A study of the sulphur amino acids of rat tissues

1. In a study of the metabolism of l-[(35)S]methionine in vivo, the labelled sulphur compounds of rat liver and brain were separated first by ion-exchange chromatography into two fractions containing (i) free sulphur amino acids such as methionine, cystathionine, cyst(e)ine and homocyst(e)ine and (ii) glutathione. 2. Two-dimensional paper chromatography with butan-1-ol-acetic acid or propionic acid-water in the first direction and 80% acetone or acetone-ethyl methyl ketone-water in the second direction was found superior to other solvent systems for separating the sulphur amino acids. 3. At 10min. after injection of [(35)S]methionine only a small part of the (35)S was found combined in free methionine or other free sulphur amino acids. 4. Evidence was obtained of the presence of adenosyl[(35)S]methionine and adenosyl[(35)S]homocysteine in perchloric acid extracts of rat liver and brain. 5. The trans-sulphuration pathway was active in brain as well as in liver.     

Transsulphuration and methylation of homocysteine in control and mutant human fibroblasts

The ability of human skin-fibroblasts in monolayer culture to carry out transsulphuration and remethylation of homocysteine has been tested. The conversion of homocyst(e)ine to cyst(e)ine and methionine was studied in control and mutant cells by incubation for 16 h with l-[³⁵S]homocystine. Labelled cysteic acid and methionine sulphone were found in hydrolysates of oxidized cell proteins. The quantities found were dependent on the time of incubation and were used as a measure of cyst(e)ine and methionine formation, respectively. In control cells, labelled cyst(e)ine and labelled methionine were found. In cystathionine β-synthase-deficient cell lines, labelled cyst(e)ine formation was reduced, while labelled methionine formed was similar to that of controls, indicating the role of transsulphuration in the formation of cyst(e)ine observed in control cells. In a 5,10-methylenetetrahydrofolate reductase-deficient cell line, labelled methionine formation was reduced, indicating the role of N-5-methyltetrahydrofolate-requiring methylation of homocysteine in the formation of methionine observed in control cells.     

Methionine metabolism and ethylene formation in etiolated pea stem sections

Stem sections of etiolated pea seedlings (Pisum sativum L. cv. Alaska) were incubated overnight on tracer amounts of l-[U-(14)C]methionine and, on the following morning, on 0.1 millimolar indoleacetic acid to induce ethylene formation. Following the overnight incubation, over 70% of the radioactivity in the soluble fraction was shown to be associated with S-methylmethionine (SMM). The specific radioactivity of the ethylene evolved closely paralleled that of carbon atoms 3 and 4 of methionine extracted from the tissue and was always higher than that determined for carbon atoms 3 and 4 of extracted SMM.Overnight incubation of pea stem sections on 1 millimolar methionine enhanced indoleacetic acid-induced ethylene formation by 5 to 10%. Under the same conditions, 1 millimolar homocysteine thiolactone increased ethylene synthesis by 20 to 25%, while SMM within a concentration range of 0.1 to 10 millimolar did not influence ethylene production. When unlabeled methionine or homocysteine thiolactone was applied to stem sections which had been incubated overnight in l-[U-(14)C]methionine, the specific radioactivity of the ethylene evolved was considerably lowered. Application of unlabeled SMM reduced the specific radioactivity of ethylene only slightly.     

Studies on flavonoid metabolism. Metabolism of (+)-[14C]catechin in the rat and guinea pig

1. The fate of (+)-[U-(14)C]catechin and (+)-[ring A-(14)C]catechin has been studied in the guinea pig and rat. 2. (+)-[U-(14)C]Catechin was shown to give rise to labelled phenolic acids, labelled phenyl-gamma-valerolactones and (14)CO(2). 3. (+)-[ring A-(14)C]-Catechin did not give rise to labelled phenolic acids, but labelled phenyl-gamma-valerolactones were detected together with a higher proportion of (14)CO(2). 4. Administered [(14)C]delta-(3-hydroxyphenyl)-gamma-valerolactone gave rise to labelled m-hydroxyphenylpropionic acid in the rat whereas administered [(14)C]m-hydroxyphenylpropionic acid gave rise to a compound yielding labelled m-hydroxybenzoic acid on hydrolysis. 5. The distribution of radioactivity in the urine and faeces of (+)-[(14)C]catechin-fed animals is described; a high proportion of residual radioactivity was found in urine that had been exhaustively extracted with diethyl ether.     

