Acidic Isoenzymes Research Articles

Inducible trehalase enzyme activity of Morchella conica Persoon mycelium

Morchella conica Pers. strains of the study were isolated from fruit bodies collected in ash-mixed forests. At first, the strains were cultured on potato dextrose agar (PDA), then on modified Murashige and Skoog (MS) solid agar media. A normal-growing strain was chosen for the trehalase induction experiments. During the trehalase induction treatment, mycelia were grown in liquid culture containing different concentrations of trehalose. After the induction period of trehalase enzymes, physiological state of the mycelium and the oxidative stress were monitored in the vegetative mycelia by measuring the change of the malondialdehyde content, superoxide dismutase enzyme activity, the fresh and dry weight. The examined Morchella conica strain utilized the trehalose properly. The rising amount of the trehalose triggered the increase of the mycelial trehalase enzyme activity. Our results clearly proved that both neutral and acidic trehalase isoenzyme activity of the Morchella conica mycelium are inducible and are playing important role in the utilization of external trehalose.

Application of Brassica napus hairy root cultures for phenol removal from aqueous solutions

Phenolic compounds present in the drainage from several industries are harmful pollutants and represent a potential danger to human health. In this work we have studied the removal of phenol from water using Brassica napus hairy roots as a source of enzymes, such as peroxidases, which were able to oxidise phenol. These hairy roots were investigated for their tolerance to highly toxic concentrations of phenol and for the involvement of their peroxidase isoenzymes in the removal of phenol. Roots grew normally in medium containing phenol in concentrations not exceeding 100 mg l −1, without the addition of H 2O 2. However, roots were able to remove phenol concentrations up to 500 mg l −1, in the presence of H 2O 2, reaching high removal efficiency, within 1 h of treatment and over a wide range of pH (4–9). Hairy roots could be re-used, at least, for three to four consecutive cycles. Peroxidase activity gradually decreased to approximately 20% of the control, at the fifth cycle. Basic and near neutral isoenzymes (BNP) decreased along time of recycling while acidic isoenzymes (AP) remained without changes. Although both group of isoenzymes would be involved in phenol removal, AP showed higher affinity and catalytic efficiency for phenol as substrate than BNP. In addition, AP retained more activity than BNP after phenol treatment. Thus, AP appears to be a promising isoenzyme for phenol removal and for application in continuous treatments. Furthermore, enzyme isolation might not be necessary and the entire hairy roots, might constitute less expensive enzymatic systems for decontamination processes.

Analysis of chitinase isoenzymes in sorghum seedlings inoculated with Fusarium thapsinum or F. proliferatum

An investigation was conducted on the chitinase isoenzymes in various tissues of 1-week-old sorghum seedlings that had been inoculated with the fungal pathogens Fusarium thapsinum and Fusarium proliferatum. Electrophoresis of non-denatured extracts on SDS-PAGE gels revealed at least 11 bands of activity. Using native PAGE gels, seven acidic isoenzymes and four basic isoenzymes were identified. Two acidic chitinases were expressed constitutively and their activity did not differ between control and inoculated seedlings. The activity of the other acidic isoenzymes increased in all tissues following inoculation. The activities of both inducible and constitutive acidic chitinase isoenzymes were higher in the scutellum than in other tissues. Inducible basic chitinases increased only in the roots and shoots after infection, while the activity of basic chitinases decreased in the seed and scutellum. The pattern of chitinase isoenzymes from dormant caryopses was different from that in seed-derived tissues in the seedling. Generally, seedling tissues infected with Fusarium fungi had higher levels of acidic isoenzymes and lower levels of basic isoenzymes than dormant caryopses. The findings were consistent with hypotheses that acidic chitinases are primarily involved in active defence responses, while basic chitinases are part of pre-formed defence mechanisms.

Biochemical and ecological characterization of two peroxidase isoenzymes from the mangrove, Rhizophora mangle

