Simultaneous determination of selenomethionine enantiomers in biological fluids by stable isotope dilution gas chromatography-mass spectrometry

A method for the stereoselective determination of D- and L-enantiomers of selenomethionine in mouse plasma was developed using gas chromatography-mass spectrometry with selected-ion monitoring (GC-MS-SIM). DL-[(2)H(3,)(82)Se]selenomethionine was used as analytical internal standard to account for losses associated with the extraction, derivatization and chromatography. Selenomethionine enantiomers in mouse plasma were purified by cation-exchange chromatography using BondElut SCX cartridge and derivatized with HCl in methanol to form methyl ester followed by subsequent N-acylation with optically active (+)-α-methoxy-α-trifluoromethylphenylacetyl chloride to form diastereomeric amide. Quantification was performed by SIM of the molecular-related ions of the diastereomers on the chemical ionization mode. The intra- and inter-day precision for D- and L-selenomethionine spiked to mouse plasma gave good reproducibility with relative standard deviation of 3% and 3% for D-selenomethionine and 6% and 3% for L-selenomethionine, respectively. The estimated amounts were in good agreement with the actual amounts spiked, the intra- and inter-day relative error being 5% and 2% for D-selenomethionine and 2% and 1% for L-selenomethionine, respectively. The present method is sensitive enough to determine pharmacokinetics of selenomethionine enantiomers.     

Quantitation of total homocysteine, total cysteine, and methionine in normal serum and urine using capillary gas chromatography-mass spectrometry

Total homocysteine, total cysteine, and methionine have been extracted and partially purified from serum and urine using reduction with 2-mercaptoethanol followed by cation-exchange chromatography and anion-exchange chromatography. The t-butyldimethylsilyl derivatives were prepared and analyzed using capillary gas chromatography-mass spectrometry with selected ion monitoring. The addition of DL-[3,3,3',3',4,4,4',4'-2H8]homocystine, DL-[3,3,3',3'-2H4]cystine, and L-[methyl-2H3]methionine to the starting samples prior to the reduction of all disulfides, including the deuterated internal standards, with 2-mercaptoethanol makes it possible to quantitate all three amino acids. Normal ranges for total homocysteine, total cysteine, and methionine have been determined in human and rat serum and in human urine.     

Simultaneous determination of methionine sulfoxide and methionine in blood plasma using gas chromatography-mass spectrometry

Methionine sulfoxide is an oxidation product of methionine with reactive oxygen species via 2-electron-dependent mechanism. Such oxidants can be generated from activated neutrophils; therefore, methionine sulfoxide can be regarded as a biomarker of oxidative stress in vivo. We describe here a method for the simultaneous determination of methionine sulfoxide and methionine in blood plasma using gas chromatography-mass spectrometry with isotopically labeled compounds as internal standards. This method comprises the inclusion of [Me-13C, Me-2H(3)]methionine sulfoxide and [Me-13C, Me-2H(3)]methionine into plasma, the removal of plasma proteins using acetonitrile, the purification of amino acids with cation-exchange chromatography, and the derivatization of methionine sulfoxide and methionine to their corresponding tert-butyldimethylsilyl derivatives using N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide. Quantitation was performed by electron impact mode. The levels of methionine sulfoxide in healthy human blood plasma were 4.0 +/- 1.0 microM (means +/- SD, n = 8), indicating that approximately 10% of methionine is detected as the oxidized form in healthy human plasma. The ratio of methionine sulfoxide in total methionine increased with treatment of human blood with phorbol 12-myristate 13-acetate, while this ratio remained constant in plasma from alloxan-induced hyperglycemic rabbits. These results indicate that this method is applicable for plasma samples and methionine sulfoxide can represent oxidative stress caused by nonradical oxidation in vivo.     

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.     

