J. Carlos Aledo

Identification of two human glutaminase loci and tissue-specific expression of the two related genes.

Abstract. Glutaminolysis is initiated by either of two isoforms, K- and L-types, of the enzyme phosphate-activated glutaminase. The chromosomal localization, genomic organization, and the tissue-specific expression of the genes have been investigated in the human by using isoform-specific cDNA probes. Results obtained from radiation hybrid mapping experiments assigned the K-glutaminase gene to human Chromosome (Chr) 2, and a second locus for l-glutaminase in Chr 12 was identified. Southern blot analysis with the L-cDNA probe showed hybridization to a single restriction fragment, while four to seven fragments were found to hybridize to the K-cDNA probe. The distribution of human glutaminase expression was also investigated: the L-cDNA probe detected a single band of 2.4 kb in liver, brain, and pancreas, whereas a single transcript of approximately 4.4 kb was detected in kidney, brain, heart, placenta, lung, and pancreas by using the K-cDNA probe. This work provides evidence that the human liver and kidney glutaminase isozymes are encoded by separate genes located on different chromosomes; furthermore, the expression pattern in human tissues revealed for both isoenzymes differs notably from the paradigm based upon the isoenzyme distribution in rats.

The C-terminus of human glutaminase L mediates association with PDZ domain-containing proteins1

The enzyme glutaminase in brain is responsible for the synthesis of neurotransmitter glutamate. We used the two‐hybrid genetic selection system in yeast to look for interactors of glutaminase in human brain. We have identified two proteins containing PDZ domains, α1‐syntrophin and a glutaminase‐interacting protein, named GIP, that showed association with human glutaminase L, as deduced from specificity test of the two‐hybrid system. The complete GIP cDNA clone has 1315 nucleotides with a 372‐base open reading frame encoding a 124‐amino acids protein. Glutaminase associates with both PDZ proteins through its C‐terminal end; mutagenesis of single amino acids revealed the sequence ‐ESXV as essential for the interaction. These data suggest the possibility that PDZ domain‐containing proteins are involved in the regulation of glutaminase in brain.

Expression of the scaffolding PDZ protein glutaminase-interacting protein in mammalian brain

A human brain cDNA clone coding for a novel PDZ‐domain protein of 124 amino acids was previously isolated in our laboratory. The protein was termed glutaminase‐interacting protein (GIP), because it interacts with the C‐terminal region of the human L‐type glutaminase (LGA). The pattern of expression and functions of GIP in brain are completely unknown, so its significance remains undefined. Here we describe the expression of GIP mRNA and protein in mammalian brain. Northern blot analysis revealed that GIP mRNA was ubiquitous in most regions of human brain but was particularly abundant in spinal cord. The presence of the protein in rat and monkey brain was studied at the regional, cellular, and subcellular level by immunocytochemistry. The protein was found to be present in both neurons and astrocytes, with a cytosolic and mitochondrial subcellular localization. Double immunofluorescence labeling with anti‐GIP and anti‐LGA antibodies using confocal microscopy revealed colocalization of both proteins in astrocyte cell processes and their perivascular end feet. Electron microscopy of rat brain neurons revealed GIP immunoreactivity concentrated also in the nuclear envelope and the plasma membrane. The multiple locations for GIP in mammalian brain are in agreement with known protein interaction partners reported for this PDZ protein. The findings presented here support a role of GIP as an important scaffold in both astrocytes and neurons and point toward astrocytic processes and perivascular end feet as plausible anatomical substrates for interaction with glutaminase.

The ATP Paradox Is the Expression of an Economizing Fuel Mechanism

The strong negative correlation between glycolytic flux and intracellular ATP concentration observed in yeast has long been an intriguing and counterintuitive phenomenon, which has been referred to as the ATP paradox. Herein, using principles of irreversible thermodynamics it was shown that if the ATP-consuming pathways are more sensitive to extracellular glucose than glycolysis, then upon glucose addition glycolysis performance can switch from an efficient working regime to a dissipative regime, and vice versa, depending on glucose availability. The efficient regime represents a good compromise between high output power and low dissipation, whereas the dissipative working regime offers a higher output power although at a high glucose cost. The physiological and evolutionary implications of this switch strategy are discussed.