ABSTRACTThis study examines phenolic peroxidase (POX) in Rhizophora mangle L. leaves in order to assess its role in phenolic manipulation and H2O2 scavenging. Sun‐exposed and understorey leaves experiencing varying degrees of nutrient stress were analysed from an oligotrophic cay off the coast of Belize. POX activity was unaffected by growth environment, but increased throughout leaf development and persisted through senescence and after abscission. Histochemical analyses indicated POX activity throughout leaf tissues, especially in the apoplast. Phenolics were similarly broadly distributed. Two isoenzymes of POX were partially characterized with pIs of 4.1 and 6.3 and masses of 65.5 and 54.3 kDa, respectively. The larger, more acidic isoenzyme showed especially high heat stability, showing no reduced activity after 24 h at 60 °C. Rhizophora mangle POX oxidized quercetin preferentially, and, to a lesser extent, coniferyl alcohol, caffeic acid, chlorogenic acid, and p‐coumaric acid. It did not oxidize ascorbate, but ascorbate could act as a secondary electron donor in the presence of a phenolic substrate and H2O2. However, because quercetin and other aglycones were not present in R. mangle leaves, and because POX showed no activity with the most abundant leaf flavonoid, rutin, it was concluded that detoxification of H2O2 is secondary to the other roles of POX in manipulation of phenolics.

Glutathione S-transferase in the developmental stages of the insect Apis mellifera macedonica

We investigated the pattern of glutathione S-transferase (GST) activity in the course of the development of Apis mellifera macedonica. GST activity is present in all developmental stages of A. mellifera macedonica. The highest activity towards the substrate 1-chloro-2,4-dinitrobenzene (CDNB) is found in the adult stage and the lowest in the egg. The kinetic characteristics of the whole enzyme change as the insect develops. Significant changes are observed in substrate specificity, inhibitor sensitivity and V max. The number of isoenzymes and their rate of expression vary as the insect develops. However, two main isoenzymes are present in all developmental stages, one in the alkaline area and the other in the acidic. While in the larval stage the acidic isoenzyme is expressed at a slightly higher rate (52.2% over 47.8% for the alkaline isoenzyme), in the adult stage, the rate is reversed dramatically (13.24% and 84.2%, respectively).

Alterations in the pattern of peroxidase isoenzymes and transient increases in its activity and in H2O2levels take place during the dormancy cycle of grapevine buds: the effect of hydrogen cyanamide

Polymorphism of peroxidase (Px) and changes in its activity and in H2O2 content were studied in buds of grapevine during dormancy. Three isoforms of Px were detected in bud-extracts, two basic and one acidic, however, the pattern of Px isoenzyme changed with the progress of dormancy. Thus, basic Px isoenzymes disappeared from extracts previous to the onset of bud-break, while acidic isoenzymes remained relatively unaltered throughout the whole dormancy period. Furthermore, transient increases in the activity of Px and in the content of H2O2 occurred previous to endodormancy release, when buds were fully dormant. Hydrogen cyanamide (H2CN2), a potent bud breaking agent in grapevines advanced as expected bud-break, but also advanced the occurrence of Px and H2O2 peaks and the changes in Px isoenzymes pattern. The results suggests that H2O2 could function as a signalling molecule inducing endodormancy release, and changes in Px polymorphism could be a useful marker to study endo/ecodormancy phase transition in buds of grapevines.

Tomato (Lycopersicon esculentum cv. pera) hairy root cultures: Characterization and changes in peroxidase activity under NaCl treatment

Hairy root cultures of Lycopersicon esculentum L. Mill ev. Pera were established by infection of leaf explants with Agrobacterium rhizogenes LBA 9402. The pattern of peroxidase isoenzymes in these tissues was similar to that of roots excised from tomato plants grown in hydroponic cultures. Hairy root cultures may be an appropriate system to analyze the peroxidase involvement in the response of isolated roots to salt stress, avoiding the problem of wounding or changes in hormone levels observed in roots excised from plants. The cultures of hairy roots allowed the evaluation of changes in peroxidase patterns not only in the tissue but also in the culture medium. Hairy roots were subcultured in Murashige and Skoog liquid medium with or without 100 mM NaCl to investigate the evolution of growth, total peroxidase activity of the tissue and culture medium, and changes in the peroxidase isoenzyme patterns under each condition of growth. Control cultures showed a growth index higher than those reported for other hairy root cultures, and it was even higher in the presence of 100 mM NaCl. The total peroxidase activity in the tissue was similar for control and salt-treated roots. Even when the total peroxidase activity of the medium decreased under salt treatment, NaCl induced secretion of a highly basic peroxidase and inhibition of the secretion of some acidic isoenzymes. These changes may explain the physiological role of these enzymes in the response to salt stress that we will possibly establish through a future study of the biochemical properties of those peroxidases.

Effect of chitosan on peroxidase activity and isoenzyme profile in hairy root cultures of Armoracia lapathifolia.