Methylation of newly synthesized ribonucleic acid by isolated rat liver nuclei. Characterization of the ribonucleic acid synthesized by nuclei from starved animals

1. Nuclei from rat liver incubated with S-adenosyl[methyl-(14)C]methionine incorporated radioactivity into RNA and into lipid and protein. 2. All of the labelled RNA was extracted from the nuclei with trichloroacetic acid at 90 degrees C. 3. The [(14)C]methyl-group incorporation into the hot-trichloroacetic acid extract was 30% inhibited by the addition of actinomycin D (100mug/mg of DNA) or by the omission of CTP, GTP and UTP. 4. Assuming that the main substrate for this triphosphate-dependent methylation was newly synthesized precursor rRNA containing one methyl group/30 uridylate residues, it was calculated that approx. 60% of the [(14)C]UMP incorporated under similar conditions represented precursor rRNA synthesis. 5. In agreement with this, low concentrations of actinomycin D (approx. 1mug/mg of DNA) sufficient to abolish the triphosphate-dependent incorporation of [(14)C]methyl group inhibited 68% of the [(14)C]UMP incorporation. 6. The incorporation of [(14)C]UMP by nuclei from starved animals decreased progressively with increasing periods of starvation, whereas the triphosphate-dependent [(14)C]methyl-group incorporation was not further decreased after 1 day of starvation. 7. This suggests that precursor rRNA synthesis decreased within 1 day whereas other species of RNA were affected only after longer periods of starvation.     

Different metabolic recycling of the lipid components of exogenous sulphatide in human fibroblasts.

Cultured human fibroblasts were fed with two differently labelled sulphatide molecules [one labelled on C-3 of the sphingosine (Sph) moiety [( Sph-3H]sulphatide), the second on C-1 of stearic acid [( stearoyl-14C]sulphatide)], and the intracellular metabolic fate of radioactivity was monitored. Incorporated radioactivity was almost all recovered in the total lipid extract, regardless of the labelling position of the added sulphatide; however, large differences in the level of incorporation occurred among labelled glycosphingolipids. For example, sphingomyelin was present as the major radiolabelled lipid after [Sph-3H]-sulphatide incubation, but was detectable only in trace amounts after [stearoyl-14C]sulphatide administration; in the latter case the radioactivity was located predominantly in glycerophospholipids. From this finding it can be inferred that the free long-chain base (sphingosine) that originates from lysosomal catabolism of sulphatide is mainly, and quite specifically, utilized for sphingomyelin biosynthesis, whereas the ceramide moiety is not; conversely the fatty acid released from ceramide is non-specifically re-utilized for phospholipid biosynthesis.     

Metabolism of 2,4',5-trichlorobiphenyl: role of the intestinal microflora in the formation of bronchial-seeking methyl sulphone metabolites in mice

Groups of germ-free and conventional mice were treated with 2,4',5-trichlorobiphenyl (triCB) and [35S]cysteine or [35S]methionine, respectively. Control animals received the labelled amino acids only. Conventional mice accumulated significantly more extractable radioactivity both in lung and kidney tissues when compared to germ-free mice. The extracted radioactivity in lung and kidney tissues was shown to be due to the accumulation of methyl-[35S]sulphonyl-triCB. The low radioactivity in lungs of the germ-free mice was also shown to be due to the accumulation of small amounts of the sulphones. The results indicate an involvement of the intestinal flora in the formation of methyl sulphone metabolites of triCB.     

