The orientation of phosphate activated glutaminase in the inner mitochondrial membrane of synaptic and non-synaptic rat brain mitochondria

Roberg, Bjørg; Torgner, Ingeborg Aa.; Kvamme, Elling

doi:10.1016/0197-0186(95)00018-4

Cited by 55 publications

(43 citation statements)

References 40 publications

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“…Our findings as follows, indicate that the functionally active PAG both in kidney [44] and brain [9] mitochondria is localized on the outer face of the inner mitochondrial The PAG activity is comparable to that previously reported [9,14,22,44,46,47] Antibodies against kidney and liver PAG (similar amounts of enzyme activity in each well, except for liver, where 20 lg of protein was applied) Fig. 1 Immunoblots of rat astrocytes, human neuroblastoma cells, rat liver mitochondria and pig kidney mitochondria.…”

Section: Localization Of Pagmentioning

confidence: 93%

“…These compounds affect the binding of phosphate which is the main activator of PAG. There are many activators and inhibitors of PAG, and the effect on the purified enzyme [2,7], is sometimes different from that on PAG in homogenates, slices, isolated mitochondria and synaptosomes [8][9][10][11]. Interesting activators are calcium, enhancing the effect of phosphate [12][13][14], some members of the tricarboxylic acid cycle [10,11] and thyroxine, acetyl aspartate and acetyl glutamate [15,16].…”

Section: Introductionmentioning

confidence: 99%

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Novel Form of Phosphate Activated Glutaminase in Cultured Astrocytes and Human Neuroblastoma Cells, PAG in Brain Pathology and Localization in the Mitochondria

et al. 2008

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A novel form of phosphate activated glutaminase (PAG), catalyzing the synthesis of glutamate from glutamine, has been detected in cultured astrocytes and SH-SY5Y neuroblastoma cells. This enzyme form is different from that of the kidney and liver isozymes. In these cells we found high enzyme activity, but no or very weak immunoreactivity against the kidney type of PAG, and no immunoreactivity against the liver type. PAG was also investigated in brain under pathological conditions. In patients with Down's syndrome the immunoreactivity in the frontoparietal cortex was significantly reduced. The findings leading to our conclusion of a functionally active PAG on the outer face of the inner mitochondrial membrane are discussed, and a model is presented.

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Section: Localization Of Pagmentioning

confidence: 93%

Section: Introductionmentioning

confidence: 99%

Novel Form of Phosphate Activated Glutaminase in Cultured Astrocytes and Human Neuroblastoma Cells, PAG in Brain Pathology and Localization in the Mitochondria

et al. 2008

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“…Whole rat forebrain tissue (f) and purified mitochondria (m) were solubilized in SDS and fractionated by SDS-PAGE (10-20% gradient gels), blotted onto nitrocellulose and probed with the above antibodies to EAAT3 (c) and with antibodies (0.2 lg/ml anti-B12; antibody ID: Ab,360) to another glutamate transporter (EAAT2, b). Note that the mitochondrial preparation (Roberg et al 1995) is highly purified as antibodies to EAAT2, which is present in plasma membranes, do not label the mitochondrial preparation. Further, as shown in c, EAAT3 is present in the whole brain extract (labeled band at around 70 kDa), but is undetectable in the mitochondrial extract (m).…”

Section: The Optimal Controlsmentioning

confidence: 99%

Specificity controls for immunocytochemistry

2006

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Antibodies have been in widespread use for more than three decades as invaluable tools for the specific detection of proteins or other molecules in biological samples. In spite of such a long experience, the field of immunocytochemistry is still troubled by spurious results due to insufficient specificity of antibodies or procedures used. The importance of keeping a high standard is increasing because massive sequencing of entire genomes leads to the identification of numerous new proteins. All the identified proteins and their variants will have to be localized precisely and quantitatively at high resolution throughout the development of all species. Consequently, antibody generation and immunocytochemical investigations will be done on a large scale. It will be economically important to secure an optimal balance between the risk of publishing erroneous data (which are expensive to correct) and the costs of specificity testing. Because proofs of specificity are never absolute, but rather represent failures to detect crossreactivity, there is no limit to the number of control experiments that can be performed. The aims of the present paper are to increase the awareness of the difficulties in proving the specificity of immunocytochemical labeling and to initiate a discussion on optimized standards. The main points are: (1) antibodies should be described properly, (2) the labeling obtained with an antibody to a single epitope needs additional verification and (3) the investigators should be required to outline in detail how they arrive at the conclusion that the immunocytochemical labeling is specific.

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“…Finally, it is important to note that presence of brain GA in the cytoplasm has also been suggested by subcellular fractionation and immunocytochemistry studies (32,35,36). Nevertheless, the relevance of extramitochondrial GA to Gln-Glu metabolism requires further investigation and is presently unknown (Figure 2).…”

Section: Glutaminase Isoenzymes In Mammalian Brainmentioning

confidence: 99%

Brain glutaminases

Márquez

Martín-Rufián

Segura

et al. 2010

BioMolecular Concepts

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Glutaminase is considered as the main glutamate producer enzyme in brain. Consequently, the enzyme is essential for both glutamatergic and gabaergic transmissions. Glutaminederived glutamate and ammonia, the products of glutaminase reaction, fulfill crucial roles in energy metabolism and in the biosynthesis of basic metabolites, such as GABA, proteins and glutathione. However, glutamate and ammonia are also hazardous compounds and danger lurks in their generation beyond normal physiological thresholds; hence, glutaminase activity must be carefully regulated in the mammalian brain. The differential distribution and regulation of glutaminase are key factors to modulate the metabolism of glutamate and glutamine in brain. The discovery of novel isoenzymes, protein interacting partners and subcellular localizations indicate new functions for brain glutaminase. In this short review, we summarize recent findings that point consistently towards glutaminase as a multifaceted protein able to perform different tasks. Finally, we will highlight the involvement of glutaminase in pathological states and its consideration as a potential therapeutic target.

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The orientation of phosphate activated glutaminase in the inner mitochondrial membrane of synaptic and non-synaptic rat brain mitochondria

Cited by 55 publications

References 40 publications

Novel Form of Phosphate Activated Glutaminase in Cultured Astrocytes and Human Neuroblastoma Cells, PAG in Brain Pathology and Localization in the Mitochondria

Novel Form of Phosphate Activated Glutaminase in Cultured Astrocytes and Human Neuroblastoma Cells, PAG in Brain Pathology and Localization in the Mitochondria

Specificity controls for immunocytochemistry

Brain glutaminases

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