Hairy root cultures of Armoracia lapathifolia established by infection with Agrobacterium rhizogenes LBA 9402 present a level and isoenzyme pattern of peroxidases (POD) comparable to nontransformed roots. Elicitation with chitosan (10, 50, and 100 mg/L) was used in order to improve POD production. Total POD activity increased about 170% after 48 h of treatment with chitosan 100 mg/L. Elicitation effect on soluble and ionically cell-wall-bound POD fractions of A. lapathifolia hairy roots was analyzed. POD activity of the ionically cell-wall-bound protein fraction increased in the presence of chitosan in a dose-response manner. No effect on soluble POD fractions was observed, but the isoenzyme pattern analyzed by isoelectrofocusing showed an increase of an acidic isoenzyme (pI = 3.4) after the elicitation treatment. The ionically cell-wall-bound protein fraction showed only basic isoenzymes, with an increase of an isoenzyme of pI = 8.7, after the elicitation treatment.

Changes in peroxidase activity and isoperoxidase pattern during strawberry ( Fragaria × ananassa) callus development

Summary Changes in peroxidase (EC. 1.11.1.7) activity and isoperoxidase pattern were analysed during strawberry ( Fragaria × ananassa ) callus development. Peroxidase activity per g fresh weight using 4-methoxy-α-naphthol, 3,3′,5,5′-tetramethyl bencidine, coniferyl alcohol or ferulic acid as substrates was highest at 40 days of culture, which coincided with the exponential phase of growth. While the specific peroxidase activity with the first three substrates was highest during the exponential growth phase, the highest specific enzymatic activity with ferulic acid as substrate was reached after 80 days of culture when callus growth had ceased. Several isoperoxidases were constitutively expressed during the complete callus growth cycle, unlike a group of acidic isoperoxidases, which were only present when callus had ceased growing. The high reactivity of the acidic isoperoxidases with ferulic acid as substrate, as judged by the k 1 and k 3 values of the peroxidase catalytic cycle, strongly suggests that these acidic isoenzymes are mainly involved in the formation of diferuloyl bridges between non-cellulosic components of the cell wall, thus contributing to cell wall rigidification, which, in turn, is partly responsible for the cessation of growth.

Sensory and endocrine characteristics of the avian pineal organ.

The avian pineal organ represents a transitional type between a photosensory organ of lower vertebrates and the endocrine gland of mammals and shows remarkable changes in its innervation and structure during ontogeny. In the avian pineal organ the progressive reduction of the pinealofugal component and the spectacular increase in pinealopetal sympathetic innervation occur in parallel. In domestic fowl the number of intrapineal AChE-positive (afferent) neurons decreases rapidly during ontogenetic development, whereas the sympathetic innervation becomes more prominent. Furthermore, the end vesicle of the pineal organ is an anatomical entity fully separated from the brain in the adult domestic fowl, as observed in some mammalian pineals. The avian pineal organ contains several types of photoreceptors with different photopigments and the synthesis of melatonin, the pineal hormone, is controlled by light. Immunoreactivity for photopigments is reduced during the posthatching development of chicken, whereas neuron-specific enolase (NSE)-immunoreactive pinealocytes increase remarkably in number in the end-vesicle of the domestic fowl with age, followed by a gradual expansion toward the proximal portion. NSE is the most acidic isoenzyme of the glycolytic enzyme enolase and is useful as a cytoplasmic marker of neurons and neuroendocrine tissue. The above-mentioned findings reflect the sequence of changes leading from pineal sense organs to pineal gland. The demonstration of melatonin receptors in a variety of avian peripheral tissues suggest a possible direct action of melatonin on the physiological functions of different organ systems in response to internal and external stimuli.

Fruit development in Capsicum annuum: changes in capsaicin, lignin, free phenolics, and peroxidase patterns.

Pepper fruits, of Capsicum annuum cv. Padron, undergo changes in content of capsaicinoids, lignin, and free phenolics during the maturation process. Although capsaicinoids increase with development, the maximal levels of free phenolics and lignin are observed during the early stages of development. A decrease of peroxidase activity was observed during maturation, and this was related with a decrease in other physiological parameters studied, namely chlorophylls and pH. Subcellular fractionation studies reveal that most peroxidase activity is localized in the soluble fraction throughout development. The changes in the peroxidase activity were accompanied by changes in the different isoenzymes. Acidic isoenzymes increased whereas the basic isoenzymes decreased over the same period, and the changes in these isoenzymes were related with capsaicin metabolism.

