Two isoenzymes of malate dehydrogenase were isolated and partially purified from the midge larva Glyptotendipes barbipes. Differential centrifugation followed by cellulose acetate and polyacrylamide gel electrophoresis revealed one isoenzyme associated with the mitochondrial fraction and another form found only in the cytoplasm.

Thesis (Ph. D.)--University of Oregon, 1999. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 67-71). Also available for download via the World Wide Web; free to University of Oregon users. Address: http://wwwlib.umi.com/cr/uoregon/fullcit?p9948022.

Recent investigations on the crude pigeon liver extracts have shown the presence of an enzyme which catalyzes the oxidative decarboxylation of malate to pyruvate and carbon dioxide. The name malic enzyme has been given to this enzyme to distinguish it from malic dehydrogenase. Under aerobic conditions the addition of ferrous sulfate resulted in inhibition of the action of malic enzyme. Subsequent investigation showed that the inhibition obaerved was not due to ferrous ions, but due to ferric ions which were contaminating the ferrous sulfate. It was also shown that under aerobic conditions ferrous ions were rapidly oxidized to ferric ions. Investigation of this oxidation of ferrous ions proved the reaction to be non-enzymatic, and to require the presence of malate at a pH of 5.0. The presence of pyruvate also caused oxidation of ferrous ions to ferric ions, but at a much slower rate. The oxidation of ferrous to ferric ions was inhibited by the addition of glutathione, It is thermodynamically possible that this observed inhibition was due to the oxidation of ferrous ions to ferric ions by the -SH group of glutathione. Three possible sites of inhibition exist. The conversion of DPN to TPN, the reduction of pyruvate to lactate, and the oxidative decarboxylation of malate. The first two reactions showed no inhibition in the presence of ferric ions while the latter was inhibited. Varying concentrations of malate indicated that the reaction is not one of competition for malate by the ferric ions and the malic enzyme. The gradual increasing of the enzyme concentration in a reaction flask inhibited with ferric ions reveals a point where the inhibition is overcome. Increasing the enzyme concentration beyond this point resulted in a constant increase in carbon dioxide evolution per unit enzyme increase up to the point where the availability of the substrate effected the rate of reaction. The addition of glutathione to the reaction inhibited with low concentrations of ferric ions caused a decrease in the observed inhibition. It is speculated that the affected enzyme possesses functional groups which are oxidized by ferric ions, inactivating the enzyme and limiting the reaction. In the presence of glutathione, the ferric ions would be reduced to ferrous ions causing a decrease in inhibition.

Ergogenic L-citrulline and malate are amino acids used in specific combination to effect muscular endurance during athletic performances. Purpose: The study aimed to investigate the ergogenic properties of citrulline-malate (CM) during a resistance training protocol. Methods: Utilizing a randomized, counterbalanced, double blind study, fifteen trained males completed a resistance training protocol once using placebo (PL) and once with CM (8.0g). Results: CM supplementation increased repetitions in chin-ups, reverse chin-ups, push-ups, and total trial repetitions. Blood lactate was significantly increased post-exercise compared to pre-exercise, but was not significantly different between CM and placebo. Further, a significant interaction effect was revealed for systolic blood pressure, a significant condition effect for diastolic blood pressure, and a significant time effect for HR. Post-hoc analysis revealed that SBP responses were more elevated in the placebo condition during recovery. Conclusion: Collectively, these novel findings suggest CM increases muscular endurance during upper body resistance exercise.

