Substrate Channeling via a Transient Protein-Protein Complex: The case of D-Glyceraldehyde-3-Phosphate Dehydrogenase and L-Lactate Dehydrogenase

BackgroundD-Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and L-lactate dehydrogenase (LDH) can form a complex that can regulate the major metabolic pathways, however, the exact mechanism remains unknown. We analyzed a possibility of NADH-channeling from GAPDH-NADH complex to LDH isozymes using enzymes from different cells.ResultsEnzyme-kinetics and NADH-binding studies showed that LDH can use GAPDH-NADH complex as a substrate. LDH activity with GAPDH-NADH complex was challenged with anti-LDH antibodies to show that the channeled and the diffusive reactions always take place in parallel. The channeling path is dominant only in assays with limiting free-NADH concertation that mimic cytosolic conditions. Analytical ultracentrifugation showed that the channeling does not require a high affinity complex. Molecular dynamics calculations and protein-protein interaction studies showed that LDH and GAPDH can form a leaky channeling complex only at subsaturating NADH concentrations. The interaction sites are conserved between LDH isozymes from heart and muscle, and between GAPDH molecules from rabbit and yeast cells. Positive electric fields between the NAD(H) binding sites on LDH and GAPDH tetramers, showed that NAD(H)-channeling within the LDH-GAPDH complex, can be an extension of NAD(H)-channeling between the adjacent subunits in each tetramer.ConclusionsIn the case of a transient (GAPDH-NADH)-LDH complex, the relative contribution from the channeled and the diffusive paths depends on the overlap between off-rates for the transient (GAPDH-NADH)-LDH complex and off-rates for the GAPDH-NADH complex. Molecular evolution or metabolic engineering protocols can exploit substrate channeling for metabolic flux control by fine-tuning substrate-binding affinity for the key enzymes in the competing reaction paths.Highlights- Substrate channeling molecular mechanism can regulate energy production and aerobic and anaerobic metabolism in cells- LDH and GAPDH can form a channeling complex only at sub-saturating NADH concentration- Channeled and diffusive paths always compete and take place in parallel- NADH channeling does not require a high-affinity complex- NADH channeling within GAPDH-LDH complex is an extension of NAD(H) channeling within each tetramer- Allosteric modulation of NADH binding affinity in GAPDH tetramer can regulate NAD(H) channeling

Isozymes of malate-and lactate dehydrogenase of Astyanax bimaculatus as biomarkers of environmental impact

The activity of certain isozymes may serve as biomarkers of specific physiological conditions of living organisms. The present work aimed to evaluate malate dehydrogenase (MDH) and lactate dehydrogenase (LDH) isozymes from the hepatic, branchial and renal tissues of the fish Astyanax bimaculatus as biochemical markers of environmental changes in the Una River Basin (Taubaté, SP, Brazil). For this study, the specimens were collected in water bodies located in three sampling sites of the basin, called P1 (Itaim stream), P2 (a stream along the Remédios municipal road) and P3 (a small lake near the Dr. José Luiz Cembranelli municipal road). Physicochemical analysis of the water from these sites indicated that P1 was the less polluted site, P2 presented high levels of electrolytes in water, and P3 presented the worst water quality among the sampling sites. The isoenzymes were separated by native-PAGE and identified by their activity on the polyacrylamide gel. The results indicated that four isozymes of MDH occur in the liver of these fish, and the activities of MDH-1 and MDH-4 were detected only in fish from P2, suggesting that these isoforms have potential as biomarkers of the presence of high levels of electrolytes in water. In the kidney, MDH-1 was detected in fish from P2 and P3, but it was not detected in those from P1, suggesting that the presence of this isozyme may be a biomarker of low-quality water. The gill MDH and LDH isozyme profiles of all tissues examined showed some similarities between individuals from the three collection sites, indicating that they are not suitable as biomarkers of the environmental conditions of these sites.

Lactate Dehydrogenase Inhibition: Biochemical Relevance and Therapeutical Potential

Lactate dehydrogenase (LHD) is a key enzyme of anaerobic metabolism in almost all living organisms and it is also a functional checkpoint for glucose restoration during gluconeogenesis and single-stranded DNA metabolism. This enzyme has a well preserved structure during evolution and among the species, with little, but sometimes very useful, changes in the amino acid sequence, which makes it an attractive target for the design and construction of functional molecules able to modulate its catalytic potential and expression. Research has focused mainly on the selection of modulator especially as far as LDH isozymes (especially LDH-5) and lactate dehydrogenases of Plasmodium falciparum (pfLDH) are concerned. This review summarizes the recent advances in the design and development of inhibitors, pointing out their specificity and therapeutic potentials.

Urinary Lactate Dehydrogenase Activity and Its Isozyme Patterns in Kawasaki Disease

Abnormal urinary findings, such as sterile pyuria, proteinuria, and microscopic hematuria, are often seen in the acute phase of Kawasaki disease (KD). We investigated the potential significance of urinary lactate dehydrogenase (U-LDH) activity and its isozyme patterns in KD. Total U-LDH activity and its isozymes (U-LDH1-5) levels were compared among 120 patients with KD, 18 patients with viral infection (VI), and 43 patients with upper urinary tract infection (UTI) and additionally compared between intravenous immunoglobulin (IVIG) responders (n=89) and nonresponders (n=31) with KD. Total U-LDH activity was higher in KD (35.4±4.8 IU/L, P<0.05) and UTI patients (66.0±8.0 IU/L, P<0.01) than in VI patients (17.0±6.2 IU/L). In the isozyme pattern analysis, KD patients had high levels of U-LDH1 and U-LDH2, while UTI patients had high levels of U-LDH3, U-LDH4, and U-LDH5. Furthermore, IVIG nonresponders of KD had significantly higher levels of total U-LDH activity (45.1±4.7 IU/L, P<0.05), especially U-LDH1 and U-LDH2 (P<0.05), than IVIG responders (32.0±2.8 IU/L). KD patients have increased levels of total U-LDH activity, especially U-LDH-1 and U-LDH2, indicating a unique pattern of U-LDH isozymes different from that in UTI patients.

