Multiple Molecular Forms of Human Heart Cytoplasmic Aspartate Aminotransferase

1982 ◽  
Vol 62 (3) ◽  
pp. 337-339 ◽  
Author(s):  
F. Y. Leung ◽  
A. R. Henderson
Keyword(s):  
Isoelectric Focusing ◽  
Aspartate Aminotransferase ◽  
Human Liver ◽  
Human Heart ◽  
Specific Activity ◽  
Molecular Forms ◽  
Aminotransferase Activity ◽  
Multiple Molecular Forms ◽  
Isoelectric Points

1. Cytoplasmic aspartate aminotransferase was isolated and purified from human heart with a final specific activity of 236 units/mg of protein. 2. Three distinct peaks of aspartate aminotransferase activity were detected by isoelectric focusing with isoelectric points of 5.46, 5.60 and 5.71. Two minor subforms were also noted as shoulder patterns with pI 5.2 and 5.8. 3. These electrophoretic characteristics are similar to previous findings of multiple molecular forms detected in human liver and erythrocytes.

Comparative Isoelectric Focusing Properties of Plasminogen Activators From Porcine and Human Tissues

1979 ◽  
Author(s):  
Edmond Cole ◽  
Rose Snopko
Keyword(s):  
Isoelectric Focusing ◽  
Human Heart ◽  
Plasminogen Activators ◽  
Ph Gradient ◽  
Molecular Forms ◽  
Porcine Heart ◽  
Fibrin Plate ◽  
Isoelectric Points ◽  
Focusing Properties ◽  
Porcine Tissues

Plasminogen activators were isolated from porcine heart, lung and kidney and from human heart and lung. Highly purified porcine heart activator has been shown to have plasminogen activating properties different from human urinary urokinase (UK). The tissue and urinary activators were subjected to isoelectric focusing in granulated dextran gel (Ultradex) over the pH gradient range 3.5 to 11.0, and activator activity was measured by bovine fibrin plate assay. Electrofocusing of Abbott UK revealed isoelectric forms at pI 8.6, 8.8, 9.2, 9.4 and above 9.8, while Serono UK gave forms at 8.2 and 8.6. In contrast, activators from porcine tissues commonly focused at about 7.3, although occasional preparations showed non-symmetrical activity profiles, and focusing of these preparations in a pH gradient 5 to 9 revealed major PI forms at 7.2 and 7.7. Activators from human heart and lung were more closely related to activators from porcine tissues than to UK. Since UK preparations probably contain proteolytically degraded molecules, tissue activators with lower isoelectric points may represent the native molecular forms. Further evidence also indicates that a more catalytically efficient form of tissue activator may be composed of an acceleratory protein and an amidase complexed to a plasminogen activator molecule. (Supported by USPHS NIH Grant HL-15817)

Amino Acid Metabolism in Plants. III. Purification and Some Properties of a Multispecific Aminotransferase Isolated from Bushbean Seedlings (Phaseolus vulgaris L.)

1972 ◽  
Vol 50 (7) ◽  
pp. 813-829 ◽  
Author(s):  
J. C. Forest ◽  
F. Wightman
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Aspartic Acid ◽  
Aspartate Aminotransferase ◽  
Soluble Protein ◽  
Specific Activity ◽  
Aromatic Amino Acids ◽  
Total Activity ◽  
Aminotransferase Activity ◽  
Aromatic Aminotransferase

The development of aromatic aminotransferase activity was examined in cotyledons, roots, and shoots of bushbean seedlings growing under light or dark conditions for the first 2 weeks after germination. All three aromatic amino acid – α-ketoglutarate aminotransferase activities were found to have similar patterns of development in comparable organs grown under the two environmental conditions, and the changes in levels of activity appeared unrelated to variations in the endogenous amounts of free aromatic amino acids in the organs of these seedlings. The highest total activity for all three transamination reactions was found in the shoots of light-grown seedlings after 14 days, whereas the aminotransferases showing highest specific activity were found in roots of both kinds of seedlings after 8 days of growth. The intracellular distribution of the three aromatic aminotransferase activities and of aspartate aminotransferase activity was investigated by differential centrifugation of root homogenates. Only a total of 10% of these two activities was found in the two particulate fractions; the soluble protein in the final supernatant fraction accounted for almost 90% of the total aromatic and aspartate aminotransferase activities.The aromatic aminotransferase in the soluble protein fraction from seedling roots was purified about 600-fold by pH precipitation, ammonium sulfate fractionation, and Sephadex chromatography, and the recovery obtained was 30–35% based on total activity. It was observed that the specific activity for aspartate–α-ketoglutarate aminotransferase increased proportionally to the increase in aromatic aminotransferase activities during the different steps of purification. Gel electrophoresis of the purified fraction revealed only one protein band which corresponded to the product-specific stained band for the three aromatic aminotransferase activities assayed on other gels. The molecular weight of the purified aminotransferase was found to be about 128 000 daltons and its Stokes radius was calculated to be 43 ± 3 Å. The pH optima for the three aromatic aminotransferase activities and for aspartate aminotransferase activity were all found to be 8.5. The purified enzyme showed no specific requirement for pyridoxal phosphate and an examination of its amino acid substrate specificity revealed that it was able to catalyze transamination of L-aspartic acid, L-phenylalanine, L-tyrosine, and L-tryptophan when α-ketoglutarate was provided as amino group acceptor. The enzyme was also found to catalyze transamination of L-glutamic acid when oxaloacetate was used as amino group acceptor, but neither pyruvate nor glyoxylate were utilized as amino acceptors for transamination of any of the amino acids examined. The enzyme was found to catalyze transamination of aspartic acid with much greater velocity than its rate of reaction with any of the three aromatic amino acids, and the inclusion of aspartic acid in a reaction medium at equimolar concentration with any one of the three aromatic amino acids resulted in strong inhibition of the aromatic aminotransferase activity of the enzyme. All the evidence indicates that the soluble protein fraction purified from bushbean roots contained only one aminotransferase which was able to catalyze the transamination of five L-amino acids. The demonstration of the substrate multispeciftcity of this pure enzyme represents the first evidence for a multispecific aminotransferase in plants.