Analysis of [2H7]methionine, [2H4]methionine,methionine, [2H4]homocysteine and homocysteine in plasma by gas chromatography–mass spectrometry to follow the fate of administered [2H7]methionine

Homocysteine plays a key role in several pathophysiological conditions. To assess the methionine–homocysteine kinetics by stable isotope methodology, we developed a simultaneous quantification method of [²H₇]methionine, [²H₄]methionine, methionine, [²H₄]homocysteine and homocysteine in rat plasma by gas chromatography–mass spectrometry (GC–MS). [¹³C]Methionine and [¹³C]homocysteine were used as analytical internal standards to account for losses associated with the extraction, derivatization and chromatography. For labeled and non-labeled homocysteine measurements, disulfide bonds between homocysteine and other thiols or proteins were reduced by dithiothreitol. The reduced homocysteine and methionine species were purified by cation-exchange chromatography and derivatized with isobutyl chlorocarbonate in water–ethanol–pyridine. Quantification was carried out by selected ion monitoring of the molecular-related ions of N(O,S)-isobutyloxycarbonyl ethyl ester derivatives on the chemical ionization mode. The intra- and inter-day precision of the assay was less than 6% for all labeled and non-labeled methionine and homocysteine species. The method is sensitive enough to determine pharmacokinetics of labeled methionine and homocysteine.     

Gas chromatographic–mass spectrometric determination of α-ketoisocaproic acid and [H7]α-ketoisocaproic acid in plasma after derivatization with N-phenyl-1,2-phenylenediamine

A method for determination of α-ketoisocaproic acid (KIC) and [4,5,5,5,6,6,6-²H₇]α-ketoisocaproic acid ([²H₇]KIC) in rat plasma was developed using gas chromatography–mass spectrometry-selected ion monitoring (GC–MS-SIM). [5,5,5-²H₃]α-Ketoisocaproic acid ([²H₃]KIC) was used as an analytical internal standard to account for losses associated with the extraction, derivatization and chromatography. The keto acids were extracted by cation-exchange chromatography using BondElut SCX cartridge and derivatized with N-phenyl-1,2-phenylenediamine to form N-phenylquinoxalinone derivatives. Quantitation was performed by SIM of the respective molecular ions at m/z 278, 281 and 285 for the derivatives of KIC, [²H₃]KIC and [²H₇]KIC on the electron impact method. The limit of detection was found to be 70 fmol per injection (S/N=3) and the limit of quantitation for [²H₇]KIC was around 50 nM in rat plasma. Endogenous KIC concentrations in 50 μl of rat plasma were measured with relative intra- and inter-day precision of 4.0% and 3.3%, respectively. The intra- and inter-day precision for [²H₇]KIC spiked to rat plasma in the range of 0.1 to 10 μM gave good reproducibility with relative standard deviation (RSD) of 6.5% and 5.4%, respectively. The intra- and inter-day relative errors (RE) for [²H₇]KIC were less than 6.4% and 3.8%, respectively. The method was applied to determine the plasma concentration of [²H₇]KIC after an intravenous administration of [²H₇]KIC in rat.     

Gas chromatographic–mass spectrometric determination of α-ketoisocaproic acid and [2H7]α-ketoisocaproic acid in plasma after derivatization with N-phenyl-1,2-phenylenediamine

A method for determination of α-ketoisocaproic acid (KIC) and [4,5,5,5,6,6,6-²H₇]α-ketoisocaproic acid ([²H₇]KIC) in rat plasma was developed using gas chromatography–mass spectrometry-selected ion monitoring (GC–MS-SIM). [5,5,5-²H₃]α-Ketoisocaproic acid ([²H₃]KIC) was used as an analytical internal standard to account for losses associated with the extraction, derivatization and chromatography. The keto acids were extracted by cation-exchange chromatography using BondElut SCX cartridge and derivatized with N-phenyl-1,2-phenylenediamine to form N-phenylquinoxalinone derivatives. Quantitation was performed by SIM of the respective molecular ions at m/z 278, 281 and 285 for the derivatives of KIC, [²H₃]KIC and [²H₇]KIC on the electron impact method. The limit of detection was found to be 70 fmol per injection (S/N=3) and the limit of quantitation for [²H₇]KIC was around 50 nM in rat plasma. Endogenous KIC concentrations in 50 μl of rat plasma were measured with relative intra- and inter-day precision of 4.0% and 3.3%, respectively. The intra- and inter-day precision for [²H₇]KIC spiked to rat plasma in the range of 0.1 to 10 μM gave good reproducibility with relative standard deviation (RSD) of 6.5% and 5.4%, respectively. The intra- and inter-day relative errors (RE) for [²H₇]KIC were less than 6.4% and 3.8%, respectively. The method was applied to determine the plasma concentration of [²H₇]KIC after an intravenous administration of [²H₇]KIC in rat.     

