Anionic Isozyme Research Articles

Purification and characterization of the individual glutathione S-transferases from sheep liver

The glutathione S-transferases (EC 2.5.1.18) have been purified to electrophoretic homogeneity from 105,000 g supernatant of sheep liver homogenate by employing a combination of gel filtration on Sephadex G-150 and affinity chromatography on S-hexylglutathione-linked Sepharose-6B columns. Approximately 70% of the original glutathione S-transferase activity toward 1-chloro-2,4-dinitrobenzene and glutathione peroxidase activity toward cumene hydroperoxide could be recovered by this purification method. Of particular importance in developing this procedure was the fact that the enzyme preparation obtained after affinity column chromatography represented all the isozymes of sheep liver glutathione S-transferases. Further purification by CM-cellulose and DEAE-cellulose column chromatography resolved the glutathione S-transferases into seven distinct cationic isozymes designated C-1, C-2, C-3, C-4, C-5, C-6, and C-7 and five overlapping anionic transferases designated A-1, A-2, A-3, A-4, and A-5, respectively, in the order of their elution from the ion-exchange columns. The sodium dodecyl sulfate SDS-gel electrophoretic data on subunit composition revealed that cationic enzymes are composed of two subunits with an identical M r of 24,000 whereas a predominant subunit with M r of 26,000 was observed in all anionic isozyme peaks except A-1. Cationic isozymes accounted for approximately 98% of the total peroxidase activity associated with the glutathione S-transferase whereas only A-1 of the anionic isozymes displayed some peroxidase activity. Isozyme C-4 was found to be the most abundant glutathione S-transferase in the sheep liver. Characterization of the individual transferases by their specificity toward a number of selected substrates, subunit composition, and isoelectric points showed some similarities to those patterns for human liver glutathione S-transferases.

Isolation and characterization of an anionic glutathione [formula omitted]-transferase from rat liver cytosol

An anionic glutathione S -transferase representing approximately 20% of the total glutathione S -transferase protein and 10% of the total transferase activity toward 1-chloro 2,4-dinitrobenzene has been purified to homogeneity from the 105,000 x g supernatant of rat liver homogenate. The SDS gel electrophoretic data on subunit composition revealed that the anionic isozyme is composed of two subunits with an identical M r of 26,000. The K m values for 1-chloro 2,4-dinitrobenzene and reduced glutathione were determined to be 0.94 mM and 0.23 mM respectively. A significant amount of glutathione peroxidase activity toward cumene hydroperoxide is associated with the new isozyme.

Molecular weight and net charge of peroxidase isozymes in F1 hybrids between L and S flax genotrophs.

In the F1 hybrids between Durrant's L and Durrant's S flax genotrophs, the relative mobilities of the anionic peroxidase isozymes were essentially the same as those in the L parent. The isozymes in both parents and their F1's were compared over a range of acrylamide gel concentrations, with plots of log relative mobility against gel concentration. Plots of comparative mobility, relative to the internal standard hemoglobin, against concentration were also examined. Both approaches provided evidence that apparent molecular weight modifications underlay the shift in mobility between the parents and the resemblance of the F1's to L, the parent which was homozygous for the dominant alleles controlling the mobility shift for at least two of the isozymes.

COMPARATIVE THERMAL STABILITY OF PEROXIDASE ISOZYMES FROM TWO FLAX GENOTROPHS

The thermal stability of peroxidase isozymes was examined in vitro in Linum usitatissimum L. Extracts of main stem tissues of the L and S genotrophs produced by Durrant were heat treated over a range of temperatures and times. Isozymes in treated extracts were separated electrophoretically, and peak areas for the four main anionic isozymes, together with their relative mobilities (Rms), were recorded. Peak areas supplied estimates of relative activities. Short duration treatments at 60° and 70 °C demonstrated differences in thermal stability between isozymes and produced changes in Rm. With prolonged treatment at 40 °C, the thermal stability of one isozyme differed from those of the other three. This isozyme was known to have a higher molecular weight than the others. In addition, prolonged treatment at 40 °C demonstrated increased thermal stability of the three lower molecular weight isozymes of genotroph S compared to those of L.