Multiple molecular forms of orotidylate decarboxylase from human liver

1977 ◽  
Vol 17 (2) ◽  
pp. 128-140 ◽  
Author(s):  
Mary T. Campbell ◽  
Neil D. Gallagher ◽  
William J. O'Sullivan
Keyword(s):  
Human Liver ◽  
Molecular Forms ◽  
Multiple Molecular Forms

Distribution of the multiple molecular forms of glucose-6-phosphate dehydrogenase in different physiological states

1979 ◽  
Vol 57 (5) ◽  
pp. 396-401 ◽  
Author(s):  
Hsiao-Lin Chang ◽  
Darold Holten ◽  
Rom Karin
Keyword(s):  
Enzyme Activity ◽  
Mammary Gland ◽  
Rat Liver ◽  
Specific Activity ◽  
Molecular Forms ◽  
Polyacrylamide Gels ◽  
Multiple Molecular Forms ◽  
Pregnancy And Lactation ◽  
Enzyme Specific Activity ◽  
Glucose 6 Phosphate Dehydrogenase

The distribution of the multiple molecular forms of rat liver and mammary gland glucose-6-phosphate dehydrogenase was determined by electrophoresis on 5% polyacrylamide gels. In both of these organs, changes in the distribution of enzyme activity among the several forms was slight even when approximately 20- to 40-fold changes in enzyme specific activity were achieved by fasting-refeeding experiments (for liver) or during pregnancy and lactation (for mammary gland), it was concluded that the induction of glucose-6-phosphate dehydrogenase in these two organs occurs without any major redistribution among the multiple molecular forms of this enzyme.

scholarly journals A Discussion of Enzyme Reference Materials: Applications and Specifications

1973 ◽  
Vol 19 (1) ◽  
pp. 5-9 ◽  
Author(s):  
Charles F Fasce ◽  
Robert Rej ◽  
William H Copeland ◽  
Raymond E Vanderlinde
Keyword(s):  
Enzyme Activity ◽  
Reference Materials ◽  
Aspartate Aminotransferase ◽  
Specific Activity ◽  
High Specific Activity ◽  
Aminotransferase Activity ◽  
Precision Control ◽  
Clinical Laboratories ◽  
Assay Conditions

Abstract Clinical laboratories estimating enzyme activity in serum are using commercial lyophilized sera for four major purposes. These uses—as a standard and for intermethod, intramethod, and precision control—are segregated, and specifications for each deployment are examined in terms of requirements for the enzyme material: freedom from interfering or indicator enzymes and chromogens; high specific activity; inclusion of optimal cofactor concentrations; commutability, human properties and source; the presence of a single isoenzyme; and stability. The effects of serum matrix and variable assay conditions on the utility of enzyme materials are analyzed. Specifications differ for each enzyme material application. The compatibility of commercial lyophilized sera containing aspartate aminotransferase activity with several cited specifications is assessed

Multiple Molecular Forms of Avian Aldolases. V. Purification and Molecular Properties of Chicken (Gallus domesticus) Liver Aldolase

1971 ◽  
Vol 49 (6) ◽  
pp. 647-657 ◽  
Author(s):  
Ronald R. Marquardt
Keyword(s):  
Molecular Weight ◽  
Specific Activity ◽  
Diffusion Constant ◽  
Sedimentation Coefficient ◽  
Sedimentation Velocity ◽  
Gallus Domesticus ◽  
Molecular Forms ◽  
Velocity Analysis ◽  
Multiple Molecular Forms ◽  
Stokes Radius

Aldolase (fructose-1,6-diphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) was purified from chicken liver. The enzyme was shown to be homogeneous according to the following criteria: purification to a constant specific activity following sequential chromatography on carboxymethyl-Sephadex and Sephadex G-200, electrophoresis on cellulose acetate strips, sedimentation velocity analysis, absence of 10 other glycolytic enzymes, and immunodiffusion in agar.The sedimentation coefficient (s°20w 8.0), Stokes radius (47 Å), diffusion constant (D°20w 4.0 × 10−7 cm2/s), and molecular weight (160 000) are similar to those of rabbit liver aldolase and the muscle and brain enzymes from both chickens and rabbits.

