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.     

The control of ribonucleic acid synthesis in bacteria. The synthesis and stability of ribonucleic acid in rifampicin-inhibited cultures of Escherichia coli

A study was made of the kinetics of labelling of the stable ribonucleic acids (rRNA+tRNA) and the unstable mRNA fraction in cultures of Escherichia coli M.R.E.600, inhibited by the addition of 0.1g of rifampicin/l. Labelling was carried out by adding either [2-¹⁴C]- or [5-³H]-uracil as an exogenous precursor of the cellular nucleic acids. From studies using DNA RNA hybridization, the kinetics of the synthesis and degradation of mRNA was followed in the inhibited cultures. Although a considerable proportion of the mRNA labelled in the presence of rifampicin decayed to non-hybridizable products, about 25% was stabilized beyond the point where protein synthesis had finally ceased. It therefore seems unwise to extrapolate the results of studies on mRNA stability in rifampicin-inhibited cultures to the situation existing in the rate of steady growth, where there appears to be little, if any, stable messenger. The kinetics of labelling of RNA in inhibited cultures indicated that the clapsed time from the addition of rifampicin to the point at which radioactivity no longer enters the total cellular ribonucleic acids is a measure of the time required to polymerize a molecule of rRNA. At 37°C, in culture grown in broth, glucose–salts or lactate salts media, exogenous [2-¹⁴C]uracil entered rifampicin-inhibited cells and was incorporated into RNA for 2 3min after the antibiotic was added. Taking this time as that required to polymerize a complete chain of 23S rRNA, the polymerization rate of this fraction in the three media was 25, 22 and 19 nucleotides added/s to the growing chains. Similar experiments in cultures previously inhibited by 0.2g of chloramphenicol/l showed virtually identical behaviour. This confirmed the work of Midgley & Gray (1971), who, by a different approach, showed that the polymerization rate of rRNA in steadily growing and chloramphenicol-inhibited cultures of E. coli at 37°C was essentially constant at about 22 nucleotides added/s. It was thus confirmed that the rate of polymerization of at least the rRNA fraction in E. coli is virtually unaffected by the nature of the growth medium and therefore by bacterial growth rate.     

A rapid quantitative method for measuring UTP, UDP, and UMP in the picomole range.

A procedure for the determination of picomole amounts of uracil nucleotides is described. The key reaction is the condensation of UTP and [¹⁴C]glucose 1-phosphate catalyzed by uridine 5′-diphosphoglucose pyrophosphorylase yielding UDP-[¹⁴C]glucose. The product is determined by selective adsorption onto charcoal in the presence of 0.8 m Trizma Base. UDP is measured as UTP after its conversion in an incubation with excess ATP and nucleoside diphosphate kinase. Similarly, UMP is analyzed after it is converted to UDP by nucleoside monophosphate kinase. The uracil nucleotide content of germinated wheat embryos had been determined with this method.     

Studies on the regulation of leucine catabolism: Mechanism responsible for oxidizable substrate inhibition and dichloroacetate stimulation of leucine oxidation by the heart

In the absence of any other oxidizable substrate, the perfused rat heart oxidizes [1-¹⁴C]leucine to ¹⁴CO₂ at a rapid rate and releases only small amounts of α-[1-¹⁴C]ketoisocaproate into the perfusion medium. The branched-chain α-keto acid dehydrogenase complex, assayed in extracts of mitochondria prepared from such perfused hearts, is very active. Under such perfusion conditions, dichloroacetate has almost no effect on [1-¹⁴C]leucine oxidation, α-[1-¹⁴C]ketoisocaproate release, or branched-chain α-keto acid dehydrogenase activity. Perfusion of the heart with some other oxidizable substrate, e.g., glucose, pyruvate, ketone bodies, or palmitate, results in an inhibition of [1-¹⁴C]leucine oxidation to ¹⁴CO₂ and the release of large amounts of α-[1-¹⁴C]ketoisocaproate into the perfusion medium. The branched-chain α-keto acid dehydrogenase complex, assayed in extracts of mitochondria prepared from such hearts, is almost completely inactivated. The enzyme can be reactivated, however, by incubating the mitochondria at 30 °C without an oxidizable substrate. With hearts perfused with glucose or ketone bodies, dichloroacetate greatly increases [1-¹⁴C]leucine oxidation, decreases α-[1-¹⁴C]ketoisocaproate release into the perfusion medium, and activates the branched-chain α-keto acid dehydrogenase complex. Pyruvate may block dichloroacetate uptake because dichloroacetate neither stimulates [1-¹⁴C]leucine oxidation nor activates the branched-chain α-keto acid dehydrogenase complex of pyruvate-perfused hearts. It is suggested that leucine oxidation by heart is regulated by the activity of the branched-chain α-keto acid dehydrogenase complex which is subject to interconversion between active and inactive forms. Oxidizable substrates establish conditions which inactivate the enzyme. Dichloroacetate, known to activate the pyruvate dehydrogenase complex by inhibition of pyruvate dehydrogenase kinase, causes activation of the branched-chain α-keto acid dehydrogenase complex, suggesting the existence of a kinase for this complex.     

