MDH protein interactions and phosphorylation: Engaging students with a novel scientific theme using CUREs

The metabolon theory describes the protein interactions of enzymes regulating a metabolic pathway. Through these interactions proteins can share substrates through direct channeling. One set of enzymes that are involved in metabolon complexes are citrate synthase and malate dehydrogenase. This study attempts to understand the interactions regulating the metabolon of malate dehydrogenase (MDH) and citrate synthase (CS), which are integral intermediate metabolic enzymes. Using size exclusion chromatography and sedimentation, previous studies identified the interaction between CS and MDH to be isoform specific, but no further work has been conducted to obtain affinity values or the biophysical nature of the interaction. Codon optimized His tagged recombinant proteins were expressed and purified using affinity chromatography. These enzymes include: glyoxysomal malate dehydrogenase (gMDH), human cytosolic malate dehydrogenase (MDH1), human mitochondrial malate dehydrogenase (MDH2), and human mitochondrial citrate synthase (CS). We analyzed the protein interactions using fast thermal melts which further explain the thermal stability of the the dimerization of the metabolons. We first show that protein concentration did not induce oligomerization of CS or any MDH isoform, indicating that any changes are not from MDH or CS homodimerization. Next, we investigated specific CS‐MDH interactions using fast thermal melts. The results show that from the initial melting temperature of CS, the addition of mitochondrial MDH had a greater shift in its melting temperature than the addition of cytosolic MDH, by 3–4°C. The melting point of CS was not altered in the presence of the plant MDH isoform. This indicates a specific and dramatic set of contacts for mitochondrial MDH and CS. However unlike the information found in the literature there was still a measurable shift in melting point when cytosolic MDH was incubated with CS. Overall this shows for the first time a quantifiable interaction between proteins in a well‐established pathway that will allow the further dissection of these interactions.Support or Funding InformationSupport for this project was from NSF REU 146045This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Malate dehydrogenases of leaf tissue from Spinacia oleracea: Properties of three isoenzymes

Nuclei, mitochondria, microsomes and cytosol were obtained from rat liver homogenates in sucrose solution by centrifugal fractionation. Dise electrophoresis exhibits the existence of two separately migrating malate dehydrogenases in these particulate fractions. One of them is preferably found in the mitochondria, the other one in the cytosol. The two malate dehydrogenase separated by disc electrophoresis are probably identical with the mitochondrial and cytoplasmic malate dehydrogenases differentiated earlier by other authors observing their different reaction kinetics with oxaloacetate.In starvation the malate dehydrogenase activity in the cytoplasm decreased, whereas that in the mitochondria increased. The increase in mitochondrial activity is prevented by actinomycin. The decrease of cytoplasmic malate dehydrogenase caused by starvation is aggravated by actinomycin. All these effects are similar if the malate dehydrogenase activities are expressed as units per g liver fresh weight, per g body weight or per mg protein content in the extract.The regulation of malate dehydrogenase in the mitochondria is discussed as repression by carbohydrates (or a metabolite originating from carbohydrates). The increase of the enzyme in starvation can therefore be understood as “derepression.” As was found by other authors [2, 16, 17a], mitochondrial malate dehydrogenase participates in gluconeogenesis in the liver; thus a repression of this enzyme by carbohydrates appears “useful for the cell economy”. Probably only the fraction of malate dehydrogenase activity additionally arising in starvation is necessary for gluconeogenesis, whereas the mitochondrial malate dehydrogenase present in normal nutrition is functioning for carbohydrate catabolism in the citric acid cycle.

Membrane-bound malate dehydrogenase in mitochondria from the alga Cyanidium caldarium

