Nuclear Localization of L-type Glutaminase in Mammalian Brain

Lucı́A Olalla,Antonia Gutiérrez,José A Campos,Zafar U Khan,Francisco J Alonso,Juan A Segura,Javier Márquez,J Carlos Aledo

doi:10.1074/jbc.c200373200

Abstract

In mammals, there are two different genes encoding for glutaminase isoforms, named liver (LGA) and kidney (KGA) types. LGA has long been believed to be present only in liver mitochondria from adult animals. However, we have recently reported the presence of LGA mRNA in human brain. We now describe the expression of LGA mRNA in the brain of other mammals (cow, mouse, rabbit, and rat) and in different areas of human brain as assessed by Northern blot analysis. The presence of mRNA encoding for this isoform in rat brain was further confirmed by reverse transcriptase-PCR cloning and sequencing. Although it has been well accepted that glutaminase is a mitochondrial enzyme, using newly generated isoform-specific antibodies, we have found a differential intracellular immunolocalization of both glutaminase isoforms in rat and monkey brain. In both species, KGA protein was present in mitochondria, whereas LGA protein was localized in nuclei. Furthermore, subcellular fractionation and Western blot analysis revealed that brain LGA was enriched in nuclei where it was catalytically active. Nuclear glutaminase exhibited a kinetic behavior that resembles that of the liver-type enzyme with regard to the low phosphate concentration requirement; however, nuclear glutaminase was susceptible to glutamate inhibition, a property that is absent in the rat liver enzyme.

Highlights

Glutamate is the major excitatory neurotransmitter in mammalian brain and has been implicated in a plethora of physiological and pathological processes
The transcripts of both isoforms possess very different molecular sizes (ϳ5 and 2.4 –2.8 kb for the KGA and LGA mRNA, respectively); this fact rules out the possibility that the results presented in Fig. 1 may be due to cross-hybridization between the LGA probe and the KGA mRNA and vice versa
Because this result was in conflict with the generally accepted idea that, at least in rat, the expression of LGA is restricted to the adult liver, we further investigated the presence of LGA mRNA in rat brain by RT-PCR

Summary

Introduction

Glutamate is the major excitatory neurotransmitter in mammalian brain and has been implicated in a plethora of physiological and pathological processes. There are two different genes encoding for GA, known as liver (LGA) and kidney (KGA) types [3]. Both enzymatic forms of GA display different kinetic and molecular characteristics [4]. We have generated isoform-specific antibodies and cDNA probes to study the expression of both glutaminase isoforms in the brain of diverse mammalian species. Our results suggest a nuclear localization of the LGA protein, which is catalytically active and displays kinetic properties different from both the liver and kidney glutaminases

Methods

Results

Conclusion