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.     

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.     

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.     

Distributions of aspartate aminotransferase and malate dehydrogenase activities in rat retinal layers

Aspartate aminotransferase (AAT), an enzyme interconverting glutamate and aspartate, has been suggested to be a marker for glutamatergic and/or aspartatergic neurons. However, AAT, glutamate, and aspartate are also involved in cellular metabolism, e.g., the malate-aspartate shuttle. To investigate the extent to which AAT might be involved in these several functions in retina, the distribution of AAT activity in rat retinal layers was compared to that of malate dehydrogenase (MDH), an enzyme of aerobic metabolism proposed to be physically complexed with AAT in the malate-aspartate shuttle mechanism. The distribution of AAT activity in retinal layers closely paralleled that of MDH (correlation coefficient AAT versus MDH = 0.93). AAT activity was proportionately higher than MDH in the photoreceptor inner segments, containing a high density of mitochondria, and in the outer plexiform layer (OPL), containing photoreceptor terminals and bipolar and horizontal cell processes. The amount of total AAT activity in the inner segments related to the mitochondrial isoenzyme is almost twice that in the other layers tested, including the OPL. The correlation between AAT and MDH activities is consistent with AAT involvement in retinal energy metabolism, although other functions, such as neurotransmission, are possible.     

In vitro import into mitochondria of the precursor of mitochondrial aspartate aminotransferase

The import of the precursor of mitochondrial aspartate aminotransferase was reconstituted in vitro with isolated mitochondria thus corroborating the earlier conclusion of a post-translational uptake. The higher Mr precursor was synthesized in a reticulocyte lysate programmed with free polysomes from chicken liver. After incubation with intact mitochondria from chicken heart about 50% of the precursor was converted to the mature form in a time-dependent process, its rate being a function of the amount of mitochondria added. The same amount of precursor was processed to the mature form on addition of a mitochondrial extract. No conversion to the mature enzyme took place when the precursor was incubated with intact mitochondria in the presence of the uncoupling agent carbonyl cyanide m-chlorophenylhydrazone or of the chelator o-phenanthroline which penetrates the mitochondrial inner membrane. In contrast, the chelator bathophenanthroline disulfonate which does not diffuse into the mitochondrial matrix did not inhibit the appearance of the mature form. The results indicate that that precursor must pass through an energized inner mitochondrial membrane before it is processed by a chelator-sensitive protease in the mitochondrial matrix. Excess mature mitochondrial aspartate aminotransferase did not compete with the precursor for its uptake into mitochondria. Mature mitochondrial aspartate aminotransferase is an alpha 2-dimer with Mr = 2 X 45,000. Both the precursor synthesized in a rabbit reticulocyte lysate and the precursor accumulated in the cytosol of carbonyl cyanide m-chlorophenylhydrazone-treated chicken embryo fibroblasts were found to exist as homodimer or hetero-oligomer and high Mr complexes (Mr greater than 300,000).     

Distribution of activities of aspartate aminotransferase isoenzymes and malate dehydrogenase in guinea pig retinal layers

Distributions of activity of the cytosolic (cAAT) and mitochondrial (mAAT) isoenzymes of aspartate aminotransferase and of malate dehydrogenase (MDH) were determined in guinea pig retinal layers. The distribution of total AAT activity (tAAT = cAAT + mAAT) and of mAAT activity correlated well (r = 0.88-0.91) with the distribution of MDH activity. mAAT activity was highest in the inner segments of the photoreceptors; there was a greater than twelve-fold difference between activity in that layer and in the inner retinal layers. cAAT activity was also highest in the inner segments, but the difference between the activity in the inner segments and the other layers was not nearly as great as with mAAT. cAAT activity was also relatively high in the outer nuclear layer, outer plexiform layer, and part of the inner plexiform layer. The high activity of cAAT, mAAT, and MDH in the inner segments indicates that all of these enzymes are involved in metabolic reactions related to energy production and/or to photoreceptive processes in the outer segments and, therefore, that the enzymes are probably involved in energy-related metabolism at synapses. However, other functions, including those related to neurotransmission, are not excluded.     

