Recognition of partially-folded mitochondrial malate dehydrogenase by GroEL. Steady and time-dependent emission anisotropy measurements

The binding of partially-folded mitochondrial malate dehydrogenase (mMDH) to GroEL was assessed by steady and nanosecond emission spectroscopy. Partially-folded intermediates of mMDH show significant residual secondary structure when examined by CD spectroscopy in the far UV. They bind the extrinsic fluorescent probe ANS and the protein-ANS complexes display a rotational correlation time of 19 ns. Similar rotational correlation time (phi = 18.6 ns) was determined for partially-folded species tagged with anthraniloyl. GroEL recognizes partially-folded species with a K(D) approximately 60 nM. The rotational correlation time of the complex, i.e., GroEL-mMDH-ANT, approaches a value of 280 ns in the absence of ATP. Reactivation of mMDH-ANT by addition of GroEL and ATP brings about a significant decrease in the observed rotational correlation time. The results indicate that partially-folded malate dehydrogenase is rigidly trapped by GroEL in the absence of ATP, whereas addition of ATP facilitates reactivation and release of folded conformations endowed with catalytic activity.     

Lactate and malate dehydrogenase and alpha-esterases in oligospermia

Lactate dehydrogenase (LDH), malate dehydrogenase, and alpha-esterase were studied electrophoretically in a total of 99 semen samples obtained from normal, vasectomized, oligospermic, and infertile males. The enzymatic patterns were compared with total sperm count and percentage sperm motility. Lactate dehydrogenase X was absent in semen samples from oligospermic as well as vasectomized males. An extra LDH band (between the second and third LDH bands) was detectable in samples which had a low sperm count but higher motility (80 to 90%). This extra band was absent in samples with higher sperm count regardless of the level of percentage motility, suggesting that the extra band possibly may be related to motility factors of low sperm count but not normal sperm count. The fastest moving alpha-esterase bands were absent in samples from oligospermic or vasectomized males. These bands were also absent in samples from infertile patients having a very low sperm count. It is not clear whether the fast-moving esterase bands are related to sperm count or possible differences in hormonal levels of individuals with low or zero sperm count samples. The patterns of malate dehydrogenase did not differ in individuals with zero, low, or normal sperm count.     

Physical properties and chemical compositions of cytoplasmic and mitochondrial malate dehydrogenase from Physarum polycephalum

The malate dehydrogenase isoenzymes from Physarum polycephalum have been purified to homogeneity as confirmed by gel filtration chromatography, polyacrylamide gel disc electrophoresis and analytical ultracentrifugation. Certain physical and chemical parameters of the malate dehydrogenase isoenzymes reported here include sedimentation, molecular weight and subunit molecular weight. Most unique of the differences between the isoenzymes were the widely separate isoelectric points of 9.83 for mitochondrial malate dehydrogenase and 6.14 for the supernatant malate dehydrogenase. The amino acid analyses of each form were done revealing the isoenzymes were unquestionably unique proteins differing in the content of ten amino acids.     

Kinetic parameters of lactate dehydrogenase in liver and gastrocnemius determined by three quantitative histochemical methods

We determined the Michaelis constant (K(m)) and maximal velocity (Vmax) of lactate dehydrogenase (LDH) in periportal hepatocytes and skeletal muscle fibers by three different histochemical assay methods. Unfixed sections of mouse liver and gastrocnemius were incubated at 37C either on substrate (L-lactate)-containing agarose gel films or in aqueous assay media with and without 18% polyvinyl alcohol (PVA) as a tissue protectant. The absorbances of the formazan final reaction products were continuously measured at 584 nm in the cytoplasm of individual cells as a function of incubation time, using an image analysis system. The kinetic parameters of purified rabbit skeletal muscle LDH incorporated into polyacrylamide gel sections were similarly determined. The intrinsic initial velocities (vi) of LDH, corrected for "nothing dehydrogenase," were determined as described in the previous article. The Km and Vmax were calculated from Hanes plots of s/vi on L-lactate concentration (s). The Km values obtained with three assay methods were similar and in the range of 21.1-21.9 mM for pure LDH, 8.62-13.5 mM for LDH in mouse periportal hepatocytes, and 13.3-17.9 mM for LDH in mouse skeletal muscle fibers. The Vmax values determined on agarose gel substrate films and in aqueous assay media without PVA were in good agreement but were 53-65% lower when 18% PVA was included in the medium. These results indicate that catalytic center activity kcat of LDH is retarded by the high viscosity of PVA media because PVA hardly inhibited the enzyme. The K(m) values of LDH determined histochemically in skeletal muscle fibers and periportal hepatocytes were respectively three to five times and two to three times higher than those determined biochemically. These differences may be due to interactions of LDH with intracellular components.     

