tyrosine into the C-terminal position of α-tubulin of rat brain cytosol was 10-fold higher for non-assembled than for assembled tubulin. The incorporation into tubulin from disassembled microtubules was higher than into non-assembled tubulin; therefore, the low incorporation into microtubules was not due to a lower acceptor capacity of their tubulin constituent.
[¹⁴C]Tyrosine was released from assembled and non-assembled [¹⁴C]tyrosinated tubulin by the action of an endogenous carboxypeptidase. Release from non-assembled tubulin was shown by incubating a tubulinyl-[¹⁴C]tyrosine preparation in the presence of CaCl₂ at a concentration that abolished microtubule formation. Release from microtubules was inferred from the observation that the percentages of [¹⁴C]tyrosine released and the decrease of the specific radioactivity of the recovered microtubules were practically identical and did not change after a 10-fold dilution of the incubated microtubules.
[³H]Phenylalanine was released from a preparation of tubulinyl-[³H]phenylalanine also by an enzymatic activity.
The capacity of a tubulin preparation to incorporate tyrosine was increased 43% by pre-treatment with endogenous carboxypeptidase.
Tubulin tyrosinated in vitro was assembled to the same extent as native tubulin. After a mixture of tubulinyl-[¹⁴C]tyrosine and tubulinyl-[³H]phenylalanine was partially assembled, the ratio of ¹⁴C/³H found in the microtubules was the same as in the non-assembled tubulin fraction.     

Alanyl turnover from lipoteichoic acid to teichoic acid in Staphylococcus aureus

Abstract The metabolism of d -alanyl substituents of lipoteichoic acid (LTA) and teichoic acid was studied in Staphylococcus aureus . Double labelling with [³H]glycerol and d -[¹⁴C]alanine revealed that during the chase LTA was stable whereas its ¹⁴C label rapidly decreased. Half-time comparison indicated an enzyme- rather than a base-catalyzed process. Correlated with the loss of [¹⁴C]alanine from LTA was an increase of the radioactivity in wall-linked alanine ester which, after hydrolysis with HF, proved to be linked to teichoic acid. These results suggest that LTA-alanine is the donor for alanine esterification of teichoic acid. In connection with previous data we hypothesize that the loss of alanine from LTA is compensated by de novo incorporation.     

THE BIOSYNTHESIS OF CHOLESTEROL AND OTHER STEROLS BY BRAIN TISSUE

Abstract— The distribution of ¹⁴C into several subcellular fractions of adult rat brain was studied as a function of time, following intracerebral injection of [2-¹⁴C]mevalonic acid. As expected from previous studies, the microsomal fraction was indicated as the site of sterol biosynthesis. The myelin fraction showed a marked and early uptake of ^I4C-labelled, digitonin-precipitable material. This was assumed to be a non-enzymic uptake of sterol intermediates. The mitochondrial fraction exhibited a rapid uptake of ¹⁴C-labelled, nonsaponifiable material, but a very slow accumulation of ¹⁴C-labelled, digitonin-precipitable product. Examination of the nonsaponifiable ¹⁴C-fractions by TLC showed a rapid appearance of labelled 4-desmethyl sterols in the microsomal fraction. The myelin fraction selectively retained 4,4'-dimethyl sterol but seemed to release this with time, possibly to be further metabolized by the microsomes. Examination of [¹⁴C]digitonin-precipitable material by the dibromide method showed that although labelled 4-desmethyl sterol appeared quite early, cholesterol itself was formed slowly in all fractions.     