by Agnes, Lo Shuk Yee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves [27-33] (2nd gp.)). / Chapter PART 1: --- Human Heart cDNA Library Sequencing / Chapter A) --- Introduction of human heart cDNA library sequencing / Chapter A.1 --- Human genome project / Chapter A.2 --- The aim of human genome project / Chapter A.3 --- Automatic sequencing / Chapter A.4 --- Cycle sequencing reaction / Chapter A.5 --- Human heart cDNA library sequencing project / Chapter B) --- Methods and materials / Chapter (I) --- Preparation of plating bacterial-Y1090 / Chapter (II) --- Plating the bacteriophage with blue-white visual selection / Chapter (III) --- Amplification of bacteriophage cDNA clones by PCR / Chapter (IV) --- Purification and quantitation of PCR products / Chapter (V) --- Cycle DNA sequencing of PCR products / Chapter (VI) --- Casting the sequencing gel / Chapter (VII) --- Sequencing by Pharmacia LKB A.L.F. DNA Sequencer / Chapter (VIII) --- Editing and saving the DNA sequence / Chapter (IX) --- Sending the DNA sequence to Genbank by E-mail / Chapter (X) --- Usage of the Genbank database / Chapter C) --- Results / Chapter D) --- Discussions / Chapter D.1 --- Application of human genomic project / Chapter D.2 --- Interpretation of the sequencing results / Chapter D.3 --- Quality of cDNA libraries and representation of mRNA population / Chapter D.4 --- "Gene expression profile in three different organs-heart, brain and liver" / Chapter D.5 --- Population study of the cDNA library / Chapter D.6 --- Isolation of a large number of novel genes by substraction cDNA library / Chapter D.7 --- Screening method to find out the complete coding sequence of interesting genes / Chapter D.8 --- Technical problems encountered and managed / Chapter PART 2: --- Identification of human cytosolic malate dehydrogenase by large scale human heart cDNA library sequencing / Chapter CHAPTER 1: --- Introduction of malate dehydrogenase / Chapter 1.1 --- Malate dehydrogenase--Kreb's cycle enzyme / Chapter 1.2 --- Two stereospecific forms of dehydrogenase / Chapter 1.3 --- NAD-binding domain / Chapter 1.4 --- The active site / Chapter 1.5 --- Comparison of surface properties between cMDH and mMDH / Chapter 1.6 --- N-terminal region and mitochondrial import / Chapter 1.7 --- Subunit-subunit interactions / Chapter 1.8 --- Physiological importance of malate dehydrogenase / Chapter 1.9 --- Secondary structure-total 11 β-strands and 9 α-helixes / Chapter 1.10 --- Objectives of the thesis / Chapter CHAPTER 2: --- Cloning and sequence analysis of human cytosolic malate dehydrogenase (hcMDH) / Chapter 2.1 --- Cloning of human cytosolic malate dehydrogenase (hcMDH) / Chapter 2.1.1 --- Methods and materials / Chapter 2.1.1.1 --- Cloning full length of hcMDH into expression vector pAED4 / Chapter 2.1.1.2 --- Preparation of competent cell-JM109 for transformation / Chapter 2.1.1.3 --- Minipreparation of plasmid DNA / Chapter 2.1.1.4 --- Midi-preparation of bacteriophage λDNA by QIAGEN´ёØ / Chapter 2.1.1.5 --- Titration of bacteriophage λ of human adult heart cDNA library / Chapter 2.1.1.6 --- Preparation of soft-agarose lysates / Chapter 2.1.1.7 --- Elution of DNA from agarose gel by GENECLEAN´ёØ / Chapter 2.1.2 --- Results / Chapter 2.1.3 --- Discussions / Chapter 2.2 --- Sequence analysis of human cytosolic malate dehydrogenase (hcMDH) / Chapter 2.2.1 --- Methods and materials: Autoread sequencing / Chapter (I) --- Annealing of primer to double-stranded template / Chapter (II) --- Sequencing / Chapter 2.2.2 --- Results and discussions / Chapter 2.3 --- Amino acids and protein structure analysis of cMDH / Chapter CHAPTER 3 : --- "Protein expression, partial purification and folding experiments of human cytosolic malate dehydrogenase (hcMDH)" / Chapter 3.1 --- Protein expression of hcMDH in E. coli / Chapter 3.1.1 --- Methods and materials / Chapter 3.1.1.1 --- Protein expression induced by IPTG / Chapter 3.1.1.2 --- Isoelectric focusing (IEF)-two dimensional gel electrophoresis / Chapter (I) --- First dimensional electrofocusing / Chapter (II) --- The second dimension SDS-PAGE electrophoresis / Chapter (III) --- Sample preparation / Chapter 3.1.2 --- Results / Chapter 3.1.3 --- Discussions / Chapter 3.1.3.1 --- The properties of expressed protein of hcMDH / Chapter 3.1.3.2 --- T7 expression system / Chapter 3.1.3.3 --- Strong φ 10 