Oscillations and bistability predicted by a model for a cyclical bienzymatic system involving the regulated isocitrate dehydrogenase reaction

We analyze the dynamics of a bienzymatic system consisting of isocitrate dehydrogenase (IDH, EC. 1.1.1.42), which transforms NADP+ into NADPH, and of diaphorase (DIA, EC 1.8.1.4), which catalyzes the reverse reaction. Experimental evidence as well as a theoretical model showed the possibility of a coexistence between two stable steady states in this reaction system G.M. Guidi et al. Biophys. J. 74 (1998) 1229-1240[, owing to the regulatory properties of IDH. Here we extend this analysis by considering the behavior of the model proposed for the IDH-DIA bienzymatic system in conditions where the system is open to an influx of its substrates isocitrate and NADP+ and to an efflux of all metabolic species. The analysis indicates that in addition to different modes of bistability (including mushrooms and isolas), sustained oscillations can be observed in such conditions. These results point to the isocitrate dehydrogenase reaction coupled to diaphorase as a suitable candidate for further experimental and theoretical studies of bistability and oscillations in biochemical systems. The results obtained in this particular bienzymatic system bear on other enzymatic systems possessing a cyclical nature, which are known to play significant roles in a variety of metabolic and cellular regulatory processes.     

Bistability in the isocitrate dehydrogenase reaction: an experimentally based theoretical study.

The enzyme isocitrate dehydrogenase (IDH, EC 1.1.1.42) can exhibit activation by one of its products, NADPH. This activation is competitively inhibited by the substrate NADP+, whereas NADPH competes with NADP+ for the catalytic site. Experimental observations briefly presented here have shown that if IDH is coupled to another enzyme, diaphorase (EC 1.8.1.4), which transforms NADPH into NADP+, the system can attain either one of two stable states, corresponding to a low and a high NADPH concentration. The evolution toward either one of these stable states depends on the time of addition of diaphorase to the medium containing IDH and its substrate NADP+. We present a theoretical and numerical analysis of a model for the IDH-diaphorase bienzymatic system, based on the regulatory properties of IDH. The results confirm the occurrence of bistability for parameter values derived from the experiments. Depending on the total concentration of NADP+ plus NADPH and the concentration of IDH, the system can either admit a single steady state or display bistability. We obtain an expression for the critical time t*, before which diaphorase addition leads to the lower steady state and after which addition of the enzyme leads to the upper steady state of NADPH. The analysis is extended to the case where the second substrate of IDH, isocitrate, is consumed in the course of the reaction without being regenerated. Bistability occurs only as a transient phenomenon in these conditions.     

A tale of two subunits: how the neomorphic R132H IDH1 mutation enhances production of αHG

Heterozygously expressed single-point mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2, respectively) render these dimeric enzymes capable of producing the novel metabolite α-hydroxyglutarate (αHG). Accumulation of αHG is used as a biomarker for a number of cancer types, helping to identify tumors with similar IDH mutations. With IDH1, it has been shown that one role of the mutation is to increase the rate of conversion from αKG to αHG. To improve our understanding of the function of this mutation, we have detailed the kinetics of the normal (isocitrate to αKG) and neomorphic (αKG to αHG) reactions, as well as the coupled conversion of isocitrate to αHG. We find that the mutant IDH1 is very efficient in this coupled reaction, with the ability to form αHG from isocitrate and NADP(+). The wild type/wild type IDH1 is also able to catalyze this conversion, though it is much more sensitive to concentrations of isocitrate. This difference in behavior can be attributed to the competitive binding between isocitrate and αKG, which is made more favorable for αKG by the neomorphic mutation at arginine 132. Thus, each partial reaction in the heterodimer is functionally isolated from the other. To test whether there is a cooperative effect resulting from the two subunits being in a dimer, we selectively inactivated each subunit with a secondary mutation in the NADP/H binding site. We observed that the remaining, active subunit was unaffected in its associated activity, reinforcing the notion of each subunit being functionally independent. This was further demonstrated using a monomeric form of IDH from Azotobacter vinelandii, which can be shown to gain the same neomorphic reaction when a homologous mutation is introduced into that protein.     