THE INCORPORATION OF DOUBLE-LABELLED ACETATE INTO GLUTAMATE AND RELATED AMINO ACIDS FROM ADULT MOUSE BRAIN: COMPARTMENTATION OF AMINO ACID METABOLISM IN BRAIN

Abstract– We have determined the incorporation of [³H]-, [1-¹⁴C]- and [2-¹⁴C]acetate into glutamate, glutamine and aspartate of the adult mouse brain. All these three acetates were incorporated more extensively into glutamine than into glutamate. This has been reported by several authors for each of these labelled acetates in separate experiments. It was shown that [³H, 2-¹⁴C]acetate can be used to obtain an acetate labelling ratio analogous to the previously used [2-¹⁴C]acetate/[1-¹⁴C]acetate labelling ratio. From these acetate labelling ratios of glutamine and glutamate conclusions can be deduced about the dynamic relationship of these amino acids with each other and with the tricarboxylic acid cycle.
A fairly large isotope effect between acetate and glutamate was observed. As this isotope effect is very likely caused by the citrate synthase reaction, it can be argued that citrate synthase involved in the conversion of labelled acetate into glutamate is far out of equilibrium in vivo. Comparing our data with literature data, the possibility can be suggested that citrate synthase in the acetate metabolizing compartment is in situ kinetically distinct from citrate synthase in other compartments of the brain.     

Phosphatidylcholine molecular species involved in Γ-linolenic acid biosynthesis in microsomes from borage seeds

Boraginaceae seeds are particularly rich in Γ -linolenic acid (6,9,12-octadecatrienoic acid, Γ -18:3). In microsomes, the analysis of phosphatidylcholine (PC) molecular species by HPLC led to identification of 15 different molecular species; among them 4 contained Γ -18:3, mostly at position 2 of sn -glycerol. Time courses of acylation and desaturation in PC molecular species were examined when [¹⁴C]oleoyl-CoA or [¹⁴C]linoleoyl-CoA was provided as substrates to isolated microsomes. With [¹⁴C]oleoyl-CoA or [¹⁴C]linoleoyl-CoA and in the absence of NADH, 3 main labelled PC molecular species were found: 18:2/[¹⁴C]18:1, 16:0/[¹⁴C]18:1 and 18:1/[¹⁴C]18:1. When NADH was present in the incubation medium, the fatty acids were progressively desaturated by the Δ12- and Δ6-desaturases successively (with [¹⁴C]oleoyl-CoA as precursor) or by the Δ6-desaturase alone (with [¹⁴C]linoleoyl-CoA as precursor). In both types of experiments, 7 final desaturation products in microsomes were evidenced; among them, 3 contained radioactive Γ -18:3, i.e . 18:2/[¹⁴C] Γ -18:3, 18:1/[¹⁴C] Γ -18:3 and 16:0/[¹⁴C] Γ -18:3. While the Δ12-desaturase had no specificity for position on the glycerol backbone, labelled Γ -linolenic acid was recovered exclusively in the sn -2 position.     

THE BIOSYNTHESIS OF CHOLESTEROL AND OTHER STEROLS BY BRAIN TISSUE

Abstract— The distribution of [¹⁴C]labelled material into subcellular fractions of 30-day-old rat brain was studied as a function of time, following intracerebral injection of [2-¹⁴C] mevalonic acid. As in the adult and 15-day-old brain, the microsomal fraction was indicated as the site of sterol synthesis. Unlike the 15-day-old animal, the myelin fraction from the 30-day-old rat was the predominately labelled fraction at 2 weeks after injection of the animal. Significant amounts of [¹⁴C]cholesterol were not present until about 4 h after injection. In order to ascertain whether different populations of cholesterol were being labelled, depending on the age of the animal injected, we compared the labelling of myelin and non-myelin components in animals injected at 15 or at 30 days of age, and sacrificed, respectively, from 14 to 29 days or from 1 to 28 days after injection. Our results indicated that there was an apparent shift of labelled sterol from non-myelin to myelin fractions at about 37–44 days of age.     