promoter / Chapter 3.1.3.4 --- E.coli BL21 host cell / Chapter 3.2 --- Partial purification and folding experiments of hcMDH / Chapter 3.2.1 --- Methods and materials / Chapter 3.2.1.1 --- Partial purification of hcMDH expressed protein / Chapter (I) --- Preparation of supernatant from E.coli crude extract / Chapter (II) --- Ion-exchange column chromatography / Chapter (III) --- Affinity chromatography / Chapter (IV) --- Gel filtration on a Sepharose CL-6B column / Chapter 3.2.1.2 --- Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) / Chapter 3.2.1.3 --- Staining the protein gel by the Coomassie Blue R-250 method / Chapter 3.2.1.4 --- Staining the protein gel by the Silver staining Method / Chapter 3.2.1.5 --- Quantitation of protein by the Bradford Method / Chapter 3.2.1.6 --- Native gel electrophoresis / Chapter 3.2.1.7 --- Malate dehydrogenase MDH enzyme staining method / Chapter 3.2.1.8 --- Malate dehydrogenase MDH enzyme assay / Chapter 3.2.1.9 --- Fast protein liquid chromatography (FPLC) / Chapter 3.2.1.10 --- Protein folding experiment / Chapter 3.2.1.11 --- Eukaryotic expression of hcMDH / Chapter 3.2.2 --- Results / Chapter 3.2.2.1 --- Partial purification by chromatography / Chapter 3.2.2.2 --- Native gel / Chapter 3.2.2.3 --- FPLC / Chapter 3.2.2.4 --- To aid folding of protein by adding NADH / Chapter 3.2.2.5 --- Eukaryotic expression / Chapter 3.2.3 --- Discussions / Chapter 3.2.3.1 --- Purification of malate dehydrogenase MDH / Chapter 3.2.3.2 --- "Methods for visualizing dehydrogenase enzymes, e.g. malate dehydrogenase" / Chapter 3.2.3.3 --- The presence of unfold hcMDH protein in bacteria / Chapter 3.2.3.4 --- Folding of protein by heat shock protein GroE / Chapter 3.2.3.5 --- Eukaryotic expression / Chapter CHAPTER 4: --- Master screening of single base change by PCR-SSCP (Single Strand Conformational Polymorphism) / Chapter 4.1 --- Theory of SSCP / Chapter 4.2 --- Methods and materials / Chapter 4.3 --- Results / Chapter 4.4 --- Discussions / Chapter 4.4.1 --- The procedure of SSCP / Chapter 4.4.2 --- An alternative quick detection method for polymorphism of hcMDH at position 565--by automatic sequencing / Chapter 4.4.3 --- Other detection methods-- RNA-PCR and ddF / Chapter 4.4.4 --- Parameters affecting sensitivity of SSCP / Chapter 4.4.5 --- Application of SSCP / Chapter CHAPTER 5: --- Southern hybridization and In situ hybridization / Chapter 5.1 --- Southern blot analysis of human cytosolic malate dehydrogenase (hcMDH) / Chapter 5.1.1 --- Methods and materials / Chapter (I) --- Transfer genomic DNA to Nylon membrane / Chapter (II) --- Synthesis of radiolabelling cDNA probe / Chapter (III) --- Pre-hybridization and hybridization reaction / Chapter 5.1.2 --- Results / Chapter 5.1.3 --- Discussions / Chapter 5.2 --- In situ hybridization / Chapter 5.2.1 --- Methods and materials / Chapter (I) --- Preparation of Dig labelling probe by random primed labelling / Chapter (II) --- Estimating the yield of Dig-labelled nucleic acids / Chapter (III) --- Denaturation and hybridization of the hcMDH probe with animal tissues / Chapter (IV) --- Color development of the tissue / Chapter 5.2.2 --- Results / Chapter 5.2.3 --- Discussions / Chapter 5.2.3.1 --- Cellular distribution of hcMDH / Chapter 5.2.3.2 --- The principle of in situ hybridization / Chapter 5.2.3.3 --- Specimen preparation / Chapter 5.2.3.4 --- Hybridization conditions / Chapter 5.2.3.5 --- "Ontogeny of MDH in rabbit fetal brain, heart and lung" / Appendixes: / "Appendix I: 531 random cDNA clones from clone no. J950 to K951 in human heart cDNA library sequencing project. The name of clones, accession number, the length of the partial sequence and percentage of match are listed" / Appendix II: The new accession no. of Novel clones in Genbank / "Appendix III: The enzymatic reaction, molecular weigth, specific activity and Michaelis constants of different sources of malate dehydrogenase" / Appendix IV: The full sequence of nucleic acids and amino acids of human cytosolic malate dehydrogenase hcMDH. Accession no. of hcMDH is U20352 in Genbank / Appendix V: Nucleotide sequences of the mouse cMDH gene / Appendix VI: Nucleotide sequences of the mouse mMDH gene / Appendix VII: Structural organization of the mouse cytosolic malate dehydrogenase and its comparison with that of the mouse mitochondrial malate dehydrogenase gene