Subunit interactions of yeast NAD+-specific isocitrate dehydrogenase

Yeast mitochondrial NAD(+)-specific isocitrate dehydrogenase is an octamer composed of four each of two nonidentical but related subunits designated IDH1 and IDH2. IDH2 was previously shown to contain the catalytic site, whereas IDH1 contributes regulatory properties including cooperativity with respect to isocitrate and allosteric activation by AMP. In this study, interactions between IDH1 and IDH2 were detected using the yeast two-hybrid system, but interactions between identical subunit polypeptides were not detected with this or other methods. A model for heterodimeric interactions between the subunits is therefore proposed for this enzyme. A corollary of this model, based on the three-dimensional structure of the homologous enzyme from Escherichia coli, is that some interactions between subunits occur at isocitrate binding sites. Based on this model, two residues (Lys-183 and Asp-217) in the regulatory IDH1 subunit were predicted to be important in the catalytic site of IDH2. We found that individually replacing these residues with alanine results in mutant enzymes that exhibit a drastic reduction in catalysis both in vitro and in vivo. Also based on this model, the two analogous residues (Lys-189 and Asp-222) of the catalytic IDH2 subunit were predicted to contribute to the regulatory site of IDH1. A K189A substitution in IDH2 was found to produce a decrease in activation of the enzyme by AMP and a loss of cooperativity with respect to isocitrate. A D222A substitution in IDH2 produces similar regulatory defects and a substantial reduction in V(max) in the absence of AMP. Collectively, these results suggest that the basic structural/functional unit of yeast isocitrate dehydrogenase is a heterodimer of IDH1 and IDH2 subunits and that each subunit contributes to the isocitrate binding site of the other.     

NAD(+)-dependent isocitrate dehydrogenase. Cloning, nucleotide sequence, and disruption of the IDH2 gene from Saccharomyces cerevisiae

NAD(+)-dependent isocitrate dehydrogenase from Saccharomyces cerevisiae is composed of two nonidentical subunits, designated IDH1 (Mr approximately 40,000) and IDH2 (Mr approximately 39,000). We have isolated and characterized a yeast genomic clone containing the IDH2 gene. The amino acid sequence deduced from the gene indicates that IDH2 is synthesized as a precursor of 369 amino acids (Mr 39,694) and is processed upon mitochondrial import to yield a mature protein of 354 amino acids (Mr 37,755). Amino acid sequence comparison between S. cerevisiae IDH2 and S. cerevisiae NADP(+)-dependent isocitrate dehydrogenase shows no significant sequence identity, whereas comparison of IDH2 and Escherichia coli NADP(+)-dependent isocitrate dehydrogenase reveals a 33% sequence identity. To confirm the identity of the IDH2 gene and examine the relationship between IDH1 and IDH2, the IDH2 gene was disrupted by genomic replacement in a haploid yeast strain. The disruption strain expressed no detectable IDH2, as determined by Western blot analysis, and was found to lack NAD(+)-dependent isocitrate dehydrogenase activity, indicating that IDH2 is essential for a functional enzyme. Overexpression of IDH2, however, did not result in increased NAD(+)-dependent isocitrate dehydrogenase activity, suggesting that both IDH1 and IDH2 subunits are required for catalytic activity. The disruption strain was unable to utilize acetate as a carbon source and exhibited a 2-fold slower growth rate than wild type strains on glycerol or lactate. This growth phenotype is consistent with NAD(+)-dependent isocitrate dehydrogenase performing an essential role in the oxidative function of the citric acid cycle.     

Structure of the monomeric isocitrate dehydrogenase: evidence of a protein monomerization by a domain duplication

NADP(+)-dependent isocitrate dehydrogenase is a member of the beta-decarboxylating dehydrogenase family and catalyzes the oxidative decarboxylation reaction from 2R,3S-isocitrate to yield 2-oxoglutarate and CO(2) in the Krebs cycle. Although most prokaryotic NADP(+)-dependent isocitrate dehydrogenases (IDHs) are homodimeric enzymes, the monomeric IDH with a molecular weight of 80-100 kDa has been found in a few species of bacteria. The 1.95 A crystal structure of the monomeric IDH revealed that it consists of two distinct domains, and its folding topology is related to the dimeric IDH. The structure of the large domain repeats a motif observed in the dimeric IDH. Such a fusional structure by domain duplication enables a single polypeptide chain to form a structure at the catalytic site that is homologous to the dimeric IDH, the catalytic site of which is located at the interface of two identical subunits.     

Cloning and characterization of the gene encoding the IDH1 subunit of NAD(+)-dependent isocitrate dehydrogenase from Saccharomyces cerevisiae.

