Enzyme localization in the anaerobic mitochondria of Ascaris lumbricoides.

Mitochondria from the muscle of the parasitic nematode Ascaris lumbricoides var. suum function anaerobically in electron transport-associated phosphorylations under physiological conditions. These helminth organelles have been fractionated into inner and outer membrane, matrix, and intermembrane space fractions. The distributions of enzyme systems were determined and compared with corresponding distributions reported in mammalian mitochondria. Succinate and pyruvate dehydrogenases as well as NADH oxidase, Mg(++)-dependent ATPase, adenylate kinase, citrate synthase, and cytochrome c reductases were determined to be distributed as in mammalian mitochondria. In contrast with the mammalian systems, fumarase and NAD-linked "malic" enzyme were isolated primarily from the intermembrane space fraction of the worm mitochondria. These enzymes are required for the anaerobic energy-generating system in Ascaris and would be expected to give rise to NADH in the intermembrane space. The need for and possible mechanism of a proton translocation system to obtain energy generation is suggested.

The single translation product of the FUM1 gene (fumarase) is processed in mitochondria before being distributed between the cytosol and mitochondria in Saccharomyces cerevisiae.

The yeast mitochondrial and cytosolic isoenzymes of fumarase, which are encoded by a single nuclear gene (FUM1), follow a unique mechanism of protein subcellular localization and distribution. Translation of all FUM1 messages initiates only from the 5'-proximal AUG codon and results in a single translation product that contains the targeting sequence located within the first 32 amino acids of the precursor. All fumarase molecules synthesized in the cell are processed by the mitochondrial matrix signal peptidase; nevertheless, most of the enzyme (80 to 90%) ends up in the cytosol. The translocation and processing of fumarase are cotranslational. We suggest that in Saccharomyces cerevisiae, the single type of initial translation product of the FUM1 gene is first partially translocated, and then a subset of these molecules continues to be fully translocated into the organelle, whereas the rest are folded into an import-incompetent state and are released by the retrograde movement of fumarase into the cytosol.

Active enzyme sedimentation of pig heart fumarase.

Active band sedimentation studies of pig heart fumarase indicate that the enzyme is predominantly tetrameric at enzyme concentrations between 0.0125 and 0.25 mg/ml and at a fumarate concentration of 2.5 mM. At enzyme concentrations of 0.25--1.0 mg/ml and fumarate concentrations known to activate and inhibit the enzyme, the sedimentation band of fumarase becomes disperse and indicates the presence of polymers greater than tetramers.

Purification and structural comparisons of the cytosolic and mitochondrial isoenzymes of fumarase from pig liver.

A method has been developed for the purification of cytosolic and mitochondrial isoenzymes of fumarase from total homogenates of pig liver. Separation of the isoenzymes from one another was achieved using chromatofocusing. The isoenzymes were pure as judged by production of single bands on electrophoresis in the presence of sodium dodecyl sulphate; they appeared to have identical or very similar subunit molecular weights. The isoenzymes differed in electrophoretic properties under nondenaturing conditions. One-dimensional peptide maps of fragments produced from the two isoenzymes by chemical cleavage at cysteine residues were identical; maps obtained after digestion with the V8 proteinase from Staphylococcus aureus showed small differences at short times of digestion which could have reflected variations in rates of hydrolysis rather than structural differences. However, two-dimensional peptide maps of digests obtained by treatment of the isoenzymes with trypsin followed by chymotrypsin had 58 peptides in common, but showed two peptides unique to the mitochondrial isoenzyme and five peptides unique to the cytosolic form. Using the dansylation procedure, the mitochondrial isoenzyme was shown to have N-terminal alanine and the cytosolic form to have N-terminal glutamic acid or glutamine. We conclude that the isoenzymes of fumarase are identical over nearly all of their amino acid sequences but differ at their N-termini; the extent of these differences is yet to be established. These results are consistent with the claim (Edwards, Y.H. and Hopkinson,D.A. (1979) Ann. Human Genet. Lond. 42, 303-313) that the isoenzymes are determined at the same genetic locus, but they raise interesting questions about the biosynthesis of the isoenzymes.

Purification, characterization and preliminary X-ray study of fumarase from Saccharomyces cerevisiae.