SYNTHESIS OF GLYCOPROTEINS AND GANGLIO-SIDES IN DEVELOPING RAT BRAIN

Abstract— Intracerebral injections of radioactive fucose into developing rats resulted in specific labelling of the brain glycoproteins in their fucose moieties. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate revealed that the radioactive glycoproteins were very heterogeneous with regard to molecular weight. A procedure utilizing [³H]fucose and [¹⁴C]fucose together with double-label counting techniques was developed for comparing the electrophoretic patterns of newly synthesized glycoproteins from different samples of tissue. By the use of this procedure we showed that the incorporation of radioactive fucose into the glycoproteins of high mol. wt. was relatively greater in the brains of 5-day-old rats than in those of 25-day-old rats. Intracerebral injection of N -[ Ac -³H]acetyl- d -mannosamine resulted in a high degree of specificity for the labelling of sialic acid moieties in glycoproteins and gangliosides. The ratio of the d.p.m. of N -[³H]acetylmannosamine incorporated into glycoproteins to the d.p.m. incorporated into gangliosides was higher in 5-day-old rats than in 15- or 25-day-old rats. Experiments in which 15-day-old rats were injected with a mixture of [¹⁴C]fucose and N -[³H]acetylmannosamine showed that there were differences in the relative degrees of incorporation of the two radioactive precursors into the various glycoproteins. The greatest incorporation of [¹⁴C]fucose relative to that of N- [³H]acetylmannosamine occurred in some of the glycoproteins of smaller mol. wt.     

IN VIVO FORMATION AND CATABOLISM OF [14C]HISTAMINE IN MOUSE BRAIN

Abstract— The formation of histamine in brain was studied in mice injected with l -[¹⁴C]-histidine (ring 2-¹⁴C) intravenously (i.v.) or intracerebrally; [¹⁴C]histamine appeared rapidly and exhibited a rapid rate of turnover. Drugs known to block various pathways of histamine catabolism were tested for effects on brain–[¹⁴C]histamine and [¹⁴C]-methyl-histamine in mice given (1) [¹⁴C]histamine i.v., (2) [¹⁴C]histamine intracerebrally, and (3) l -[¹⁴C]histidine i.v. Blood-borne histamine did not enter brain; brain histamine was formed locally by decarboxylation of histidine Methylhistamine did cross the blood-brain barrier. Methylation was the major route of histamine catabolism in mouse brain and some of the methylhistamine formed was destroyed by monoamine oxidase. No evidence for catabolism by the action of diamine oxidase was found.     

Interrelationships of Ubiquinone and Sterol Syntheses in Cultured Cells of Neural Origin

Abstract: Ubiquinone synthesis has been studied in cultured C-6 glial and neuroblastoma cells by utilizing an inhibitor, 3-β-(2-diethylaminoethoxy) androst-5-en-17-one hydrochloride (U18666A), of cholesterol biosynthesis. Exposure of C-6 glial cells to nanomolar quantities of U18666A caused a marked inhibition of total sterol synthesis from [¹⁴C]acetate or [³H]mevalonate within minutes. A 95% inhibition was apparent after a 3-h exposure to 200 ng/ml of U18666A. These observations, together with studies of the incorporation of radioactivity from the two precursors into cholesterol, desmosterol, lanosterol, and squalene, indicated that although the most sensitive site to inhibition by U18666A is desmosterol reduction to cholesterol, a major site of inhibition is demonstrable at a more proximal site, perhaps squalene synthetase. As a consequence of the latter inhibition, exposure of C-6 glial cells to U18666A caused a marked stimulation of incorporation of [¹⁴C]acetate or [³H]mevalonate into ubiquinone. Over a wide range of U18666A concentrations, the increase in ubiquinone synthesis was accompanied by an approximately similar decrease in total sterol synthesis. Whereas in the absence of U18666A only approximately 7% of the radioactivity incorporated from [³H]mevalonate into isoprenoid compounds was found in ubiquinone, in the presence of the drug approximately 90% of incorporated radioactivity was found in ubiquinone. The reciprocal effects of U18666A on ubiquinone and sterol syntheses were apparent also in the neuronal cells. The data thus demonstrate a tight relationship between ubiquinone and sterol biosyntheses in cultured cells of neural origin. In such cells ubiquinone synthesis is exquisitely sensitive to the availability of isoprenoid precursors derived from the cholesterol biosynthetic pathway.     

