Cationic Isoenzyme Research Articles

Purification of two peroxidase isoenzymes of Aloe barbadensis which oxidize p-coumaric acid

Using a combination of hydrophobicity and ion-exchange chromatography methods, one cationic (p I 9.0) and one anionic (p I 4.5) peroxidase (donor: hydrogen-peroxide oxidoreductase; EC 1.11.1.7) isoenzymes of Aloe barbadensis have been purified (the cationic peroxidase to homogeneity as judged by SDS-PAGE analysis and microsequencing). This allowed us to initiate the investigation of individual catalytic properties to be related to their respective functions in vivo. The two peroxidases have an optimal activity at pH 6.0. Apparent affinities for H 2O 2 range between 0.01 and 0.14 mM depending on the phenolic substrate and the isoenzyme. The apparent K m values for the phenolics ( p-coumaric acid and hydroquinone) are some 25-fold lower in the anionic (around 0.02 mM) than in the cationic (around 0.77 and 0.34 mM, respectively) isoenzyme. The possible functions of the activities are discussed.

Peroxidase Activity and Isoperoxidase Pattern in Tobacco Leaves Infected with Tobacco Necrosis Virus and Other Viruses Inducing Necrotic and Non‐Necrotic Alterations

AbstractThe level of peroxidase activity was greatly enhanced in tobacco leaves infected by tobacco necrosis virus (TNV) and other viruses which induce necrotic symptoms (TMV, ToMV and PVYN). The intensity was related to the age of the leaves infected: absent or neglible in mature leaves and very pronounced in young growing infected leaves. On the contrary, changes in peroxidase activity were negligible when the infection was provoked by viruses which do not produce necrotic reactions (TMV and PVYO). Analysis of the peroxidase isoenzymes, pattern in tobacco leaves infected by TNV and other necrosis‐inducing viruses revealed in all cases, a slight increase in anionic (pl 3.5–3.7) and a considerable increase in moderately anionic isoenzymes particularly the pl 4.6 isoenzyme which in TNV and PVYN‐infected leaves reached levels up to 21 and 72 times the healthy control values. A considerable increase in the cationic (pl9.3–8.8) isoenzymes and the appearance of one moderately cationic isoenzyme (pl 8.2) was also detected. In leaf extracts from‐virus‐infected tobacco leaves with nonnecrotic response, no, or negligible alterations on the isoenzyme pattern were detected. However, infection by a fungal parasite (Erisyphe cichoracearum), which established a fully compatible, non‐necrotic, interaction with tobacco leaves, like the necrosis‐inducing viruses, changed the isoperoxidase pattern. The data suggest the necrotic alterations and associated changes in the peroxidase activity and isoperoxidase pattern in virus‐infected leaves are not clearly related.

Spectroscopic investigations on the highly purified lactoperoxidase Fe(III)-heme catalytic site

Purification of the lactoperoxidase (LPO) major cationic isoenzyme was significantly improved by the use of preparative chromatographic and electrophoretic methods combined with analytical electrophoretic techniques and image processing. A detailed report is given of the experimental procedure. Furthermore, electron paramagnetic resonance has played a fundamental role in evaluating the enzyme purity against lactoferrin and minor LPO isoenzyme components in setting the final steps of the purification. With the aim to completely clarify the Fe(III)-heme high-spin nature of the native LPO, two samples of lactoperoxidase, LPO1 and LPO2 ( RZ = 0.95) from farm and commercial milk, respectively, were purified and characterized in particular by electron paramagnetic resonance (EPR) spectroscopy, in comparison with a commercial preparation (LPOs). The LPO1 EPR spectrum, at physiological pH, is clearly indicative of the presence of an iron(III)-heme high-spin catalytic site in the native enzyme. On the contrary, in the LPO2 spectrum a thermal equilibrium between high- and low-spin iron(III)-heme species is present. The low-spin component of the spectrum has been assigned to an LPO-NO − 2 adduct due to the presence of some nitrite impurities originating either from commercial unpasteurized milk or from external sources. The LPOs EPR spectrum shows the presence of some spurious lines in the g ≊ 6 and 4 regions due to the minor LPO isoenzyme components and to lactoferrin, respectively. The LPO EPR spectra previously reported in the literature contain a variable number of spurious lines in the g ≊ 4 and 2 regions as a consequence of lactoferrin impurity and LPO low-spin adducts with endogenous or exogenous anions. Furthermore, the interaction of LPO with its native substrate (the thiocyanate anion), which previously was shown by NMR and EPR (at high substrate concentration) spectroscopies, has been confirmed by EPR at low temperature and low substrate concentration and by optical spectroscopy at room temperature and high substrate concentration as a function of pH. The LPO activity at optimum pH ( ≊ 4–5) has been measured in phosphate and acetate buffer using as an oxidizable substrate the system dimethylamino benzoic acid 3-methyl-2-benzothiazolinone hydrazone hydrochloride monohydrate (DMAB-MBTH), which was considered a good chromogen for other peroxidases such as HRP and zucchini peroxidases. The LPO vs. SCN − activity at optimum pH ( ≊5.5) has been measured in phosphate and acetate buffer. Kinetic studies in the presence of the DMAB-MBTH chromogen show a noncompetitive inhibition between hydrogen peroxide and thiocyanate, in particular at optimum SCN − substrate activity around pH 5.5. Spectroscopic results together with activity and kinetic data seem to indicate the insertion of thiocyanate as a ligand bridge between Fe(III)-heme and N 3H imidazole of the distal histidine to give a small Fe(III) interaction at room temperature, which became stronger at low temperature. Previous NMR and preliminary EPR measurements suggested the heme pocket site close to the Fe(III) ion as the SCN − binding site.