Simultaneous purification by affinity chromatography of rat liver mitochondrial aspartate aminotransferase and malate dehydrogenase and electrophoretic properties

Mitochondrial aspartate aminotransferase and malate dehydrogenase were purified to homogeneity from rat liver by the use of aspartate-coupled Sepharose, ion exchange, and Blue Sepharose chromatography. This procedure permits rapid preparation of these enzymes. The pI of each enzyme was determined and anomalous electrophoretic properties of aspartate aminotransferase were described.     

The anomalous kinetics of coupled aspartate aminotransferase and malate dehydrogenase. Evidence for compartmentation of oxaloacetate.

The optimum cofactor requirements for triacylglycerol biosynthesis in rat adipose-tissue homogenates containing mitochondrial, microsomal and cytosolic fractions were investigated. In general the optimum concentrations of cofactors for triacylglycerol biosynthesis were found to differ from those for total fatty acid esterification. The results provided further evidence for the key role of phosphatidate phosphohydrolase in the regulation of triacylglycerol biosynthesis. Albumin was included in the incubation medium to permit the use of concentrations of added fatty acids that would swamp the effects of endogenous fatty acids. The addition of albumin had little effect on the incorporation of palmitic acid and stearic acid into lipids including triacylglycerols. By contrast, a critical concentration of albumin (about 60 muM) was required before incorporation of oleic acid or linoleic acid into triacylglycerols occurred. The system was used to study the incorporation of different 1-14C-labelled fatty acids from a mixture of unesterified fatty acids [palmitic acid 30%; stearic acid 10%; oleic acid 40%; linoleic acid 20% (molar percentages)] separately into the positions 1,2 and 3 of triacyl-sn-glycerols. In general the stereo-specific distribution of the labelled fatty acids incorporated into triacylglycerols paralleled the normal distribution of fatty acids within rat adipose-tissue triacylglycerols, suggesting that the specificities of the relevant acyltrasferases have the major role in determining the positional distribution of fatty acids within triacylglycerols.     

The effect of sulfhydryl group reagents on the permeation of mitochondrial aspartate aminotransferase into mitochondria in vitro.

When mitochondria are incubated with radioactively labeled mitochondrial aspartate aminotransferase (EC 2.6.1.1), the enzyme is taken up into the organelles. Mersalyl and p-hydroxymercuriphenyl sulfonic acid, but not N-ethylmaleimide or ethacrynic acid, decrease the extent of this uptake. Inhibition of the uptake by low concentrations of mercurial reagents is due to blockage of a single sulfhydryl group per monomer of the enzyme. Blockage of mitochondrial thiols does not inhibit uptake of the enzyme. A single sulfhydryl group out of a total of six per monomer of the native enzyme reacts with 5,5′-dithiobis-(2-nitrobenzoic acid). This is the same sulfhydryl group that reacts with low levels of mercurial reagents with consequent inhibition of uptake of the enzyme into mitochondria but without effect on the catalytic activity. N-Ethylmaleimide does not react with this group. N-Ethylmaleimide reacts with a different sulfhydryl group with concomitant decrease in enzymic activity but with no effect on uptake of the enzyme into mitochondria. High levels of mercurial reagents similarly decrease enzymic activity. Unlike the effect on uptake into mitochondria, the inhibition by mercurial reagents of enzymic activity is not reversed by treatment with cysteine. The significance of these observations with respect to the mechanism of uptake of aspartate aminotransferase into mitochondria is discussed, and comparisons are made between the reactivities of sulfhydryl groups in rat liver aspartate aminotransferase and in the enzymes from other animals.     