A radioactive assay for acetylcholinesterase using anion-exchange disk

A radioactive assay for acetylcholinesterase is described. The assay is based on the separation of [¹⁴C]acetate from [¹⁴C]acetylcholine by differential adsorption of the former on DEAE anion-exchange disks. The procedure is simple and sensitive and eliminates the use of ion-exchange resin columns or organic extractions. Moreover, when unpurified enzyme preparations are assayed, linear steady-state kinetics can be observed with this method as contrasted to the nonlinear colorimetric method using acetylthiocholine and dithiobisnitrobenzoate. This method also permits the detection in biological samples of low levels of acetylcholinesterase activity, which is not detectable by the colorimetric method. Using the present radioactive method, cellular levels of acetylcholinesterase have been surveyed in N4TG1 neuroblastoma cells, NG108-15 neuroblastoma x glioma hybrid cells, H9c2 myoblasts, and 3T3-L1 and 3T3-C2 fibroblasts.     

Evidence that multiple species of aminoacylated transfer RNA are present in regenerating optic axons of goldfish

This study reports that 4S RNA present in regenerating optic axons of goldfish is likely to be transfer RNA. Evidence is also presented which indicates that this transfer RNA is similar to transfer RNA found in tectal cells and that its aminocylation is likely to occur both in retinal ganglion cells prior to axonal transport as well as in the axon itself. Fish with regenerating optic nerves received intraocular injections of [³H]uridine followed 4 days later by intracranial injections of [¹⁴C]uridine. Radioactive tectal 4S RNA was isolated 6 days after [³H]uridine injections and chromatographed by BD cellulose chromatography. Optical density as well as radioactivity profiles for both [¹⁴C]4S RNA (from tectal cells) and [³H]4S RNA (90% of which originated from regenerating optic axons) were found to be similar toE. coli transfer RNA optical density profiles, indicating that the intra-axonal 4S RNA is likely to be transfer RNA. Moreover, comparisons of³H/¹⁴C suggest that intra-axonal and cellular 4S RNAs are composed of similar species of transfer RNA. Results of other experiments indicated that aminoacylation of axonally transported tRNA occurs both in the retina and in optic axons subsequent to axonal transport.     

Effect of pyridoxine deficiency on nucleic acid metabolism in the rat

1. The nucleic acid metabolism in the pyridoxine-deficient rat has been investigated through studies on the incorporation of radioactivity from various isotopically labelled compounds into liver and spleen DNA and RNA. 2. In pyridoxine deficiency, the incorporation of radioactivity from sodium [¹⁴C]formate was apparently increased. The magnitude of this effect on incorporation into liver RNA and DNA and spleen RNA was approximately the same. The incorporation into spleen DNA was enhanced to a much greater degree. Administration of pyridoxine 24hr. before the rats were killed reversed the changes in incorporation of radioactivity from [¹⁴C]formate. 3. In pyridoxine deficiency, the incorporation of radioactivity from dl-[3-¹⁴C]serine, [8-¹⁴C]adenine, [Me-³H]thymidine and [2-¹⁴C]deoxyuridine was decreased. The incorporation of radioactivity from l-[Me-¹⁴C]methionine was not affected. No noteworthy differences in the effect of pyridoxine deficiency on the incorporation of radioactivity from dl-[3-¹⁴C]serine into DNA and RNA were observed, whereas the effect of the deficiency on the incorporation of radioactivity from [8-¹⁴C]adenine into spleen DNA was somewhat greater than that into spleen RNA. Administration of pyridoxine 24hr. before the rats were killed reversed the changes in incorporation of radioactivity from [3-¹⁴C]serine and [8-¹⁴C]adenine. 4. The adverse effects of pyridoxine deficiency on the biosynthesis of nucleic acids and cell multiplication are discussed in relation to the role of pyridoxal phosphate in the production of C₁ units via the serine-hydroxymethylase reaction.     

Stability of rapidly labelled messenger ribonucleic acid in Bacillus amyloliquefaciens during the phases of minimum and maximum extracellular enzyme formation.