β‐1,3‐Glucanase isoenzymes and genes in resistant and susceptible pepper (Capsicum annuum) cultivars infected with Phytophthora capsici

Infection of pepper (Capsicum annuum) plants with Phytophthora capsici led to the accumulation of β‐1,3‐glucanases in the stem tissues soon after inoculation. After the appearance of the symptoms on the pepper stems, β‐1,3‐glucanase accumulation became much more pronounced in the resistant (Smith‐5 [S‐5]) than in the susceptible (Yolo Wonder [YW]) cultivar. β‐1,3‐Glucanase activity was also detected in the control stems (uninoculated) but only in the resistant cultivar. The direct detection of β‐1,3‐glucanase on polyacrylamide gels revealed a basic isoenzyme (Rf 0.18) in the intracellular fraction (INTRA‐F), which may be associated with the expression of resistance to P. capsici since its activity was detected only in the resistant cultivar. The fact that other basic isoenzymes (Rf 0.70 and 0.61 from the INTRA‐F and Rf 0.57 from the intercellular fraction) and one acidic isoenzyme (Rf 0.70 from the intercellular fraction) were detected in both cultivars suggests that these play a less important role in the development of resistance although they appeared earlier and at greater intensity in the resistant cultivar than in the susceptible one. Using degenerate primers and reverse‐transcriptase polymerase chain reaction (RT‐PCR), β‐1,3‐glucanase product of approximately 500 bp was obtained and cloned from cv. S‐5 total RNA. This was used as a probe on northern blots of RNA extracted from both cultivars at specific times after infection. One transcript, size 2.0 kb, was detected in both cultivars, and accumulated very close to the inoculation site between 1 and 3 days after inoculation, but to a greater extent in the resistant cultivar (S‐5) than in the susceptible cultivar (YW). These data suggest that an early and rapid accumulation of β‐1,3‐glucanase transcript is associated with the defence reaction that develops in pepper stems infected with P. capsici.

Peroxidase production in vitro by Armoracia lapathifolia (horseradish)-transformed root cultures: effect of elicitation on level and profile of isoenzymes.

Transformed roots of Armoracia lapathifolia (horseradish) were established by infection with Agrobacterium rhizogenes LBA 9402. They were used as a culture system in vitro for peroxidase production in vitro, to avoid many of the problems that affect the traditional production from field-grown species of Armoracia sp. The time course of growth of these cultures showed that total peroxidase attained maximum levels at the end of the exponential growth phase. At this stage of culture, elicitation assays were performed with AgNO3 and CuSO4 as abiotic elicitors and with fungal extracts of Verticillum sp., Monodyctis cataneae and Aspergillus niger as biotic elicitors. The best results were obtained with Verticillum sp., 24 h after elicitation, with an increase of approx. 100% in peroxidase activity. The isoenzyme pattern analysed by isoelectric focusing revealed predominantly basic and acidic isoenzymes in both plant roots and transformed root cultures. Elicited samples showed a similar isoenzyme pattern with a slight increase in basic isoenzymes.

Efficient production of mannan-degrading enzymes by the basidiomycete Sclerotium rolfsii.

Sclerotium rolfsii CBS 191.62 was cultivated on a number of carbon (C) sources, including mono- and disaccharides, as well as on polysaccharides, to study the formation of different mannan-degrading enzyme activities. Highest levels of mannanase activity were obtained when alpha-cellulose-based media were used for growth, but formation of mannanase could not be enhanced by employing galactomannan as the only carbon source. Although both xylanase and cellulase formation was almost completely repressed when S. rolfsii was grown on more readily metabolizable carbohydrates, including glucose or mannose, considerable amounts of mannanase activity were secreted under these growth conditions. Enhanced mannanase production only commenced when glucose was depleted in the medium. The maximal mannanase activity of 240 IU/mL obtained in a laboratory fermentation is remarkable. Mannanase activity formed under these derepressed conditions could be mainly attributed to one major, acidic mannanase isoenzyme with a pI value of 2.75.

Identification of a Critical Phenylalanine Residue in Horseradish Peroxidase, Phe179, by Site-Directed Mutagenesis and1H-NMR: Implications for Complex Formation with Aromatic Donor Molecules