DIFFERENCES IN MOLECULAR WEIGHT BETWEEN CORRESPONDING ANIONIC PEROXIDASE ISOZYMES FROM TWO FLAX GENOTROPHS

Anionic peroxidase isozymes from main stem tissues of adult plants of two flax (Linum usitatissimum L.) genotrophs were separated using acrylamide gel electrophoresis. A range of seven acrylamide concentrations was used for the gels, enabling the effect of gel concentration on relative mobility (Rm) to be examined. The regression of log (Rm) on gel concentration was linear for two of the four main isozymes found. Differences in linear regression slope between the L and S flax genotroph isozymes suggested genotroph differences in molecular weight.

Segregation of the environmentally induced relative mobility shifts in flax genotroph peroxidase isozymes

Crosses were made in all combinations between the large (L) and small (S) flax genotrophs, and their reciprocal F1's. Total main stem peroxidase activity was measured prior to electrophoretic separation; individual anionic isozyme relative mobilities (Rm's) were obtained from electrophoresis of main stem homogenates. Both activity and Rm were examined in parent, F1, F2 and backcross generations on an individual plant basis. Segregation for isozyme Rm occurred, and indicated control over each isozyme's Rm by a simple system with a dominant allele in L and a recessive in S. For activity, dominance was absent, and there was no evidence of segregation.

Iaa oxidase and polyphenol oxidase activities of peanut peroxidase isozymes

Four anionic isozymes (A 1, A 2, A 4 and A 5) from peanut cells in suspension medium possessed IAA oxidase and polyphenol oxidase activities. The specific activities of each of the enzymes differed among the 4 isozymes. The pH optima established in these assays for peroxidase was acidic, for IAA oxidase neutral and for polyphenol oxidase alkaline. All 4 isozymes had different K m and V max for the enzyme activities of peroxidase and polyphenol oxidase. The sigmoid kinetics from the IAA oxidase assays for the isozymes probably indicates an allosteric nature.

The kinetics of synthesis and degradation of aspartate aminotransferase isozymes in rat peripheral red blood cells during cytodifferentiation.

SummaryThe kinetics of synthesis and degradation of aspartate aminotransferase (EC 2.6.1.1) isozymes were investigated in peripheral red blood cells of rats made reticulocytotic with phenylhydrazine. Immunochemical studies showed that reticulocytes contain predominantly the cationic isozyme, whereas mature erythrocytes contain only the anionic isozyme. Following initiation of phenylhydrazine treatment, the cationic isozyme increased 47-fold above its control level on Day 5, and the anionic isozyme increased threefold above its control level on the 8th day. The half-lives were 24.6 and 154.9 hr for the cationic and anionic isozyme, respectively. The faster turnover of the cationic isozyme appears to be related to its rapid rate of degradation.We thank Miss Frances Grainger for her excellent technical assistance.

Activity and relative mobility of peroxidase and esterase isozymes of flax (Linum usitatissimum) genotrophs. II. F1 hybrids and nuclear DNA reversion types.

The effects of growth of one genotype of flax in soil supplemented by either nitrogen, phosphorous and potassium (NPK) or nitrogen and potassium (NK) on its progeny produced by several generations of complete selfing were studied. The two types of progeny produced, genotroph L, induced by NPK and genotroph S induced by NK, and their F1reciprocal hybrids were examined. L and S differed in plant weight and in the activities and relative mobilities of their corresponding anionic peroxidase and esterase isozymes. No reciprocal differences were detected and the mean F1values for all characteristics were intermediate between the parents, with the exception of the peroxidase isozyme relative mobilities which displayed dominance of parent L. The genotrophs were known to exhibit a 16% difference in nuclear DNA content which could be reverted to 0%, without altering the genotroph fresh weight difference, by growing the genotrophs in lower than normal temperatures. Examination of the peroxidase and esterase isozymes of nDNA reverted genotrophs showed that, with one possible exception, there was no accompanying reversion of the activities and relative mobilities of the peroxidase or esterase isozymes.