The stability of rapidly labelled hybridizable messenger RNA in both exponential and post-exponential phase cells of Bacillus amyloliquefaciens was measured in terms of the rate of loss of its radioactivity. In the exponential phase, where 96% of the mRNA was specific for cell proteins and only 4% was exoprotein mRNA, the label was lost exponentially from the rapidly labelled hybridizable mRNA fraction with a half-life of six minutes at 30 °C. The antibiotic rifampicin, at a concentration of 10 μg/ml, had no effect on the characteristics of decay of this exponential-phase mRNA. In the post-exponential phase, where there were equal amounts of cell protein and exoprotein-specific mRNA, rapidly labelled hybridizable mRNA decayed exponentially in the presence of rifampicin (10 μg/ml), with a half-life of six minutes at 30 °C. In the absence of rifampicin the characteristics of decay were more complex. The evidence available suggested that this was due to the superimposition of a component attributable to reincorporation of degradation products of radioactive RNA on the characteristic exponential decay pattern of the post-exponential mRNA.Measurement of the stability of active mRNA, by studying the loss of ability to incorporate l-[¹⁴C]leucine into protein in the presence of rifampicin (10 μg/ml), gave half-lives of 4.5 minutes and six minutes, respectively, for exponential and post-exponential material.     

A possible mechanism of inhibition of protein synthesis by dimethylnitrosamine

1. The incorporation of [¹⁴C]leucine into liver proteins of rats was measured in vivo at various times after treatment of the animals with dimethylnitrosamine and was correlated with the state of the liver ribosomal aggregates. Inhibition of incorporation ran parallel with breakdown of the aggregates. 2. Inhibition of leucine incorporation into protein and breakdown of ribosomal aggregates were not preceded by inhibition of incorporation of [¹⁴C]orotate into nuclear RNA of the liver. 3. Evidence was obtained of methylation of nuclear RNA in the livers of rats treated with [¹⁴C]dimethylnitrosamine. 4. Zonal centrifugation analysis of radioactive, nuclear, ribosomal and transfer RNA from livers of rats treated with [¹⁴C]dimethylnitrosamine revealed labelling of all centrifugal fractions to about the same extent. 5. It is suggested that methylation of messenger RNA might occur in the livers of dimethylnitrosamine-treated rats and the possible relation of this to inhibition of hepatic protein synthesis is discussed.     

Effects of Hydroxyurea and Deoxyadenosine on the Synthesis of Deoxycytidine Phosphate and on the Uptake of Nucleosides in Excised Root Tips of Vicia faba

The effects of hydroxyurea and deoxyadenosine on the synthesis of deoxycytidine phosphate was studied by measuring the incorporation of [¹⁴C]-cytidine into acid soluble deoxycytidine phosphate in root tips of Vicia faba. Hydroxyurea and deoxyadenosine both markedly depressed the incorporation of [¹⁴C]-cytidine. Deoxyadenosine had the additional effect of inhibiting the uptake of [¹⁴C]-cytidine. Furthermore, millimolar concentrations of deoxyadenosine inhibited the uptake of micromolar concentrations of adenosine, thymidine, and deoxycytidine. The incorporation of [¹⁴C]-cytidine into RNA was only slightly affected by hydroxyurea. Deoxyadenosine inhibited the incorporation into RNA to about the same extent as the uptake of [¹⁴C]-cytidine. It is suggested that hydroxyurea reduced the incorporation of radioactive cytidine into deoxycytidine phosphate mainly by interfering with ribonucleotide reduction. The depression of [¹⁴C]-cytidine incorporation into deoxycytidine phosphate in the presence of deoxyadenosine is believed to be the result of an inhibition of both ribonucleotide reduction and [¹⁴C]-cytidine uptake.     

Fluorescent nucleotide triphosphate substrates for snake venom phosphodiesterase

The procedure described utilizes a crude cell-free extract from the yeast Saccharomyces cerevisiae as enzymatic source for the synthesis of coproporphyrin III from [¹⁴C]δ-aminolevulinic acid with a high yield of conversion (?60%). Both specific radioactivity and total radioactivity of coproporphyrin III can be adjusted fairly well. This procedure is not time consuming for yeast acellular extracts or porphyrin ester preparations. The acellular extracts can be stored frozen (?30°C) for at least 1 year without loss of enzymatic activity. The same procedure can be used for [¹⁴C]protoporphyrin preparation.     

The labelling of nucleic acids by radioactive precursors in the blue-green algae Anacystis nidulans and Synechocystis aquatilis Sanv.