No description available.

A Sinorhizobium meliloti (S. meliloti ) mutant, Rm30O49, deficient in malate dehydrogenase (MDH) activity was isolated via random Tn5tac1 mutagenesis. DNA sequence analyses revealed 60 the inaction is within the mdh gene. Rm30049 lacks MDH activity under all growth conditions, but shows increased or decreased activities of the TCA cycle enzymes 2-oxoglutarate dehydrogenase and succinate dehydrogenase in the presence or absence, respectively, of IPTG (isopropyl beta-D-thiogalactoside). The symbiotic phenotype of the mutant is an inability to fix nitrogen. Alfalfa seedlings inoculated with Rm30049 produced small white root nodules, but were chlorotic and failed to reach a wild-type shoot dry weight. Cosmid clone pDS15 was isolated by heterologous complementation of a Rhizobium leguminosarum sucD mutant by the S. meliloti pLAFR1 clone bank. This cosmid also restored MDH activity to Rm30049, and complemented the mutant growth and symbiotic phenotypes. Three Tn5 insertions isolated in pDS15 within sucA failed to complement Rm30049. DNA sequence analyses indicate that the mdh gene is part of the TCA cycle operon with sucCD, and that downstream and upstream of this, are operons encoding sucAB and sdhCDAB, respectively.

M.Sc. (Biochemistry) / Please refer to full text to view abstract

No description available.