The cell surface receptor for immunoglobulin E. Effect of tunicamycin on molecular properties of receptor from rat basophilic leukemia cells

Cell surface receptors for immunoglobulin E were isolated by repetitive affinity chromatography from rat basophilic leukemia cells biosynthetically labeled with L-[35S]methionine and D-[3H]mannose. Native immunoglobulin E receptor appeared as a very broad band in the 45,000 to 62,000 Mr region in sodium dodecyl sulfate polyacrylamide gels. However, from cells cultured in the presence of tunicamycin, a relatively narrow band with an apparent Mr of 38,000 was isolated. The 38,000 Mr band rebound to immunoglobulin E-Sepharose, was immunoprecipitated with antibodies to immunoglobulin E receptor, shared tryptic peptides with native receptor, and was labeled with L-[35S]methionine but not D-[3H]mannose, and thus appears to be immunoglobulin E receptor lacking N-linked oligosaccharides. It is demonstrated that N-linked oligosaccharides account for much of the apparent heterogeneity of native receptor in sodium dodecyl sulfate polyacrylamide gels and in two-dimensional gel electrophoresis. A receptor-associated protein with apparent Mr = 30,000, prominently labeled with L-[35S]methionine but not with D-[3H]mannose, did not have altered molecular properties when isolated from tunicamycin-cultured cells, and did not share tryptic peptides with receptor.     

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.     

Specific [3H]Piflutixol Binding to CHAPS-Solubilised Rat Striatal Preparations Involves Dopamine D-2 but Not D-1 Binding Sites

[3H]Piflutixol binding to rat striatal membrane preparations identifies both D-1 and D-2 sites. We used [3H]piflutixol to characterise those binding sites present in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)-solubilised rat striatal preparations. The specific binding of [3H]piflutixol, as defined using cis-flupenthixol, to CHAPS-solubilised rat striatal tissue was saturable and of high affinity. Specific [3H]piflutixol binding to the solubilised preparations was displaced stereoselectively by the isomers of butaclamol and to an equal extent by both cis-flupenthixol and (+/-)-sulpiride. A positive correlation was found between the capacity of a range of drugs to displace [3H]piflutixol binding and the displacement of [3H]spiperone to the same preparations. The Bmax of [3H]piflutixol binding was not different from that of [3H]spiperone binding to the same preparation. These studies suggest that, in contrast to specific binding of membrane preparations, the specific binding of [3H]piflutixol to CHAPS-solubilised preparations involves mainly D-2 sites. Specific [3H]piflutixol binding, in contrast to [3H]spiperone binding, showed only slow dissociation from soluble preparations. The binding of [3H]piflutixol to CHAPS-solubilised preparations was retained during passage through a gel filtration column. This prelabelling of solubilised striatal preparations using [3H]piflutixol may aid in the purification of CHAPS-solubilised rat striatal D-2 sites.     

Determination of the benztropine analog AHN-1055, a dopamine uptake inhibitor, in rat plasma and brain by high-performance liquid chromatography with ultraviolet absorbance detection

N-Substituted 3alpha-[bis(4'-fluorophenyl)methoxy] tropanes represent a series of novel potential cocaine abuse therapeutics. AHN-1055, a member of this series, has been assessed to be the most suitable analog for pharmacokinetic studies. A sensitive and specific high-performance liquid chromatography method was developed to quantitate AHN-1055 in rat plasma and brain tissue. Reversed-phase chromatography with ultraviolet detection (lambda=220 nm) was utilized to quantitate the eluate. Plasma or brain tissue samples were prepared by liquid-liquid extraction using hexane, followed by evaporation, reconstitution in mobile phase, and injection onto an ABZ+plus column. AHN-1055 and oxprenolol (internal standard) eluted at approximately 9.9 and 5.01 min, respectively, without any interfering peaks. The calibration curves were found to be linear in the range of 25-10000 ng/ml for plasma and 50-5000 ng/g for brain (r2> or =0.999). The intra- and inter-day variabilities were < or =10% whereas the intra- and inter-day errors were < or =8.5%. Plasma and brain recoveries of AHN-1055 were 95 and 79%, respectively. Stability studies showed plasma quality control samples to be stable through at least three freeze-thaw cycles (error<3.5%), for at least 24 h when subjected to room temperature (error<3%) and for at least 30 h after loading the processed samples onto the autosampler (error<3%). AHN-1055 stock solution was found to be stable for at least 4 months when stored at 4 degrees C (error<6%). The validated method accurately quantified AHN-1055 in plasma and brain samples collected from a pharmacokinetic study consisting of an intravenous bolus in the tail vein of adult male Sprague-Dawley rats.     