Biochemical studies on the sulphated glycosaminoglycan fraction of skin fibroblasts cultured from a patient with the Hurler syndrome

1. The metabolism of the sulphated glycosaminoglycan fraction in cultured skin fibroblasts derived from a patient with the Hurler syndrome and from a normal subject was studied. Two labelled precursors, Na(2) (35)SO(4) and d-[2-(3)H]glucose, were used and their intracellular fates during uptake and ;chase' periods were assessed after separation of sulphated glycosaminoglycans from hyaluronic acid. After 4 or 8h of exposure to culture medium containing both labels, [(35)S]sulphate incorporation into the sulphated glycosaminoglycan fraction was twofold greater in Hurler-syndrome cells than in normal cells. At the same time, the rate of incorporation of [(3)H]glucose into the sulphated glycosaminoglycan fraction was approximately the same for both cell types. Consequently, an increased (35)S/(3)H ratio (nmol of [(35)S]sulphate incorporated/nmol of [(3)H]glucose incorporated) was observed for Hurler-syndrome cells compared with normal cells. 2. The results of ;chase' experiments revealed that although the expected loss and relative retention of labelled sulphate occurred in the sulphated glycosaminoglycan fraction of normal and Hurler-syndrome cells, both cell types retained all of their radioactivity derived from [(3)H]glucose. 3. After 34h exposure to a ;corrective-factor' preparation from urine, the sulphated glycosaminoglycan content (as hexosamine and [(35)S]sulphate) of the Hurler-syndrome cells approached normal values. At the same time, there was an increase in specific radioactivity of ;corrected' Hurler-syndrome cells.     

Phospholipid methylation by intact rat Leydig cells

Phospholipid methylation by intact Leydig cells was investigated by determining the incorporation of radioactivity from [3H-methyl] methionine into phospholipids. Leydig cells incorporated significantly more radioactivity into phospholipids than did unpurified testicular cells, non-Leydig testicular cells, or red blood cells. Approximately 40% of the radioactivity was found in phosphatidylcholine, indicating that the methyltransferase pathway for the synthesis of this phospholipid is highly active in rat Leydig cells. Addition of luteinizing hormone to cells preloaded with [3H-methyl] methionine did not alter the rate of phospholipid methylation. However, phospholipid methylation by Leydig cells desensitized by the injection of human chorionic gonadotropin 1 to 7 days previously was reduced by approximately 60%. Inhibition of phospholipid methylation to 75% of normal with homocysteine thiolactone did not affect luteinizing hormone-stimulated androgen production. Further inhibition of phospholipid (and protein) methylation by treatment with homocysteine thiolactone and 3-deazaadenosine significantly reduced luteinizing hormone-stimulated androgen production. The results of this study demonstrate that the methyltransferase pathway for the synthesis of phosphatidylcholine is highly active in intact Leydig cells but is reduced in desensitized Leydig cells. There does not appear to be a close association between the activity of this pathway and the ability of luteinizing hormone to acutely stimulate androgen production.     

Studies on bone matrix glycoproteins. Incorporation of [1-14C]glucosamine and plasma [14C]glycoprotein into rabbit cortical bone

The radioactively labelled constituents present in bone matrix were compared 12 days after injection of either [(14)C]glucosamine or plasma [(14)C]glycoprotein. Both precursors are utilized in the synthesis of organic matrix by bone tissue. Cortical bone from animals injected with [(14)C]glucosamine contains radioactivity derived from glucosamine and plasma glycoproteins and all glycoprotein fractions are labelled. Plasma [(14)C]glycoprotein labels the less acidic glycoproteins to a greater extent than the more acidic components. An antibody has been raised against the less-acidic-glycoprotein fraction of bone. The latter contains a glycoprotein of alpha-mobility that appears to be concentrated specifically in bone tissue and which is present also in plasma. This alpha-glycoprotein accounts for a large proportion of the components labelled and retained in bone matrix after [(14)C]glucosamine injection.     

