Use of charcoal adsorption for the microassay of acetyl-CoA-hydrolase.

A sensitive and rapid radiochemical micromethod is described for measuring the activity of acetyl-CoA hydrolase (EC 3.1.2.1). [1-¹⁴C]Acetyl-CoA is incubated with tissue homogenates; unhydrolyzed [1-¹⁴C]acetyl-CoA is separated from the radiolabeled product, [1-¹⁴C]acetate, by adsorption to charcoal. The soluble [1-¹⁴C]acetate is measured by liquid scintillation techniques. This procedure makes it possible to measure as little as 0.2 to 0.4 nmol acetate generated per assay.     

Double-standard isotope dilution assay: I. Quantitative assay of indole-3-acetic acid

Isotope dilution analysis for the quantitation of labile compounds has been limited in applicability by the amount of sample necessary to determine specific activity. A method is described for the analysis of radiolabeled compounds which allows the direct determination of specific activity by gas chromatography. It requires the availability of the radiolabeled internal standard, as is customarily used in an isotope dilution assay, and also requires a chemically related radiolabeled compound to serve as a second internal standard. It is this second internal standard, added in known amounts, that permits quantitation of the gas chromatography. The method is illustrated by assaying indole-3-acetic acid in plant extracts using [¹⁴C]indole-3-acetic acid as the internal standard and adding [¹⁴C]indole-3-butyric acid as the second internal standard for quantitation of the gas chromatographic procedures. Used with a nitrogen-specific thermionic detector the method is selective and is sensitive at the nanogram level. The synthesis of [2-ring-¹⁴C]indole-3-butyric acid is also described.     

A general procedure for the preparation of labeled l-ascorbic acid from labeled d-glucose

A simple, three-step conversion of 1,2-O-isopropylidene-α-d-glucofuranose into l-ascorbic acid, originally described by Bakke and Theander, was used to prepare l-[4-¹⁴C]ascorbic acid from milligram amounts of d-[3-¹⁴C]glucopyranose in 28% radioisotopic yield. In addition, l-[6-¹⁴C]- and l-[U-¹⁴C]-ascorbic acid were prepared from d-[1-¹⁴C]- and d-[U-¹⁴C]-glucopyranose, respectively. The procedure is useful for the synthesis of l-ascorbic acid bearing isotopic hydrogen, carbon, or oxygen atoms at specific positions, subject only to the availability of starting material.     

A radiochemical method for determination of ethanol oxidation

A radiochemical method for the measurement of ethanol exidation by tissue preparations is described. Ethanol oxidation is determined from the production of [¹⁴C]acetaldehyde, quantified as the semicarbazone derivative, from [1-¹⁴C]ethanol. The assay is quantitative, reproducible and highly correlated with the NADH-enzymic-spectrophotometric procedure.     

Biosynthesis of p-hydroxybenzoic acid in poplar lignin

Biosynthetic pathways to p-hydroxybenzoic acid in polar lignin were examined by tracer experiments. High incorporation of radioactivity to the acid was observed when shikimic acid-[1-¹⁴C], phenylalanine-[3-¹⁴C], trans-cinnamic acid-[3-¹⁴C], p-coumaric acid-[3-¹⁴C] and p-hydroxybenzoic acid-[COOH-¹⁴C] were administered, while incorporation was low from shikimic acid-[COOH-¹⁴C], phenylalanine-[1-¹⁴C], phenylalanine-[2-¹⁴C], tyrosine-[3-¹⁴C], benzoic acid-[COOH-¹⁴C], sodium acetate-[1-¹⁴C] and d-glucose-[U-¹⁴C]. Thus p-hydroxybenzoic acid in poplar lignin is formed mainly via the pathway: shikimic acid → phenylalanine → trans-cinnamic acid → p-coumaric acid → p-hydroxybenzoic acid.     

In Vivo Pathway of Oleate and Linoleate Desaturation in Developing Cotyledons of Cucumis sativus L. Seedlings

Exogenous [1-¹⁴C]oleic acid and [1-¹⁴C]linoleic acid were taken up and esterified to complex lipids by greening cucumber (Cucumis sativus L.) cotyledons. Both ¹⁴C-labeled fatty acids were initially esterified to phosphatidylcholine prior to eventual accumulation in triacylglycerols and galactolipids. Kinetic data suggest that esterification occurs prior to desaturation and that phosphatidylcholine is the initial site of both [¹⁴C]-oleate and [1-¹⁴C]linoleate esterification and of [1-¹⁴C]oleate desaturation to [1-¹⁴C]linoleate. [1-¹⁴C]Linoleic acid was esterified more rapidly than [¹⁴C]oleic acid and its desaturation product, [1-¹⁴C]α-linolenate, occurred mainly on monogalactosyl diacylglycerol, although some was also observed on the other major acyl lipids, including phosphatidylcholine.     