The precursor of mitochondrial aspartate aminotransferase is translocated into mitochondria as apoprotein

Mitochondrial aspartate aminotransferase is synthesized on free polysomes as a higher molecular weight precursor (Sonderegger, P., Jaussi, R., Christen, P., and Gehring, H. (1982) J. Biol. Chem. 257, 3339-3345). The present study examines whether the coenzyme pyridoxal phosphate or pyridoxamine phosphate is required for the uptake of the precursor into mitochondria. Chicken embryo fibroblasts were cultured in medium prepared with and without pyridoxal. In cells grown in the presence of pyridoxal only holoform of aspartate aminotransferase and no apoenzyme was detected. Cells cultured under pyridoxal deficiency contained about 30% of apoenzyme in secondary cultures. All of this apoform was identified as mitochondrial isoenzyme. In order to differentiate whether this apoenzyme corresponded to newly synthesized protein or originated from pre-existing holoenzyme, double isotope-labeling experiments were performed. Secondary cultures of chicken embryo fibroblasts grown under pyridoxal depletion were labeled with [3H]methionine, and then pulsed with [35S]methionine. In another series of experiments, the 3H-labeled cells were pulsed with [35S]methionine in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone in order to accumulate the precursor. Subsequently, the accumulated precursor was chased into the mitochondria by addition of the carbonyl cyanide m-chlorophenylhydrazone antagonist cysteamine. The holo- and apoenzyme from the ultrasonic extract of the double-labeled cells were separated by affinity chromatography on a phosphopyridoxyl-AH-Sepharose column, immunoprecipitated, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Under both experimental conditions, the 3H/35S ratio of the apoenzyme was less than half of that of the holoenzyme. Therefore, the apoenzyme and not the holoenzyme is the first product of the precursor in the mitochondria. Apparently, the precursor of mitochondrial aspartate aminotransferase is transported into mitochondria as apoprotein and is processed there independently of the coenzyme.     

Purification and properties of the cytosolic and mitochondrial forms of aspartate aminotransferase and malate dehydrogenase from rat heart

The present work describes the purification from rat heart of the mitochondrial and cytosolic forms of the enzymes of the malate--aspartate shuttle, aspartate aminotransferase (EC 2.6.1.1) and malate dehydrogenase (EC 1.1.1.37), by a single procedure after the preparation of the original crude extract. In 10 purification steps, the four enzymes were obtained electrophoretically pure in yields ranging from 6 to 54% of their respective isoenzyme levels in the crude extract. Apoenzymes were formed from the aminotransferases by reacting them with cysteine sulfinate and dialyzing. Complete reconstitution was obtained after a brief incubation with pyridoxal phosphate. All four enzymes are dimers. The mitochondrial isoenzymes are of slightly lower molecular weight than their respective cytosolic forms. Michaelis constants and maximal velocities were derived by the use of primary and secondary plots. In general, the properties of the enzymes from rat heart are similar to the properties of the enzymes from other animal sources.     

Partitioning of malate dehydrogenase isoenzymes into glyoxysomes, mitochondria, and chloroplasts

Malate dehydrogenase isoenzymes catalyzing the oxidation of malate to oxaloacetate are highly active enzymes in mitochondria, in peroxisomes, in chloroplasts, and in the cytosol. Determination of the primary structure of the isoenzymes has disclosed that they are encoded in different nuclear genes. All three organelle-targeted malate dehydrogenases are synthesized with an amino terminal extension that is cleaved off in connection with the import of the enzyme precursor into the organelle. The sequence of the 27 amino acids of the mitochondrial transit peptide is unrelated to the 37-residue glyoxysomal transit peptide, which in turn is entirely different in sequence from the 57-residue chloroplastic transit peptide. With the exception of malate dehydrogenase and 3-ketoacyl thiolase, peroxisomal enzymes are synthesized without transit peptides and are frequently translocated into the organelle with a peroxisomal targeting signal consisting of a conserved tripeptide at the carboxy terminus of the protein. Based on the observation that this tripeptide (Ala-His-Leu) occurs in the transit peptides of glyoxysomal malate dehydrogenase and peroxisomal 3-ketoacyl thiolase, the possible significance of amino terminal transit peptides for peroxisome import is discussed.     

Concomitant purification of three porcine heart mitochondrial enzymes: citrate synthase, aspartate aminotransferase, and malate dehydrogenase

The mitochondrial enzymes citrate synthase, malate dehydrogenase, and aspartate aminotransferase were purified to homogeneity from porcine hearts by use of Bio-Rex 70, carboxymethylcellulose CM32, and Affi-Gel blue chromatography. This procedure provides relatively rapid, large-scale preparation of the three enzymes based on their differential binding to commercially available cation-exchange resins followed by a final affinity chromatography step.