NAD(+)-dependent isocitrate dehydrogenase from Saccharomyces cerevisiae is composed of two nonidentical subunits, designated IDH1 and IDH2. The gene encoding IDH2 was previously cloned and sequenced (Cupp, J.R., and McAlister-Henn, L. (1991) J. Biol. Chem. 266, 22199-22205), and in this paper we describe the isolation of a yeast genomic clone containing the IDH1 gene. A fragment of the IDH1 gene was amplified by the polymerase chain reaction method utilizing degenerate oligonucleotides based on tryptic peptide sequences of the purified subunit; this fragment was used to isolate a full length IDH1 clone. The nucleotide sequence of the IDH1 coding region was determined and encodes a 360-residue polypeptide including an 11-residue mitochondrial targeting presequence. Amino acid sequence comparison between IDH1 and IDH2 reveals a 42% sequence identity, and both IDH1 and IDH2 show approximately 32% identity to Escherichia coli NAD(P)(+)-dependent isocitrate dehydrogenase. To examine the function of the IDH1 subunit and to determine the metabolic role of NAD(+)-dependent isocitrate dehydrogenase the IDH1 gene was disrupted in a wild type haploid yeast strain and in a haploid strain lacking IDH2. The IDH1 disruption strains expressed no detectable IDH1 as determined by Western blot analysis, and these strains were found to lack NAD(+)-dependent isocitrate dehydrogenase activity indicating that IDH1 is essential for a functional enzyme. Over-expression of IDH1 in a strain containing IDH2 restored wild type activity but did not result in increased levels of activity, suggesting that both IDH1 and IDH2 are required for a functional enzyme. Growth phenotype analysis of the IDH1 disruption strains revealed that they grew at a reduced rate on the nonfermentable carbon sources examined (glycerol, lactate, and acetate), consistent with NAD(+)-dependent isocitrate dehydrogenase performing a critical role in oxidative function of the citric acid cycle. In addition, the IDH1 disruption strains grew at wild type rates in the absence of glutamate, indicating that these strains are not glutamate auxotrophs.     

A kinetic model of functioning and regulation of Escherichia coli isocitrate dehydrogenase

A Rapid Equilibrium Random Bi Ter mechanism of formation of two dead-end complexes was proposed to describe the experimental data on the functioning of E. coli isocitrate dehydrogenase (IDH). A kinetic model for the enzyme functioning was constructed, which assumes that it is regulated through reversible phosphorylation by its kinase/phosphatase, which in turn is regulated by IDH substrates and central metabolites such as pyruvate (Pyr), 3-phosphoglycerate (3-PG), and AMP. It was shown using the model that increasing the concentration of these effectors results in an increase of the active part of IDH, thus leading to an increase in the Krebs cycle flux. We predict that the ratio of the phosphorylated and free forms of IDH (IDHP/IDH) is more sensitive to AMP, NADPH, and isocitrate concentrations than to Pyr and 3-PG. The model allows a realistic prediction of changes in the IDHP/IDH ratio, which would occur under changes of biosynthetic and energetic loading of the E. coli cell.     

Qualitative analysis of a mathematical model of a open enzyme reaction

A general case of the set of two differential equations, describing an open reaction v1 leads to S v reversible E P v2 leads to, has been considered. The requirements to the character of the functions v1([S]), v2([P]) and v([S], [P]) were formulated for the case of existence and absence of alternative steady states and sustained oscillations. The formulae were derived to determine the slope of the unstable portion of the quasi-steady state characteristic. The generalized model of Monod, Wyman and Changeux has been considered as an example of v([S], [P]). It has been shown that with monotonically decreasing v1 and monotonically increasing v2, the alternative steady states and oscillations are possible only in the presence of substrate inhibition or product activation. However, under the joint action of substrate inhibition and product activation, the system will exhibit bistability rather than an oscillatory behavior. In the case of an irreversible two-substrate reaction which can be described by a similar mathematical model, inhibition by the first and second substrate is equivalent to substrate inhibition and product activation.     