Regulation of Aspartate Aminotransferase Isozymes by d-Erythrose 4-Phosphate and Glycoladehyde Phosphate, the Naturally Occurring Homologues of d-Glyceraldehyde 3-Phosphate

Abstract As part of a study of the regulation of aspartate aminotransferase isozymes (EC 2.6.1.1), we have investigated the characteristics of inhibition of the anionic and cationic isozymes by d-erythrose 4-phosphate and glycolaldehyde phosphate, the naturally occurring homologues of d-glyceraldehyde 3-phosphate. Erythrose-4-P has been found to be a time-dependent inhibitor of both the cationic and anionic isozymes. The inhibitor dissociation constants and types of inhibition were determined from residual enzyme activity after equilibration of each isozyme with the inhibitor and one substrate in the preliminary incubation mixtures. Inhibition of both isozymes by erythrose-4-P was completely competitive with respect to α-ketoglutarate and completely noncompetitive with respect to aspartate. The Ki values of the cationic isozyme were 1.44 and 0.135 mm for the pyridoxal and pyridoxamine forms, respectively. The Ki values of the anionic isozyme were 3.04 and 0.33 mm for the pyridoxal and pyridoxamine forms, respectively. Both forms of the cationic isozyme were more sensitive to inhibition than the corresponding forms of the anionic isozyme. Inhibition of aspartate aminotransferase isozymes by glycolaldehyde-P was complete and not time-dependent. Inhibition was competitive with respect to α-ketoglutarate as the variable substrate and uncompetitive with respect to aspartate as the variable substrate. The Ki values were 1.07 and 0.79 mm for the pyridoxamine form of the cationic and anionic isozymes, respectively. The characteristics of the inhibition of aspartate aminotransferase isozymes by erythrose-4-P, glyceraldehyde-3-P, and glycolaldehyde-P are not related to number of carbon atoms in these compounds. Kinetic analyses of the binding of glycolaldehyde-P and glyceraldehyde-3-P to the isozymes showed that their binding is mutually exclusive. The results suggest that glyceraldehyde-3-P and its naturally occurring homologues erythrose-4-P and glycolaldehyde-P may be involved in the regulation of gluconeogenesis in vivo by affecting the activity of the isozymes of aspartate aminotransferase.