Preparation of radioactive acetyl-l-carnitine by an enzymatic exchange reaction

A rapid method for the preparation of [1-¹⁴C]acetyl-l-carnitine is described. The method involves exchange of [1-¹⁴C]acetic acid into a pool of unlabeled acetyl-l-carnitine using the enzymes acetyl-CoA synthetase and carnitine acetyltransferase. After isotopic equilibrium is attained, radioactive acetylcarnitine is separated from the other reaction components by chromatography on Dowex 1 (Cl^?) anion exchange resin. One of the procedures used to verify the product [1-¹⁴C]acetyl-l-carnitine can be used to synthesize (3S)-[5-¹⁴C]citric acid.     

Synthesis and preliminary biological evaluation of [11C]methyl (2-amino-5-(benzylthio)thiazolo[4,5-d]pyrimidin-7-yl)-d-leucinate for the fractalkine receptor (CX3CR1)

The reference standard methyl (2-amino-5-(benzylthio)thiazolo[4,5-d]pyrimidin-7-yl)-d-leucinate (5) and its precursor 2-amino-5-(benzylthio)thiazolo[4,5-d]pyrimidin-7-yl)-d-leucine (6) were synthesized from 6-amino-2-mercaptopyrimidin-4-ol and BnBr with overall chemical yield 7% in five steps and 4% in six steps, respectively. The target tracer [¹¹C]methyl (2-amino-5-(benzylthio)thiazolo[4,5-d]pyrimidin-7-yl)-d-leucinate ([¹¹C]5) was prepared from the acid precursor with [¹¹C]CH₃OTf through O-[¹¹C]methylation and isolated by HPLC combined with SPE in 40–50% radiochemical yield, based on [¹¹C]CO₂ and decay corrected to end of bombardment (EOB). The radiochemical purity was >99%, and the specific activity (SA) at EOB was 370–1110 GBq/μmol with a total synthesis time of ～40-min from EOB. The radioligand depletion experiment of [¹¹C]5 did not display specific binding to CX₃CR1, and the competitive binding assay of ligand 5 found much lower CX₃CR1 binding affinity.     

o-phthalaldehyde for the fluorometric assay of nonprotein amino compounds.

Procedures for the preparation of UDP-N-[1-¹⁴C]acetyl-d-glucosamine and UDP-N-[1-¹⁴C]acetyl-d-galactosamine with very high specific activities are deseribed. The overall yield based on the amount of [1-¹⁴C]acetate used is greater than 80%. The N-acetyl-d-glucosamine-α-1-phosphate used in this synthesis is prepared by phosphorylation of tetraacetyl-d-N-acetylglucosamine with crystalline phosphoric acid. N-acetyl-d-glucosamine-α-1-phosphate is then deacetylated in anhydrous hydrazine with hydrazine sulfate as a catalyst. d-glucosamine-α-1-phosphate is N-acetylated with [¹⁴C]acetate using N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline as the coupling agent. The acetylated product is coverted to the UDP derivative with yeast UDP-N-acetyl-d-glucosamine pyrophosphorylase. UDP-N-[1-¹⁴C]acetylgalactosamine is prepared by acetylation of UDP-galactosamine using [1-¹⁴C]acetate and N-ethoxy-carbonyl-2-ethoxy-1,2-dihydroquinoline. UDP-galactosamine is prepared enzymatically using galactokinase and galactose-1-phosphate uridyltransferase. The labeled products, isolated and characterized by ion-exchange and paper chromatography, were active as substrates in glycosyl transferase systems.     

Formation of (-⊃-1′,2′-epi-2-cis-xanthoxin acid from a precursor of abscisic acid