Interaction between citrate synthase and malate dehydrogenase. Substrate channeling of oxaloacetate

The interactions between pig heart citrate synthase and mitochondrial malate dehydrogenase or cytosolic malate dehydrogenase were studied using the frontal analysis method of gel filtration and by precipitation in polyethylene glycol. This method showed that an interaction between citrate synthase and mitochondrial malate dehydrogenase occurred but no interaction between citrate synthase and cytosolic malate dehydrogenase. Channeling of oxaloacetate in the malate dehydrogenase and citrate synthase-coupled systems was tested using polyethylene glycol precipitates of citrate synthase and mitochondrial malate dehydrogenase, and citrate synthase and cytosolic malate dehydrogenase. The effectiveness of large amounts of aspartate aminotransferase and oxaloacetate decarboxylase, as competing enzymes for the intermediate oxaloacetate, was examined. Aspartate aminotransferase and oxaloacetate decarboxylase were less effective competitors for oxaloacetate when precipitated citrate synthase and mitochondrial malate dehydrogenase in polyethylene glycol was used at low ionic strength compared with free enzymes in the absence of polyethylene glycol or with a co-precipitate of citrate synthase and cytosolic malate dehydrogenase. Substrate channeling of oxaloacetate with citrate synthase-mitochondrial malate dehydrogenase precipitate was inefficient at high ionic strength. These effects could be explained through electrostatic interactions of mitochondrial but not cytosolic malate dehydrogenase with citrate synthase.     

Simulation of the enzyme reaction mechanism of malate dehydrogenase

A hybrid numerical method, which employs molecular mechanics to describe the bulk of the solvent-protein matrix and a semiempirical quantum-mechanical treatment for atoms near the reactive site, was utilized to simulate the minimum energy surface and reaction pathway for the interconversion of malate and oxaloacetate catalyzed by the enzyme malate dehydrogenase (MDH). A reaction mechanism for proton and hydride transfers associated with MDH and cofactor nicotinamide adenine dinucleotide (NAD) is deduced from the topology of the calculated energy surface. The proposed mechanism consists of (1) a sequential reaction with proton transfer preceding hydride transfer (malate to oxaloacetate direction), (2) the existence of two transition states with energy barriers of approximately 7 and 15 kcal/mol for the proton and hydride transfers, respectively, and (3) reactant (malate) and product (oxaloacetate) states that are nearly isoenergetic. Simulation analysis of the calculated energy profile shows that solvent effects due to the protein matrix dramatically alter the intrinsic reactivity of the functional groups involved in the MDH reaction, resulting in energetics similar to that found in aqueous solution. An energy decomposition analysis indicates that specific MDH residues (Arg-81, Arg-87, Asn-119, Asp-150, and Arg-153) in the vicinity of the substrate make significant energetic contributions to the stabilization of proton transfer and destabilization of hydride transfer. This suggests that these amino acids play an important role in the catalytic properties of MDH.     

The clinical diversity and role of chemotherapy in lymphoproliferative disorder in liver transplant recipients

Two-dimensional analyses of mitochondrial proteins of Brassica napus revealed a set of differences in patterns of mitochondrial matrix proteins isolated from different nuclear backgrounds. One of these varying proteins was identified as mitochondrial malate dehydrogenase (mMDH;EC 1.1.1.37) by homology analyses of the partial amino acid sequence. Immunological detection identified additional mMDH subunits and detected different patterns of mMDH subunits in two distinct mitochondria types although they were isolated from plants with the same nuclear genotype. These differences are also reflected in isozym patterns, whereas Southern analyses showed no alteration in genome structure. Therefore mitochondria type-specific mMDH modifications are possible.     