Malate Dehydrogenase (MDH) stands as a pivotal enzyme crucial for cellular energy metabolism, orchestrating the conversion of malate to oxaloacetate in both prokaryotic and eukaryotic organisms. This study delves into the evolutionary trajectories of MDH1 (cytoplasmic) and MDH2 (mitochondrial), offering substantial evidence supporting the archaebacterial origin of eukaryotes. The dataset spans nine groups, encompassing human MDH1 and MDH2, mammalian MDH1 and MDH2, amphibian MDH2, arthropod MDH2, amoeba MDH1, archaea, and bacteria. Protein BLAST analysis revealed significant sequence homology in mammalian MDH1, particularly among primates, underlining a close evolutionary connection. Conversely, lower eukaryotes, including amoeba, arthropods, and amphibians, exhibited marked divergence from human MDH1 and MDH2. Phylogenetic analysis unveils distinct clusters for MDH1 and MDH2, accentuating significant genetic diversity between mitochondrial and cytoplasmic MDH enzymes. Prokaryotic MDH sequences cluster with human mitochondrial MDH2, while MDH1 forms a separate cluster. Staphylococcus MDH aligns with archaeal MDH, emphasizing the diversity within bacterial MDH evolution. Protein variability analysis indicates noteworthy divergence of human MDH1 from prokaryotic MDH, while MDH2 displays comparatively lower divergence. Pairwise evolutionary divergence analysis sheds light on complex relationships among MDH protein sequences. Human MDH1 shows close evolutionary ties to mammalian MDH1, whereas MDH2 exhibits a unique pattern, aligning closely with mammalian MDH2, arthropods, and amphibians. Furthermore, MDH2 demonstrates closer proximity to bacterial MDH, supporting a bacterial origin of mitochondrial MDH. In contrast, MDH1 displays less divergence to archaeal MDH than its bacterial counterpart, endorsing an archaeal origin for cytoplasmic MDH. In conclusion, this study provides compelling support for the archaebacterial origin of eukaryotes, suggesting a bacterial endosymbiont within an archaeal host that evolved into mitochondria. It contributes valuable insights into MDH evolution, unraveling the intricate relationships and unique adaptations shaping the evolutionary history of eukaryotic cells.
 Bangladesh J Microbiol, Volume 40, Number 1, June 2023, pp 15-24

This paper addresses the question of how the staff involved in the teaching of a first-year chemistry course define the problems experienced by the students in the course. All of the staff involved in the teaching of first-year chemistry in a relatively small Australasian university agreed to be interviewed for the study. The interviews revealed not only the way these people defined the sources of student difficulty but also the ways in which they believed these could be addressed. The paper reports an analysis of these data which reveals the diversity of views held. The implications of the data analysis are explored.

Many studies of alcohol adaptation in Drosophila melanogaster have focused on the Adh polymorphism, yet the metabolic elimination of alcohol should involve many enzymes and pathways. Here we evaluate the effects of glycerol-3-phosphate dehydrogenase (Gpdh) and cytosolic malate dehydrogenase (Mdh1) genotype activity on adult tolerance to ethanol. We have created a set of P-element-excision-derived Gpdh, Mdh1, and Adh alleles that generate a range of activity phenotypes from full to zero activity. Comparisons of paired Gpdh genotypes possessing 10 and 60% normal activity and 66 and 100% normal activity show significant effects where higher activity increases tolerance. Mdh1 null allele homozygotes show reductions in tolerance. We use piggyBac FLP-FRT site-specific recombination to create deletions and duplications of Gpdh. Duplications show an increase of 50% in activity and an increase of adult tolerance to ethanol exposure. These studies show that the molecular polymorphism associated with GPDH activity could be maintained in natural populations by selection related to adaptation to alcohols. Finally, we examine the interactions between activity genotypes for Gpdh, Mdh1, and Adh. We find no significant interlocus interactions. Observations on Mdh1 in both Gpdh and Adh backgrounds demonstrate significant increases in ethanol tolerance with partial reductions (50%) in cytosolic MDH activity. This observation strongly suggests the operation of pyruvate-malate and, in particular, pyruvate-citrate cycling in adaptation to alcohol exposure. We propose that an understanding of the evolution of tolerance to alcohols will require a system-level approach, rather than a focus on single enzymes.

1. The mitochondrial malate dehydrogenase from rat liver has been purified to a state of homogeneity as judged by starch-gel electrophoresis and the cytoplasmic isoenzyme has been obtained in a partically purified state. 2. Inhibition of the isoenzymes by sulphite has been studied. 3. In mitochondria loaded with sulphite, the catalytic activity of the (partially inhibited) internal malate dehydrogenase has been measured by addition of oxaloacetate to the suspension medium and observation of the consequent decrease in fluorescence of NADH. 4. Addition of mitochondrial malate dehydrogenase to suspensions of mitochondria loaded with sulphite resulted in an increase in the level of intramitochondrial enzymic activity as measured by the above technique. Addition of the cytoplasmic isoenzyme did not result in such an increase. 5. These results show that mitochondria in suspension are permeable to the mitochondrial malate dehydrogenase but not to the cytoplasmic isoenzyme. 6. This conclusion has been confirmed by direct measurement of a decrease of enzyme activity in solution and an increase inside the mitochondria after incubation of organelles in solutions containing mitochondrial malate dehydrogenase. No such effect was observed with the cytoplasmic isoenzyme. 7. Some features of the permeation process have been studied.