The formation of glycoproteins in tissues of higher plants. Specific labelling with d-[l-14C]glucosamine

1. Radioactivity from d-[l-(14)C]glucosamine is incorporated into ethanol-insoluble compounds of high molecular weight in a number of plant tissues, including roots of corn (Zea mays), callus cells of sycamore (Acer pseudoplatanus), axenic cultures of duckweed (Lemna minor) and germinating seedlings of corn, broad bean (Vicia faba) and barley (Hordeum vulgare). 2. Except in the case of Lemna, where some of the radioactivity was recovered in glucose, hydrolysis of these ethanol-insoluble materials with acid released [(14)C]glucosamine as the major radioactive product. 3. The labelled compounds isolated from Zea roots and the Acer cells are believed to be glycoproteins rather than polysaccharides on the basis of their solubility properties, their charge characteristics and their susceptibility to hydrolysis by 0.5m-potassium hydroxide and by the proteases trypsin and Pronase. Further, radioactive peptides were isolated and purified after Pronase treatment and shown to contain glucosamine as well as a number of amino acids. 4. The experiments therefore indicate that d-[(14)C]glucosamine can be used as a specific precursor of the amino sugar units of plant as well as animal glycoproteins.     

Tissue distribution of a menthyl-conjugated oligodeoxyribonucleotide antisense to PAI-1 mRNA

The inhibitory effect of numerous analogues of PO-16, an hexadecadeoxyribonucleotide antisense to sequences -22 to -17 of PAI-1 mRNA coding for a fragment of the signal peptide, on the expression of PAI-1 in endothelial cells, and physiological consequences of the subsequently reduced PAI-1 activity tested in vitro and in vivo, were described in our previous studies. Of particular interest was PO-16 5'-O-conjugated with menthyl phosphorothioate (MPO-16R). In this work, tissue localisation of MPO-16R labelled with [(35)S] phosphorothioate at the 3'-end, was determined. [(35)S]MPO-16R and control [(35)S]MPO-16R-SENSE oligonucleotides were administered intravenously into 22 rats and organ distribution of the labelled bioconjugates was assessed after 24 and 48 h. For this purpose, tissue sections were subjected to autoradiography, and quantitated by liquid scintillation after solubilisation. Overall clearance of radioactivity was already seen after 24 h, with the radioactivity recovered mainly in the kidney and liver. A smaller fraction of radioactivity was also retained in the spleen and heart. The kidney concentration of the labelled probe was higher than that of liver by 50%. The distribution of PAI-1 mRNA in untreated rat kidney, liver, spleen and heart established by two independent techniques: Ribonuclease Protection Assay and Real-Time PCR, shows the same pattern as that observed for [(35)S]MPO-16R antisense.     

Evidence that macrophages transfer arachidonic acid and cholesterol to tissues in vivo

Our previous studies have shown that [(14)C]-labelled cholesterol (CHOL) and arachidonic acid (AA) are transferred from macrophages (Mphi) to lymphocytes (LY) when these cells are co-cultured. In this study, we investigated whether these lipids can be transferred from control and thioglycollate-elicited Mphi (THIO-elicited Mphi) to various tissues and organs in vivo. For this purpose, control and THIO-elicited Mphi were pre-treated with [(14)C]-AA and [(3)H]-CHOL and then injected into the jugular vein of adult rats. More than 75% of the radioactivity injected was found in the liver of rats treated with [(14)C]-AA labelled-Mphi either control and THIO-stimulated. The radioactivity of [(3)H]-CHOL labelled Mphi was transferred mainly to the liver (51% in the control Mphi and 23% in the thioglycollate Mphi7) but it was also found in the kidney, lung and spleen. These results support the proposition that the transfer of lipids between cells also occurs in vivo. The full significance of this phenomenon however remains to be elucidated.     

Pharmacokinetic studies of methionine in rats using deuterium-labeled methionine quantitative assessment of remethylation

The pharmacokinetics of methionine has been studied in rats by means of stable isotope methodology. After the i.v. bolus injection of [2H7]methionine (5 mg/kg body wt.), the plasma concentrations of [2H7]methionine, demethylated [2H4]homocysteine and remethylated [2H4]methionine were determined simultaneously with endogenous methionine and homocysteine by gas chromatography-mass spectrometry. The half-life for [2H7]methionine were 35.0 +/- 6.9 min. The appearance of the metabolites, [2H4]homocysteine and [2H4]methionine, in the plasma was very rapid. The fraction of [2H7]methionine that remethylated to [2H4]methionine through [2H4]homocysteine were 0.185 +/- 0.028. The administered [2H7]methionine did not influence the plasma levels of endogenous methionine and homocysteine. The present stable isotope methodology has made it possible to evaluate the pharmacokinetics of methionine, including the estimation of remethylation.     

