The use of a tritium release assay to measure 6-N-trimethyl-l-lysine hydroxylase activity: Synthesis of 6-N-[3-3H]trimethyl-dl-lysine

doi:10.1016/0003-2697(81)90349-3

Analytical Biochemistry

Volume 116, Issue 1, 1 September 1981, Pages 230-236

https://doi.org/10.1016/0003-2697(81)90349-3 Get rights and content

Abstract

6-N-[3-³H]Trimethyl-dl-lysine was synthesized from 6-N-acetyl-l-lysine by the following chemical scheme: 6-N-acetyl-l-lysine → 2-keto-6-N-acetylcaproic acid → 2-[3-³H]keto-6-N-acetylcaproic acid → 2-[3-³H]keto-6-N-acetylcaproic acid oxime → 6-N-[3-³H]acetyl-dl-lysine → dl-[3-³H]lysine → 2-N-[3-³H]formyl-dl-lysine → 2-[3-³H]formyl-6-N-trimethyl-dl-lysine → 6-N-[3-³H]trimethyl-dl-lysine. Using a 70% ammonium sulfate fraction obtained from a high-speed rat kidney supernatant, the cosubstrate and cofactor requirements for 6-N-trimethyl-l-lysine hydroxylase activity as measured by tritium release from 6-N-[3-³H]trimethyl-dl-lysine were: α-ketoglutarate, ferrous ions, l-ascorbate, and oxygen, with added catalase showing a slight but distinct stimulatory effect. On incubation with the crude rat kidney preparation, the release of tritium from 6-N-[3-³H]trimethyl-dl-lysine was linear with both time of incubation and protein concentration. Hydroxylation of 6-N-trimethyl-l-lysine, as measured by tritium release from the labeled substrate, was examined in rat kidney, heart, liver, and skeletal muscle tissues, and found to be most active in the kidney.

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^☆: This work was supported by Grant AM-21197 from the National Institutes of Health. R. Stein is a Public Health Service predoctoral fellow on a training program (5T 32 AM07329) from the National Institutes of Health. The studies represent part of a thesis to be submitted by R. Stein to the Sue Golding Graduate Division of Medical Sciences of the Albert Einstein College of Medicine in partial fulfillment for the Ph.D. degree. A preliminary report of this work was presented at the 71st Annual Meeting of the American Society of Biological Chemists held in New Orleans, La., June 1–5, 1980 (Fed. Proc.39, 2091 (Abstract 2573)).

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The use of a tritium release assay to measure 6-N-trimethyl-l-lysine hydroxylase activity: Synthesis of 6-N-[3-3H]trimethyl-dl-lysine☆

Abstract

J. Biol. Chem

J. Biol. Chem

Arch. Biochem. Biophys

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Anal. Biochem

J. Biol. Chem

Arch. Biochem. Biophys

Arch. Biochem. Biophys

Biochem. Biophys. Res. Commun

Biochim. Biophys. Acta

The use of a tritium release assay to measure 6-N-trimethyl-l-lysine hydroxylase activity: Synthesis of 6-N-[3-³H]trimethyl-dl-lysine☆