Glutamine and Glucose as Precursors of Transmitter Amino Acids: Ex Vivo Studies

Abstract: Radiolabelled glutamine and glucose were infused into lateral ventricles of rats in order to label transmitter amino acid pools in vivo . Brain regions close to the lateral ventricle (hippocampus, corpus striatum, hypothalamus) were labelled more effectively than more distant structures such as cerebral cortex or cerebellum. All regions were labelled to much the same extent over 30-150 min by [U-¹⁴C]glucose, [U-¹⁴C]glutamine, or [³H]glutamine administered alone or together in doublelabel experiments when allowance was made for any differences in precursor specific radioactivities. Slices of cerebral cortex or hippocampus from brains labelled in vivo were incubated and stimulated in vitro with veratrine (75 μ M ); tetrodotoxin (1 μ M ) was present in the control medium. Single-label experiments showed that [U-¹⁴C]- glutamine was more effective than [U-¹⁴C]glucose for labelling releasable glutamate and GABA. Double-label experiments showed that [³H]glutamine and [U-¹⁴C]- glucose given together in vivo labelled glutamate and GABA releasable in vitro to a similar extent. Both types of experiment empbasise the large contribution made by glutamine in vivo to pools of transmitter glutamate and GABA.     

THE BIOSYNTHESIS OF CHOLESTEROL AND OTHER STEROLS BY BRAIN TISSUE

Abstract— The distribution of [¹⁴C]-labeIled material into subcellular fractions of 15-day-old rat brain was studied as a function of time after intracerebral injection of [2-¹⁴C]mevalonic acid. As previously shown for adult brain, the data indicated the microsomal fraction to be the site of sterol biosynthesis. The synaptosomal fraction exhibited a marked early uptake of [¹⁴C]-nonsaponifiable material. Total radioactivity in both myelin and myelin-like fractions remained low in comparison to that in the other subcellular fractions at all time periods examined. At 2 h after injection, labelled digitonin-precipitable material was demonstrable in all subcellular fractions. Examination of the [¹⁴C]-labelled nonsaponifiable material by thin-layer chromatography indicated the rapid appearance of labelled 4-desmethyl sterol in all subcellular fractions, with the most rapid appearance in the myelin fraction, followed in decreasing order by microsomal, synaptosomal, and mitochondrial fractions. Examination of [¹⁴C] digitonin-precipitable material from each fraction by the dibromide method demonstrated that although 4-desmethyl sterol appeared quickly, the formation of cholesterol was slow in all fractions, an effect that had been reported earlier for adult brain.     

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.     

Radiolabelling of the monate moiety in the study of pseudomonic acid biosynthesis

Abstract Monic acid A was isolated from a Pseudomonas fluorescens fermentation in which pseudomonic acid A (PA) was the principal secondary metabolite. [3-¹⁴C]3-Hydroxy-3-methyl-glutaric acid (HMG) given early in the idiophase radiolabelled PA (1.1% incorporation), confirming experimentally the putative direct involvement of HMG in the biosynthesis of PA, but contributed relatively insignificant radiolabel to the monic acid extracted from the broth at the end of the fermentation. Ethionine inhibited (80%) PA biosynthesis and correspondingly reduced incorporation of [¹⁴C]HMG. In contrast, ethionine increased incorporation of [methyl-¹⁴C]methionine into PA and enhanced specific radioactivity of the antibiotic 8-fold. Ethionine inhibition of secondary metabolite methylations did not divert pseudomonate biosynthesis to give unusual analogues, implying that methylation of a putative pentaketide precursor of the monate moiety forms a vital intermediate of the pseudomonate pathway, but caused a new [¹⁴C]HMG-derived polar metabolite of biosynthetic interest to become evident.