Glutathione transferase isoenzymes from human prostate

By using affinity-chromatography and isoelectric-focusing techniques, several forms of glutathione transferase (GSTs) were resolved from human prostate cytosol. All the three major classes of GST, i.e. Alpha, Mu and Pi, are present in human prostate. However, large inter-individual variation in the qualitative and quantitative expression of different isoenzymes resulted in the samples investigated. The most abundant group of prostate isoenzymes showed acid (pI 4.3-4.7) behaviour and were classified as Pi class GSTs on the basis of their immunological and structural properties. Immunohistochemical staining of Pi class GSTs was prevalently distributed in the epithelial cells surrounding the alveolar lumen. Class Mu GSTs are also expressed, although in small amounts and in a limited number of samples, by human prostate. The major cationic isoenzyme purified from prostate, GST-9.6; (pI 9.6; apparent subunit molecular mass of 28 kDa), appears to be different from the cationic GST alpha-epsilon forms isolated from human liver and kidney as evidenced by its structural, kinetical and immunological properties. This enzyme, which accounts for about 20-30% (on protein basis) of total amount of GSTs, is expressed by only 40% of samples. GST-9.6 has the ability to cross-react in immunoblotting analysis with antisera raised against rat liver GST 2-2, rather than with antisera raised against members of human Alpha, Mu and Pi class GSTs. Although prostate GST-9.6 shows close relationship with the human skin GST pI 9.9, it does not correspond to any other known human GST.

Purification and heat stability of Cox's apple pulp peroxidase isoenzymes

SummaryThree peroxidase isoenzymes, two anionic and one cationic, were isolated from extracts of apple pulp using gel filtration and ion‐exchange chromatography. The homogeneity of the purified isoenzymes was established by isoelectric focusing and staining for peroxidase activity. The isoenzymes varied in their heat stability: the anionic isoenzymes were found to be more heat stable than the cationic isoenzyme. The measured enzymic activity of the isoenzymes was greatest when o‐dianisidine was used as the test substrate. After heating solutions of the anionic isoenzymes at 80–100°C for 2 min, up to 80% of the original enzymic activity was observed to regenerate when the heat‐treated isoenzymes were held at 30°C for 1 hr. Regeneration of enzymic activity following heat inactivation was not observed for the cationic isoenzyme.

The heat stability of purified spring cabbage peroxidase isoenzymes

Two peroxidase isoenzymes, purified from extracts of Spring cabbage, have been shown to possess different heat-stabilities. The anionic isoenzyme had an isoelectric point of 3·7 and was relatively heat stable, while the cationic isoenzyme, pI 9·9, was more readily inactivated by heat. Neither isoenzyme showed regeneration of peroxidase activity following heat inactivation.

Purification and spectral characterization of a paraperoxidase from Cucurbita pepo ripe fruits

The isolation and purification of the two major anionic (A 1) and cationic (C 1) peroxidase isoenzymes extracted from the ripe fruit of Curcubita pepo have been described. The anionic isoenzyme A 1 has spectral properties very similar to those of normal horseradish peroxidase isoenzymes. The cationic isoenzyme C 1 has a paraperoxidase nature and has been characterized by electronic and circular dichroism spectroscopy, together with several of its derivatives. The spectra suggest that native isoenzyme C 1 may exist as equilibrium mixture of high-spin and low-spin forms, but such equilibria are apparently absent in the various derivatives of the enzyme. Careful comparison of the spectra of the native isoenzyme C 1 and its reduced form with those of the corresponding cyanide derivatives seems to exclude the possibility that the paraperoxidase character could arise from a cyanide-bound form of the enzyme. The spectra of isoenzyme C 1 recorded in the presence of various substrates and H 2O 2 indicate an accumulation of compound III.