Lactate dehydrogenase activity and isoform distribution in the rat pelvic ganglion: effects of diabetes and bladder outlet obstruction

We have previously shown that the intramural motor nerves in the rat bladder can function in anoxic conditions. The present study aims to explore the distribution and activity of lactate dehydrogenase (LDH), the key enzyme for ATP generation in anoxia. The activity and isoform distribution pattern of LDH was studied in pelvic ganglia from male and female rats. A histochemical investigation showed that the LDH activity was intense in the ganglion cells, and weak in the other tissue components (nerve bundles, connective tissue). The male pelvic ganglion weighted 55% more than the female pelvic ganglion, the enzyme activity per unit ganglion weight was 60% higher and the total LDH activity was 155% higher. The isoform distribution was similar, with M4 being dominant isoform, followed by M3H. Infravesical outlet obstruction in the female rat induced a threefold increase in ganglion weight, and the total LDH activity increased twofold. In this hypertrophic female ganglion a decreased relative amount of M4, and an increased amount of MH3, was found. Diabetes in the male rat had no effect on ganglion weight or its contents and isoform distribution of LDH.     

Effects of early severe malnutrition on heart and skeletal muscle lactate dehydrogenase

Following weaning, rats from litter sizes of 15 to 20 were subjected to severe food restriction for 10 weeks, and compared to rats from litter size 5 fed ad libitum. Percent M (muscle type) lactate dehydrogenase, M and H (heart type) subunit activity and lactate dehydrogenase activity (per g wet weight) determined both electrophoretically and spectrophotometrically were all significantly lower in separated left and right ventricles of the malnourished rats. There were no differences in skeletal muscle lactate dehydrogenase activity. Following 5 weeks of ad libitum feeding, the previously malnourished rats showed large increases in body weight. Now only the right ventricle showed slight decreases in H subunit and lactate dehydrogenase activity; other measurements in the right ventricle and all in the left ventricle had returned to control levels. Skeletal muscle lactate dehydrogenase activity was not different from controls.     

Electrostatic channeling of oxaloacetate in a fusion protein of porcine citrate synthase and porcine mitochondrial malate dehydrogenase

Mitochondrial malate dehydrogenase and citrate synthase are sequential enzymes in the Krebs tricarboxylic acid cycle. We have shown [Lindbladh, C., Rault, M., Hagglund, C., Small, W. C., Mosbach, K., Bülow, L., Evans, C., and Srere, P.A (1994) Biochemistry 33, 11692-11698] that a fusion protein of yeast mitochondrial citrate synthase and yeast mitochondrial malate dehydrogenase channels oxaloacetate between the active sites. A Brownian dynamics simulation model of porcine mitochondrial enzymes of citrate synthase and malate dehydrogenase was used [Elcock, A. H., and McCammon, A. M. (1996) Biochemistry 35, 12652-12658], showing that a positive electrostatic surface potential between the active sites of the fusion protein could account for the channeling of oxaloacetate we observed with the yeast fusion protein. Since the data were established with a yeast fusion protein and the model was with porcine fusion protein, we have now prepared and studied the porcine fusion protein. The channeling of the oxaloacetate intermediate was the same for the porcine fusion protein as it was for the yeast fusion protein. This channeling behavior is eliminated at high ionic strength. A fusion protein of porcine citrate synthase and porcine cytosolic malate dehydrogenase does not exhibit any channeling of oxaloacetate. A model of the fusion protein with the cytosolic malate dehydrogenase shows no clear positive electrostatic potential surface between the two active sites, thus distinguishing it from the fusion protein with the mitochondrial malate dehydrogenase. These results establish the electrostatic nature of channeling in mitochondrial fusion proteins.     

Influence of aging and endurance training on lactate dehydrogenase in liver and skeletal muscle