Regulation of aspartate amino-transferase isozymes by glyceraldehyde-3-phosphate

This review concerns our work on glyceraldehyde-3-P as a time-dependent inhibitor of rat liver aspartate aminotransferase isozymes, and the possibility that glyceraldehyde-3-P may function as a rapidly acting regulator of aspartate aminotransferase. The d-isomer and the dl-racemate of glyceraldehyde-3-P were equally effective for each isozyme. Study of several glycolytic intermediates indicated that the conjoint presence of the free aldehyde and the phosphoryl residue was necessary for inhibition. Maximum inhibition of the anionic isozyme occurred at pH values from 8.4 to 10.3; the cationic isozyme was optimally inhibited at pH 7.4. Keto acid substrates decreased the inhibition, whereas amino acid substrates accentuated it. Inhibition of the cationic isozyme was completely competitive with respect to α-ketoglutarate ( K i = 0.98 m m ) and oxaloacetate ( K i not measurable), and completely noncompetitive with respect to l-aspartate ( K i = 0.084 m m ) and l-glutamate ( K i = 0.11 m m ). Inhibition of the anionic isozyme was mixed partially competitive-partially noncompetitive with respect to α-ketoglutarate ( K i = 1.9 m m ) and oxaloacetate ( K i = 1.5 m m ) and partially noncompetitive with respect to l-aspartate ( K i = 0.39 m m ) and l-glutamate ( K i = 0.57 m m ). These data suggest that both keto acids bind to the isozymes at a single site and compete with glyceraldehyde-3-P for that site, whereas the amino acids bind to a site other than the one for which keto acids and glyceraldehyde-3-P compete. Homologues of glyceraldehyde-3-P were also investigated. Ribose-5-P, fructose-6-P and glucose-6-P did not inhibit either isozyme since they exist as the internal hemiacetals. d-erythrose-4-P was a time-dependent inhibitor of both isozymes. Inhibition was completely competitive with respect to α-ketoglutarate, K i = 3.08 m m and 1.4 m m for the anionic and cationic isozymes, respectively, and was completely noncompetitive with respect to l-aspartate, K i = 0.334 m m and 0.135 m m for the anionic and cationic isozymes, respectively. Inhibition by glycolaldehyde-P was not time-dependent and was completely competitive with respect to α-ketoglutarate and uncompetitive with respect to l-aspartate for both isozymes. The K i values were 0.77 m m and 1.01 m m for the anionic and cationic isozymes, respectively. We propose that glyceraldehyde-3-P and its homologues inhibit aspartate aminotransferase isozymes by forming a Schiff base with one of the ϵ-amino groups of lysine at the enzymically active site. Aspartate would potentiate inhibition by converting the enzyme to the pyridoxamine form, thereby exposing a second ϵ-amino lysyl group which would react with the inhibitor. Evidence for a Schiff base was obtained by NaBH 4 reduction of apoisozymes in the presence of dl-glyceraldehyde-3-P. This prevented restoration of activity upon addition of pyridoxal-5′-P. It seems likely that the divalent phosphoryl group on the inhibitor molecule is involved in the competition at the keto acid binding site. Our results suggest the possibility that glyceraldehyde-3-P may be implicated in the regulation, in vivo, of gluconeogenesis as well as other metabolic pathways by affecting the activity of the isozymes of aspartate aminotransferase.

Effects of oxaloacetate and L-glutamate on glyceraldehyde 3-phosphate inhibition of aspartate aminotransferase isozymes as measured by a 2oxoglutarate dehydrogenase coupled assay.

A new coupled reaction for the measurement of aspartate aminotransferase activity with oxaloacetate and l‐glutamate as substrates is described. This reaction measures the appearance of the product, 2‐oxoglutarate, with 2‐oxoglutarate dehydrogenase. It avoids the high concentrations of phosphate or arsenate which have been used to catalyze tautomerization in those assays which utilize the absorbance of the enol form of oxaloacetate to measure its concentration. These salts inhibit the cationic isozyme of aspartate aminotransferase. Values for Km(oxaloacetate) and Km(l‐glutamate) were 0.037 mM and 7.8 mM, respectively, for rat liver anionic isozyme and 0.006 mM and 8.6 mM, respectively, for the cationic isozyme.Glyceraldehyde 3‐phosphate inhibition of the anionic isozyme was partially competitivepartially noncompetitive with respect to oxaloacetate and partialy noncompetitive with respect to l‐glutamate, with β= 0.31. Inhibition by glyceraldehyde‐3‐P of the cationic isozyme was non‐competitive with respect to l‐glutamate. Ki values for glyceraldehyde‐3‐P for the anionic isozyme were 0.57 mM in the presence of l‐glutamate and 1.5 mM in the presence of oxaloacetate. The Ki value for the cationic isozyme in the presence of l‐glutamate was 0.11 mM. These values are in agreement with those that were reported previously for inhibition in the presence of l‐aspartate and 2‐oxoglutarate. The similarity between the types of inhibition in both directions of transamination indicates that both keto acid substrates bind to the isozymes at a single site and compete with glyceraldehyde‐3‐P for that site. The noncompetitive nature of inhibition in the presence of amino acid substrates suggests that at least one point of their attachment is outside of the site to which the keto acids and glyceraldehyde‐3‐P bind.