Determination of rimantadine in rat plasma by liquid chromatography/electrospray mass spectrometry and its application in a pharmacokinetic study

A rapid and sensitive liquid chromatography/mass spectrometry (LC/MS) method was developed and validated for the determination of rimantadine in rat plasma. Rimantadine was extracted by protein precipitation with methanol, and the chromatographic separation was performed on a C(18) column. The total analytical run time was relatively short (4.6 min), and the limit of assay quantification (LLOQ) was 2 ng/mL using 50 microL of rat plasma. Rimantadine and the internal standard (amantadine) were monitored in selected ion monitoring (SIM) mode at m/z 180.2 and 152.1, respectively. The standard curve was linear over a concentration range from 2 to 750 ng/mL, and the correlation coefficients were greater than 0.999. The mean intra- and inter-day assay accuracy ranged from 100.1-105.0% to 100.3-104.0%, respectively, and the mean intra- and inter-day precision was between 1.3-2.3% and 1.8-3.0%, respectively. The developed assay method was successfully applied to a pharmacokinetic study in rats after oral administration of rimantadine hydrochloride at the dose of 20 mg/kg.     

In Situ Molecular Size of Agonist Dopamine D-2 Binding Sites in Rat Striatum

Specific binding of [3H]N-propylnorapomorphine [( 3H]NPA) to 3,4-dihydroxyphenylethylamine (dopamine) D-2 receptors was investigated in rat striatum in vitro. For various dopamine receptor substances, the rank order of potency to inhibit [3H]NPA binding was spiroperidol greater than or equal to NPA greater than LY 171555 greater than SCH 23390 greater than SKF 38393. A single high-affinity binding site was found in membranes prepared in either Tris-citrate buffer or imidazole buffer; the affinity constants were 0.11 and 0.76 nM, respectively. The number of receptors (33 pmol/g wet weight) was independent of whether the membranes were prepared in Tris-citrate buffer or imidazole buffer and was similar to the number of receptors estimated by [3H]spiroperidol binding to dopamine receptors. Irradiation inactivation of frozen whole rat striata showed a monoexponential loss of [3H]NPA binding sites without a change in the binding affinity. The target size of the [3H]NPA binding site was 81,000 daltons, which shows that the functional molecular entity to bind the dopamine D-2 agonist was smaller than the molecular entity to bind the dopamine D-2 antagonist [3H]spiroperidol (target size, 137,000 daltons).     

Determination of ketone body kinetics using a D-(-)-3-hydroxy[4,4,4-2H3]butyrate tracer

In studies where D-(-)-3-hydroxy[4,4,4-2H3]butyrate is employed as isotopic tracer in vivo, we have described a selected ion monitoring, gas-liquid chromatography-mass spectrometry micromethod which measures [2H3] tracer enrichment in 3-hydroxybutyrate and acetoacetate from 300-microliters blood samples. For plasma samples in the physiologic range, intra- and interassay precisions for each ketone averaged better than +/- 1% and +/- 2%, respectively. The use of the method was validated by comparing kinetic data obtained with the above tracer with simultaneous flux data obtained with conventional D-(-)-3-hydroxy[3-14C]butyrate tracer in five fasted rats.     

Dimethylsulfoniopropionate and methanethiol are important precursors of methionine and protein-sulfur in marine bacterioplankton.