The purpose of this investigation was to determine the effects of aging and endurance training on lactate dehydrogenase (LDH) activity and isozyme pattern in liver and skeletal muscle. Male Fischer 344 rats (n = 30) of three different age groups (young, 4 months; middle-aged, 12 months and old, 22 months) were trained on a treadmill at 75% running capacity for 1 h/day, five times per week for 10 weeks. Age-matched sedentary controls (n = 36) were used for comparison. Total LDH enzyme activity was measured spectrophotometrically; LDH isozymes were separated by native 5.5% polyacrylamide gel electrophoresis and quantified densitometrically. With increasing age, hepatic LDH activity decreased 28%. Old sedentary animals displayed significantly less (22%) hepatic LDH 5 than young and middle-aged animals, and significantly more (40%) hepatic LDH 4 than middle-aged animals. Training resulted in a significant decrease (38%) in total hepatic LDH activity in young rats only. Young animals displayed a significant increase in hepatic LDH 3 (28%), whereas middle-aged animals exhibited a significant decrease in hepatic LDH 3 (40%) with training. No change in total hepatic LDH activity was exhibited in middle-aged or old rats with training. Neither aging or training had a significant effect on LDH activity or isozyme pattern in extensor digitorum longus (EDL). Similarly, LDH activity was maintained in soleus with age, and isozyme pattern was only negligibly affected. We conclude that with age there is a decline in hepatic LDH activity and a decrease in the LDH 5 isozyme. Endurance training induced significant decreases in hepatic LDH activity of young animals. However, these decreases were not a result of shifts in isozymal pattern. Further, LDH activity was maintained in EDL and soleus muscle with age. Finally, endurance training did not have a significant effect on LDH activity or isozymal pattern of EDL or soleus.     

Platelet-derived growth factor beta-receptors can both promote and inhibit chemotaxis in human vascular smooth muscle cells

The effect of the three platelet-derived growth factor (PDGF) isoforms AA, AB, and BB on migration was investigated in cultured human saphenous vein smooth muscle cells. The modified Boyden chamber technique yielded efficacies BB > AB, AA = 0. However, the BB concentration-response relationship displayed a pronounced peak, occurring between 1 and 10 ng/mL, with no response above this range. Checkerboard analysis showed that the promotion of migration at low concentrations was chemotactic in nature but that the downturn was independent of gradient. Furthermore, at high concentrations BB was able to prevent chemotaxis induced by fetal calf serum and epidermal growth factor (EGF). Experiments using low concentrations of BB in combination with high concentrations of AA to saturate PDGF alpha-receptors in the presence and absence of a neutralizing antibody to alpha-receptors revealed that alpha-receptor activation induced partial inhibition of chemotaxis but this did not account for the inhibition of migration by high concentrations of BB. Despite possessing no significant chemotactic action itself, high concentrations of the AB isoform completely inhibited BB induced chemotaxis. Taken together these results suggest that the chemotactic signal induced by PDGF is dominated by PDGF beta-receptors and switches from positive at low concentrations to negative at higher concentrations. Stimulation of DNA synthesis by the three isoforms (as measured by [3H] thymidine incorporation) yielded saturable responses for the AB and BB isoforms, with similar efficacy and weak or no response for the AA isoform. Concentration-dependent patterns of tyrosine phosphorylation of certain proteins mirrored the form of the chemotactic response and suggest one possible underlying regulatory mechanism to account for the disparity between PDGF-induced chemotaxis and DNA synthesis.     

Enzymatic activity of pig heart mitochondrial malate dehydrogenase monomolecular films by surface exchange technique

A technique for studying the catalytic activity of enzymes spread as a film at an air-water interface, by exchanging the subphase under the film to remove unspread enzyme molecules, was developed, and its effectiveness was studied using surface-spread mitochondrial malate dehydrogenase. Mitochondrial malate dehydrogenase formed stable films which gave reproducible pi-A curves. The enzyme activity was measured by the oxidation rate of reduced nicotinamide adenine dinucleotide (NADH) in the presence of the substrate oxalacetic acid. Oxalacetic acid and NADH were injected into the subphase. The catalytic activity of the enzyme was dependent on the surface pressure of the film. The maximum catalytic activity was observed at a surface pressure of 4.4 dynes/cm. The activity was higher at intermediate surface pressures than at very low or very high surface pressures. A high bulk catalytic activity was observed in the unstable region, i.e., at a high degree of compression, of the film. The catalytic activity of the surface-spread enzyme was only a fraction of an equivalent amount of enzyme in solution.     

