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S Doonan

Publications and source records attributed to S Doonan.

At least 55 records · Page 3Linked to original sources
PubMed1988

Purification and structural comparisons of the cytosolic and mitochondrial fumarases from baker's yeast.

1. The cytosolic and mitochondrial fumarases (EC 4.2.1.2) from baker's yeast (Saccharomyces cerevisiae) have been purified to homogeneity. 2. Subunit molecular weights for the cytosolic and mitochondrial isoenzymes were 53,000 and 48,000 respectively. 3. Peptide maps obtained after digestion of the two isoenzymes with trypsin were almost identical but showed significant small differences. The same was true of peptide maps obtained after digestion with the glutamic acid-specific proteinase from S. aureus.

Chromatography↗
PubMed1986

Nucleotide sequence of a cDNA coding for mitochondrial fumarase from human liver.

The nucleotide sequence of a 1.46 kb cDNA, selected from a human liver library by the expression of fumarase antigenic determinants, was determined using the dideoxy chain termination method. The cDNA contained an open reading frame extending from the extreme 5'-base and coding for a protein with 468 amino acids. This protein, with the exception of an N-terminal methionine, was identified as mitochondrial fumarase. The protein showed a high degree of identity of structure with the fumarase from Bacillus subtilis (56.6%) and a fumarase from Escherichia coli (product of the fumC gene, 59.3%), and a lower degree of identity with the aspartase of E. coli (37.2%).

Amino Acid Sequence↗
PubMed1986

The complete amino acid sequences of cytosolic and mitochondrial aspartate aminotransferases from horse heart, and inferences on evolution of the isoenzymes.

We report here the complete amino acid sequences of the cytosolic and mitochondrial aspartate aminotransferases from horse heart. The two sequences can be aligned so that 48.1% of the amino acid residues are identical. The sequences have been compared with those of the cytosolic isoenzymes from pig and chicken, the mitochondrial isoenzymes from pig, chicken, rat, and human, and the enzyme from Escherichia coli. The results suggest that the mammalian cytosolic and mitochondrial isoenzymes have evolved at equal and constant rates whereas the isoenzymes from chicken may have evolved somewhat more slowly. Based on the rate of evolution of the mammalian isoenzymes, the gene-duplication event that gave rise to cytosolic and mitochondrial aspartate aminotransferases is estimated to have occurred at least 10(9) years ago. The cytosolic and mitochondrial isoenzymes are equally related to the enzyme from E. coli; the prokaryotic and eukaryotic enzymes diverged from one another at least 1.3 X 10(9) years ago.

Amino Acid Sequence↗
PubMed1985

The primary structure of mitochondrial aspartate aminotransferase from human heart.

The complete amino acid sequence of the mitochondrial aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) from human heart has been determined based mainly on analysis of peptides obtained by digestion with trypsin and by chemical cleavage with cyanogen bromide. Comparison of the sequence with those of the isotopic isoenzymes from pig, rat and chicken showed 27, 29 and 55 differences, respectively, out of a total of 401 amino acid residues. Evidence for structural microheterogeneity at position 317 has also been obtained.

Amino Acid Sequence↗
PubMed1985

The role of metal ions in the uptake of aspartate aminotransferase and malate dehydrogenase into isolated rat liver mitochondria in vitro.

To gain further insight into the mitochondrial receptor area which allows selective uptake of both purified aspartate aminotransferase and malate dehydrogenase into mitochondria, the inhibition of metal complexing agents such as bathophenanthroline and tiron on the uptake of both enzymes has been investigated. In view of the nature of the inhibition found, we propose the existence of metal ion(s) at or near the aspartate aminotransferase, but far from the malate dehydrogenase binding site.

1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium ↗
PubMed1985

Removal of an N-terminal peptide from mitochondrial aspartate aminotransferase abolishes its interactions with mitochondria in vitro.

Treatment of mitochondrial aspartate aminotransferase from rat liver with trypsin leads to specific cleavage of the bonds between residues 26 and 27, and residues 31 and 32. The proteolysed enzyme has only a small residual catalytic activity, but retains a conformation similar to that of the native form as judged by accessibility and reactivity of cysteine residues. Proteolysis abolishes the ability of the enzyme either to bind to mitochondria or to be imported into the organelles. This suggests that the N-terminal segment of the native enzyme is essential for both of these functions, at least in the model system used to study the import process.

Amino Acids↗
PubMed1985

Kinetic studies of the uptake of aspartate aminotransferase and malate dehydrogenase into mitochondria in vitro.

