Localization of l-lactate dehydrogenase in mitochondria

Kline, Edward S.; Brandt, Richard B.; Laux, Jerome E.; Spainhour, Stephen E.; Higgins, Edwin S.; Rogers, Kenneth S.; Tinsley, Steven B.; Waters, M. Gerard

doi:10.1016/0003-9861(86)90323-1

Cited by 64 publications

(53 citation statements)

References 13 publications

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“…However, Baba and Sharma [39] also evidenced the presence of LDH in the mitochondria of the rat heart and skeletal muscle using histochemical techniques. Later studies, performed by Kline et al [40] and Brandt et al [41], who employed different techniques, gave similar results. The importance of the findings is strongly connected with the hypothesis of the intracellular (intramuscular) lactate shuttle being a key element of the idea presented and discussed by Gladden [42], that lactate is an important intermediary in numerous metabolic processes, a particularly mobile fuel for aerobic metabolism, and perhaps a mediator of the redox state among various compartments both within and between cells.…”

Section: Resultssupporting

confidence: 62%

Ultracentrifugation studies of the location of the site involved in the interaction of pig heart lactate dehydrogenase with acidic phospholipids at low pH. A comparison with the muscle form of the enzyme

Terlecki

Czapińska

Hotowy

2007

Cellular and Molecular Biology Letters

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Lactate dehydrogenase (LDH) from the pig heart interacts with liposomes made of acidic phospholipids most effectively at low pH, close to the isoelectric point of the protein (pH = 5.5). This binding is not observed at neutral pH or high ionic strength. LDH-liposome complex formation requires an absence of nicotinamide adenine dinucleotides and adenine nucleotides in the interaction environment. Their presence limits the interaction of LDH with liposomes in a concentration-dependent manner. This phenomenon is not observed for pig skeletal muscle LDH. The heart LDH-liposome complexes formed in the absence of nicotinamide adenine dinucleotides and adenine nucleotides are stable after the addition of these substances even in millimolar concentrations. The LDH substrates and studied nucleotides that inhibit the interaction of pig heart LDH with acidic liposomes can be ordered according to their effectiveness as follows: NADH > NAD > ATP = ADP > AMP > pyruvate. The phosphorylated form of NAD (NADP), nonadenine nucleotides (GTP, CTP, UTP) and lactate are ineffective. Chemically cross-linked pig heart LDH, with a tetrameric structure stable at low pH, behaves analogously to the unmodified enzyme, which excludes the participation of the interfacing parts of subunits in the interaction with acidic phospholipids. The presented results indicate that in lowered pH conditions, the NADH-cofactor binding site of pig heart LDH is strongly involved in the interaction of the enzyme with acidic phospholipids.

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Section: Resultssupporting

confidence: 62%

Ultracentrifugation studies of the location of the site involved in the interaction of pig heart lactate dehydrogenase with acidic phospholipids at low pH. A comparison with the muscle form of the enzyme

Terlecki

Czapińska

Hotowy

2007

Cellular and Molecular Biology Letters

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“…Human and pig mitochondria were prepared by differential centrifugation as described previously (Beattie, 1968;Kline et al, 1986) with slight modifications (Clement and Deters, 2000). To account for the biological variability liver samples from pigs or human organs were pooled (from at least three individuals per pool).…”

Section: Methodsmentioning

confidence: 99%

“…Human and pig microsomes from liver were obtained by ultracentrifugation as described previously (Clement et al, 1996). The kidney microsomes were prepared analogously.Human and pig mitochondria were prepared by differential centrifugation as described previously (Beattie, 1968;Kline et al, 1986) with slight modifications (Clement and Deters, 2000). To account for the biological variability liver samples from pigs or human organs were pooled (from at least three individuals per pool).…”

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confidence: 99%

Characterization of in Vitro Biotransformation of New, Orally Active, Direct Thrombin Inhibitor Ximelagatran, an Amidoxime and Ester Prodrug