Multiple molecular forms of avian aldolases. I. Crystallization and physical properties of chicken (Gallus domesticus) breast muscle aldolase

1969 ◽  
Vol 47 (5) ◽  
pp. 517-526 ◽  
Author(s):  
Ronald R. Marquardt
Keyword(s):  
Molecular Weight ◽  
Specific Activity ◽  
Diffusion Constant ◽  
Sedimentation Coefficient ◽  
Double Diffusion ◽  
Glycolytic Enzyme ◽  
Gallus Domesticus ◽  
Molecular Forms ◽  
Rabbit Muscle ◽  
Multiple Molecular Forms

Aldolase (fructose 1,6-diphosphate-D-glyceraldehyde 3-phosphate lyase, EC 4.1.2.13) was purified and crystallized from chicken (Gallus domesticus) breast muscle.The crystalline enzyme is homogeneous according to the following criteria: purification to a constant specific activity, electrophoresis on cellulose acetate strips, absence of five other glycolytic enzyme activities, and immunodiffusion in agar.The sedimentation coefficient, diffusion constant, and molecular weight of the chicken enzyme are the same as for rabbit muscle aldolase. The ultraviolet spectra of the two proteins are the same. Electrophoretic comparison between the rabbit and chicken enzymes revealed a slightly different rate of migration.Antibodies directed against the pure chicken enzyme were prepared, and the reaction with pure chicken and rabbit aldolase was followed using the precipitin and double diffusion tests. A very pronounced reaction was observed between anti-serum and the chicken enzyme; the rabbit enzyme, in contrast, did not cross-react with the anti-serum.

Purification of lactate dehydrogenase isoenzyme-5 from human liver.

1981 ◽  
Vol 27 (1) ◽  
pp. 88-93 ◽  
Author(s):  
S M Pettit ◽  
D A Nealon ◽  
A R Henderson
Keyword(s):  
Enzyme Activity ◽  
Lactate Dehydrogenase ◽  
Isoelectric Focusing ◽  
Gel Electrophoresis ◽  
Human Liver ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Major Protein ◽  
Dehydrogenase Isoenzyme ◽  
Major Protein Band

Abstract We present a method for preparing human liver lactate dehydrogenase (L-lactate:NAD+ oxidoreductase; EC 1.1.1.27) isoenzyme-5 by sequential ion-exchange chromatography, general-ligand (AMP analog) affinity chromatography, and preparative isoelectric focusing. The yield ws 40%, with a 493-fold purification. The final specific activity was 458 kU per gram of protein. The preparation contained less than 0.2% of lactate dehydrogenase isoenzyme-4, was homogeneous by agarose gel electrophoresis and also by polyacrylamide gel electrophoresis at pH 8.9 and 6.9, and showed one major protein band (containing all the enzyme activity) and one minor anodic contaminant (containing no enzyme activity) by analytical isoelectric focusing. The enzyme had a mean pI value of 9.59 (SD 0.04) (n = 5) at 5 degrees C. By comparison, the pI value of a preparation of rabbit lactate dehydrogenase-5 was 9.16 (5 degrees C).

Isolation and purification of aspartate aminotransferase isoenzymes from human liver by chromatography and isoelectric focusing.

1981 ◽  
Vol 27 (2) ◽  
pp. 232-238 ◽  
Author(s):  
F Y Leung ◽  
A R Henderson
Keyword(s):  
Isoelectric Focusing ◽  
Aspartate Aminotransferase ◽  
Human Liver ◽  
Exchange Chromatography ◽  
Mitochondrial Enzymes ◽  
Cation Exchange Chromatography ◽  
Ammonium Sulfate Precipitation ◽  
Activity Maximum ◽  
Isolation And Purification ◽  
Optimum Ph

Abstract To purify cytoplasmic and mitochondrial isoenzymes of aspartate aminotransferase (EC 2.6.1.1) from human liver. We used heat treatment, ammonium sulfate precipitation, anion- and cation-exchange chromatography, affinity chromatography, and isoelectric focusing. Final preparations of the isoenzymes were homogeneous, with specific activities of 198 and 208 kU/g for the cytoplasmic and the mitochondrial enzymes, respectively. The mitochondrial isoenzyme focused as a single band with a pl value of 9.60, whereas the cytoplasmic isoenzyme had subforms with pl values of 5.22, 5.42, and 5.62 at 4 degrees C. In Tris . HCl buffer, both isoenzymes have an activity maximum at pH 7.8. In [bis(2-hydroxyethyl)amino]tris(hydroxymethyl)methane (Bistris) buffer, however, the mitochondrial isoenzyme also showed an optimum pH of 6.7.

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