Fumarase (fumarate hydratase, EC 4.2.1.2) from Saccharomyces cerevisiae has been purified to homogeneity by a method including acetone fractionation, DEAE ion-exchange and dye-sorbent affinity chromatography. The suggested method allows fumarase purification with a yield higher than 60% and may be used to obtain large enzyme quantities. The native protein consists of four subunits with a approximately 50 kDa molecular mass each and has an isoelectric point at pH 6.5 +/- 0.3. The equilibrium constant for fumarate hydration is about 4.3 (25 degrees C, pH 7.5), the Michaelis constants for fumarate and 1-malate are approximately 30 microM and approximately 250 microM, respectively. The enzyme is activated by substrates and multivalent anions, the activation seems to be of a non-competitive type. The fumarase complex with meso-tartaric acid has been crystallized by the vapor diffusion method. The unit cell parameters are a = 93.30, b = 94.05 and c = 106.07 A, space group P2(1)2(1)2(1). The unit cell contains 2 protein molecules. The crystals diffract to at least 2.6 A resolution and are suitable for X-ray structure analysis.

Purification and crystallization of fumarase C from Escherichia coli.

Fumarase C purified from Escherichia coli has been crystallized in the presence of polyethylene glycol in both a citrate buffer at pH 5.3 and a 3-(4-morpholino)-propanesulfonic acid buffer at pH 7.5 yielding two crystal forms. An orthorhombic C222(1) form was obtained in citrate at pH 5.3 and an orthorhombic I222 form was obtained in 3-(4-morpholino)-propanesulfonic acid (pH 7.5). Complete native data sets have been collected on both crystal forms: the C222(1) form is complete to 2.10 A and the I222 form is complete to 2.20 A.

Chicken fumarase. I. Purification and characterization.

Fumarase from chicken heart is purified 400 times from the crude muscle extract. The isolation procedure includes ammonium sulfate fractionations, Bio-Gel P-300 column chromatography and electrofocusings on pH-gradients from pH 3 to 10 and from pH 7 to 9. Chicken fumarase behaves as an homogeneous protein in sedimentation, diffusion and electrofocusing studies; the protein possesses a single amino-terminal residue: lysine. The analysis of the CD and ORD spectra suggests the presence of 60-65 p. cent of alpha-helix, 0 - 5 p. cent of beta-structure with the remaining portions of the protein in an unordered conformation. Chicken fumarase is found to be composed of 4 subunits of identical molecular weight (51.000) and devoid of disulfide bridges. Finally, the physicochemical properties of chicken fumarase are compared with those of the porcine enzyme.

Purification and characterization of two types of fumarase from Escherichia coli.

Two distinct types of fumarase were purified to homogeneity from aerobically grown Escherichia coli W cells. The amino acid sequences of their NH2-terminals suggest that the two enzymes are the products of the fumA gene (FUMA) and fumC gene (FUMC), respectively. FUMA was separated from FUMC by chromatography on a Q-Sepharose column, and was further purified to homogeneity on Alkyl-Superose, Mono Q, and Superose 12 columns. FUMA is a dimer composed of identical subunits (Mr = 60,000). Although the activity of FUMA rapidly decreased during storage, reactivation was attained by anaerobic incubation with Fe2+ and thiols. Studies on the inactivation and reactivation of FUMA suggested that oxidation and the concomitant release of iron inactivated the enzyme in a reversible manner. While the inactivated FUMA was EPR-detectable, through a signal with g perpendicular = 2.02 and g = 2.00, the active enzyme was EPR-silent. These results suggested FUMA is a member of the 4Fe-4S hydratases represented by aconitase. After the separation of FUMC from FUMA, purification of the former enzyme was accomplished by chromatography on Phenyl-Superose and Matrex Gel Red A columns. FUMC was stable, Fe-independent and quite similar to mammalian fumarases in enzymatic properties.

Studies on the turnover rates of cytosolic and mitochondrial fumarases in rat liver.

The turnover rates of mitochondrial and cytosolic fumarase in the rat liver were determined by injecting L-[U-14C]leucine and following the decay of specific radioactivity incorporated into immunoprecipitates from the partially purified enzymes. The half-life of mitochondrial fumarase (t 1/2 = 9.7 days) was significantly different from that of the cytosolic enzyme (t 1/2 = 4.8 days). Studies on the incorporation of radioactive leucine into fumarase in the liver under steady-state conditions showed that the rate of synthesis of this enzyme in cytosol was about 2 times higher than that in the mitochondrial enzyme. The results showed that the mitochondrial fumarase turns over considerably more slowly than the cytosolic enzyme in the rat liver. These results suggest that the turnover of two fumarases with different localizations may be under different and independent control systems. In the case of the mitochondrial fumarase, the decay curve of its specific radioactivity obtained by single injection of L-[U-14C]leucine was quite unusual. No change was observed in the specific radioactivity of the mitochondrial fumarase for about 7 days after pulse labeling, then the specific radioactivity decreased exponentially with a half-life of 9.7 days.