An Electrophoretic Study of LDH-Isozyme Distribution in Developing Embryonic and Adult Tissues of the Toad Bufo arabicus

Lactate (LDH) dehydrogenases were studied by electrophoresis to investigate the possibility of isozymic differences in different stages and organs of B. arabicus embryos, tadpoles and adults. The expression pattern of lactate dehydrogenase (LDH) isozymes was analysed using nondenaturing discontinuous polyacrylamide gel electrophoresis (PAGE). Five LDH isozymes were observed in five different adult tissues. The most cathodal isozyme LDH-5 (A4) showed the strongest activity. All embryonic stages tested displayed the anodic isozymes first. Four LDH isozymes were present in the stages 1-16 (fertilized egg-tail bud stage). The first appearance of the fifth isozyme was detected in stage 17 when the muscular response started. This isozyme showed gradual increase in intensity from stage 17 to stage 43.

Adaptations in metabolic capacity of rat soleus after paralysis

To determine whether long-term reductions in neuromuscular activity result in alterations in metabolic capacity, the activities of oxidative, i.e., succinate dehydrogenase (SDH) and citrate synthase (CS), and glycolytic, i.e., α-glycerophosphate dehydrogenase (GPD), enzyme markers were quantified in rat soleus muscles 1, 3, and 6 mo after a complete spinal cord transection (ST). In addition, the proportional content of lactate dehydrogenase (LDH) isozymes was used as a marker for oxidative and glycolytic capacities. The myosin heavy chain (MHC) isoform content of a fiber served as a marker of phenotype. In general, MHC isoforms shifted from MHC1 toward MHC2, particularly MHC2x, after ST. Mean SDH and CS activities were higher in ST than control at all time points. The elevated SDH and CS activities were indicative of an enhanced oxidative capacity. GPD activities were higher in ST than control rats at all time points. The increase in activity of SDH was larger than GPD. Thus the GPD-to-SDH (glycolytic-to-oxidative) ratio was decreased after ST. Compared with controls, total LDH activity increased transiently, and the LDH isozyme profile shifted from LDH-1 toward LDH-5, indicative of an enhanced glycolytic capacity. Combined, these results indicate that 1) the metabolic capacities of soleus fibers were not compromised, but the interrelationships among oxidative and glycolytic capacity and MHC content were apparently dissociated after ST; 2) enhancements in oxidative and glycolytic enzyme activities are not mutually exclusive; and 3) chronic reductions in skeletal muscle activity do not necessarily result in a reduced oxidative capacity.

187EFFECTS OF OXYGEN TENSION ON ESTABLISHMENT, LDH ISOZYMES, AND MRNA EXPRESSION OF MURINE EMBRYONIC STEM CELLS

Orchestrated differentiation of embryonic stem cells into specific tissues or cells will be invaluable for xenotransplantation, biomedicine, and pharmacology. However, the lack of a standardized culture environment for establishment and maintenance of cell lines has hindered the application of this technology. In other cell types, O2 concentration in the culture environment can have a profound effect on proliferation and differentiation. This study, therefore, tested the hypothesis that establishment dynamics, LDH isoforms, and mRNA expression patterns of the resulting cell lines would be affected by the O2 tension in the culture environment. Blastocysts recovered from mice (C57BL/6) uteri on Day 4 (post coitis) were placed in Speciality Medium (4500mgL−1 glucose) DMEM (plus 0.01μgmL−1 LIF) and cultured in a gas environment of 5% CO2 and either ∼20% or 5% O2. More (P&lt;0.05) blastocysts hatched and produced outgrowths (Day 4 of DMEM culture) in the low (76.7±0.1%) compared to the high (58.3±0.1%) O2 group. Although the number of cells per outgrowth was similar between groups (low=58.1±5.2, high=58.8±5.7), a smaller number of colonies in the high O2 group (9/15; 64.3%) stained positive for alkaline phosphatase relative to the low O2 group (14/15; 93.3%). Oxygen treatment had no effect on the patterns of activity of the oxioreductase, lactate dehydrogenase (LDH), in either outgrowths or established stem cell lines. Interestingly, the stem cell lines (both O2 groups) displayed multiple isoforms (Isoforms 3, 4, and 5) of LDH, whereas the outgrowths displayed only Isoform 5. In contrast, two-cell embryos and blastocysts displayed only Isoform 1, and fibroblasts displayed Isoforms 4 and 5. Expression (mRNA) profiles were developed from blastocyst outgrowths, stem cell colonies, and established stem cell lines cultured under either high or low O2 tension, using a RT-PCR for LDH (Isozymes α and β) and a key regulatory enzyme of glycolysis, phosphofructokinase-2 (PFK2; Isozyme 1 and 2). There were no differences between the high and low O2 groups in mRNA expression of LDHα in the outgrowths, or established stem cells. Expression of LDHβ was at a very low level regardless of O2 treatment or cell type. Outgrowths from the low O2 group expressed Isozyme 1 of PFK2 whereas the outgrowths from the high O2 group did not. Enhanced expression of PFK2 is suggestive of increased glycolytic capacity. Reduced O2 environment during the peri-hatching period had significant effects on the resulting embryonic stem cells, supporting the hypothesis that O2 tension can affect stem cell establishment and maintenance.