Dimerization and Bifunctionality Confer Robustness to the Isocitrate Dehydrogenase Regulatory System in Escherichia coli

An important goal of systems biology is to develop quantitative models that explain how specific molecular features give rise to systems-level properties. Metabolic and regulatory pathways that contain multifunctional proteins are especially interesting to study from this perspective because they have frequently been observed to exhibit robustness: the ability for a system to perform its proper function even as levels of its components change. In this study, we use extensive biochemical data and algebraic modeling to develop and analyze a model that shows how robust behavior arises in the isocitrate dehydrogenase (IDH) regulatory system of Escherichia coli, which was shown in 1985 to experimentally exhibit robustness. E. coli IDH is regulated by reversible phosphorylation catalyzed by the bifunctional isocitrate dehydrogenase kinase/phosphatase (IDHKP), and the level of IDH activity determines whether carbon flux is directed through the glyoxylate bypass (for growth on two-carbon substrates) or the full tricarboxylic acid cycle. Our model, which incorporates recent structural data on IDHKP, identifies several specific biochemical features of the system (including homodimerization of IDH and bifunctionality of IDHKP) that provide a potential explanation for robustness. Using algebraic techniques, we derive an invariant that summarizes the steady-state relationship between the phospho-forms of IDH. We use the invariant in combination with kinetic data on IDHKP to calculate IDH activity at a range of total IDH levels and find that our model predicts robustness. Our work unifies much of the known biochemistry of the IDH regulatory system into a single quantitative framework and highlights the importance of constructing biochemically realistic models in systems biology.     

Bistability and the ordered bimolecular mechanism.

An equation describing the instantaneous velocity of an ordered bimolecular enzymatic reaction that exhibits inhibition by substrate and product was derived. Using kinetic constant values for horse liver alcohol dehydrogenase, the velocity expression was applied to an open-reaction system. The calculated steady-state surfaces displayed regions of bistability, which further substantiates the link between substrate inhibition and multiple steady states. This general computational approach may be applied to any system that can be described by an instantaneous velocity equation.     

A model for the dynamics of bipolar disorders

Bipolar disorders are characterized by recurrent, alternating episodes of mania and depression. To examine the dynamical bases of this cyclical illness we consider a minimal model for bipolar disorders based on the observation that the two poles of the disease are mutually exclusive. We assume that the propensities to mania and depression, which are correlated with the activity of two putative neural circuits that promote, respectively, the manic or the depressive state, inhibit each other. When mutual inhibition is sufficiently strong, the model predicts bistability: the bipolar system is then either in a depressive or in a manic state and can display abrupt switches between these stable states. We consider two simple mechanisms which, when added to mutual inhibition, allow the model to pass from bistability to oscillations. Self-sustained oscillations provide a mechanism for the spontaneous, recurrent switching between mania and depression. The model can generate oscillations with a variety of waveforms, including simple periodic oscillations with comparable or unequal durations of the manic and depressive episodes, or small-amplitude oscillations around one of the two states preceding large-amplitude periodic changes in the propensities to mania or depression. The model provides a theoretical framework that covers the bipolar spectrum, i.e., cycling between the two poles of the disease, or evolution to either mania or depression or to an intermediate state without alternating between the two poles of the disease. The model accounts for the clinical observation that antidepressants can trigger the transition to mania or increase the frequency of bipolar cycling.     

Isocitrate dehydrogenase kinase/phosphatase

In Escherichia coli, isocitrate dehydrogenase (IDH) is regulated by phosphorylation. This phosphorylation cycle is catalyzed by an unusual, bifunctional protein:IDH kinase/phosphatase. IDH kinase/phosphatase is expressed from a single gene, aceK, and both activities are catalyzed by the same polypeptide. The amino acid sequence of IDH kinase/phosphatase does not exhibit the characteristics which are typical of other protein kinases, although it does contain a consensus ATP binding site. The available evidence suggests that the IDH kinase and IDH phosphatase reactions occur at the same active site and that the IDH phosphatase reaction results from the back reaction of IDH kinase tightly coupled to ATP hydrolysis. The function of the IDH phosphorylation cycle is to control the flux of isocitrate through the glyoxylate bypass. This pathway is essential for growth on acetate because it prevents the quantitative loss of the acetate carbons as CO2 in the Krebs' cycle. IDH kinase/phosphatase monitors general metabolism by responding to the levels of a wide variety of metabolites, many of which activate IDH phosphatase and inhibit IDH kinase. The ability of IDH kinase/phosphatase to monitor general metabolism allows. the IDH phosphorylation cycle to compensate for substantial perturbations of the system, such as a 15-fold overproduction of IDH. The significance of the cellular level of IDH kinase/phosphatase has also been evaluated. The level of this protein is in great excess of that required for steady-state growth on acetate. In contrast, IDH kinase/phosphatase is, in some cases, rate-limiting for the dephosphorylation of IDH which results when preferred carbon sources are added to cultures growing on acetate.     

