Chris Doumen

Kinetic analysis of dynamic 13C NMR spectra: Metabolic flux, regulation, and compartmentation in hearts

Control of oxidative metabolism was studied using 13C NMR spectroscopy to detect rate-limiting steps in 13C labeling of glutamate. 13C NMR spectra were acquired every 1 or 2 min from isolated rabbit hearts perfused with either 2.5 mM [2-13C]acetate or 2.5 mM [2-13C]butyrate with or without KCl arrest. Tricarboxylic acid cycle flux (VTCA) and the exchange rate between alpha-ketoglutarate and glutamate (F1) were determined by least-square fitting of a kinetic model to NMR data. Rates were compared to measured kinetics of the cardiac glutamate-oxaloacetate transaminase (GOT). Despite similar oxygen use, hearts oxidizing butyrate instead of acetate showed delayed incorporation of 13C label into glutamate and lower VTCA, because of the influence of beta-oxidation: butyrate = 7.1 +/- 0.2 mumol/min/g dry wt; acetate = 10.1 +/- 0.2; butyrate + KCl = 1.8 +/- 0.1; acetate + KCl = 3.1 +/- 0.1 (mean +/- SD). F1 ranged from a low of 4.4 +/- 1.0 mumol/min/g (butyrate + KCl) to 9.3 +/- 0.6 (acetate), at least 20-fold slower than GOT flux, and proved to be rate limiting for isotope turnover in the glutamate pool. Therefore, dynamic 13C NMR observations were sensitive not only to TCA cycle flux but also to the interconversion between TCA cycle intermediates and glutamate.

Altered metabolite exchange between subcellular compartments in intact postischemic rabbit hearts.

To examine metabolic regulation in postischemic hearts, we examined oxidative recycling of 13C within the glutamate pool (GLU) of intact rabbit hearts. Isolated hearts oxidized 2.5 mmol/L [2-13C]acetate during normal conditions (n = 6) or during reperfusion after 10 minutes of ischemia (n = 5). 13C-Nuclear magnetic resonance spectra were acquired every 1 minute. Kinetic analysis of 13C incorporation into GLU provided both tricarboxylic acid (TCA) cycle flux and the interconversion rate (F1) between the TCA cycle intermediate, alpha-ketoglutarate (alpha-KG), and the largely cytosolic GLU. The rate-pressure product in postischemic hearts was 46% of normal (P < .05). No difference in substrate utilization occurred between groups, with acetate accounting for 92% of the carbon units entering the TCA cycle at the citrate synthase step. TCA cycle flux in postischemic hearts was normal (normal hearts, 10.7 mumol.min-1.g-1; postischemic hearts, 9.4 mumol.min-1.g-1), whereas F1 was 72% lower at 2.9 +/- 0.4 versus 10.2 +/- 2.5 mumol.min-1.g-1 (mean +/- SE) in normal hearts (P < .05). From additional hearts perfused with 2.5 mmol/L [2-13C]acetate plus supplemental 5 mmol/L glucose, any potential differences in endogenous carbohydrate availability were proved not to account for the reduced rate alpha-KG and GLU exchange, which remained depressed in postischemic hearts. However, specific activities of the transaminase enzyme, catalyzing chemical exchange of alpha-KG and GLU, were the same, and transaminase flux was 100 mumol.min-1.g-1 in postischemic hearts versus 68 mumol.min-1.g-1 in normal hearts. Normal transaminase activity and the increased flux in postischemic hearts are contrary to the reduced F1. The findings indicate reduced metabolite transport rates across the mitochondrial membranes of stunned myocardium, particularly through the reversible alpha-KG-malate carrier.

Dehydrogenase regulation of metabolite oxidation and efflux from mitochondria in intact hearts.