Segregation of two glutaminase isoforms in islets of Langerhans

Despite the importance of glutamatergic signalling in the co-ordination of hormone secretion, the identity of the enzyme for the production of glutamate in beta-cells is still unresolved. We have found that the endocrine pancreas co-expresses two isoforms of GA (glutaminase), denoted as kidney-type (KGA) and liver-type (LGA), with a complementary cellular pattern of expression. Whereas KGA was mainly present in alpha-cells, LGA was very abundant in beta-cells. This spatial segregation may have important functional implications, facilitating a differential regulation of glutamate production in insulin- and glucagon-secreting cells.

Early differential expression of two glutaminase mRNAs in mouse spleen after tumor implantation

The influence of progressive tumor growth on phosphate-activated glutaminase (PAG) expression in splenocytes from mice bearing Ehrlich ascites carcinoma cells was investigated. Implantation of Ehrlich ascites tumor cells in the peritoneal cavity of mice led to a 2.3-fold stimulation of spleen PAG activity 48 h later. Four days after tumor implantation the glutaminase activity had returned to nearly basal value and remained at this level throughout the tumor development. Northern blot analysis indicated that two species of glutaminase mRNA were expressed in the spleen, which showed a differential expression pattern during the first 2 days after tumor implantation. The abundance of the transcript of higher electrophoretic mobility (approximately 3 kb) constantly increased over the first 2 days of tumor growth. The mRNA of lower electrophoretic mobility (approximately 6 kb) peaked at 12 h after tumor implantation and returned to control values at 48 h. These results demonstrate that tumor has the capability of altering glutaminase expression in the host spleen.

Energy Diagrams for Enzyme-Catalyzed Reactions: Concepts and Misconcepts.

Despite the utility that energy diagrams have as a teaching and learning tool, a survey of their use, in seven popular Biochemistry textbooks, reveals that there is certain confusion around this topic. In our opinion, this confusion arises from the reluctance of authors to consider and indicate the conditions under which the reaction being represented occurs. For an enzyme‐catalyzed reaction, it should be stressed that, under conditions where the overall reaction is spontaneous, each elementary step must exhibit a negative free energy change, and this must be properly reflected in the progression profile of the reaction.

Metabolic pathways: does the actual Gibbs free‐energy change affect the flux rate?

Energy dissipation fulfils an important function in metabolism driving the flux of matter through metabolic pathways. The flux rate is a multivariable function. One of such variables affecting the flux is the actual free energy change. A particular case of metabolic pathway is analysed to illustrate this fact, using only general enzymic and thermodynamic concepts.

UPREGULATION OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE MRNA IN THE SPLEEN OF TUMOR-BEARING MICE

The influence of tumor implantation on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels and stability was determined in the spleen of tumor-bearing mice. While GAPDH mRNA levels were not altered in skeletal muscle, kidney and liver from tumor-bearing mice, tumor implantation led to a 5.6-fold increase in the levels of splenic GAPDH mRNA. An enhanced message stability was observed in splenocytes from tumor-bearing animals, suggesting the involvement of post-transcriptional mechanisms in the selective GAPDH mRNA accumulation after tumor implantation. The GAPDH activity/glycolytic flux ratio was 18.5 in the spleen of healthy mice. Therefore, the three-fold increase in the glycolytic flux observed after tumor implantation could hardly justify the necessity for the upregulation of GAPDH.

Corrigendum to: The C-terminus of human glutaminase L mediates association with PDZ domain-containing proteins (FEBS 24464): [FEBS Letters 488 (2001) 116–122]

An unfortunate error occurred in the ¢rst sentence of Section 2.1. This sentence should read: The N-terminal (N-PAG) and C-terminal (C-PAG) baits were generated by inserting into the pEG202 (pLexA) vector [15] the coding sequences for amino acid residues 68^392 and 347^602, respectively, of the human glutaminase full-length cDNA cloned from ZR-75 breast cancer cells [11]. In Fig. 1 the box representing the N-bait, the number 1 should be substituted by 68.