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

1987 ◽  
Vol 65 (3) ◽  
pp. 239-244 ◽  
Author(s):  
Charalampos Mavrides ◽  
Guylaine Nadeau
Keyword(s):  
Malate Dehydrogenase ◽  
Crude Extract ◽  
Aspartate Aminotransferase ◽  
Rat Heart ◽  
Pyridoxal Phosphate ◽  
Single Procedure ◽  
Purification And Properties ◽  
Michaelis Constants ◽  
Malate Aspartate Shuttle ◽  
Original Crude

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.

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

1985 ◽  
Vol 33 (7) ◽  
pp. 624-630 ◽  
Author(s):  
C D Ross ◽  
D A Godfrey
Keyword(s):  
Energy Metabolism ◽  
Correlation Coefficient ◽  
Malate Dehydrogenase ◽  
Aspartate Aminotransferase ◽  
Horizontal Cell ◽  
Plexiform Layer ◽  
The Other ◽  
Retinal Layers ◽  
Cell Processes ◽  
Malate Aspartate Shuttle

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.

The preparation of the cytoplasmic and mitochondrial forms of malate dehydrogenase and aspartate aminotransferase from pig heart by a single procedure

1974 ◽  
Vol 57 (2) ◽  
pp. 432-451 ◽  
Author(s):  
Beat E. Glatthaar ◽  
Gary R. Barbarash ◽  
Barbara E. Noyes ◽  
Leonard J. Banaszak ◽  
Ralph A. Bradshaw
Keyword(s):  
Malate Dehydrogenase ◽  
Aspartate Aminotransferase ◽  
Single Procedure

Interlaboratory Proficiency, Intermethod Comparison, and Calibrator Suitability in Assay of Serum Aspartate Aminotransferase Activity

1975 ◽  
Vol 21 (8) ◽  
pp. 1141-1158 ◽  
Author(s):  
Robert Rej ◽  
C F Fasce ◽  
Raymond E Vanderlinde
Keyword(s):  
Malate Dehydrogenase ◽  
Aspartate Aminotransferase ◽  
Continuous Flow ◽  
Pyridoxal Phosphate ◽  
Diazonium Salt ◽  
Assay Method ◽  
Kinetic Assay ◽  
Mean Values ◽  
Cytoplasmic Enzyme ◽  
Calibration Materials

Abstract Sources of variation in assays of aspartate aminotransferase (EC 2.6.1.1) activity were examined in an interlaboratory survey and through an examination of materials used as calibration materials in these assays. Four highly stable lyophilized specimens containing human cytoplasmic enzyme, with activities of 0, 22, 46, and 96 U/liter at 30 °C and optimal substrate concentrations, were assayed by 319 laboratories. Mean values obtained on these specimens by laboratories using 2,4-dinitrophenylhydrazine kits varied among manufacturers and deviated from values expected from this procedure. The average coefficient of variation (CV) with these kits was > 20%. Automated continuous-flow procedures with use of diazonium salt showed the best precision (av CV, < 10%). However, the automated continuous-flow mAlate Dehydrogenase/Nadh Coupled Method Produced An Average Cv > 20%. Results from each of the automated methods were related to a reference malate dehydrogenase/NADH coupled continuous kinetic assay method by temperature relationships alone. Mean values from manual diazonium salt procedures were 1.7-fold greater than similar reference values (av CV was 18%). The higher results were attributed to the use of poorly-defined units and to an artifact caused by chromophore stabilizers in this procedure when aqueous samples are used. The average CV in continuous kinetic methods varied among kit manufacturers, ranging from 6 to 28% for the specimen of highest activity. Variations in results were much larger at 366 nm than at 340 nm. Interassay relationships of these methods are presented. Concentrations of pyruvate in commercially available calibration materials differed between manufacturers, varied in stability, and deviated from the expected concentration. For some colorimetric assays the precision attained on reported absorbance values for the enzyme specimens was of the same order of magnitude as that for pyruvate standards. Other sources of error are revealed by the interlaboratory survey. The value of commercially available sources of enzyme activity as calibration or control materials was assessed by evaluating the following properties: activity at suboptimal concentrations of L-aspartate or 2-oxoglutarate, temperature effects, preincubation lability owing to aspartate and phosphate, pyridoxal phosphate saturation, contamination with glutamate dehydrogenase, and manufacturer's rated activity. These properties are compared to those of human cytoplasmic enzyme in a human serum matrix.

scholarly journals ACTIVITY OF MALATE-ASPARTATE SHUTTLE ENZYMES OF BULLS AND BOARS REPRODUCTIVE ORGANS AND EPIDIDIMAL SPERMATOZOA

2020 ◽  
Vol 21 (2) ◽  
pp. 86-89
Author(s):  
N. V. Kuzmina ◽  
D. D. Ostapiv ◽  
O. I. Chajkovska ◽  
R. D. Ostapiv ◽  
O. P. Panych
Keyword(s):  
Malate Dehydrogenase ◽  
Aspartate Aminotransferase ◽  
Protein Concentration ◽  
Tissue Specificity ◽  
Testicular Tissue ◽  
Reproductive Organs ◽  
The Body ◽  
White Breed ◽  
Dairy Breed ◽  
Malate Aspartate Shuttle