Organic sulfur compounds are present in all aquatic systems, but their use as sources of sulfur for bacteria is generally not considered important because of the high sulfate concentrations in natural waters. This study investigated whether dimethylsulfoniopropionate (DMSP), an algal osmolyte that is abundant and rapidly cycled in seawater, is used as a source of sulfur by bacterioplankton. Natural populations of bacterioplankton from subtropical and temperate marine waters rapidly incorporated 15 to 40% of the sulfur from tracer-level additions of [(35)S]DMSP into a macromolecule fraction. Tests with proteinase K and chloramphenicol showed that the sulfur from DMSP was incorporated into proteins, and analysis of protein hydrolysis products by high-pressure liquid chromatography showed that methionine was the major labeled amino acid produced from [(35)S]DMSP. Bacterial strains isolated from coastal seawater and belonging to the alpha-subdivision of the division Proteobacteria incorporated DMSP sulfur into protein only if they were capable of degrading DMSP to methanethiol (MeSH), whereas MeSH was rapidly incorporated into macromolecules by all tested strains and by natural bacterioplankton. These findings indicate that the demethylation/demethiolation pathway of DMSP degradation is important for sulfur assimilation and that MeSH is a key intermediate in the pathway leading to protein sulfur. Incorporation of sulfur from DMSP and MeSH by natural populations was inhibited by nanomolar levels of other reduced sulfur compounds including sulfide, methionine, homocysteine, cysteine, and cystathionine. In addition, propargylglycine and vinylglycine were potent inhibitors of incorporation of sulfur from DMSP and MeSH, suggesting involvement of the enzyme cystathionine gamma-synthetase in sulfur assimilation by natural populations. Experiments with [methyl-(3)H]MeSH and [(35)S]MeSH showed that the entire methiol group of MeSH was efficiently incorporated into methionine, a reaction consistent with activity of cystathionine gamma-synthetase. Field data from the Gulf of Mexico indicated that natural turnover of DMSP supplied a major fraction of the sulfur required for bacterial growth in surface waters. Our study highlights a remarkable adaptation by marine bacteria: they exploit nanomolar levels of reduced sulfur in apparent preference to sulfate, which is present at 10(6)- to 10(7)-fold higher concentrations.     

A deuterium surface coil NMR study of the metabolism of D-methionine in the liver of the anesthetized rat

The hepatic metabolism of deuteriated D-methionine has been studied in the intact, anesthetized rat using 2H NMR spectroscopy. The rate of formation of the principal labeled metabolite, [methyl-2H3]sarcosine, from the D-[methyl-2H3]methionine precursor was found to be as rapid as the rate observed previously in NMR studies of the hepatic metabolism of L-methionine. Similarly, rates of clearance of labeled methionine from the liver, formation of N-trimethyl-labeled metabolites, and labeling of the HDO pool were all found to be similar to the rates observed in the L-methionine studies. In contrast, all of these metabolic transformations are strongly inhibited by pretreatment of the rats with sodium benzoate, an inhibitor of D-amino acid oxidase. In vivo 2H NMR studies of sodium benzoate treated rats given L-[methyl-2H3]-methionine exhibit a much more rapid formation of [methyl-2H3]sarcosine than rats given the D enantiomer, consistent with the expectation that the sodium benzoate does not interfere with either the formation of S-adenosylmethionine or the subsequent transmethylation of glycine. However, the rates of methionine clearance and formation of deuteriated water are markedly reduced in this study relative to rats receiving the labeled D- or L-methionine without sodium benzoate pretreatment. These results indicate that subsequent to the initial oxidative deamination of the labeled D-methionine, the reamination to give L-methionine is rapid compared with the further degradation of the alpha-keto acid. Thus, the results are consistent with a dominant contribution of the glycine/sarcosine shuttle to the metabolism of excess D- or L-methionine.     

Uptake of amino acids and nucleic acid precursors by regenerating rat liver

1. At 0.5-1.0h after partial hepatectomy the intracellular acid-soluble fraction of rat liver took up twice as much radioactivity from [(3)H]orotic acid, [(3)H]uridine and [(3)H]thymidine as did similar fractions from sham-operated animals. This increase in penetration was not prevented by adrenalectomy or actinomycin, both of which decreased precursor uptake into nuclear RNA at this time. The increase in entry was still shown by thymidine at 22h after operation, at the height of the S period. Adenine penetration was not increased 1h after partial hepatectomy. 2. Plasma concentrations of ornithine and possibly methionine, tyrosine and lysine were raised 1.5h after partial hepatectomy. [(3)H]Lysine entry into regenerating liver at this time was increased by 60%; [(3)H]valine uptake was unaffected. Intracellular amounts of tyrosine, phenylalanine and ornithine in the liver were also increased. 3. The relation of these events to the start of liver regeneration is discussed.     

