Trichur K. Sundaram

Purification of bacterial malate dehydrogenases by selective elution from a triazinyl dye affinity column

Abstract A technique of selective elution from affinity adsorbent columns has been devised for the purification of malate dehydrogenase ( L -malate:NAD+ oxidoreductase, EC 1.1.1.37) from a number of mesophilic and thermophilic bacteria. It is dependent on the combined action of both NAD+ and L -malate, the two reactants in the malate dehydrogenase system. These two reactants individually or NADH or oxaloacetate alone at its equilibrium concentration in the malate dehydrogenase system cannot effect elution of the enzyme. Evidence is presented suggesting that malate dehydrogenase is eluted as a ternary complex with NAD+ and L -malate. Using agarose-linked Procion Red HE3B as the affinity adsorbent, the purification method consists of a single step for some malate dehydrogenase species and two easy steps for the other species. It is rapid and efficient, producing malate dehydrogenase homogeneous by several criteria in good yield.

Monomeric malate synthase from a thermophilic Bacillus: Molecular and kinetic characteristics

Abstract The molecular weight of malate synthase purified from a thermophilic Bacillus was determined to be 62,000 by sedimentation equilibrium methods, confirming the value obtained earlier by the gel filtration technique. This enzyme and its homologs from other bacteria, which are all monomeric proteins with molecular weights of approximately 60,000. therefore differ from the considerably larger and multimeric malate synthases from yeast, Neurospora crassa , and other eucaryotic microorganisms and plants. Amino acid analysis reveals the thermophile synthase to be relatively rich in glutamic acid and to have a higher content of arginine in comparison with the yeast enzyme. The Bacillus enzyme is an acidic protein with an isoelectric pH of 4.6 and has two sulfhydryl groups titratable with 5,5′-dithiobis(2-nitrobenzoic acid). Its parameters indicative of its overall hydrophobicity and of levels of helicity and turn, which were deduced from the amino acid composition, lie well within the range recorded for a number of mesophile and thermophile enzymes. However, the level of β-sheet structure is considerably lower than that calculated for the yeast synthase; this supports a trend recently observed for certain other thermophile proteins. The synthase isolated from the thermophilic Bacillus appears to be homogeneous by several criteria, although upon electrophoresis in the native state in polyacrylamide it yields two protein bands that are both enzymatically active. Several kinetic characteristics of this enzyme are also reported.

Cloning sequencing and over-expression of Escherichia coli malate dehydrogenase

SummaryThe malate dehydrogenase gene (mdh) from E. coli W3899 has been cloned and its nucleotide sequence determined. A comparison of the DNA sequence presented here and other E. coli mdh gene sequences reveals a number of important differences including an “apparent” frame shift in the C-terminal region of the gene. The mdh gene was shown to be expressed from its own transcription promotor at a very high level (50% soluble cell protein) in this system and a simple single step procedure for purification of the gene product in large quantities is described.

A facile method for the isolation of porcine heart mitochondrial malate dehydrogenase by affinity elution chromatography on Procion Red HE3B

A quick, simple method has been devised for isolating pig heart mitochondrial malate dehydrogenase in apparently homogeneous state and good yield. It entails the adsorption of the enzyme to agarose-linked Procion Red HE3B and specific elution of a ternary complex consisting of the malate dehydrogenase, NAD+, and L-malate.

Binding behaviour of pyruvate carboxylase from Bacillus stearothermophilus on Sepharose—avidin

The specific affinity of avidin for biotin [l] affords a potentially powerful facility for the affinity chromatography of biotin-containing carboxylases. The extreme tightness of this non-covalent binding has, however, presented problems with regard to the elution of these enzymes from agrose-avidin columns [2] . To our knowledge, no whole, undissociated biotin-containing carboxylase has yet been isolated in satisfactory yield from such columns. We report here the specific binding of pyruvate carboxylase (EC 6.4.1.1) from Bucillus stearothermophilus to Sepharose--avidin and its elution in relatively good yield in an undissociated, enzymitally active state.

Action of surfactants on porcine heart malate dehydrogenase isoenzymes and a simple method for the differential assay of these isoenzymes.

The cationic surfactant, cetyl (hexadecyl) trimethylammonium bromide (CTAB), completely inactivates porcine heart cytoplasmic malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) at concentrations (of surfactant) which do not affect the activity of the mitochondrial isoenzyme. These concentrations are close to, or higher than, the critical micelle concentration of CTAB. An increase in the ionic strength of the medium significantly retards the CTAB-induced inactivation of the cytoplasmic enzyme. The enzyme is also markedly protected against CTAB inactivation by NADH; L-malate on its own has no effect but a combination of NADH and L-malate affords greater protection than NADH alone. The CTAB inactivation is not reversed by dilution of the surfactant. The highly selective action of CTAB on the two malate dehydrogenases, which correlates well with their electrostatic charges, has been exploited for a simple and reliable differential assay of these isoenzymes. The anionic surfactant, sodium dodecyl sulphate (SDS), at concentrations well below the critical micelle concentration, inactivates both isoenzymes, but the mitochondrial enzyme is significantly more sensitive than its cytoplasmic counterpart. There is thus some correlation, though not as strong as with CTAB, between SDS inactivation and the charges of the two malate dehydrogenases. An increase in ionic strength has opposite effects on the two isoenzymes: the mitochondrial enzyme becomes more resistant and the cytoplasmic enzyme less so. Both isoenzymes are rendered more resistant to SDS by the inclusion of NADH. Inactivation of the enzymes caused by short exposure to SDS is largely reversed by dilution of the detergent, but longer exposure leads to progressive irreversible loss of activity. NADH very effectively protects the isoenzymes against irreversible inactivation. It is likely that a reversible phase of inactivation precedes an irreversible phase and that in the former phase SDS acts competitively with NADH. Both malate dehydrogenases possess considerable resistance to the nonionic detergent, Triton X-100.