Tomato shoots and avocado mesocarp supplied with (±)-[2-¹⁴C]-5-(1,2-epoxy-2,6,6-trimethylcyclohexyl)-3-methylpenta-cis-2-trans-4-dienoic acid metabolize it into (+)-abscisic acid and a more polar material that was isolated and identified as (?)-epi-1′(R),2′(R)-4′(S)-2-cis-xanthoxin acid. The (+)-1′(S),2′(S)-4′(S)-2-cis-xanthoxin acid recently synthesized from natural violaxanthin, has the 1′,2′-epoxy group on the opposite side of the ring to that of the 4′(S)-hydroxyl group and the compound is rapidly converted into (+)-abscisic acid. The 1′,2′-epoxy group of (?)-1′,2′-epi-2-cis-xanthoxin acid is on the same side of the ring as the 4′(S) hydroxyl group: the compound is not metabolized into abscisic acid. The configuration of the 1′,2′-epoxy group probably controls whether or not the 4′(S) hydroxyl group can be oxidized. (+)-2-cis-Xanthoxin acid is probably not a naturally occurring intermediate because a ‘cold trap’, added to avocado fruit forming [¹⁴C]-labelled abscisic acid from [2-¹⁴C]mevalonate, failed to retain [¹⁴C] label.     

A simple and sensitive assay for dopamine-beta-hydroxylase

A simple, rapid, and highly sensitive radiochemical assay for measuring the activity of dopamine-β-hydroxylase in tissues and serum is described. Enzyme activity is detected by converting [1-¹⁴C]tyramine to [1-¹⁴C]octopamine which is then subjected to periodate cleavage to form [¹⁴C]form-amide. This radiolabeled product is oxidized to ¹⁴CO₂ by addition of permanganate and the ¹⁴CO₂ is trapped and counted. The assay is simple and sensitive, it can linearly detect enzyme in all tissues with a wide range of activity, it uses maximal concentration of substrate, and it requires the addition of only one concentration of EMI to block endogenous inhibitor(s) in different tissues or enzyme concentrations.     

Biosynthesis of (+)-Tartaric Acid from l-[4-C]Ascorbic Acid in Grape and Geranium

The metabolic fate of l-[4-¹⁴C]ascorbic acid has been examined in the grape (Vitis labrusca L.) and lemon geranium (Pelargonium crispum L. L'Hér. cv. Prince Rupert) under conditions comparable to data from l-[1-¹⁴C]ascorbic acid and l-[6-¹⁴C]ascorbic acid experiments. In detached grape leaves and immature berries, l-[4-¹⁴C]ascorbic acid and l-[1-¹⁴C]ascorbic acid were equivalent precursors to carboxyl labeled (+)-tartaric acid. In geranium apices, l-[4-¹⁴C]ascorbic acid yielded internal labeled (+)-tartaric acid while l-[6-¹⁴C]ascorbic acid gave an equivalent conversion to carboxyl labeled (+)-tartaric acid. These findings clearly show that two distinct processes for the synthesis of (+)-tartaric acid from l-ascorbic acid exist in plants identified as (+)-tartaric acid accumulators. In grape leaves and immature berries, (+)-tartaric acid synthesis proceeds via preservation of a four-carbon fragment derived from carbons 1 through 4 of l-ascorbic acid while carbons 3 through 6 yield (+)-tartaric acid in geranium apices.     

Biogenesis of betalamic acid

When d,l-dopa-[1-¹⁴C] and -[2-¹⁴C] was fed to yellow flower buds of Portulaca grandiflora betalamic-[¹⁴C] acid was obtained. The labeled betalamic acid was converted to ¹⁴C-labeled betanin in order to obtain a stable substance which could be recrystallized to a radio-pure sample. Decarboxylation of the radiopure betanin obtained from the sequence using dopa-[1-¹⁴C] indicated that the ¹⁴C-carboxyl group of dopa corresponded to a ¹⁴C-carboxyl group in betanin and hence in betalamic acid. The shape of the ORD curve for the naturally occurring betalamic acid was the same as that recorded for a sample of [S]-betalamic acid derived by degradation of betanin. These data support the hypothesis that betalamic acid is formed in vivo by an oxidative cleavage of l-dopa and that it is an intermediate in the biogenesis of other betalains from dopa.     

The function of the pentose phosphate pathway in Phaseolus mungo hypocotyls

The metabolism of d-gluconate-[1-¹⁴C] and -[6-¹⁴C] by segments from etiolated hypocotyls of Phaseolus mungo has been studied. The release of ¹⁴CO₂ from gluconate-[1-¹⁴C] was greater than that from gluconate-[6-¹⁴C] in all parts of hypocotyls examined. Incorporation of the radioactivity from gluconate-[6-¹⁴C] into RNA, lignin and aromatic amino acid fractions was greater in the upper (younger) part of the hypocotyls. Incorporation into sugars was greater in the lower (more mature) parts.     