The functional and structural significance of Phe179 of horseradish peroxidase isoenzyme C (HRP C) has been investigated by site-directed mutagenesis. This residue is located in a structurally variable insertion between helices F and G, a motif unique to peroxidases of higher plants. Results obtained for three recombinant enzymes, with Phe179 substituted by Ala, His, or Ser, provide the first demonstration of the importance of this side chain for the binding of aromatic donor molecules. Experimental parameters for direct comparison with the wild-type enzyme were obtained by extensive solution state characterization using both optical and 1H-NMR spectroscopy. Significant chemical shift variations for resonances associated with the exposed heme edge, notably heme methyl C18H3 and heme propionate C17(1)H2, were recorded in NMR spectra of both the resting and cyanide-ligated states of the three Phe179 mutants. Furthermore, comparison of NOE connectivities in NOESY spectra of cyanide-ligated wild-type and mutant enzymes enabled the elusive assignment of the aromatic side chain in close proximity to heme methyl C18H3 to be made to Phe179. Replacement of Phe179 by Ala resulted in an 80-fold decrease in the binding affinity of the cyanide-ligated enzyme for benzhydroxamic acid, with a Kd value similar to that determined for cyanide-ligated HRP A2 (an acidic isoenzyme with valine at position 179). The binding affinity of Phe179-->Ser was similarly decreased, while that of Phe179-->His was partially restored relative to wild-type HRP C. Cyanide-ligated Phe179-->His HRP C exhibited a unique pH-dependent spectral transition associated with a pKa value of 6.5 +/- 0.2, assigned to the His179 side chain. Two closely related enzyme forms exhibiting different affinities for benzhydroxamic acid were observed at neutral pH and above, indicating that the protonation state of His179 gave rise to microheterogeneity in the aromatic donor molecule binding site.

The isomerization of glutamate dehydrogenase in response to lead toxicity in maize

Maize (Zea mays) was cultivated on lead-adultrated soil up to 600 mg(Pb) kg-1. At maturity, the maize seeds were harvested. The glutamate dehydrogenase (GDH) was fractionated to its isoenzyme population by Rotofor isoelectric focusing (IEF). The increasing Pb concentration progressively enhanced the more acidic isoenzymes (pI 6.3 - 6.5), and at the same time suppressed the less acidic isoenzymes (pI 7.3 - 7.8) and at the 600 mg(Pb) kg-1(soil) only the most acidic couple of isoenzymes (pI 6.3, and 6.5) were detectable. The NH4+ Km values of the GDH increased progressively from 6.2 in the control to 100 mM and the total glutathione content of maize seeds from 60 to 240 nmol g-1 in the 600 mg(Pb) kg-1(soil) treated maize. The orderly, and sequential isomerization of GDH in response to Pb suggests that the enzyme functions as a sensor in the monitoring of environmentally induced stress.

Antioxidant and glutathione-associated enzymes in Wilms' tumour after chemotherapy.

The present study demonstrates the activities of antioxidant and glutathione-associated enzymes and the level of glutathione in Wilms' tumour (nephroblastoma) samples after chemotherapy (mainly actinomycin D and vincristine). We observed higher activity of superoxide dismutase in Wilms' tumour compared to adjacent morphologically unchanged kidney. On the other hand, in this tumour lower activities of catalase and the glutathione-associated enzymes glutathione synthetase, gamma-glutamyl transpeptidase, glutathione reductase and total glutathione S-transferases (GST) were found. Using isoelectric focusing we separated different forms of GST in tested tissues and revealed lower activities of the basic enzymes in Wilms' tumour, which may be responsible for the decrease of total GST activity. Moreover, we found the acidic isoenzymes to be the predominant class of GST in nephroblastoma. In Wilms' tumours with unfavourable histology a high activity of these isoenzymes together with a high level of GSH were observed. We suggest that these parameters may participate in the known phenomenon of anticancer drug resistance of tumours with unfavourable histology.

A comparative study of the purity, enzyme activity, and inactivation by hydrogen peroxide of commercially available horseradish peroxidase isoenzymes A and C

Horseradish peroxidase (HRP) is a commercially important enzyme that is available from a number of supply houses in a variety of grades of purity and isoenzymic combinations. The present article describes a comparative study made on nine HRP preparations. Six of these samples were predominantly composed of basic HRP, pl 8.5, and three of acidic HRP, pl 3.5. Two of the basic preparations were of lower purity than the others. The apparent molar catalytic activity of basic HRP with 0.5 mMABTS and 0.2 mM H(2)O(2) was around 950 s(-1) (about 770 s(-1) for the less pure samples) and with a 5 mM guaiacol and 0.6 mM H(2)O(2) was about 180 s(-1) for all the samples. A similar value (approximately 1000 s(-1)) was observed for acidic HRP but only at higher concentrations of ABTS (20 mM). With 20 mM guaiacol the molar catalytic activity of the acid isoenzyme was 65 s(-1). The apparent K(M) for ABTS of the acidic isoenzyme was 4 mM whereas for the basic isoenzyme it was 0.1 mM. All the enzymes were inactivated by H(2)O(2) when it was supplied as the only substrate. Under these conditions the partition ratio (r = number of catalytic cycles given by the enzyme before its inactivation), apparent dissociation constant (K(l)), and apparent rate constant of inactivation (k(inact)) were about twice as large for the acidic samples (1350, 2.6 mM, 9 x 10(-3) s(-1)) as for the basic (650, 1.3 mM, 5 x 10(-3) s(-1)). The apparent catalytic constant (k(cat)) was 3-4 times larger, and the efficiency of catalysis (k(cat)/K(l)) was double for the acidic isoenzyme, but the efficiency of inactivation (k(inact)/K(l)) was similar. The data obtained provide useful information for those using HRP isoenzymes for biotechnological applications (e.g., biosensors, bioreactors, or assays).