To test how α-ketoglutarate dehydrogenase (α-KGDH) activity influences the balance between oxidative flux and transmitochondrial metabolite exchange, we monitored these rates in isolated mitochondria and in perfused rabbit hearts at an altered kinetics ( K m) of α-KGDH for α-ketoglutarate (α-KG). In isolated mitochondria, relative K mdropped from 0.23 mM at pH = 7.2 to 0.10 mM at pH 6.8 ( P < 0.05), and α-KG efflux decreased from 126 to 95 nmol ⋅ min-1 ⋅ mg-1. In intact hearts, K m was reduced with low intracellular pH, while matching control workload and respiratory rate with increased Ca2+(pHi = 7.20, perfusate CaCl2 = 1.5 mM; pHi = 6.89, perfusate CaCl2 = 3 ± 1 mM). Sequential13C nuclear magnetic resonance spectra from hearts oxidizing [2-13C]acetate provided tricarboxylic acid cycle flux and the exchange rate between α-KG and cytosolic glutamate ( F 1). Tricarboxylic acid cycle flux was 10 μmol ⋅ min-1 ⋅ g-1in both groups, but F 1 fell from a control of 9.3 ± 0.6 to 2.8 ± 0.4 μmol ⋅ min-1 ⋅ g-1at low K m. The results indicate that increased activity of α-KGDH occurs at the expense of α-KG efflux during support of normal workloads.

Substrate preferences of the heart mitochondria of the horseshoe crab Limulus polyphemus

Abstract 1. 1. Tightly coupled mitochondria were isolated from the longitudinal hearts of Limulus polyphemus, the horseshoe crab. 2. 2. Succinate and α-ketoglutarate were oxidized at the highest rate while active malate and fumarate utilization was only observed in the presence of small amounts of pyruvate. 3. 3. The mitochondria showed a relatively high respiratory activity with pyruvate and proline. 4. 4. Palmityol-1-carnitine was a poor substrate. 5. 5. The respiratory data, coupled with the enzyme profile on crude extracts of the hearts, are indicative of a carbohydrate-based metabolism.

cDNA identification, comparison and phylogenetic aspects of lombricine kinase from two oligochaete species

Creatine kinase and arginine kinase are the typical representatives of an eight-member phosphagen kinase family, which play important roles in the cellular energy metabolism of animals. The phylum Annelida underwent a series of evolutionary processes that resulted in rapid divergence and radiation of these enzymes, producing the greatest diversity of the phosphagen kinases within this phylum. Lombricine kinase (EC 2.7.3.5) is one of such enzymes and sequence information is rather limited compared to other phosphagen kinases. This study presents data on the cDNA sequences of lombricine kinase from two oligochaete species, the California blackworm (Lumbriculus variegatus) and the sludge worm (Tubifex tubifex). The deduced amino acid sequences are analyzed and compared with other selected phosphagen kinases, including two additional lombricine kinase sequences extracted from DNA databases and provide further insights in the evolution and position of these enzymes within the phosphagen kinase family. The data confirms the presence of a deleted region within the flexible loop (the GS region) of all six examined lombricine kinases. A phylogenetic analysis of these six lombricine kinases clearly positions the enzymes together in a small subcluster within the larger creatine kinase (EC 2.7.3.2) clade.

Variable intron/exon structure in the oligochaete lombricine kinase gene.

Lombricine kinase is an annelid enzyme that belongs to the phosphagen kinase family of which creatine kinase and arginine kinase are the typical representatives. The enzymes play important roles in the cellular energy metabolism of animals. Biochemical, physiological and molecular information with respect to lombricine kinase is limited compared to other phosphagen kinases. This study presents data on the cDNA sequences of lombricine kinase from two smaller oligochaetes, Enchytraeus sp. and Stylaria sp. The deduced amino acid sequences are analyzed and compared with other selected phosphagen kinases. The intron/exon structure of the lombricine kinase gene was determined for these two species as well as two additional oligochaetes, Lumbriculus variegatus and Tubifex tubifex, and compared with available data for annelid phosphagen kinases. The data indicate the existence of a variable organization of the proposed 8-intron/9-exon gene structure. The results provide further insights in the evolution and position of these enzymes within the phosphagen kinase family.