Fluorometric determination of isocitrate dehydrogenase (EC 1.1.1.42; 1; NADP+ dependent) in ruminant milk

The enzyme isocitrate dehydrogenase (EC 1.1.1.42; 1; NADP⁺ dependent) located in the mammary cell cytosol mediates the synthesis of the majority of reducing equivalents for the energetically demanding milk fat and cholesterol synthesis in mammary cell cytosol. The present article presents a novel fluorometric method for quantification of the activity of this enzyme (IDH) in ruminant milk without pretreatment of the sample. Further, 493 goat milk samples – harvested before, during and after a nutritional restriction – were analysed for IDH activity i) with addition of extra substrate (isocitrate), and ii) with the intrinsic isocitrate solely. The IDH activity ranged from 0.22 to 15.4 units [nano moles product/(ml * min)] (un-supplemented) and from 0.22 to 45.6 units (isocitrate supplemented). The IDH activity increased considerably in milk during the nutritional restriction period concomitant with the increase in the metabolite isocitrate concentration and somatic cell count and returned to the initial level shortly after restriction period. The present ‘high through-put’ analytical method may be beneficial in future studies to phenotype modifications in mammary energy metabolism and milk fat synthesis, for which IDH activity may be a biomarker.     

Coupling oscillations and switches in genetic networks

Switches (bistability) and oscillations (limit cycle) are omnipresent in biological networks. Synthetic genetic networks producing bistability and oscillations have been designed and constructed experimentally. However, in real biological systems, regulatory circuits are usually interconnected and the dynamics of those complex networks is often richer than the dynamics of simple modules. Here we couple the genetic Toggle switch and the Repressilator, two prototypic systems exhibiting bistability and oscillations, respectively. We study two types of coupling. In the first type, the bistable switch is under the control of the oscillator. Numerical simulation of this system allows us to determine the conditions under which a periodic switch between the two stable steady states of the Toggle switch occurs. In addition we show how birhythmicity characterized by the coexistence of two stable small-amplitude limit cycles, can easily be obtained in the system. In the second type of coupling, the oscillator is placed under the control of the Toggleswitch. Numerical simulation of this system shows that this construction could for example be exploited to generate a permanent transition from a stable steady state to self-sustained oscillations (and vice versa) after a transient external perturbation. Those results thus describe qualitative dynamical behaviors that can be generated through the coupling of two simple network modules. These results differ from the dynamical properties resulting from interlocked feedback loops systems in which a given variable is involved at the same time in both positive and negative feedbacks. Finally the models described here may be of interest in synthetic biology, as they give hints on how the coupling should be designed to get the required properties.     

Isocitrate binding at two functionally distinct sites in yeast NAD+-specific isocitrate dehydrogenase

Yeast NAD(+)-specific isocitrate dehydrogenase (IDH) is an octamer containing two types of homologous subunits. Ligand-binding analyses were conducted to examine effects of residue changes in putative catalytic and regulatory isocitrate-binding sites respectively contained in IDH2 and IDH1 subunits. Replacement of homologous serine residues in either subunit site, S98A in IDH2 or S92A in IDH1, was found to reduce by half the total number of holoenzyme isocitrate-binding sites, confirming a correlation between detrimental effects on isocitrate binding and respective kinetic defects in catalysis and allosteric activation by AMP. Replacement of both serine residues eliminates isocitrate binding and measurable catalytic activity. The putative isocitrate-binding sites of IDH1 and IDH2 contain five identical and four nonidentical residues. Reciprocal replacement of the four nonidentical residues in either or both subunits (A108R, F136Y, T241D, and N245D in IDH1 and/or R114A, Y142F, D248T, and D252N in IDH2) was found to be permissive for isocitrate binding. This provides further evidence for two types of binding sites in IDH, although the authentic residues have been shown to be necessary for normal kinetic contributions. Finally, the mutant enzymes with residue replacements in the IDH1 site were found to be unable to bind AMP, suggesting that allosteric activation is dependent both upon binding of isocitrate at the IDH1 site and upon the changes in the enzyme normally elicited by this binding.     