Doctor of Philosophy / Food Science Institute / Melvin C. Hunt / Several experiments were conducted to evaluate factors affecting myoglobin redox forms stability and detection of myoglobin redox forms using near infrared (NIR) spectroscopy. In experiment 1, we investigated the relationship between metmyoglobin (MMb) reduction and oxidation of malate to α-ketoglutarate with regeneration of reduced nicotinamide adenine dinucleotide (NADH) via malate dehydrogenase (MDH). Our specific objectives for this experiment were: (1) to examine the interaction of malate and MDH to reduce MMb in vitro, (2) to determine the influence of pH, temperature, NAD[superscript]+, and malate concentration on MDH enzyme activity and MMb reduction, and (3) to determine the effects of malate on NADH generation, metmyoglobin reducing activity, and color stability using beef muscles (Longissimus lumborum, Psoas major, and Semitendinosus) extracts. We observed that, nonenzymatic reduction of horse MMb in vitro in a malate-MDH-NADH system increased with increasing NAD[superscript]+ and L-malate concentrations. Our findings further confirmed that reduction of MMb in beef extract was NAD[superscript]+ and malate concentration dependent (p < 0.05). A model system was described for studying mechanisms of enzymatic reduction of metmyoglobin reduction as a means to improve meat color and the results support the hypothesis that malate can replenish NADH via MDH activity, ultimately resulting in stabilizing myoglobin redox chemistry.In experiment 2, we assessed the ability of mitochondrial and cytoplasmic malate dehydrogenase present in postrigor bovine skeletal muscle to utilize malate as fuel for NADH regeneration and MMb reduction via the malate-NAD-MMb system. Furthermore, addition of lactate to beef mitochondrial and cytoplasmic isolates was evaluated to determine if interactions between malate and lactate increased MMb reduction. Addition of malate to isolated beef mitochondrial and cytoplasmic isolates at pH 7.2 increased (p < 0.05) MMb reduction. MMb reduction resulting from the addition of malate and lactate was equal or greater than MMb reduction resulting from malate alone. The findings from this study provided evidence that mitochondria and cytoplasmic proteins isolated from beef skeletal muscles of different metabolic origin differ substantially in their enzymatic composition. Malate-MDH assisted-MMb reduction using Mitochondrial and cytoplasmic isolates from the three beef skeletal muscles exhibited substantial differences in enzymatic compositions and their ability to reduce MMb in vitro. Differences were also observed in the enzymatic characteristics of MDH-assisted-MMb among the three beef muscles. In experiment 3, we investigated the effects of three glycolytic and tricarboxylic acid cycle metabolites on myoglobin redox forms and their in influence on meat color stability. Eighteen combinations of malate (M), lactate (L), and pyruvate (P) were added to beef Longissimus lumborum, Psoas major, and Semitendinosus muscle homogenates to study their effects on metmyoglobin formation during incubation at 25 °C. Changes in surface color at 0, 2, 4, 8, and 12 hrs were evaluated using refecto-spectrophotometry [both L*a*b* and wavelengths specific for MMb]. Results from this study suggests that at 2% concentrations level of the individual metabolites (M, L, or P), the most effective metabolite at retarding MMb formation was L > M > P in the ST, and M > L > P in the PM and LL muscles. MMB was reduced most effectively with combination of metabolites where M+L > M+P > L+P. Enhancement of meat with these metabolites can effectively extend color life of postrigor meat apparently by providing more reducing conditions for myoglobin, thus increasing myoglobin redox form stability. Experiment 4 was conducted to determine how near-infrared (NIR) tissue oximeter measurements of post-rigor beef skeletal muscle relate with the more established methods of quantifying myoglobin redox states. Surface color differences were created by packaging steaks in vacuum (VAC), 80% O[subscript]2 and 20% CO[subscript]2 modified atmosphere packaging (HiOx MAP), polyvinyl chloride film overwrap (PVC), and HiOx MAP converted to PVC (HiOx-PVC) after 2 days. Changes in surface color and sub-surface pigments during display (0,2, 4, 10, and 15 days at 2 °C) were characterized by using a reflectance-spectrophotometer and a near-infrared tissue oximeter, respectively. Fiber orientation, storage, and packaging affected (p < 0.05) color, total pigment, deoxymyoglobin, and oxymyoglobin content. Tissue oximetry measurements appear to have potential for real-time monitoring of myoglobin redox forms and oxygen status of packaged meat, but fiber orientation needs to be controlled. In experiment 5, we investigated the response of frequency-domain multidistance (FDMD) NIR tissue oximetry for detecting absolute amounts of myoglobin (Mb) redox forms and their relationship to meat color stability. Four packaging formats were used to create different blends of Mb redox forms and meat colors during display. Changes in surface color and subsurface pigment forms during simulated display (0, 2, 4, and 10 d at 2 °C) were evaluated using surface reflecto-spectrophotometry (both L*a*b* and specific wavelengths) and FDMD NIR tissue oximetry. Data for both methods of direct measurement of oxymyoglobin and deoxymyoglobin were strongly related and accounted for 86 to 94% of the display variation in meat color. Indirect estimates of metmyoglobin ranged from r[superscript]2 = 59 to 85%. It appears that NIR tissue oximetry has potential as a noninvasive, rapid method for the assessment of meat color traits and may help improve our understanding of meat color chemistry in post-rigor skeletal muscle.