End group analysis of the cytosolic and mitochondrial fumarases from rat liver.

Some molecular properties of the cytosolic and mitochondrial fumarases were compared. The carboxyl(C)-terminal amino acid of both the cytosolic and mitochondrial fumarases of rat liver cell was identified as leucine by using carboxypeptidase (CPase) A. As the amino(N)-terminal amino acid of both the cytosolic and mitochondrial fumarases could not be identified by the dansyl chloride method or by the cyanate method, the N-termini of these two fumarases seems to be masked. Both fumarases, after S-carboxymethylation, were completely digested with pronase E and CPase A and B, and the amino acids with blocked amino group were analyzed by high voltage paper electrophoresis and amino acid analysis after acid hydrolysis of these amino acid derivatives. The N-termini of the mitochondrial and cytosolic fumarases were identified as pyroglutamic acid and N-acetylalanine, respectively. To compare the primary structures of the two fumarases in detail, each fumarase was digested with an arginine-specific protease or cleaved with cyanogen bromide. The electrophoretic profiles of the digests of these fumarases were indistinguishable from each other.

Intracellular distribution of fumarase in various animals.

The subcellular distribution of fumarase was investigated in the liver of various animals and in several tissues of the rat. In the rat liver, fumarase was predominantly located in the cytosolic and mitochondrial fractions, but not in the peroxisomal fraction. The amount of fumarase associated with the microsomes was less than 5% of the total enzyme activity. The investigation of the intracellular distribution of hepatic fumarase of the rat, mouse, rabbit, dog, chicken, snake, frog, and carp revealed that the amount of the enzyme located in the cytosol was comparable to that in the mitochondria of all these animals. The subcellular distribution of the enzyme in the kidney, brain, heart, and skeletal muscle of rat, and in hepatoma cells (AH-109A) was also investigated. Among these tissues, the brain was the only exception, having no fumarase activity in the cytosolic fraction, and the other tissues showed a bimodal distribution of fumarase in the cytosol and the mitochondria. The mitochondrial fumarase was predominantly located in the matrix. About 10% of the total fumarase was found in the outer and inner membrane, although it was unclear whether this fumarase was originally located in these fractions. No fumarase activity was detected in the intermembranous space.

Continuous aqueous phase extraction of proteins: automated on-line monitoring of fumarase activity and protein concentration.

Automated assay techniques are described for on-line measurements of fumarase activity and total protein concentration, including in-line sample dilution and sample multiplexing during continuous aqueous phase extraction. Fumarase was determined by following the conversion of L-malate to fumurate at a wavelength of 250 nm, while the protein assay was based on the Biuret reaction. Actual assay times of 2 and 4 min were achieved for the fumarase and protein measurements, respectively, with an effective measurement cycle time of 2 min. Standard deviations of c. 3.2 and 2% of the measured values were calculated for the enzyme and protein values, respectively. The assay system was coupled to a computer to allow on-line data visualization and storage.

[Optimization of culture media and conditions for cultivating Erwinia sp., a producer of fumarase and aspartase]. / Optimizatsiia sredy i uslovii kul'tivirovaniia produtsenta fumarazy i aspartazy Erwinia sp.

Mathematical methods of experimental design were used to determine the optimal concentrations of nutrient medium components, aeration conditions, and pH providing for the maximum biomass yields, as well as fumarase and aspartase activities, during submerged cultivation of Erwinia sp. The data showed that different concentrations of carbon source (molasses) and pH of the nutrient medium were required to reach the maximum yields of fumarase and aspartase. Calculations suggested that the combination of these optimized factors would result in 3.2-, 3.4-, and 3.8-fold increases in the Erwinia sp. biomass, aspartase activity, and fumarase activity yields, respectively. The experimental data were consistent with these estimates to a 80% accuracy.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of recombinant human fumarase.

Human fumarase (HsFH) is a well-known citric acid cycle enzyme and is therefore a key component in energy metabolism. Genetic studies on human patients have shown that polymorphisms in the fumarase gene are responsible for diseases such as hereditary leiomyomatosis and renal cell cancer. As a first step in unravelling the molecular basis of the mechanism of fumarase deficiency in genetic disorders, the HsFH gene was cloned in pET-28a, heterologously expressed in Escherichia coli, purified by nickel-affinity chromatography and crystallized using the vapour-diffusion technique. X-ray diffraction experiments were performed at a synchrotron source and the structure was solved at 2.1 Šresolution by molecular replacement.