Synthesis and radiobiological applications of [35S]l-homocysteine thiolactone

[³⁵S]l-Homocysteine thiolactone ([³⁵S]l-HCTL) was synthesized and its biodistribution evaluated as a potential brain radioprotective agent and as a tissue hypoxia marker. Drug uptake in mouse brain exceeded that in s.c. tumor 3 h post injection only. Multiple indicator dilution experiments in the rabbit heart indicate that membrane permeability of [³⁵S]l-HCTL does not limit its usefulness as a hypoxia marker. In addition, a positive correlation was observed between regional coronary blood flow and myocardial content of [³⁵S]adenosylhomocysteine formed from [³⁵S]homocysteine and adenosine.     

Conversion of methionine to homocysteine thiolactone in liver

Since hemocysteinemia is associated with arteriosclerosis, the conversion of methione to homocysteine thiolactone was studied in guinea pig liver in vivo. 60 min after intraperitoneal injection of [¹⁴C]methione, [¹⁴C]homocystein thiolactone was found to constitute 9.1% ± 0.2 of the lipid bound ¹⁴C and 20% ± 1.0 of the acid soluble ¹⁴C. This conversion is the first step of a new pathway by which the sulfur of methionine is transferred to phosphoadenosine phosphosulfate.     

Preparative labeling of proteins with [35S]methionine.

The most common technique for preparative labeling of proteins with radioisotopes for experimental purposes utilizes 125I. This isotope has certain limitations, including the emission of gamma- and X-irradiation, the release of gaseous 125I2 from solutions of Na 125I, and the potential for concentration of 125I in thyroid glands. We have discovered a means for labeling proteins rapidly and simply with [35S]methionine. The technique is applicable to a wide variety of proteins. Antibodies labeled by our technique remain functional.     

Labeling of proteins with [35S]methionine and/or [35S]cysteine in the absence of cells

Incubation of [35S]methionine and [35S]cysteine with bovine albumin, globulin, catalase, hemoglobin, or human globulin resulted in incorporation of the 35S label into each of these proteins. Trichloroacetic acid (TCA) precipitation revealed that the percentage of label incorporated ranged from 1 to 15%. The 35S labeling was resistant to dissociation by reducing SDS-PAGE, prolonged dialysis against 4 M urea, heating, TCA precipitation, and dilution by gel filtration. The labeling effect was more efficient with [35S]cysteine than [35S]methionine. Incubation of 35S label with proteins differing in methionine and cysteine content revealed no requirement for sulfur-containing amino acids in the target protein. Protein carboxymethylation reduced but did not prevent 35S label incorporation. Amino acid analysis of labeled proteins revealed that the radioactive label was not consistently associated with an individual amino acid. Differences in the ability of various proteins to spontaneously label with these amino acids suggest caution in the interpretation of metabolic labeling experiments and the necessity for inclusion of additional controls. Alternatively, our experience indicates a potentially useful method for labeling proteins in the absence of cells.     

Nonenzymatic labeling of proteins by preparations of [35S]methionine

A rapid, simple, and sensitive radiochemical assay for the measurement of purine or pyrimidine nucleoside kinases (EC 2.7.1.-) is described. The substrate (thymidine, deoxyuridine, deoxycytidine, deoxyguanosine, deoxyadenosine, uridine, cytidine, and adenosine) is separated from the product (the respective 5′-nucleotide) on neutral alumina columns which retain the nucleotides but not the nucleosides. The nucleotides are recovered by elution with 0.4 m sodium phosphate buffer, pH 7.6.     

Labelling of proteins with [35S]methionine in monolayer cultures of rat hepatocytes

Optimal conditions for the labelling of proteins with [35S]methionine in monolayers of rat hepatocytes have been established. The ability to incorporate the radioactive amino acid was constant for at least 26 h and independent of whether the medium was buffered with CO2/HCO3 or with 4-(2-hydroxyethyl)-1-piper-azineethanesulphonic acid (Hepes). Preincubation in methionine-free medium for up to 30 min yielded increasing, and from 60 to 180 min decreasing, rates of incorporation. An apparent Km value of 0.06 mM was obtained for the incorporation reaction in cells preincubated for 40 min.     

The chemical synthesis of high specific-activity [35S]adenosylhomocysteine

The study of the family of transmethylases, critical to normal cellular function and often altered in cancer, can be facilitated by the availability of a high specific-activity S-adenosylhomocysteine. We report the two-step preparation of [35S]adenosylhomocysteine from [35S]methionine at a specific activity of 1420 Ci/mmol in an overall yield of 24% by a procedure involving demethylation of the [35S]methionine to [35S]homocysteine followed by condensation with 5'-chloro-5'-deoxyadenosine. The ease of the reactions, ready availability and low cost of the reagents and high specific-activity and stability of the product make the procedure an attractive one with many uses, and superior to current methodology.     

A comparison of the uptake of [75Se]selenite, [75Se]selenomethionine and [35S]methionine by tissues of ewes and lambs.