Conversion of labeled substrates to sugars, cell wall polysaccharides, and tartaric Acid in grape berries

[U-¹⁴C]Sucrose, myo-[U-¹⁴C]inositol, [6-¹⁴C]- and [U-¹⁴C]glucuronate, UDP-[U-¹⁴C]glucuronate, [U-¹⁴C]gluconate, and l-[1-¹⁴C]ascorbic acid were fed into grape berries, Vitis labrusca L. cv. Delaware, at intervals throughout the ripening process and incorporation of ¹⁴C into several metabolites was studied.     

Application of enzymatically synthesized short-chain-length hydroxy fatty acid coenzyme A thioesters for assay of polyhydroxyalkanoic acid synthases

Various hydroxyacyl coenzyme A (CoA) thioesters were synthesized from the corresponding hydroxyalkanoic acid (such as e.g. [3-¹⁴C]d-(–)-hydroxybutyric acid, [1-¹⁴C]d-lactic acid, [1-¹⁴C]l-lactic acid, etc.) and from acetyl-CoA employing the propionate CoA transferase of Clostridium propionicum. Preparative isolation of the thioesters on hydrophobic matrices and analysis by HPLC are reported. These thioesters were subjected to a radiometric or a spectrometric assay of polyhydroxyalkanoic acid (PHA) synthase activity. The latter was based on the release of CoA from, for example, d-(–)-3-hydroxybutyryl-CoA, which was detected spectroscopically at 412 nm by reduction of 5,5-dithiobis(2-nitrobenzoic acid) and provided a convenient assay of poly(3-hydroxybutyrate) synthase. When [1-¹⁴C]lactyl-CoA was used as substrate in a PHA synthase assay employing crude extracts obtained from various wild-type strains, [1-¹⁴C]lactyl-CoA was used as a substrate at a rate that was only less than 10^–4 of the rate than with [3-¹⁴C]d-(–)-3-hydroxybutyryl-CoA or was negligible. One exception was a recombinant strain of Escherichia coli, which overexpressed the PHA synthase complex of Chromatium vinosum and which used [1-¹⁴C]d-lactyl-CoA as substrate at a relatively high rate. Correspondence to: A. Steinbüchel     

5-Hydroxylevulinic acid,a new intermediate in the biosynthesis of protoanemonin

Administration of 5-hydroxy[1-¹⁴C]-and [4-¹⁴C]levulinic acid to Helleborus foetidus led to the isolation of [1-¹⁴C]- and [4-¹⁴C]protoanemonin, respectively. There was also incorporation of radioactivity into the four glucosides ranunculin, isoranunculin, ranuncoside and ranunculoside. Acid hydrolysis of radioactive ranuncoside gave labelled 5-hydroxylevulinic acid (HKV). A study of the incorporation of various ¹⁴C-labelled tracers into protoanemonin suggested that HKV is formed in higher plants by a new reduction of 2-ketoglutarate (2-KG) without free 4,5-dioxovalerate (DOVA) as an intermediate. A scheme for the biosynthesis of the antibiotic protoanemonin and its glucosidic precursors is proposed. It is shown that 5-(β-d-glucopyranosyloxy)levulinic acid could be the genuine precursor of all the compounds studied.     

Hydrolysis of novel diacylglycerol and phorbol diesters by serum lipase

Rat serum, active in the hydrolysis of the tumor-promoting phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA), was examined with regard to lipid interferences of [³H]TPA hydrolysis and enzyme substrate specificity. The enzymatic hydrolysis of TPA could be enhanced 8-fold, ever crude serum, by using a lipid-free acetone powder of rat serum. Addition of lipid to the lipid-free acetone powder produced potent inhibition of TPA hydrolysis. The inclusion of multilamallar liposomes resulted in similar inhibition, and isolation of liposomes by high-speed centrifugation showed that 95% of the radiolabeled TPA was associated with the fatty pellet. Substrate specificity studies demonstrated that the serum activity hydrolyzes the long-chain ester of TPA and the long-chain primary acyl group of diacylglycerols. TPA was hydrolyzed at approximately twice the rate of dioleoylglycerol; however, the most reactive substrates were those synthetic analogs of diacylglycerol containing a short-chain ester group at the sn-2 position. Palmitic acid was liberated from [1-¹⁴C]palmitoyl-2-acetyl-sn-glycerol and [1-¹⁴C]palmitoyl-2-butyryl-sn-glycerol at 120- and 33-tinies the rate of TPA hydrolysis, respectively. Lipase resistant 1-hexadecyl-2-[³H]acetylglycerol was also used as substrate, but the sn-2 ester moiety showed poor lability. The diacylglycerol analogs are new lipase substrates and, in view of their similarities to the fatty acyl portion of TPA, it is thought that these compounds could serve as protein kinase C activators.     