Biosynthesis of ethylene from methionine in aminoethoxyvinylglycine-resistant avocado tissue

This study was conducted to determine if aminoethoxyvinylglycine (AVG) insensitivity in avocado (Persea americana Mill., Lula, Haas, and Bacon) tissue was due to an alternate pathway of ethylene biosynthesis from methionine. AVG, at 0.1 millimolar, had little or no inhibitory effect on either total ethylene production or [(14)C] ethylene production from [(14)C]methionine in avocado tissue at various stages of ripening. However, aminoxyacetic acid (AOA), which inhibits 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (the AVG-sensitive enzyme of ethylene biosynthesis), inhibited ethylene production in avocado tissue. Total ethylene production was stimulated, and [(14)C]ethylene production from [(14)C]methionine was lowered by treating avocado tissue with 1 millimolar ACC. An inhibitor of methionine adenosyltransferase (EC 2.5.1.6), l-2-amino-4-hexynoic acid (AHA), at 1.5 millimolar, effectively inhibited [(14)C]ethylene production from [(14)C]methionine in avocado tissue but had no effect on total ethylene production during a 2-hour incubation. Rates of [(14)C]AVG uptake by avocado and apple (Malus domestica Borkh., Golden Delicious) tissues were similar, and [(14)C]AVG was the only radioactive compound in alcohol-soluble fractions of the tissues. Hence, AVG-insensitivity in avocado tissue does not appear to be due to lack of uptake or to metabolism of AVG by avocado tissue. ACC synthase activity in extracts of avocado tissue was strongly inhibited (about 60%) by 10 micromolar AVG. Insensitivity of ethylene production in avocado tissue to AVG may be due to inaccessibility of ACC synthase to AVG. AVG-resistance in the avocado system is, therefore, different from that of early climacteric apple tissue, in which AVG-insensitivity of total ethylene production appears to be due to a high level of endogenous ACC relative to its rate of conversion to ethylene. However, the sensitivity of the avocado system to AOA and AHA, dilution of labeled ethylene production by ACC, and stimulation of total ethylene production by ACC provide evidence for the methionine --> SAM --> ACC --> ethylene pathway in avocado and do not suggest the operation of an alternate pathway.     

The efficiency of methionine incorporation from isoaccepting species of tRNAMet into rabbit globin in an homologous reticulocyte lysate system.

Rabbit globin alpha and beta chains were labeled with [3H]leucine, and with [35S] -methionine from reticulocyte tRNAMet isoacceptors using a rabbit reticulocyte cell-free synthesis system. [35S]Methionine from the three tRNAMet species isolated by RPC-5 chromatography was incorporated into internal positions of both alpha and beta globin. The initiator tRNA, tRNAIMet, exhibited very low efficiency for incorporating methionine internally, while tRNAIIMet was four times more efficient than tRNAIIIMet. Amino acid analysis of the tryptic peptides of the labeled globins revealed that all three isoacceptors incorporated methionine into the normal methionine peptides. Similar studies with Escherichia coli [35S]Met-tRNAfMet showed a 3-fold increase over the reticulocyte initiator tRNA in its capacity to incorporate methionine into the internal positions of rabbit globin.     

Biosynthesis and characterization of adrenocorticotropic hormone, alpha-melanocyte-stimulating hormone, and an NH2-terminal fragment of the adrenocorticotropic hormone/beta-lipotropin precursor from rat pars intermedia.

Isolated intermediate lobe cells from 40 rat pituitaries were incubated for 3 h with [35S]methionine + [3H]-phenylalanine, [35S]methionine, [3H]valine, and [3H]leucine. The cell extracts were purified by carboxymethyl-cellulose chromatography (CMC) and the fraction eluting with ovine adrenocorticotropic hormone (ACTH) was further purified either by another CMC under the same conditions or by high performance liquid chromatography (HPLC). Microsequencing of the product from the second CMC allowed the identification of a peptide containing methionine 4 and phenylalanine 7, as expected for the NH2 terminus of ACTH. Purification by HPLC of a similar peptide obtained from the three other incubations gave three main raoactive peaks which were further characterized by their migration rates on polyacrylamide gels, molecular weight, and microsequencing. Results indicated that intact ACTH (residues 1-39) is present in extracts of rat intermediate lobe, but in very small quantities (less than 1% of the beta-endorphin content). ACTH is probably broken down into smaller fragments, e.g. alpha-melanocyte-stimulating hormone (alpha-MSH) (ACTH, 1-13) and corticotropin-like intermediate lobe peptide (CLIP) (ACTH, 18-39). These studies also revealed with existence of a peptide having identical sequence with the (N-1) terminus of the ACTH/lipotropin (LPH) precursor.