THE EFFECT OF METHIONINE SULPHOXIMINE ON THE INCORPORATION OF LABELLED GLUCOSE, ACETATE, PHENYLALANINE AND PROLINE INTO GLUTAMATE AND RELATED AMINO ACIDS IN THE BRAINS OF MICE

—The incorporation of radioactivity from labelled glucose, acetate, phenylalanine and proline into glutamate, aspartate and glutamine was measured in mice treated with methionine sulphoximine and in the control animals. The labelled precursors were injected and their incorporation determined before the onset of convulsions. The incorporation of radioactivity from labelled glucose into the dicarboxylic amino acids was reduced, in particular the incorporation into glutamine. The incorporation of radioactivity from labelled acetate and phenylalanine into glutamate and aspartate was increased by methionine sulphoximine, while the incorporation into glutamine was not changed very much. The labelling of glutamine, relative to glutamate, was reduced with all precursors, indicating that glutamine synthetase was inhibited in vivo by methionine sulphoximine. It is very likely that methionine sulphoximine affects many aspects of energy metabolism in brain; in particular the metabolism of glucose seems to be inhibited, while the rate of conversion of substrates other than glucose seems to be increased.     

Suitability of L-[35S]methionine for studying the biosynthesis of the polypeptides of mouse liver endoplasmic reticulum membrane fractions in vivo.

The use of L-[35S]methionine (500-700 Ci/mmol (1 Ci = 37 GBq) for labelling the polypeptides of liver rough (R) and smooth (S)endoplasmic reticulum (ER) membrane fractions in vivo was studied. Adult mice were injected intraperitoneally with 400 muCi of the isotope and killed at various times (2'min to 24 h) thereafter. RER and SER fractions were prepared, stripped of ribosomes, and treated with Triton X-100 to remove intravesicular contents. Sufficient radioactivity was present in individual aliquots (75 microgram protein) of the ER membrane fractions to permit their analysis by fluorography after separation by electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. By 3 min, although the majority of the labelled components were of intravesicular origin, some 12 membrane polypeptides were labelled in the RER fraction (including one corresponding in migration to cytochrome P-450); some 6 of these latter polypeptides were labelled to a lesser degree in the SER membrane fraction at this time. By 5 min, the patterns of radioactive polypeptides of the RER and SER fractions (including both membrane and intravesicular components) were identical. By 7 min, some 28 labelled membrane polypeptides were detectable in the total microsomal membrane. Analysis of the 24-h samples revealed that all the membrane polypeptides seen by staining with Coomassie blue were visualised by fluorography. Other studies revealed the applicability of the approach used for producing highly labelled cell sap and serum proteins. The overall results demonstrate the suitability of L-[35S]methionine administered in vivo for producing mouse liver ER membrane polypeptides of relatively high radioactivity and are consistent with a rapid conversion of RER to SER by ribosome detachment or membrane flow.     

Inhibition of ethylene biosynthesis by aminoethoxyvinylglycine and by polyamines shunts label from 3,4-[C]methionine into spermidine in aged orange peel discs

The flux of radioactivity from 3,4-[(14)C]methionine into S-adenosyl-l-methionine (SAM), 1-aminocyclopropane-1-carboxylic acid (ACC), spermine, and spermidine while inhibiting conversion of ACC to ethylene by 100 millimolar phosphate and 2 millimolar Co(2+) was studied in aged peel discs of orange (Citrus sinensis L. Osbeck) fruit. Inhibition up to 80% of ethylene production by phosphate and cobalt was accompanied by a 3.3 times increase of label in ACC while the radioactivity in SAM was only slightly reduced. Aminoethoxyvinylglycine (AVG) increased the label in SAM by 61% and reduced it in ACC by 47%. Different combinations of standard solution, in which putrescine or spermidine were administered alone or with AVG, demonstrated clearly that inhibition of ethylene biosynthesis-at the conversion of SAM to ACC-by AVG, exogenous putrescine or exogenous spermidine, stimulated the incorporation of 3,4-[(14)C]methionine into spermidine.     