Kinetic measurements of the uptake of native mitochondrial aspartate aminotransferase and malate dehydrogenase into mitochondria in vitro were carried out. The uptake of both the enzymes is essentially complete in 1 min and shows saturation characteristics. The rate of uptake of aspartate aminotransferase into mitochondria is decreased by malate dehydrogenase, and vice versa. The inhibition is exerted by isoenzyme remaining outside the mitochondria rather than by isoenzyme that has been imported. The thiol compound beta-mercaptoethanol decreases the rate of uptake of the tested enzymes; inhibition is a result of interaction of beta-mercaptoethanol with the mitochondria and not with the enzymes themselves. The rate of uptake of aspartate aminotransferase is inhibited non-competitively by malate dehydrogenase, but competitively by beta-mercaptoethanol. The rate of uptake of malate dehydrogenase is inhibited non-competitively by aspartate aminotransferase and by beta-mercaptoethanol. beta-Mercaptoethanol prevents the inhibition of the rate of uptake of malate dehydrogenase by aspartate aminotransferase. These results are interpreted in terms of a model system in which the two isoenzymes have separate but interacting binding sites within a receptor in the mitochondrial membrane system.

Animals↗
PubMed1985

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.

Amino Acid Sequence↗
PubMed1985

The subcellular distribution of fumarase isozymes in rat liver.

Between 13 and 25% of the fumarase activity of rat liver was found to be cytosolic in origin the remainder being localised in the mitochondria. Electrophoretic analysis on cellulose acetate showed that mitochondria do not contain detectable levels of cytosolic isozyme or vice versa.

Animals↗
PubMed1984

Partial amino-acid sequence and cysteine reactivities of cytosolic aspartate aminotransferase from horse heart.

Cytosolic aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) from horse heart has five cysteine residues, two of which can be titrated with 5,5'-dithiobis(2-nitrobenzoid acid) in the native enzyme with no impairment of catalytic activity. The rate of modification is unaffected by the presence of substrates. Reaction with N-ethylmaleimide leads to loss of catalytic activity, the rate of inactivation being increased by the presence of substrates. Peptides containing 361 amino-acid residues (about 88% of the total number in the protein) have been isolated and aligned by comparison with the known sequence of the isotopic isoenzyme from pig heart. In the regions compared, 342 of the residues are identical. Hence, assuming that those regions are representative of the whole, then the cytosolic isoenzymes from horse and from pig have about 95% identity of structure. Uniquely among the mammalian cytosolic aspartate aminotransferases so far examined, the enzyme from horse heart is acetylated at the N-terminus.

Amino Acid Sequence↗
PubMed1984

Structural and genetic relationships between cytosolic and mitochondrial isoenzymes.

The most common type of genetic relationship between cytosolic and mitochondrial isoenzymes will probably be found to be divergent evolution from a common ancestral form. This is firmly established for the aspartate aminotransferases and less directly so in other cases. The two isoenzymes of aspartate aminotransferase have evolved at roughly equal rates at the level of total amino acid sequence but certain limited surface regions of the mitochondrial form have been much more highly conserved than corresponding regions in the cytosolic protein; these regions probably play a role in topogenesis of the mitochondrial isoenzyme. It is of interest that nearly all mitochondrial proteins are initially synthesised as precursors of molecular weight greater than the mature forms. In the case of aspartate aminotransferase, and possibly of other such isoenzymes, the N-terminus of the mature protein is nearly coincident with that of the cytosolic isoenzyme. Hence during evolution either the gene for the mitochondrial isoenzyme has gained an extra coding region for this N-terminal extension or, less likely, the structural gene for the cytosolic form has suffered a sizeable terminal deletion. Cytosolic and mitochondrial superoxide dismutases have not shared a common ancestral form as shown by the fact that their primary structures are completely unrelated. On the other hand, the mitochondrial and prokaryotic enzymes are clearly related. There is now, however, evidence to suggest that some prokaryotes possess a copper/zinc enzyme related to the eukaryotic cytosolic form. Hence the possibility arises that primitive prokaryotes possessed both proteins. The copper/zinc superoxide dismutase has been retained in the cytosol of eukaryotic cells and a few bacterial species.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Transport of proteins into mitochondria.