Kinetics of the inhibition of aspartate aminotransferase isozymes by DL-glyceraldehyde 3-phosphate.

The inhibitor dissociation constants and types of inhibition of the isozymes of aspartate aminotransferase from rat liver by the time‐dependent inhibitor dl‐glyceraldehyde 3‐phosphate were determined from residual enzyme activity after equilibration of each isozyme with the inhibitor and one substrate in the preliminary incubation mixtures.The results showed that the inhibition of the cationic isozyme was completely competitive with respect to α‐ketoglutarate and completely noncompetitive with respect to l‐aspartate. The pyridoxamine form of the cationic isozyme was approximately 10 times more sensitive to inhibition by dl‐glyceraldehyde 3‐phosphate (Ki= 0.084 mM) than the pyridoxal form (Ki= 0.98 mM). Inhibition of the anionic isozyme was mixed partially competitive‐partially noncompetitive with respect to α‐ketoglutarate and partially noncompetitive with respect to l‐aspartate. The pyridoxamine form of the anionic isozyme was approximately 5 times more sensitive to inhibition by dlDL‐glyceraldehyde 3‐phosphate (Ki= 0.39 mM) than the pyridoxal form (Ki= 1.9 mM). Both forms of the cationic isozyme were more sensitive to inhibition than the corresponding forms of the anionic isozyme.The degree to which inhibition of the anionic isozyme was partial was assessed by determining the interaction constants α and β. These were α= 6.9 and β= 0.32, and indicated that the anionic isozyme, when fully combined with dl‐glyceraldehyde 3‐phosphate, retained 32% of its catalytic capacity. The cationic isozyme was completely inhibited with α=∞ and β= 0.Comparison of the Ki values for the pyridoxamine and pyridoxal forms of the isozymes of aspartate aminotransferase with the concentrations of d‐glyceraldehyde 3‐phosphate reported for perfused liver and for incubated erythrocytes suggests that inhibition may occur in vivo.

Time-dependent Inhibition of Aspartate Aminotransferase Isozymes by dl-Glyceraldehyde 3-Phosphate

Abstract dl-Glyceraldehyde 3-phosphate in low concentrations has been found to be a time-dependent inhibitor of aspartate aminotransferase (EC 2.6.1.1) isozymes. The cationic isozyme is more susceptible than the anionic isozyme to this inhibition. The l-isomer of glyceraldehyde 3-phosphate was as effective as the d-isomer for each isozyme. Study of various glycolytic intermediates indicated that the conjoint presence of the free aldehyde group and the phosphoryl residue of glyceraldehyde 3-phosphate was necessary for inhibition. The extent of inhibition was dependent on the duration of preliminary incubation, the concentration of glyceraldehyde-3-P, and the pH of the preliminary incubation mixture, but was independent of the concentration of enzyme. Half-maximum inhibition of each isozyme was obtained between pH 6.5 and 6.7. Maximum inhibition of the anionic isozyme was obtained at pH values of 8.4 and above, while the cationic isozyme was optimally inhibited at pH 7.4. The presence of α-ketoglutarate in the preliminary incubation mixture decreased inhibition while aspartate accentuated it. After 30 min of preliminary incubation with glyceraldehyde-3-P, dialysis of the anionic isozyme against buffer, aspartate, α-ketoglutarate, or pyridoxal 5-phosphate resulted in a substantial release of inhibition. In the case of the cationic isozyme, substantial release of inhibition was obtained only by dialysis against α-ketoglutarate. The addition of α-ketoglutarate to either the anionic or cationic isozyme-inhibitor preliminary incubation mixtures released inhibition, but only in the case of the anionic isozyme was the extent of release dependent on the duration of preliminary incubation. Prolonged incubation of this isozyme with glyceraldehyde-3-P resulted in the appearance of a second phase of inhibition. The mechanism of the inhibition of the isozymes by glyceraldehyde-3-P is discussed.