Locations of the regulatory sites for isocitrate dehydrogenase kinase/phosphatase

Isocitrate dehydrogenase (IDH)(1) of Escherichia coli is regulated by a bifunctional protein, IDH kinase/phosphatase. In this paper, we demonstrate that the effectors controlling these activities belong to two distinct classes that differ in mechanism and in the locations of their binding sites. NADPH and isocitrate are representative members of one of these effector classes. NADPH inhibits both IDH kinase and IDH phosphatase, whereas isocitrate inhibits only IDH kinase. Isocitrate can "activate" IDH phosphatase by reversing product inhibition by dephospho-IDH. Mutations in icd, which encodes IDH, had parallel effects on the binding of these ligands to the IDH active site and on their effects on IDH kinase and phosphatase, indicating that these ligands regulate IDH kinase/phosphatase through the IDH active site. Kinetic analyses suggested that isocitrate and NADPH prevent formation of the complex between IDH kinase/phosphatase and its protein substrate. AMP, 3-phosphoglycerate, and pyruvate represent a class of regulatory ligands that is distinct from that which includes isocitrate and NADPH. These ligands bind directly to IDH kinase/phosphatase, a conclusion which is supported by the observation that they inhibit the IDH-independent ATPase activity of this enzyme. These effector classes can also be distinguished by the observation that mutant derivatives of IDH kinase/phosphatase expressed from aceK3 and aceK4 exhibited dramatic changes in their responses to AMP, 3-phosphoglycerate, and pyruvate but not to NADPH and isocitrate.     

Kinetic and physiological effects of alterations in homologous isocitrate-binding sites of yeast NAD(+)-specific isocitrate dehydrogenase.

Yeast NAD(+)-specific isocitrate dehydrogenase is an allosterically regulated octameric enzyme composed of four each of two homologous but nonidentical subunits designated IDH1 and IDH2. Models based on the crystallographic structure of Escherichia coli isocitrate dehydrogenase suggest that both yeast subunits contain isocitrate-binding sites. Identities in nine residue positions are predicted for the IDH2 site whereas four of the nine positions differ between the IDH1 and bacterial enzyme sites. Thus, we speculate that the IDH2 site is catalytic and that the IDH1 site may bind but not catalytically alter isocitrate. This was examined by kinetic analyses of enzymes with independent and concerted replacement of residues in each yeast IDH subunit site with the residues that differ in the other subunit site. Mutant enzymes were expressed in a yeast strain containing disrupted IDH1 and IDH2 loci and affinity-purified for kinetic analyses. The primary effects of various residue replacements in IDH2 were reductions of 30->300-fold in V(max) values, consistent with the catalytic function of this subunit. In contrast, replacement of all four residues in IDH1 produced a 17-fold reduction in V(max) under the same assay conditions, suggesting that the IDH1 site is not the primary catalytic site. However, single or multiple residue replacements in IDH1 uniformly increased half-saturation concentrations for isocitrate, implying that isocitrate can be bound at this site. Both subunits appear to contribute to cooperativity with respect to isocitrate, but AMP activation is lost only with residue replacements in IDH1. Overall, results are consistent with isocitrate binding by IDH2 for catalysis and with isocitrate binding by IDH1 being a prerequisite for allosteric activation by AMP. The effects of residue substitutions on enzyme function in vivo were assessed by analysis of various growth phenotypes. Results indicate a positive correlation between the level of IDH catalytic activity and the ability of cells to grow with acetate or glycerol as carbon sources. In addition, lower levels of activity are associated with increased production of respiratory-deficient (petite) segregants.     

Isozyme patterns in callus cultures and in plants regenerated from calli ofCereus peruvianus (Cactaceae)

Electrophoretic patterns for isocitrate dehydrogenase (IDH; EC 1.1.1.42), acid phosphatase (ACP; EC 3.1.3.2), peroxidase (PER; EC 1.11.1.7), and esterase (EST; EC 3.1.1.1) isozymes were determined inCereus peruvianus tissues and used as markers of genetic uniformity of calli and of the plants regenerated from callus cultures. One IDH, six ACP, six PER, and six EST isozymes were induced in cultured callus tissues in medium containing three 2,4-dichlorophenoxyacetic acid and kinetin combinations. Four ACP, two PER, and three EST isozymes were still present in all regenerated plantsin vitro and therefore can be used as markers of theC. peruvianus plants regenerated from callus tissues. The differential patterns of ACP and IDH isozymes and the similar zymograms for PER and EST isozymes presented by callus tissues were used in a comparison of callus tissues cultured for 2 years. The comparative analysis of zymograms within each enzyme system indicated a mean heterogeneity coefficient of 0.33 forC. peruvianus calli cultured for 2 years. Because of the isozyme variations, which developed in culture medium and were transferred to the regenerated plants, the IDH, ACP, PER, and EST enzyme systems can be considered to be good markers for investigating possible genetic variations in plant populations ofC. peruvianus obtainedin vitro from callus culture.This research was supported by the CNPq