Biotin subunits of acetyl CoA carboxylase and pyruvate carboxylase from a thermophilic Bacillus.

Abstract Two biotin-containing polypeptides of molecular weights 140,000 and 22,000 have been identified by gel electrophoresis in a sodium dodecyl sulfate-denatured extract of cells of a thermophilic Bacillus. These polypeptides can be separated from each other by either gel filtration through Sepharose 6B or affinity chromatography on a Sepharose-avidin column. The larger polypeptide is renatured under appropriate conditions to yield enzymically active pyruvate carboxylase. Enzyme reconstitution experiments show that the smaller polypeptide is a component of acetyl CoA carboxylase. The biotin subunits of these two carboxylases are thus distinct from, and dissimilar to, each other. The demonstration that a pyruvate carboxylase-deficient mutant of the Bacillus contains the smaller, but not the larger, polypeptide corroborates this conclusion.

Stability and immunological cross-reactivity of malate dehydrogenases from mesophilic and thermophilic sources.

The thermostability in vitro of dimeric and tetrameric malate dehydrogenases [S)-malate:NAD+ oxidoreductase, EC 1.1.1.37) from mesophilic and thermophilic bacteria shows a good correlation to the growth temperature of the source organism but no consistent relationship to enzyme subunit structure. The thermophile malate dehydrogenases are, in general, more resistant to the surfactants, sodium dodecyl sulphate (SDS) and hexadecyltrimethylammonium bromide, and to the denaturants, guanidinium chloride and urea, than their mesophilic counterparts, with the dimer in each thermal class being more resistant to the chemical perturbants than the tetramer. Sedimentation analysis suggests that denaturation of the malate dehydrogenases by acid-periodate or SDS produces discrete subunits, whereas denaturation by guanidinium chloride followed by carboxymethylation yields ill-defined protein species. SDS and acid-periodate were therefore preferred to generate denatured malate dehydrogenases for use as immunogens and antigens. The native malate dehydrogenases exhibit immunological cross-reactivity only when they are in the same oligomeric form and derived from closely related species, which may, however, be from different thermal classes. Taking immunological cross-reactivity as an indicator of structural similarity, this supports the idea that the thermophilic trait evolved independently within each phyletic line. With denatured malate dehydrogenases as immunogens and antigens, cross-reactivity is manifested between all the malate dehydrogenases examined. This suggests that appreciable primary structural homology exists between the malate dehydrogenases, whether dimeric or tetrameric, from thermophiles and mesophiles and from various taxa.

Regulatory characteristics of phosphoenolpyruvate carboxylase from the extreme thermophile, Thermus aquaticus.

Phosphoenolpyruvate carboxylase from the extremely thermophilic bacterium, Thermus aquaticus YT-1, exhibits a virtually absolute requirement for acetyl CoA and there is strong positive cooperativity in the interaction of this activator with the enzyme. Several tricarboxylic acid cycle intermediates inhibit the enzyme. These findings suggest an anaplerotic role for the enzyme and an allosteric modulation of its activity by acetyl CoA and tricarboxylic acid cycle intermediates.

Anaplerotic Enzymes of Acetate and Pyruvate Metabolism: Distinctive Characteristics in Bacillus Stearothermophilus

Until recently, the unusual thermostability of proteins from thermophilic organisms was expected to be the consequence of gross peculiarities in their structure, not shared by their mesophilic counterparts (11). However, in a large number of instances, enzymes from thermophiles have appeared similar to cognate mesophile enzymes in most properties. This has led to the recognition that the structural peculiarity underlying the stability of thermophile proteins may be subtle (11). Indeed it has been argued on theoretical grounds that very small-structural changes could account for the observed differences in stability between thermophile and mesophile proteins (14). Moreover the degree of thermal resistance can vary from one thermophile protein to another, and it is probable that in the evolution of thermophiles the balancing of the two requirements of thermostability and catalytic efficiency of their proteins, possibly mutually conflicting, was a key factor. Thus it is likely that no single general mechanism will explain the stability of all thermophile proteins. There is a case therefore, for examining a wide variety of thermophile enzymes for unusual characteristics. Our study of isocitrate lyase (Ls-isocitrate glyoxylate-lyase, EC 4.1.3.1), malate synthase (L-malate glyoxylate-lyase [CoA-acetylating], EC 4.1.3.2) and pyruvate carboxylase (pyruvate: CO2 ligase (ADP), EC 6.4.1.1), all enzymes involved in the generation of C4 compounds to meet the anaplerotic (6)needs of metabolism, reveals distinctive features in the first two enzymes and a possibly distinctive feature in the carboxylase, ether than thermostability, that are not apparently possessed by their counterparts in mesophiles.