The fate of selenium, given as Na2(75)SeO3, or [75Se]selenomethionine, and of [35S]methionine administered intravenously to ewes and lambs, has been examined. The main intention was to follow the incorporation of selenium into protein in a number of tissues, including liver and kidney, and to measure the extent of that incorporation of selenoamino acid, particularly with respect to the administration of selenite. The ewes chosen were lactating ewes with lambs at foot, and the lambs were animals which had been weaned on to fodder low in selenium and were recovering from white muscle disease with selenium therapy. These two experimental situations were chosen as they offered conditions under which selenium incorporation might be considered to be maximal. Entry of isotope into milk was rapid and was greater when 75Se was given as the selenoamino acid than as selenite. In both ewes and lambs greater amounts of activity, derived from selenite, were bound to plasma proteins than to the proteins of milk. This was particularly evident in samples taken some hours after administration. This ability of the plasma to bind selenium was demonstrated by alkaline dialysis. Small, though significant amounts of selenium, derived from Na2(75)SeO3, were incorporated as selenoamino acids into the proteins of liver, kidney and pancreas, as well as into the proteins of milk and plasma. In ewes, both selenomethionine and selenocystine were identified chromatographically in enzyme digests of defatted liver and kidney. Some differences occurred in the distribution of labelled compounds in organs from lactating ewes and recovering lambs. The incorporation of selenium into protein is discussed briefly in relation to the recent findings of an association between selenium and the enzyme glutathione peroxidase.     

The role of homocysteine thiolactone in some of human diseases

In the present article we discuss the most recent data regarding the role of homocysteine, its cyclic thioester--homocysteine thiolactone and the process of protein N-homocysteinylation in human disease. The protective role of thiolactonase/paraoxonase enzyme, carried on high density lipoproteins (HDL) in human blood, as well as the influence of structural modifications on HDL function are discussed. We also describe the effect of vitamin therapy (folic acid, vitamins: B6, B12) used for lowering the homocysteine level in humans as well.     

Incorporation of the sulfur of L-[35S]methionine into the biotin molecule by intact cells of Rhodotorula glutinis

The source of sulfur for biotin in microorganisms was studied. Using intact cells of Rhodotorula glutinis AKU 4847, L-methionine was much more effective for the synthesis of biotin from dethiobiotin than various other sulfur compounds tested. The reaction was carried out in the presence of L-[³⁵S]methionine. The radioactive biotin synthesized was isolated from the reaction mixture by a procedure involving cation- and anion-exchange column chromatographies, avidin treatment and membrane filtration, and then identified by radiochromatography and bioautography with Lactobacillus arabinosus. It was thus shown that the sulfur of methionine was incorporated into the biotin molecule by R. glutinis.     

The metabolism of l-serylglycine O[35S]-sulphate in the rat

1. The preparation of potassium l-serylglycine O-sulphate and the corresponding ³⁵S-labelled ester is described. 2. Intraperitoneal injection of potassium l-serylglycine O[³⁵S]-sulphate to rats results in about 75% of the radioactivity of the dose appearing in the urine within 48hr. Almost 72% of the radioactivity recovered in the urine was in the form of inorganic [³⁵S]sulphate. 3. Analysis of urines by paper chromatography showed the presence of unchanged l-serylglycine O[³⁵S]-sulphate and several other unidentified ³⁵S-labelled materials. 4. It has been established that micro-organisms of the gastrointestinal tract do not play any significant role in the production of inorganic [³⁵S]sulphate from the injected ester. 5. l-Serylglycine O-sulphate was hydrolysed by crude dipeptidase preparations from rat kidney and intestine to yield l-serine O-sulphate and glycine as the sole products.     

The metabolism of potassium dodecyl [35S]-sulphate in the rat

The metabolic fate of potassium dodecyl [(35)S]sulphate was studied in rats. Intraperitoneal and oral administration of the ester into free-ranging animals were followed by the excretion of the bulk of the radioactivity in the urine within 12hr., approximately 17% being eliminated as inorganic [(35)S]sulphate. Similar results were obtained in experiments in which potassium dodecyl [(35)S]sulphate was injected intravenously into anaesthetized rats with bile-duct and ureter cannulae. Analysis of urinary radioactivity revealed the presence of a new ester sulphate (metabolite A). This metabolite was isolated, purified and subsequently identified as the sulphate ester of 4-hydroxybutyric acid by paper, thin-layer and gas chromatography, by paper electrophoresis and by comparison of its properties with those of authentic butyric acid 4-sulphate. The identity of the metabolite was confirmed by isotope-dilution experiments. When either purified metabolite A or authentic potassium butyric acid 4[(35)S]-sulphate was administered to free-ranging rats the bulk of the radioactivity was eliminated unchanged in the urine within 12hr., approx. 20% of the dose appearing as inorganic [(35)S]sulphate. Whole-body radioautography and isolated-liver-perfusion experiments implicated the liver as the major site of metabolism of potassium dodecyl [(35)S]sulphate. It is suggested that butyric acid 4-sulphate probably arises by omega-oxidation of dodecyl sulphate to a fatty acid-like compound, which is then degraded by beta-oxidation.