Summary The labelling of nucleic acids of growing cells of the blue-green algae Anacystis nidulans and Synechocystis aquatilis by radioactive precursors has been studies. A. nidulans cells most actively incorporate radioactivity from [2-¹⁴C]uracil into both RNA and DNA, while S. aquatilis cells incorporate most effectively [2-¹⁴C]uracil and [2-¹⁴C]thymine.Deoxyadenosine does not affect incorporation of label from [2-¹⁴C]thymidine into DNA, but weakly inhibits [2-¹⁴C]thymine incorporation into both nucleic acids and significantly suppresses the incorporation of [2-¹⁴C]uracil.The radioactivity from [2-¹⁴C]uracil and [2-¹⁴C]thymine is found in RNA uracil and cytosine and DNA thymine and cytosine. The radioactivity of [2-¹⁴C]thymidine is incorporated into DNA thymine and cytosine. These results and data of comparative studies of nucleic acid labelling by [2-¹⁴C]thymine and [5-methyl-¹⁴C]thymine suggest that the incorporation of thymine and thymidine into nucleic acids of A. nidulans and S. aquatilis is accompanied by demethylation of these precursors. In this respect blue-green algae resemble fungi and certain green algae.     

Ribonucleic Acid Synthesis in Plant Mitochondria: Effects of Some Inhibitors and Cyclic Nucleotides

Mitochondria isolated from 48-h germinating Vigna sinensis (L.) Savi can incorporate [³H]uridine into acid-insoluble material. The incorporation is highly sensitive to rifampicin and partially so to ethidium bromide, two specific inhibitors of template function. The inhibitory effect of rifampicin can be partly counteracted by cyclic 3′:5′-AMP but not by cyclic 3′:5′-GMP, if they are allowed to interact with the synthetic system before the treatment with rifampicin. This indicates that cyclic AMP and rifampicin compete for a common site on the RNA polymerase responsible for DNA-dependent RNA synthesis. Inhibition by ethidium bromide is unaffected by prior nucleotide interaction with the system.     

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.     

Measurement of intracellular collagen degradation

Intracellular degradation of newly synthesized collagen is quantitated by incubating fibroblasts with [¹⁴C]proline and determining the percentage of total [¹⁴C]hydroxyproline that is present in a low molecular weight fraction. Several problems make this difficult. (1) Commercial [¹⁴C]proline is often contaminated with [¹⁴C]hydroxyproline and must be purified before use. (2) Salt and [¹⁴C]proline interfere with the determination of [¹⁴C]hydroxyproline in the low molecular weight fraction and must be removed by preparative ion-exchange chromatography. (3) Epimerization of trans- to cis-hydroxyproline during acid hydrolysis is variable and must be taken into account. (4) Loss of [¹⁴C]hydroxyproline during processing varies; [³H]hydroxyproline can be used as an internal measure of recovery, even though tritium may be lost during hydrolysis. An analytic cation-exchange resin is used for the final quantitation of [¹⁴C]hydroxyproline in the low and high molecular weight fractions. With these methods, degradation of newly synthesized collagen can be determined with a precision of ± 3%.     

Influence of cyclic 3′,5′-adenosine monophosphate on uracil uptake by rifampicin treatedEscherichia coli cells

Summary Incubation of cells from a wild type strain ofE. coli with 0.3 mg/ml rifampicin for 15 minutes lead to a complete inhibition of RNA synthesis measured as the uracil incorporation into the trichloroacetic acid insoluble fraction. In these rifampicin-treated cells [¹⁴C]uracil incorporation tended to decrease during a further incubation at 37°. Addition of cyclic AMP increased the inactivation of the system responsible for [¹⁴C]uracil uptake. The cyclic nucleotide effect seems to be specific since ATP or 5AMP did not increase such inactivation.Dedicated to ProfessorLuis F. Leloir on the occasion of his 70th birthday.     

A yeast endoribonuclease stimulated by Novikoff hepatoma small nuclear RNAs U1 and U2

Using [³H]m⁷Gppp[¹⁴C]RNA-poly(A) from yeast as a substrate, an endoribonuclease has been detected in enzyme fractions derived from a high salt wash of ribonucleoprotein particles of $\text{Saccharomyces}\text{cerevisiae}$. The [³H]m⁷Gppp[¹⁴C]RNA-poly(A) seems to be a preferred substrate since other polyribonucleotides are hydrolyzed more slowly, if at all. The enzyme is inhibited by ethidium bromide, but fully double-stranded polyribonucleotides are not hydrolyzed. The hydrolysis of [³H]m⁷Gppp[¹⁴C]RNA-poly(A) is stimulated about 2.5-fold by the addition of small nuclear RNAs U1 and U2 of Novikoff hepatoma cells. Results show that the stimulation involves an interaction of the labeled RNA with the small nuclear RNA.     