Characterization of the Kinetics of Cardiac Cytosolic Malate Dehydrogenase and Comparative Analysis of Cytosolic and Mitochondrial Isoforms

MALATE DEHYDROGENASE ISOZYMES IN PLANTS: PREPARATION, PROPERTIES, AND BIOLOGICAL SIGNIFICANCE

The nucleotide sequence corresponding to codons for the 17-amino acid residues in the presumed targeting presequence for yeast mitochondrial malate dehydrogenase was removed by oligonucleotide-directed mutagenesis of the isolated gene (MDH1). Integrative transformation was used to insert the "leaderless" gene (mdhl-) into the MDH1 chromosomal locus of a strain containing a disrupted MDH1 gene. Expression of the mature form of malate dehydrogenase as a primary translation product was verified by demonstrating that the mature form is synthesized in mdhl- cells at the same rate as the precursor form in MDH1 cells in the presence of carbonyl cyanide m-chlorophenylhydrazone and by comparison of in vitro translation products of RNAs from mdhl- and MDH1 cells. Expression of mdhl- restores total cellular malate dehydrogenase activity to levels comparable to those in wild type cells and reverses the phenotype associated with strains containing MDH1 disruptions by restoring wild type rates of growth in media containing acetate as a carbon source. Immunochemical analyses and enzyme assays show comparable levels of malate dehydrogenase in the matrix fractions from mitochondria isolated from mdhl- and MDH1 cells and give no evidence for accumulation of the mature enzyme in the cytosol of mdhl- cells. These results indicate that the presequence for malate dehydrogenase is not essential for efficient mitochondrial localization or function in yeast.

Malate dehydrogenase fractions of the chick embryo were demonstrated after starch gel electrophoresis of homogenates of liver, brain and spleen. A total of seven malate dehydrogenase fractions were observed to occur in the chick embryo in an organ specific pattern. Treatment of the homogenates with urea, sodium chloride-sodium phosphate, and p-chloromercuribenzoate prior to electrophoresis revealed that only three distinct malate dehydrogenase-active proteins were presence. Two of these proteins exhibited properties similar to those previously reported for the supernatant malate dehydrogenase and mitochondrial malate dehydrogenase of other species. Becuase of the differing properties of chick malate and lactate dehydrogenase it is concluded that the molecular basis for malate dehydrogenase isozymes is different from that reported for lactate dehydrogenase isozymes.

<p><span lang="EN-US"><span style="font-family: Times New Roman; font-size: medium;">Chemistry is often perceived as being a dry and boring science subject, probably because of traditional teaching method, static textbook, lack of interactivity and difficulty in visualization of the abstract chemical concepts. E-learning resources (e-resources) and the utilization of technology have become one of the major trends in education, replacing the conventional textbook and traditional talk and chalk teaching method. A study was undertaken to assess students’ perceptions of the impact of the e-resources and technology on various aspects of their learning. Data were collected through a questionnaire involving 28 undergraduates, who were taking first-year Chemistry course in Taylor’s University, Malaysia. A Moodle e-Learning platform was made available to the students, where the students can assess e-resources anytime and anywhere. Innovative technologies such as mobile clicker, simulation and augmented reality (AR) were integrated into classroom instruction. Most of the students found the e-resources provided useful and well prepared them for the assessments in the course. Their understanding and ability to answer questions had improved. They were motivated to study the subject because it was more interactive and engaging. Overall findings showed that these e-resources and innovative technologies gave positive impact on students learning in chemistry.</span></span></p>

Recently, we found that the activity of malate dehydrogenase (MDH), one of the key enzymes in the biosynthesis of lipids, is regulated in Drosophila melanogaster by juvenile hormone (JH) and JH analogues. Here we report that the response of MDH to JH, however, depends on the developmental stage of the animal in relation to its endogenous titre of ecdysteroids. During the interecdysial period of the last instar, when endogenous ecdysteroids are low, MDH rapidly responded to JH by increasing activity, while little or no response was monitored in wandering (post-feeding) larvae, i.e., after a pulse of ecdysteroids. Activity of MDH in ecd1 and su(f)ts67g, two ecdysteroid-deficient mutants of Drosophila, increased during a temperature shift to 29 degrees C as compared to wild-type controls, and was sensitive to administration of JH. In contrast, when ecd1 or su(f)ts67g larvae during restrictive conditions were fed on a 20-hydroxyecdysone diet, JH was unable to increase the activity of the MDH enzyme. However, when JH was applied prior to 20-hydroxyecdysone feeding, the activity of MDH was significantly stimulated. Two phases of MDH response to JH, dependent on ecdysteroids, could be observed. The first phase was short and independent of RNA and protein syntheses. But activation of MDH in later stages of the response was inhibited by actinomycin D and cycloheximide, indicating that MDH might be regulated by JH at the transcriptional and/or translational level.

Electrophoretic patterns of malate and lactate dehydrogenases in a freshwater catfish