Effects of phosphate and other anions on measurement of the activities of the isozymes of rat liver aspartate aminotransferase

When the activity of the isozymes of rat liver aspartate aminotransferase is measured in Tris-HCl buffer at pH 7.4, the addition of phosphate or sulfate ions inhibits the cationic isozyme and slightly accelerates the anionic isozyme. Addition of chloride ions counteracts the effect of sulfate and phosphate on the cationic isozyme. At low concentrations of α-keto-glutarate, inhibition of the anionic isozyme by phosphate is competitive, and inhibition of the cationic isozyme is of mixed type. When mixtures of the isozymes are assayed in phosphate buffer, low values for the activity of the cationic isozyme are obtained. Thus the anionic fraction is overestimated.

Kinetics and Electrophoretic Properties of the Isozymes of Aspartate Aminotransferase from Pig Heart

Abstract Cationic aspartate aminotransferase from pig heart was completely destroyed by heating to 75° for 20 min in the purification procedure of Jenkins, Yphantis, and Sizer (11). Chromatography of the partially purified enzyme preparation on carboxymethyl cellulose according to the method of Henson and Cleland (8) yielded two enzyme fractions both of which migrated toward the anode upon starch gel electrophoresis. These two fractions were also kinetically and immunochemically indistinguishable from each other. Pig heart mitochondrial aspartate aminotransferase migrated toward the cathode upon starch gel electrophoresis. It differed both kinetically and immunochemically from the anionic isozyme of pig heart.

Isozymes of aspartate aminotransferase in tissues and blood of man

Available literature on the characteristics of alanine and aspartate aminotransferases has been briefly reviewed. Our own studies have concerned themselves with the isozymes of aspartate aminotransferase in the tissues and blood of man. The anionic (supernatant) and the cationic (mitochondrial) isozymes of liver and heart have been prepared, and antisera to each of the isozymes from heart have been produced. The values for K m( l-aspartate ) were the same for the anionic isozyme of heart or liver and were distinctly higher than the values that were obtained for the cationic components. Conversely, the values for K m( α-ketoglutarate) were the same for the anionic isozyme of heart or liver and were distinctly lower than the values that were obtained for the cationic components. Antiserum to each of the two isozymes from heart inhibited the homologous isozyme from heart or liver specifically and practically completely. Hemolysates of washed human mature erythrocytes yielded, in general, one band, anionic in migration. This component had values for K m( l-aspartate ) and K m( α-ketoglutarate) and immunochemical characteristics that were essentially the same as those obtained for the anionic component of liver or heart. The observation that a cationic band was present in the blood of an individual after blood donation led to the study of experimentally produced reticulocytosis in rabbits. The total erythrocyte aspartate aminotransferase activity increased five- to sixfold, and a cationic mitochondrial isozyme appeared. The relationship of the electrophoretic pattern of aspartate transaminase in human tissues to the electrophoretic pattern in the serum was also studied. Starch paste electrophoresis was performed on homogenates of forty specimens from ten different tissues and two tumors. The aminotransferase was distributed into two main areas, one in the region of the gamma globulin and the other in the alpha-beta globulin region. The serum aspartate aminotransferase in four normal persons and in twenty patients with neoplastic disease with elevated serum enzyme activity was completely confined to one area—the alpha-beta globulin region. Fourteen patients also exhibited a cationic component in the serum. The appearance of this component appeared to be associated with an acute phase of the patient's disease and its disappearance with the subsidence of this phase. The general role of the cationic, mitochondrial and the anionic, supernatant isozymes in regulation of cell metabolism is discussed briefly.

Glutamic-Oxaloacetic Transaminases in Reticulocytes and Erythrocytes

In rabbits, mature erythrocytes contain only the anionic isozyme of glutamic-oxaloacetic transaminase. Reticulocytosis induced by massive bleeding or by treatment with acetylphenylhydrazine is characterized by a five- to sixfold increase in red-cell transaminase and the appearance of a cationic transaminase isozyme that apparently resides in the mitochondrial fraction of reticulocytes.