Overexpression of cytosolic malate dehydrogenase (MDH2) causes overproduction of specific organic acids in Saccharomyces cerevisiae

Saccharomyces cerevisiae accumulates L-malic acid through a cytosolic pathway starting from pyruvic acid and involving the enzymes pyruvate carboxylase and malate dehydrogenase. In the present study, the role of malate dehydrogenase in the cytosolic pathway was studied. Overexpression of cytosolic malate dehydrogenase (MDH2) under either the strong inducible GAL10 or the constitutive PGK promoter causes a 6- to 16-fold increase in cytosolic MDH activity in growth and production media and up to 3.7-fold increase in L-malic acid accumulation in the production medium. The high apparent Km of MDH2 for L-malic acid (11.8 mM) indicates a low affinity of the enzyme for this acid, which is consistent with the cytosolic function in the enzyme and differs from the previously published Km of the mitochondrial enzyme (MDH1, 0.28 mM). Under conditions of MDH2 overexpression, pyruvate carboxylase appears to be a limiting factor, thus providing a system for further metabolic engineering of L-malic acid production. The overexpression of MDH2 activity also causes an evaluation in the accumulation of fumaric acid and citric acid. Accumulation of fumaric acid is presumably caused by high intracellular L-malic acid concentrations and the activity of the cytosolic fumarase. The accumulation of citric acid may suggest the intriguing possibility that cytosolic L-malic acid is a direct precursor of citric acid in yeast.     

Differential gene expression in multilocus isozyme systmes of the developing green sunfish

The patterns of expression of eight multilocous isozyme systems were investigated in the differentiated adult tissues and the early embryonic stages (0-210 hours after fertilization) of the green sunfish, Lepomis cyanellus. Enzymes encoded by approximately 23 gene loci were resolved by starch-gel electrophoresis and detected by specific histochemical staining. The developmental patterns of these isozyme systems appear to be the result of the diffential expression of the multiple gene loci. Isozymic forms of glucoseophosphate isomerase (GPI-A2), malate dehydrogenase (MDH-A2), and creatine kinase (CK-C2) were present in most differentiated tissues, in the unfertilized eggs, and in all stages of embryonic development. Closely homologous forms of these isozymes (GPI-B2, MDH-B2, and CK-A2) were expressed predominantly in skeletal muscle and were first detected at around the time of hatching (38-42 hours). The similar temporal and spatial patterns of gene expressions for the GPI, LDH, MDH, and CK loci suggest that the duplicates loci encoding enzymes, diverged in their regulation to patterns of differential gene expression which are similar for each enzyme system.     

Tissue-specific responses of branched-chain alpha-ketoacid dehydrogenase activity in metabolic acidosis

In adrenalectomized rats, acidosis does not increase whole-body leucine oxidation unless a physiologic amount of glucocorticoids (dexamethasone) is also provided; an equivalent dose of dexamethasone without acidosis does not change leucine catabolism. Because the influences of acidification and glucocorticoids on branched-chain amino acid metabolism in specific organs are unknown, the function of branched-chain alpha-ketoacid dehydrogenase (BCKAD), the rate-limiting enzyme in branched-chain amino acid catabolism, in adrenalectomized rat skeletal muscle and liver, the two major tissues that degrade branched-chain amino acid was measured. In muscle of acidotic adrenalectomized rats receiving dexamethasone, basal and total BCKAD activities were increased 2.6- (P < 0.05) and 2.8-fold (P < 0.05), respectively. Neither acidosis nor dexamethasone alone increased these activities. BCKAD E1alpha subunit mRNA in muscle of acidotic rats given dexamethasone was increased 1.89-fold (P < 0.05) in parallel with the change in BCKAD activity; BCKAD E2 subunit mRNA was increased by acidosis, dexamethasone, or a combination of both stimuli. In contrast, basal BCKAD activity in liver of rats with acidosis or dexamethasone was nearly threefold lower (P < 0.05) and changes in enzyme activity reflected reduced subunit mRNA. Thus, there are reciprocal, tissue-specific changes in BCKAD function in response to acidosis.     

Shortening velocity is different in longitudinal and circular muscle layers of the rabbit urethra

The aim of the study was to investigate whether the functional difference between circular and longitudinal muscles in the female rabbit urethra is reflected in their shortening properties and lactate dehydrogenase (LDH) activity. For mechanical experiments the preparations were chemically skinned to avoid influence of membrane-related mechanisms and to enable maximal activation. Force velocity relations and the maximal shortening velocity (v(max)) were determined using the isotonic quick-release method. The v(max) was three times higher in longitudinal muscle. LDH isoform pattern was determined on agarose gels. The M-subunit, favourable for lactate formation, constituted 70% of the total in both types of muscle. There was no difference in the LDH isoform pattern despite the marked difference in v(max). We conclude that the difference in v(max) reflects differences in the contractile machinery itself. These mechanical characteristics are advantageous for the role of the circular as a tonic muscle contracting during bladder filling, and the longitudinal as a phasic muscle active in opening up the urethra during micturition.     