Glutathione-S-transferase activity and isoenzyme levels measured by two methods in ovarian cancer, and their value as markers of disease outcome.

A study has been carried out to investigate the cellular distribution and levels of glutathione-S-transferase isoenzymes (GST), acidic (pi), basic (alpha) and neutral (mu), in ovarian tumour biopsies, and to measure GST activity in the same tumour specimens. Two methods of assessing isoenzyme levels (immunohistochemistry and Western blot) were compared. Well-known important clinicopathological features were correlated with response to treatment, overall survival and progression-free survival for each of 97 patients from whom biopsies had been obtained. The glutathione-S-transferase isoenzyme levels were also correlated with overall and progression-free survival, and with the important clinicopathological features. As expected, there was a significant correlation between FIGO stage, histological grade of tumour, amount of residual disease after staging laparotomy, response to chemotherapy, and both overall and progression-free survival. Glutathione-S-transferase isoenzyme levels (acidic, basic and neutral) measured by Western blot were not found to be significantly correlated with any of the clinicopathological parameters tested. Using the immunohistochemistry method of detection there was a correlation between the GST acidic isoenzyme level and the amount of residual disease remaining after initial debulking surgery (higher levels were detected in the group with no residual disease, P=0.034), and also between the GST acidic isoenzyme level and the type of chemotherapy regimen used. Higher levels of the acidic isoenzyme were present in tumour biopsies taken from the patient group who had received a combination regimen (cyclophosphamide, carboplatin, ifosfamide and doxorubicin). The neutral and basic GST isoenzyme levels were not significantly correlated with any of the clinicopathological parameters. None of the GST isoenzyme levels were significantly correlated with response to treatment, overall survival or progression-free survival (using either method of detection). Similarly, glutathione transferase activity showed no significant correlation with prognosis or survival.

Catalase Is Differentially Expressed in Dividing and Nondividing Protoplasts.

Based on our previous results that peroxidase is induced in dividing tobacco protoplasts but it is not expressed in the nondividing grapevine (Vitis vinifera L.) protoplasts during culture (C.I. Siminis, A.K. Kanellis, K.A. Roubelakis-Angelakis [1993] Physiol Plant 87: 263-270), we further tested the hypothesis that oxidative stress may be implicated in the recalcitrance of plant protoplasts. The expression of catalase, a major defense enzyme against cell oxidation, was studied during isolation and culture of mesophyll protoplasts from the recalcitrant grapevine and regenerating tobacco (Nicotiana tabacum L.). Incubation of tobacco leaf strips with cell wall-degrading enzymes resulted in a burst of catalase activity and an increase in its immunoreactive protein; in contrast, no such increases were found in grapevine. The cathodic and anodic catalase isoforms consisted exclusively of subunits [alpha] and [beta], respectively, in tobacco, and of subunits [beta] and [alpha], respectively, in grapevine. The catalase specific activity increased only in grapevine protoplasts during culture. The ratio of the enzymatic activities to the catalase immunoreactive protein declined in dividing tobacco protoplasts and remained fairly constant in nondividing tobacco and grapevine protoplasts during culture. Also, in dividing tobacco protoplasts the de novo accumulation of the catalase [beta] subunit gave rise to the acidic isoenzymes, whereas in nondividing tobacco and grapevine protoplasts, after 8 d in culture, only the basic isoenzymes remained due to de novo accumulation of the [alpha] subunit. The pattern of catalase expression in proliferating tobacco leaf cells during callogenesis was similar to that in dividing protoplasts. The different responses of catalase expression in dividing and nondividing tobacco and grapevine mesophyll protoplasts may indicate a specificity of catalase related to induction of totipotency.