Clement¹,

Lopian²

2003

Drug Metab Dispos

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This article is available online at http://dmd.aspetjournals.org ABSTRACT:N-Hydroxylated amidines (amidoximes) can be used as prodrugs of amidines. The prodrug principle was developed in our laboratory for pentamidine and had been applied to several other drug candidates. One of these compounds is melagatran, a novel, synthetic, low molecular weight, direct thrombin inhibitor. To increase the poor oral bioavailability due to its strong basic amidine functionality selected to fit the arginine side pocket of thrombin, the less basic N-hydroxylated amidine was used in addition to an ethyl ester-protecting residue. The objective of this investigation was to study the reduction and the hydrolytic metabolism of ximelagatran via two mono-prodrugs (N-hydroxy-melagatran and ethyl-melagatran) to melagatran by in vitro experiments. New high-performance liquid chromatography methods were developed to analyze all four compounds. The biotransformation of ximelagatran to melagatran involving the reduction of the amidoxime function and the ester cleavage could be demonstrated in vitro by microsomes and mitochondria from liver and kidney of pig and human, and the kinetic parameters were determined. So far, one enzyme system capable of reducing N-hydroxylated structures has been identified in pig liver microsomes, consisting of cytochrome b 5 , NADH-cytochrome b 5 reductase, and a P450 isoenzyme of the subfamily 2D. This enzyme system also reduces ximelagatran and N-hydroxy-melagatran. The participation of recombinant human CYP1A2, 2A6, 2C8, 2C9, 2C19, 2D6, and 3A4 with cytochrome b 5 and b 5 reductase in the reduction can be excluded. In summary, ximelagatran and N-hydroxy-melagatran are easily reduced by several enzyme systems located in microsomes and mitochondria of different organs.Melagatran is the active form of the novel, oral thrombin inhibitor ximelagatran (Gustafsson et al., 2001). Melagatran has suboptimal bioavailability because of the presence of a carboxylic acid, a secondary amine, and an amidine residue resulting in a charged molecule at physiological pH values. The prodrug ximelagatran was developed with a view to improving absorption. Ximelagatran comprises an ethyl ester group in place of the carboxylic acid and an N-hydroxyamidine group in place of the amidine. After protonation of amidines at the double-bonded nitrogen, cations are formed that are highly stabilized by mesomerism. Amidines are very strong bases (Albert et al., 1948) and protonated under physiological conditions. By introduction of an oxygen atom in the amidine functional group, the basicity is lowered by 5 pK a value. Amidoximes have been used as prodrugs for certain amidine-containing drugs, including pentamidine derivatives (Clement, 1993) and sibrafiban (Weller et al., 1996). It has been shown that certain amidoximes are rapidly reduced in vitro and in vivo to the amidines (Clement et al., 1988(Clement et al., , 1992Hauptmann et al., 1988). More recently, it has been demonstrated that the model compound benzamidoxime is reduced by microsom...

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“…In 1978, Mole et al (30) determined that rat heart homogenates could oxidize lactate 20% more rapidly than pyruvate, whereas with homogenates of white skeletal muscle lactate and pyruvate, oxidation rates were essentially the same. In the 1980s, Brandt, Kline, and colleagues (4,24) demonstrated presence of LDH in rat liver, kidney, and heart mitochondria. Furthermore, they showed that isolated liver mitochondria were capable of oxidizing lactate at least as fast as pyruvate (24).…”

Section: Discussionmentioning

confidence: 99%

“…In the 1980s, Brandt, Kline, and colleagues (4,24) demonstrated presence of LDH in rat liver, kidney, and heart mitochondria. Furthermore, they showed that isolated liver mitochondria were capable of oxidizing lactate at least as fast as pyruvate (24). They interpreted their results as permitting the lactate shuttle (4).…”

Section: Discussionmentioning

confidence: 99%

Colocalization of MCT1, CD147, and LDH in mitochondrial inner membrane of L6 muscle cells: evidence of a mitochondrial lactate oxidation complex