Subcellular distribution of aspartate aminotransferase isoenzymes in chicken heart: A quantitative study

1. 1. The content of the two aspartate aminotransferase isoenzymes in isolated mitochondria and in the cytosolic fraction from chicken heart was determined by radioimmunoassays. 2. 2. The cationic isoenzyme was found to be associated with the mitochondrial fraction; its content measured in the cytosolic fraction was within the range of that of contaminating mitochondrial marker enzymes. 3. 3. The anionic isoenzyme was found exclusively in the cytosolic fraction, in mitochondria a content of <0.05% of the total was measured. 4. 4. Thus, in birds, the anionic and the cationic isoenzyme of aspartate aminotransferase show the same strict intracellular heterotopism as found previously in mammals.

A re-evaluation of the retrograde axonal transport of horseradish peroxidase isoenzymes by semi-quantitative cytochemical analysis

Retrograde axonal transport of 2 horseradish peroxidase (HRP) isoenzymes, the cationic isoenzyme C (HRP-C) and anionic isoenzyme A (HRP-A), was compared using the trigeminal innervation of the cornea in the rabbit. The effects of various fixatives, cytochemical reaction solutions and intracellular decay on the enzymatic activity and cytochemical visualization of both HRP-C and HRP-A were first evaluated quantitatively. Spectrophotometric assays showed that HRP-C and HRP-A respectively retained 92% and 84% of their initial activity after exposure to a fixative containing 1% paraformaldehyde and 0.5% glutaraldehyde for 1 h at 20°. The specific activity of HRP-A was 26% that of HRP-C when assayed in a solution containing 0.5 mg/ml diaminobenzidine and 0.01% H 2O 2 in 0.15 m citrate buffer, pH 5.1. By killing the rabbits at different times following injection of HRP into the corneas, the intracellular half-life of HRP-C was estimated to be 40–52 h and HRP-A 8–12 h. After taking all of these differences into consideration, corrected concentrations of the 2 isoenzymes were compared for retrograde axonal transport. Results showed that both label intensity (scored according to 4 classes) and total number of labeled cell profiles in animals injected with HRP-C was significantly greater than in animals injected with HRP-A. These data are consistent with evidence from previous in vivo and in vitro studies demonstrating a certain selectivity of both uptake and retrograde axonal transport of HRP isoenzyme C that does not appear to be simple ‘fluid-phase’ internalization. The methodology and precautionary steps taken in this study in obtaining a semi-quantitative evaluation of HRP cytochemical visualization should be of general use in a variety of histochemical studies.

Pyrrolooxygenase isoenzymes from wheat germ

Both porphobilinogen oxygenase and skatole pyrrolooxygenase of wheat germ have isoenzyme forms of different charge. The more cationic isoenzymes were eluted from DEAE-cellulose with 10 mM Tris-HCl buffer (pH 7.6) and the less cationic were eluted with 50 mM NACl in the same buffer. The former had almost twice as many free amino groups (per mg of protein) as the latter. The more cationic isoenzyme was more sensitive to chelating agents and to acid treatment. They were differently inhibited by sodium dodecyl treatment and by temperature inactivation. Porphobilinogen oxygenase isoenzymes showed different activities with different buffers and also differed in their kinetics.

The Significance of Malate Dehydrogenase Isoenzymes in Human Blood Platelets

Two MDH isoenzymes were detected in the homogenate of normal human blood platelets. According to their properties the cationic isoenzyme is compartmentalized in the mitochondria, the anionic one belongs to the cytoplasma. In spite of the few mitochondria in human blood platelets the proportion of the cationic enzyme is relatively high. Both of these enzymes could belong to a transfer system for malate transport across the mitochondrial membrane. As human blood platelets do not contain creatine phosphate a system like that could be of significance for platelet function.

Serum levels of the mitochondrial isoenzym of aspartate aminotransferase in myocardial infarction and muscular dystrophy.

Summary. The cationic (mitochondrial) isoenzyme of aspartate aminotransferase is a normal constituent of plasma. After myocardial infarction the seru~ level usually rises, reaching a peak 24 to 48 h later, and returns to normal more slowly than does the total aspartate aminotransferase activity. The serum level was found to be raised in all 6 cases of muscular dystrophy tested.