There is still much that is obscure concerning the transport of proteins into or through the mitochondrial membrane systems. In addition, as pointed out previously, it is unlikely that the details of the process are the same for proteins destined for different compartments of the organelle. A brief summary of the process for matrix proteins might be as follows: The proteins are synthesized on free polysomes as precursors of higher molecular weight than the native forms. These precursors are liberated into the cell cytosol and subsequently translocated into the mitochondria. This timing might be different in yeast under some circumstances, synthesis being completed in association with the mitochondria. The precursors interact with a receptor in the outer mitochondrial membrane interaction being mediated by the presequences of the precursors. The presequences therefore act as addressing signals as well as possibly playing a role in one or all of (a) solubilization of precursors, (b) prevention of premature assembly into multimeric structures, or (c) maintenance of nonnative configurations required for transport. Interaction occurs with a second receptor, this time in the inner membrane of the mitochondria, interaction being with multiple sites in the polypeptide chain. Transport across the inner membrane then occurs, this transport depending on a transmembrane electrochemical gradient of which the proton component is the essential part. Transport is accompanied or followed by proteolysis of the prepiece, and formation of the native structure. While steps 1 and 2 of this sequence can be considered well established, the remaining steps are still poorly understood or purely hypothetical. Nevertheless, this sequence of events is consistent with known facts about the process and provides a framework for future investigations.

Animals↗
PubMed1983

Uptake of malate dehydrogenase into mitochondria in vitro. Some characteristics of the process.

1. It was previously shown [Passarella, Marra, Doonan & Quagliariello (1980) Biochem. J. 192, 649-658] that, when mitochondrial malate dehydrogenase from rat liver is incubated with sulphite-loaded mitochondria from the same source, uptake of the enzyme occurs, as judged by a fluorimetric assay of intramitochondrial enzyme activity. Confirmation of sequestration of the enzyme inside the organelles is provided by its proteinase-resistance after uptake. 2. Enzyme uptake into mitochondria is inhibited by enzyme treatment with mersalyl at concentrations that do not affect its catalytic activity. 3. Enzyme uptake is energy-dependent, as shown by inhibition of the process by carbonyl cyanide p-trifluoromethoxyphenylhydrazone and by antimycin. ATP and oligomycin, on the other hand, both stimulate the process, but stimulation by ATP is inhibited by oligomycin. These results suggest that uptake depends on maintenance of transmembrane ion gradient rather than direct ATP involvement. 4. Measurements of delta psi by means of the 'redistribution signal' probe safranine suggest no dependence of malate dehydrogenase uptake on membrane potential. 5. Comparison of the effects of the ionophores valinomycin, nonactin, gramicidin and nigericin shows that uptake depends on maintenance of a transmembrane pH gradient.

Adenosine Triphosphate↗
PubMed1983

Primary structure of aspartate aminotransferase from horse heart and comparison with that of other homotopic and heterotopic isoenzymes.

Sulphydryl groups of mitochondrial aspartate aminotransferase from horse heart were titrated with 5,5'-dithiobis (2-nitrobenzoic acid). From analysis of peptic peptides, 378 amino acid residues (94.3% of the total) in the protein were identified. The results of amino acid sequence analysis are compared with those of cytosolic and mitochondrial aspartate aminotransferases from other sources.

Amino Acid Sequence↗
PubMed1982

Uptake of aspartate aminotransferase into mitochondria in vitro depends on the transmembrane pH gradient.

1. The effects of various inhibitors of electron transport and of oxidative phosphorylation and the effects of ionophores on the uptake of native aspartate aminotransferase into mitochondria were investigated. 2. Both antimycin and cyanide completely inhibited the uptake of the enzyme. On the other hand, uptake was stimulated to ATP and by oligomycin; however, the stimulation by ATP is inhibited by oligomycin. 3. The effects of ionophores of the valinomycin type in media containing K+ ions depended on the conditions used. Valinomycin alone stimulated the uptake of the enzyme, but in the presence of phosphate ions uptake was abolished. Nonactin was without effect at a low K+ concentration, but was stimulatory at 100 mM-KCl. Gramicidin also stimulated the uptake process. 4. Nigericin completely abolished uptake of aspartate aminotransferase into mitochondria. 5. The uptake of te enzyme was decreased by 18% in the absence of inhibitors or ionophores when the external pH was increased from 6.9 to 7.6. 6. These results indicate that ATP is not directly involved in the uptake of aspartate aminotransferase into mitochondria, neither is there a requirement for a cation gradient. Rather the uptake depends on the maintenance of a pH gradient across the mitochondrial inner membrane.

Adenosine Triphosphate↗