Hashimoto¹,

Hussien²,

Brooks³

2006

American Journal of Physiology-Endocrinology and Metabolism

201

211

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Results of previous studies suggested a role of mitochondria in intracellular and cell-cell lactate shuttles. Therefore, by using a rat-derived L6 skeletal muscle cell line and confocal laser-scanning microscopy (CLSM), we examined the cellular locations of mitochondria, lactate dehydrogenase (LDH), the lactate-pyruvate transporter MCT1, and CD147, a purported chaperone protein for MCT1. CLSM showed that LDH, MCT1, and CD147 are colocalized with the mitochondrial reticulum. Western blots showed that cytochrome oxidase (COX), NADH dehydrogenase, LDH, MCT1, and CD147 are abundant in mitochondrial fractions of L6 cells. Interactions among COX, MCT1, and CD147 in mitochondria were confirmed by immunoblotting after immunoprecipitation. These findings support the presence of a mitochondrial lactate oxidation complex associated with the COX end of the electron transport chain that might explain the oxidative catabolism of lactate and, hence, mechanism of the intracellular lactate shuttle. lactate transport; cell culture; confocal laser-scanning microscopy; immunocytochemistry; immunoprecipitation ONCE THOUGHT TO BE FORMED as the result of a lack of oxygen, lactate is now known to be formed continuously in the presence of oxygen (6). On the basis of isotope tracer, arterial-venous difference mass balance, and biopsy studies (2, 8) we now know that working skeletal muscle is not only the major site of lactate production but also the major site of its removal, mainly via oxidation. Lactate shuttling between sites of formation and disposal represents a carbon source for oxidation and gluconeogenesis and a vehicle for cell-cell signaling via redox changes (6). The carboxylic acids lactate and pyruvate are exchanged across lipid bilayer membranes by facilitated proton-linked transport (34, 35, 37) involving a family of monocarboxylate transport (MCT) proteins (15). Of the now recognized superfamily of solute transporters (18), the first four isoforms are lactate-pyruvate transporters. The first isoform discovered (MCT1) (15) is thought to facilitate cellular lactate uptake and oxidation (2,6,12) and is abundant in sarcolemmal and mitochondrial membranes of striated muscle and other cells, including neurons (7,10,28,29). In contrast, the MCT2 isoform, a high-affinity pyruvate transporter (5,14,27), is found predominantly in peroxisomes and is highly expressed in liver (29). In adult mammalian muscle, MCT2 appears in sarcolemmal and subsarcolemmal domains of oxidative muscle fibers, but MCT2 is not abundant compared with MCT1 (20). MCT3 appears to be specialized for function in the basal lateral membrane of retinal epithelium (40). MCT4, the other isoform expressed in muscle, is associated with sarcolemmal membranes of fast-twitch fibers (20). As part of the cell-cell lactate shuttle, sarcolemmal MCTs are thought to facilitate tissue and cellular lactate exchange and use.With the accumulating mass of physiological (2, 8) and other data showing tissue lactate exchange and wide diversity of tissue MCT expression (6, 19), there a...

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Localization of l-lactate dehydrogenase in mitochondria

Cited by 64 publications

References 13 publications

Ultracentrifugation studies of the location of the site involved in the interaction of pig heart lactate dehydrogenase with acidic phospholipids at low pH. A comparison with the muscle form of the enzyme

Ultracentrifugation studies of the location of the site involved in the interaction of pig heart lactate dehydrogenase with acidic phospholipids at low pH. A comparison with the muscle form of the enzyme

Characterization of in Vitro Biotransformation of New, Orally Active, Direct Thrombin Inhibitor Ximelagatran, an Amidoxime and Ester Prodrug

Colocalization of MCT1, CD147, and LDH in mitochondrial inner membrane of L6 muscle cells: evidence of a mitochondrial lactate oxidation complex

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