The pathway from arachidonic to docosapentaenoic acid (20:4n-6 to 22:5n-6) and from eicosapentaenoic to docosahexaenoic acid (20:5n-3 to 22:6n-3) studied in testicular cells from immature rats

The concentration-dependent metabolism of 1-¹⁴C-labelled precursors of 22:5n-6 and 22:6n-3 was compared in rat testis cells. The amounts of [¹⁴C]22- and 24-carbon metabolites were measured by HPLC. The conversion of [1-¹⁴C]20:5n-3 to [3-¹⁴C]22:6n-3 was more efficient than that of [1-¹⁴C]20:4n-6 to [3-¹⁴C]22:5n-6. At low substrate concentration (4 μM) it was 3.4 times more efficient, reduced to 2.3 times at high substrate concentration (40 μM). The conversion of [1-¹⁴C]22:5n-3 to [1-¹⁴C]22:6n-3 was 1.7 times more efficient than that of [1-¹⁴C]22:4n-6 to [1-¹⁴C]22:5n-6 using a low, but almost equally efficient using a high substrate concentration. When unlabelled 20:5n-3 was added to a cell suspension incubated with [1-¹⁴C]20:4n-6 or unlabelled 22:5n-3 to a cell suspension incubated with [1-¹⁴C]22:4n-6, the unlabelled n-3 fatty acids strongly inhibited the conversion of [1-¹⁴C]20:4n-6 or [1-¹⁴C]22:4n-6 to [¹⁴C]22:5n-6. In the reciprocal experiment, unlabelled 20:4n-6 and 22:4n-6 only weakly inhibited the conversion of [1-¹⁴C]20:5n-3 and [1-¹⁴C]22:5n-3 to [¹⁴C]22:6n-3. The results indicate that if both n-6 and n-3 fatty acids are present, the n-3 fatty acids are preferred over the n-6 fatty acids in the elongation from 20- to 22- and from 22- to 24-carbon atom fatty acids. In vivo the demand for 22-carbon fatty acids for spermatogenesis in the rat may exceed the supply of n-3 precursors and thus facilitate the formation of 22:5n-6 from the more abundant n-6 precursors.     

Oxidation of Glucose,Ribose, Alanine,and Glutamate by Leishmania braziliensis panamensis1

The metabolism of [1-¹⁴C]- and [6-¹⁴C]glucose, [1-¹⁴]ribose, [1-¹⁴C]- and [U-¹⁴C]alanine, and [1-¹⁴C]- and [5-¹⁴C]glutamate by the promastigotes of Leishmania braziliensis panamensis was investigated in cells resuspended in Hanks' balanced salt solution supplemented with ribose, alanine, or glutamate. The ratio of ¹⁴CO₂ produced from [1-¹⁴C]glucose to that from [6-¹⁴C]glucose ranged from about two to six, indicating appreciable carbon flow through the pentose phosphate pathway. A functional pentose phosphate pathway was further demonstrated by the production of ¹⁴CO₂ from [1-¹⁴C]ribose although the rate of ribose oxidation was much lower than the rate of glucose oxidation. The rate of ¹⁴CO₂ production from [1-¹⁴C]glucose was almost linear with time of incubation, whereas that of [6-¹⁴C]glucose accelerated, consistent with an increasing rate of flux through the Embden-Meyerhof pathway during incubation. Increasing the assay temperature from 26°C to 34°C had no appreciable effect on the rates or time courses of oxidation of either [1-¹⁴C]- or [6-¹⁴C]glucose or of [1-¹⁴C]ribose. Both alanine and glutamate were oxidized by L. b. panamensis, and at rates comparable to or appreciably greater than the rate of oxidation of glucose. The ratios of ¹⁴CO₂ produced from [1-¹⁴C]- to [U-¹⁴C]alanine and from [1-¹⁴C]- to [5^-14C]glutamate indicated that these compounds were metabolized via a functioning tricarboxylic acid cycle and that most of the label that entered the tricarboxylic acid cycle was oxidized to carbon dioxide. Heating the cultures for 6 or 12 h at 34°C, which converts the promastigotes into an ellipsoidally shaped intermediate form, decreased the rates of oxidation of glucose, alanine, and glutamate. The oxidation of glutamate decreased by about 50% and 70% after a 6-h or 12-h heat treatment, respectively. Returning the heated cultures to 26°C initiated a reversion to the promastigote form and recovery of the rate of glucose oxidation, but glutamate oxidation did not return to control levels by 19 h at 26°C.