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Glutamine catabolism by heart muscle. Properties of phosphate-activated glutaminase.

ABSTRACT: 1. Rat heart homogenates catabolized glutamine in the presence of rotenone, an inhibitor of the respiratory chain. 2. The reaction was markedly stimulated by phosphate and inhibited by glutamate, but not greatly affected by ammonia. 3. Glutamine breakdown was enhanced by lactate, malate, citrate and ATP, and almost completely blocked by 1 mM-N-ethylmaleimide. 4. The Km for glutamine measured in homogenates supplemented with either 50 mM- or 100 mM-phosphate and at pH 7.3 or 8.0 was about 4 mM, whereas the Vmax was larger at higher anion concentrations and at the more alkaline pH. 5. Activity of glutaminase was 3-fold greater in isolated mitochondria than in muscle homogenates. Distribution of the activity was the same as that of a mitochondrial marker, cytochrome c oxidase. 6. Properties of glutaminase with respect to its dependence on the concentrations of phosphate, glutamine and H+ were the same in intact and broken mitochondria and very similar to those in homogenates. However, the specific activity of the enzyme was considerably smaller in frozen-thawed than in intact organelles. 7. It is concluded that heart mitochondria possess a kidney-type phosphate-activated glutaminase that can serve as a source of myocardial glutamate.

SUBMITTER: Nelson D

PROVIDER: S-EPMC1130817 | biostudies-other | 1992 Mar

REPOSITORIES: biostudies-other

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Project description:Ischemic episodes are a leading cause of death worldwide with limited therapeutic interventions. The current study explored mitochondrial phosphate-activated glutaminase (GLS1) activity modulation by PKCβII through GC-MS untargeted metabolomics approach. Mitochondria were used to elucidate the endogenous resistance of hippocampal CA2-4 and dentate gyrus (DG) to transient ischemia and reperfusion in a model of ischemic episode in gerbils. In the present investigation, male gerbils were subjected to bilateral carotids occlusion for 5 min followed by reperfusion (IR). Gerbils were randomly divided into three groups as vehicle-treated sham control, vehicle-treated IR and PKCβII specific inhibitor peptide βIIV5-3-treated IR. Vehicle or βIIV5-3 (3 mg/kg, i.v.) were administered at the moment of reperfusion. The gerbils hippocampal tissue were isolated at various time of reperfusion and cell lysates or mitochondria were isolated from CA1 and CA2-4,DG hippocampal regions. Recombinant proteins PKCβII and GLS1 were used in in vitro phosphorylation reaction and organotypic hippocampal cultures (OHC) transiently exposed to NMDA (25 μM) to evaluate the inhibition of GLS1 on neuronal viability. PKCβII co-precipitates with GAC (GLS1 isoform) in CA2-4,DG mitochondria and phosphorylates GLS1 in vitro. Cell death was dose dependently increased when GLS1 was inhibited by BPTA while inhibition of mitochondrial pyruvate carrier (MPC) attenuated cell death in NMDA-challenged OHC. Fumarate and malate were increased after IR 1h in CA2-4,DG and this was reversed by βIIV5-3 what correlated with GLS1 activity increases and earlier showed elevation of neuronal death (Krupska et al., 2017). The present study illustrates that CA2-4,DG resistance to ischemic episode at least partially rely on glutamine and glutamate utilization in mitochondria as a source of carbon to tricarboxylic acid cycle. This phenomenon depends on modulation of GLS1 activity by PKCβII and remodeling of MPC: all these do not occur in ischemia-vulnerable CA1.

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Glutamine catabolism by heart muscle. Properties of phosphate-activated glutaminase.

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