Regulation of rat 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Role of the NH2-terminal region

The role of the NH2-terminal region of the liver and skeletal muscle 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatases was investigated, as well that of a mutant of the liver isoform lacking the first 22 amino acids, by the overexpression of these enzymes in Escherichia coli and the comparison of their kinetic properties. The muscle isoform and the deletion mutant had Km values for fructose 6-phosphate which were 50- and 20-fold higher, respectively, than that of the liver isoform, and the bisphosphatase maximal velocity of the liver deletion mutant was 4-fold higher than that of the native liver isoform. Phosphorylation of the liver isoform increased bisphosphatase activity by 2-3-fold and the Km for fructose 6-phosphate of the 6-phosphofructo-2-kinase by 10-15-fold, but these kinetic effects were greatly diminished for the deletion mutant despite equivalent phosphorylation by cAMP-dependent protein kinase. Arg-173 of the skeletal muscle isoform was found to be functionally equivalent to the residue corresponding to the essential fructose 6-phosphate binding residue of the liver kinase domain, Arg-195. The results suggest that 1) the NH2-terminal regions of the liver and skeletal muscle isoforms are important determinants of fructose 6-phosphate affinity, and 2) the initial 22 amino acids of the liver isoform exert an inhibitory influence on the bisphosphatase and mediate, at least in part, the response of both activities of the enzyme to cAMP-dependent phosphorylation.     

Malate dehydrogenase from the green gliding bacterium Chloroflexus aurantiacus is phylogenetically related to lactic dehydrogenases

The gene encoding malate dehydrogenase (MDH) from Chloroflexus aurantiacus was cloned, sequenced, and analyzed. The mdh gene corresponded to a polypeptide of 309 amino acids with a molecular mass of 32,717 Da. The primary structure and the coenzyme-binding domain showed a high degree of similarity to lactate dehydrogenase (LDH), whereas the conserved amino acids that participate in substrate binding were those typical of MDHs. Using PCR techniques, the mdh gene was cloned in the expression vector pET 11a, and large amounts of active C. aurantiacus MDH were produced in Escherichia coli after induction with isopropyl beta-D-thiogalactoside. The expressed enzyme thus obtained was purified and retained full activity at 55 degrees C. High levels of expression of mdh were also observed when the gene and its flanking sequences were cloned into pUC18/19, indicating that the putative sigma 70 promoter sequences found upstream of the C. aurantiacus mdh functioned in E. coli. When these sequences were deleted, the expression in E. coli was reduced dramatically.     

In vivo tissue specific modulation of rat insulin receptor gene expression in an experimental model of mineralocorticoid excess

Insulin receptor (IR) gene expression at the mRNA level was investigated in hindlimb skeletal muscle, epididymal adipose tissue and in the liver of rats exposed to prolonged in vivo administration of deoxycorticosterone acetate (DOCA). Following treatment, plasma insulin levels were reduced while glucose levels increased compared to values in control rats. DOCA-treated animals showed an increase in blood pressure and a reduction in body weight. This treatment also induced hypokalemia and decreased plasma protein levels. Sodium levels were unaffected. Moreover, no differences in DNA and protein content or in the indicator of cell size (protein/DNA) were observed in the skeletal muscle or adipose tissue of animals. In contrast, there was a clear increase in the protein and DNA contents of the liver with no change in the indicator of cell size. Northern blot assays revealed 2 major IR mRNA species of approximately 9.5 and 7.5 Kb in the 3 tissues from control animals. DOCA treatment induced no change in the levels of either RNA species in skeletal muscle. However, a decrease of approximately 22% was detected in the levels of both species in adipose tissue whereas the liver showed an increase of 64%. These results provide the first evidence for an in vivo tissue-specific modulation of IR mRNA levels under experimental conditions of mineralocorticoid excess.