Preparation of radioactive Tay-Sachs ganglioside labeled in the sialic acid moiety

A procedure is described for the preparation of Tay-Sachs ganglioside specifically labeled in the sialic acid portion of the molecule. Rat brain gangliosides were labeled biosynthetically by the intracranial injection of N-acetyl-(3)H-D-mannosamine. Radioactive gangliosides were isolated and selectively degraded with bacterial neuraminidase and rat liver beta-galactosidase to Tay-Sachs ganglioside-(3)H. Radioactivity in the labeled product was confined to the N-acetyl-neuraminic acid portion of the molecule.     

Glutathione specifically labeled with isotopes

A procedure for synthesis of glutathione selectivity labeled with isotopes is described. A strain of Escherichia coli enriched in its content of gamma-glutamylcysteine synthetase and glutathione synthetase by recombinant DNA techniques is immobilized in a carrageenan matrix and treated with toluene to render the cells more permeable to the substrates. The immobilized cell matrix is incubated with a mixture containing the appropriately labeled amino acid, the other amino acid constituents of glutathione, ATP, and acetylphosphate. The radiolabeled product is isolated by column chromatography.     

Preparation of some labeled glycosphingolipids by catalytic addition of tritium

Three experiments are described in which seven tritium-labeled glycosphingolipids were prepared by catalytic addition of tritium gas to unsaturated centers. These compounds include glucosyl ceramide, galactosyl ceramide, lactosyl ceramide (cytolipin H), N-acetylgalactosaminyl (β1 → 3) galactosyl (α1 → 3) galactosyl (β1 → 4) glucosyl ceramide (cytolipin R), and three species of gangliosides: G_M1 (G₄), G_M3 (G₆), and G_D1a (G₃).     

Preparation of tritium labeled beta-endorphin suitable for studying brain receptor interactions

The opioid peptide (porcine) β-endorphin has been tritiated using reductive methylation to prepare a derivative containing mainly [³H]dimethyllysine. The tritiated β-endorphin has a specific activity of 9.8 Ci/mmol and is stable for an extended period of time. The labeled peptide binds reversibly to rat brain membrane preparations with a dissociation constant of 0.4 ± 0.1 nM and a receptor content of 23 ± 2 pmol/g brain. Under the conditions used, there is evidence for only one class of receptors. The technique employed for tritium labeling of β-endorphin should also be applicable to various other peptides including α-endorphin, γ-endorphin, and C′-fragment that have been found in brain and pituitary.     

Synthesis of tritium labeled cortoic acids

A procedure is described for the microsynthesis and purification of the high specific activity tritium labeled cortisol metabolites, 20 alpha- and 20 beta-cortolic acids and 20 alpha- and 20 beta-cortolonic acids.     

Directed biosynthesis of prostaglandin F2 alpha multiple labeled with tritium

By selecting specific activators of PGF reductase and determining their optimal ratio, we have performed the directed biosynthesis of PGF2 alpha with a 45-50% yield. With the use of [5,6,8,9,11,12,14,15-3H8]arachidonic acid, multiple-labelled PGF2 alpha with molar radioactivity 6.1 TBe/mmol has been synthesised.     

Isolation of the tritium labeled subunit II of cytochrome oxidase

Radioactively labelled subunit II of cytochrome oxidase is isolated after irradiation of molecular crystals of the enzyme with thermally activated tritium.     

Enzymatic semisynthesis of insulin specifically labelled with tritium at position B-30

We have synthesized porcine insulin labelled with tritium at position B-30 using enzyme-catalysed formation of a peptide bond. The resulting insulin derivative has the label in the expected position and is biologically active. We have tested our procedure to prepare batches up to 50 muCi of tritiated insulin at a specific radioactivity of up to 1.14 Ci/mmol.     

Radiometric assays of N-acetylglucosaminylphosphotransferase and alpha-N-acetylglucosaminyl phosphodiesterase with substrates labeled in the glucosamine moiety

The assay of fibroblast and leukocyte-N-acetylglucosaminylphosphotransferase with alpha-methylmannoside acceptor and commercially available UDP-[3H or 14C]N-acetylglucosamine donor was modified to yield low background and consequently high sensitivity and reliability comparable to those obtained with the synthetically made [beta-32P]UDP-N-acetylglucosamine donor. This was achieved by an additional elution step that removed free [3H or 14C]N-acetylglucosamine which appeared to be the breakdown product responsible for the high background. In addition, the [3H or 14C]N-acetylglucosamine-1-phospho-6-alpha-methylmannoside product of the transfer reaction was then isolated and, following desalting, could serve as a substrate for the assay of alpha-N-acetylglucosaminyl phosphodiesterase. Cell preparations of patients with I-cell disease and pseudo-Hurler polydystrophy demonstrated severe to moderate deficiency of transferase activity and normal phosphodiesterase activity toward the respective substrates labeled with 3H or 14C in the glucosamine moiety.     

Synthesis of nucleosides labeled with tritium at the 5'-carbon atom of the ribose residue

Chemical and enzymatic syntheses of [5'-3H]adenosine, [5'-3H]guanosine, and [5'-3H]uridine have been developed. The reduction of beta-D-ribo-pentadialdo-1,4-furanosyl derivatives of corresponding bases is used in the chemical synthesis. The maximum molar activity of the labelled products was 220 TBk/mol in reactions with [3H]NaBH4 and 370-740 TBk/mol in reactions with gaseous tritium. The enzymatic synthesis was performed by the rebosylation of heterocyclic bases with nucleoside phosphorylase and [5'-3H]uridine as a ribosyl donor. Nucleoside phosphorylase is proposed to be used in the immobilized form to avoid the decrease of molar activity. Nucleosides labelled with tritium both in ribosyl and heterocyclic moieties were synthesised enzymatically.