Properties and submitochondrial localization of pig and rat renal phosphate-activated glutaminase

Abstract

Two pools of phosphate-activated glutaminase (PAG) were separated from pig and rat renal mitochondria. The partition of enzyme activity corresponded with that of the immunoreactivity and also with the postembedding immunogold labeling of PAG, which was associated partly with the inner membrane and partly with the matrix. The outer membrane was not labeled. PAG in intact mitochondria showed enzymatic characteristics that were similar to that of the membrane fraction and also mimicked that of the polymerized form of purified pig renal PAG. PAG in the soluble fraction showed properties similar to that of the monomeric form of purified enzyme. It is indicated that the pool of PAG localized inside the inner mitochondrial membrane is dormant due to the presence of high concentrations of the inhibitor glutamate. Thus the enzymatically active PAG is assumed to be localized on the outer face of the inner mitochondrial membrane. The activity of this pool of PAG appears to be regulated by compounds in the cytosol, of which glutamate may be most important.

Brain Edema in Acute Liver Failure

“…PAG activity was determined by the method of Roberg et al 43 Briefly, frontal cortical tissue was homogenized in 10 volumes (w/v) of buffer containing 250 mmol/L D-mannitol, 70 mmol/L sucrose, 10 mmol/L HEPES, (pH 8.6). Homogenates (100 l) were incubated at 37°C for 1 hour in 30 mmol/L glutamine, 1 mmol/L potassium EDTA, and 150 mmol/L potassium phosphate buffer.…”

Induction and Targeting of the Glutamine Transporter SN1 to the Basolateral Membranes of Cortical Kidney Tubule Cells during Chronic Metabolic Acidosis Suggest a Role in pH Regulation

“…Antibodies against SN1 and PAG previously have been characterized extensively and were used as described (12)(13)(14). Other antibodies were obtained commercially for aquaporin-1 (AQP1; Alpha-Diagnostics, San Antonio, TX), AQP2 (Santa Cruz Biotechnology, Santa Cruz, CA), Calbindin (Abcam, Cambridge, UK), and Tamm-Horsfall glycoprotein (Chemicon International, Temecula, CA), URO-8 (Bioquote Limited, North Yorkshire, UK) or as a gift for phosphate-independent glutaminase (PIG; Dr. Huseby, University of Tromsø, Norway).…”

“…PAG protein Western blots with quantification was performed with ECL plus as described (13,14). Immunoperoxidase and immunofluorescence staining was carried out with the biotin-streptavidin-peroxidase/DAB system and immunofluorescence-coupled secondary antibodies, respectively.…”

“…Glutamine hydrolysis was assayed at 37°C and pH 8.6 as described previously (14). Briefly, the homogenates (in 250 mM d-mannitol, 70 mM sucrose, 10 mM HEPES, and protease inhibitors [pH 7.4]) were incubated in 30 mM l-glutamine, 150 mM potassium-phosphate, and 10 mM HEPES/Tris after preincubation of the homogenates with 1 mM acivicin (AT-125), an inhibitor of PIG enzyme activity (16) for 30 min on ice.…”

Glitazones regulate glutamine metabolism by inducing a cellular acidosis in MDCK cells

“…Thus an important point to establish from the perspective of this model is the intracellular localization of the alanine aminotransferase. Furthermore, if the functional glutaminase activity is indeed within the cytosolic compartment (23,32), glutamate formed within the cytosol would be effectively disrupted from subserving functions localized to this compartment, including matrix synthesis, and be directed into the mitochondrial pathway. Noteworthy is that not all investigators view the mitochondrial glutaminase as being functional within the cytosolic compartment and instead assign it to the mitochondrial matrix (21) or matrix surface of the inner mitochondrial membrane (12).…”