The activity of malate-aspartate shuttle enzymes in the reproductive organs and epididimal sperm of bulls and boars was studied. The research was conducted on bulls of the Ukrainian black-spotted dairy breed (n = 5; aged 14 - 16 months) and boars of the Great White breed (n = 3; aged 10 - 12 months). After slaughter, the testes and testicular appendages were removed and the spermatozoa were washed with 0.9% sodium chloride solution. Testicular and epididimal tissues were homogenized and centrifuged. Aspartate aminotransferase (AST), malate dehydrogenase (MDG) and protein concentration were determined in the supernatant and epididimal sperm. The specie and tissue specificity of aspartate aminotransferase and malate dehydrogenase activity have been established. In particular, in bulls, the activity of AST in the tissues of the testis, head and body of the epididymis of bulls is almost the same (116.5 - 118.3 nmol/min×mg of protein) and on 18.5 - 19.7% (р<0,05) higher in the tail of the appendix. In this case, MDG activity in the reproductive organs shows wavy changes: in the testicular tissue is high (1.96 ± 0.15 nmol / min × mg of protein), and in the epididymis: in the head is reduced (3.5 times ; p <0,001), increased in the body (p <0,05) and decreased again in the tail. In the sperm of bulls, the activity of AST from the head of the appendix - 69.3 ± 8.06 nmol / min × mg of protein, from the body - increased (p <0.05) and again (p <0.001) decreased from the tail of the appendix. MDG activity in sperm from the head of the epididymis was 0.50 ± 0.04 nmol/min×mg of protein, lower by 38.0% (p <0.05) from the body and 50.0% (p <0.001) higher in the tail. In boars, the activity of AST in testicular tissue - 20.3 ± 5.22 nmol / min × mg of protein and higher in the appendix: 45.6% - in the head, 59.0% - in the body and 64.0 % - in the tail. Similarly, the activity of MDG in testicular tissue was 0.22 ± 0.02 nmol / min × mg of protein, increases by 40.6% in the head and remained at the same level in the body of the appendix, and in the tail tissue above 2, 5 - 2.7 times (p <0.001). In bovine germ cells, the activity of AST and MDG from the body of the appendix was, respectively, 102.3 ± 6.1 and 13.20 ± 0.15 nmol / min × mg of protein, lowered twice (p <0,001) and 10.7 % in the head and more than four times (p <0,001) and 61,2% (p <0,001) in the tail of the appendix.

Comparison of the precursor and mature forms of rat heart mitochondrial malate dehydrogenase

Biochemistry ◽  
1987 ◽  
Vol 26 (1) ◽  
pp. 128-134 ◽  
Author(s):  
Paula M. Grant ◽  
Steven L. Roderick ◽  
Gregory A. Grant ◽  
Leonard J. Banaszak ◽  
Arnold W. Strauss
Keyword(s):  
Malate Dehydrogenase ◽  
Rat Heart ◽  
Mitochondrial Malate Dehydrogenase

scholarly journals Aspartate aminotransferase. The effects of ionic concentration on kinetic constants of both isoenzymes

1968 ◽  
Vol 106 (3) ◽  
pp. 581-586 ◽  
Author(s):  
T. R. C. Boyde
Keyword(s):  
Aspartate Aminotransferase ◽  
Specific Effect ◽  
Ionic Concentration ◽  
Phosphate Concentration ◽  
Kinetic Constants ◽  
Sharp Minimum ◽  
Ionic Concentrations ◽  
Michaelis Constants ◽  
Added Salt

1. The Michaelis constants for both isoenzymes for both substrates depend strongly on ionic concentration, being approximately proportional to phosphate concentration over considerable ranges. This is probably an effect of anions only. 2. In the absence of added salt, Km (2-oxoglutarate) (anionic isoenzyme) is so small as to be indeterminate. 3. Km (l-aspartate) (anionic isoenzyme) passes through a sharp minimum at about 3·3mm-phosphate. It is not clear whether this is a specific effect of phosphate. 4. Both substrates are inhibitory at sufficiently low ionic concentrations. 5. A modified graphical procedure is described for the derivation of the kinetic constants.

The C-S lysis of L-cysteine conjugates by aspartate and alanine aminotransferase enzymes

1995 ◽  
Vol 14 (5) ◽  
pp. 422-427 ◽  
Author(s):  
Peter J Gaskin ◽  
Harriet J Adcock ◽  
Lorraine D Buckberry ◽  
Paul H Teesdale-Spittle ◽  
P Nicholas Shawl
Keyword(s):  
Aspartate Aminotransferase ◽  
Alanine Aminotransferase ◽  
Pyridoxal Phosphate ◽  
Enzyme Inhibitor ◽  
Potential Interaction ◽  
Direct Inhibition ◽  
Porcine Heart ◽  
Lyase Activity ◽  
Oxyacetic Acid ◽  
Dependent Mechanism

One biotransformation pathway which is responsible for the generation of mutagenic and cytotoxic metabolites is that of the C-S lysis (CSL) of L-cysteine conjugates. Thirteen cysteine S-conjugates, synthesised in our labora tories, were incubated with porcine heart aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT), and the C-S lyase activity for each enzyme-sub strate combination was determined. ASAT and ALAT were shown to exhibit CSL activity. It was also demonstrated that this activity was inhibited in the presence of the pyri doxal phosphate (PLP)-dependent enzyme inhibitor amino(oxyacetic acid) (AOAA) confirming the pyridoxal phosphate dependent mechanism by which C-S lysis is known to take place. Since the activities of these enzymes are used as biomarkers for the assessment of organ dam age, the potential interaction of L-cysteine conjugates with them may suppress their activity through direct inhibition.

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