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.     

The mechanism of dextransucrase action: Biosynthesis of branch linkages by acceptor reactions with dextran

Dextransucrase from Leuconostoc mesenteroides B-512 catalyzes the polymerization of dextran from sucrose. The resulting dextran has 95% α-1 → 6 linkages and 5% α-1 → 3 branch linkages. A purified dextransucrase was insolubilized on Bio-Gel P-2 beads (BGD, Bio-Gel-dextransucrase). The BGD was labeled by incubating it with a very low concentration of [¹⁴C]sucrose or it was first charged with nonlabeled sucrose and then labeled with a very low concentration of [¹⁴C]sucrose. After extensive washings with buffer, the ¹⁴C label remained attached to BGD. This labeled material was previously shown to be [¹⁴C]dextran and was postulated to be attached covalently at the reducing end to the active site of the enzyme. When the labeled BGD was incubated with a low molecular weight nonlabeled dextran (acceptor dextran) all of the BGD-bound label was released as [¹⁴C]dextran whereas essentially no [¹⁴C]dextran was released when the labeled BGD was incubated in buffer alone under comparable conditions. The released [¹⁴C]dextran was shown to be a slightly branched dextran by hydrolysis with an exodextranase. Acetolysis of the released dextran gave 7.3% of the radioactivity in nigerose. Reduction with sodium borohydride, followed by acid hydrolysis, gave all of the radioactivity in glucose, indicating that the nigerose was exclusively labeled in the nonreducing glucose unit. These results indicated that [¹⁴C]dextran was being released from BGD by virtue of the action of the low molecular weight dextran and that this action gave the formation of a new α-1 → 3 branch linkage. A mehanism for branching is proposed in which a C₃-OH on an acceptor dextran acts as a nucleophile on C₁ of the reducing end of a dextranosyl-dextransucrase complex, thereby displacing dextran from dextransucrase and forming an α-1 → 3 branch linkage. It is argued that the biosynthesis of branched linkages does not require a separate branching enzyme but can take place by reactions of an acceptor dextran with a dextranosyl-dextransucrase complex.     

Synthesis of Deoxyglucose-1-Phosphate, Deoxyglucose-1,6-Bisphosphate, and Other Metabolites of 2-Deoxy-D-[14C]Glucose in Rat Brain In Vivo: Influence of Time and Tissue Glucose Level

Abstract: Abstract: When the kinetics of interconversion of deoxy[¹⁴C]glucose ([¹⁴C]DG) and [¹⁴C]DG-6-phosphate ([¹⁴C]DG-6-P) in brain in vivo are estimated by direct chemical measurement of precursor and products in acid extracts of brain, the predicted rate of product formation exceeds the experimentally measured rate. This discrepancy is due, in part, to the fact that acid extraction regenerates [¹⁴C]DG from unidentified labeled metabolites in vitro. In the present study, we have attempted to identify the ¹⁴C-labeled compounds in ethanol extracts of brains of rats given [¹⁴C]DG. Six ¹⁴C-labeled metabolites, in addition to [¹⁴C]DG-6-P, were detected and separated. The major acid-labile derivatives, DG-1-phosphate (DG-1-P) and DG-1,6-bisphosphate (DG-1,6-P₂), comprised ?5 and ?10–15%, respectively, of the total ¹⁴C in the brain 45 min after a pulse or square-wave infusion of [¹⁴C]DG, and their levels were influenced by tissue glucose concentration. Both of these acid-labile compounds could be synthesized from DG-6-P by phosphoglucomutase in vitro. DG-6-P, DG-1-P, DG-1,6-P₂, and ethanol-insoluble compounds were rapidly labeled after a pulse of [¹⁴C]DG, whereas there was a 10–30-min lag before there was significant labeling of minor labeled derivatives. During the time when there was net loss of [¹⁴C]DG-6-P from the brain (i.e., between 60 and 180 min after the pulse), there was also further metabolism of [¹⁴C]DG-6-P into other ethanol-soluble and ethanol-insoluble ¹⁴C-labeled compounds. These results demonstrate that DG is more extensively metabolized in rat brain than commonly recognized and that hydrolysis of [¹⁴C]DG-1-P can explain the overestimation of the [¹⁴C]DG content and underestimation of the metabolite pools of acid extracts of brain. Further metabolism of DG does not interfere with the autoradiographic DG method.