Methionine Recycling in Brain: A Role for Folates and Vitamin B-12

Abstract: The recycling of methionine via homocysteine was measured in vivo in brain. After constant intravenous infusions (5 h) of both [³H-methyl] methionine and [³⁵S]methionine into rats, the ratios of [³H-methyl]methionine to [³⁵S]methionine in liver, brain and plasma were determined, Similar experiments were performed in rabbits, except that the [³H-methyl]- and [³S]methionine were injected intraventricularly. If the methyl group of methionine was removed with the formation of homocysteine and then replaced by another (unlabeled) methyl group, the specific activity of the [³H-methyl]methionine would decrease more than that of [³⁵S]methionine; i.e., the ratio of [³H-methyl]- to [³⁵S]methionine in the tissue would decline. The results showed that the ratios of [³H-methyl]- to [³⁵S]methionine in liver and brain were less than the same ratio in plasma in the rats. The comparable ratios in the brain and CSF of rabbits were less than the ratio in the injectate. Since brain contains only one enzyme capable of remethylating homocysteine to methionine, the vitamin B-12–dependent methyltetrahydrofolate-homocysteine methyltransferase (EC 2.1.1.13), our results for methionine recycling via homocysteine in brain strongly support the activity of this enzyme in brain in vivo.     

Effects of analogues of ethanolamine and choline on phospholipid metabolism in rat hepatocytes

1. Analogues of ethanolamine and choline were incubated with different labelled precursors of phospholipids and isolated hepatocytes and the effects on phospholipid synthesis were studied. 2. 2-Aminopropan-1-ol and 2-aminobutan-1-ol were the most efficient inhibitors of [(14)C]ethanolamine incorporation into phospholipids, whereas the incorporation of [(3)H]choline was inhibited most extensively by NN-diethylethanolamine and NN-dimethylethanolamine. 3. When the analogues were incubated with [(3)H]glycerol and hepatocytes, the appearance of (3)H in unnatural phospholipids indicated that they were incorporated, at least in part, via CDP-derivatives. The distribution of [(3)H]glycerol among molecular species of phospholipids containing 2-aminopropan-1-ol and 1-aminopropan-2-ol was the same as in phosphatidylethanolamine. In other phospholipid analogues the distribution of (3)H was more similar to that in phosphatidylcholine. 4. NN-Diethylethanolamine stimulated both the conversion of phosphatidylethanolamine into phosphatidylcholine and the incorporation of [Me-(14)C]methionine into phospholipids. Other N-alkyl- or NN-dialkyl-ethanolamines also stimulated [(14)C]methionine incorporation, but inhibited the conversion of phosphatidylethanolamine into phosphatidylcholine. This indicates that phosphatidyl-NN-diethylethanolamine is a poor methyl acceptor, in contrast with other N-alkylated phosphatidylethanolamines. 5. These results on the regulation of phospholipid metabolism in intact cells are discussed with respect to the possible control points. They also provide guidelines for future experiments on the manipulation of phospholipid polar-headgroup composition in primary cultures of hepatocytes.     

The role of vitamin B12 in the metabolism of Euglena gracilis var. bacillaris

1. The concentrations of RNA, DNA and protein are decreased in cells of Euglena gracilis var. bacillaris grown on suboptimum concentrations of vitamin B(12). 2. The addition of vitamin B(12) to deficient cells stimulates the incorporation of [(14)C]formate into the above cell components as well as into thymine of DNA and serine and methionine of protein. 3. In a cell-free system from vitamin B(12)-deficient cells, the incorporation of labelled formate into thymidylate is decreased to a greater extent with uridine than with deoxyuridine as the substrate. 4. The addition of unlabelled glutamate dilutes the radioactivity incorporated into thymine from labelled formate. 5. These results are interpreted to mean that, in DNA synthesis, vitamin B(12) has a greater role in the reduction of ribotides to deoxyribotides than in the reduction of formate to thymine methyl and that the vitamin B(12)-dependent conversion of glutamate into beta-methylaspartate also contributes to thymine synthesis.