使用L-[1-¹⁴C]亮氨酸进行蛋白质合成测定时的酮异己酸污染误差。

Ketoisocaproate contamination errors in protein synthesis determinations using L[1-14C]leucine.

作者信息

López Hellin J, Schwartz S, Garlick P J, Rosell M, Arbós M A, Andreu A L, García E

机构信息

Unitat de Recerques Metabòliques S. Grisolía, Hospital Vall d'Hebron, Barcelona, Spain.

出版信息

J Biochem Biophys Methods. 1993 Aug;27(1):57-63. doi: 10.1016/0165-022x(93)90068-y.

DOI:10.1016/0165-022x(93)90068-y

PMID:8409211

Abstract

Protein synthesis rate determinations in vivo using L-[1-14C]-leucine may be underestimated because of contamination by radioactive ketoisocaproate (KIC) resulting from leucine metabolism. The aim of this work was to set up a reliable method to determine the KIC/leucine radioactivity ratio in protein-free homogenates, and to apply it to study the extent of the protein synthesis ratio error due to KIC contamination. Cation-exchange chromatography using Dowex AG 50W-X8 resin was used to separate KIC from leucine, eluting KIC with water and leucine with 4 M ammonia. The errors found in the protein synthesis ratio were 6.20% in liver and 2.34% in jejunum.

摘要

使用L-[1-14C]-亮氨酸进行体内蛋白质合成速率测定时，可能会因亮氨酸代谢产生的放射性酮异己酸（KIC）污染而被低估。本研究的目的是建立一种可靠的方法来测定无蛋白匀浆中KIC/亮氨酸的放射性比值，并将其应用于研究KIC污染导致的蛋白质合成比值误差程度。使用Dowex AG 50W-X8树脂进行阳离子交换色谱法，将KIC与亮氨酸分离，用水洗脱KIC，用4M氨水洗脱亮氨酸。在肝脏中发现的蛋白质合成比值误差为6.20%，在空肠中为2.34%。