High malic enzyme activity in tumor cells and its cross-reaction with antipigeon liver malic enzyme serum

Summary Rabbit antibodies against pigeon liver malic enzyme (EC 1.1.1.40) were prepared. The antiserum gave single precipitation line with crude pigeon liver extract. Cross reaction was observed with partially purified malic enzyme or crude extract from chicken liver. Positive cross reaction was also observed with the concentrated cytosolic fraction of two human carcinoma cell lines which were demonstrated to contain high malic enzyme activity. All other proteins examined did not react with the antibodies. When purified pigeon liver malic enzyme was mixed with the antiserumin vitro, a time-dependent inactivation of the enzyme activity was observed. Protection of the enzyme activity against antiserum inactivation was afforded by NADP⁺ orL-malate. Metal Mn²⁺ gave little protection.     

Chicken Intestinal Alkaline Phosphatase : I. The kinetics and thermodynamics of reversible inactivation II. Reactivation by zinc ions

Purified chicken intestinal alkaline phosphatase is active at pH 8 to 9, but becomes rapidly inactivated with change of pH to 6 or less. Also, a solution of the inactivated enzyme at pH 4.5 rapidly regains its activity at pH 8. In the range of pH 6 to 8 a solution of purified alkaline phosphatase consists of a mixture of active and inactive enzyme in equilibrium with each other. The rate of inactivation at lower pH and of reactivation at higher pH increases with increase in temperature. Also, the activity at equilibrium in the range of pH 6 to 8 increases with temperature so that a solution equilibrated at higher temperature loses part of its activity on cooling, and vice versa, a rise in temperature shifts the equilibrium toward higher activity. The kinetics of inactivation of the enzyme at lower pH and the reactivation at higher pH is that of a unimolecular reaction. The thermodynamic values for the heat and entropy of the reversible inactivation and reactivation of the enzyme are considerably lower than those observed for the reversible denaturation of proteins. The inactivated enzyme at pH 4 to 6 is rapidly reactivated on addition of Zn ions even at pH 4 to 6. However, zinc ions are unable to replace magnesium ions as cocatalysts for the enzymatic hydrolysis of organic phosphates by alkaline phosphatase.     

Inactivation of jack bean urease by N-ethylmaleimide: pH dependence,reversibility and thiols influence

N-Ethylmaleimide (NEM) was studied as an inactivator of jack bean urease at 25 °C in 20 mM phosphate buffer, pHs 6.4, 7.4, and 8.3. The inactivation was investigated by incubation procedure in the absence of a substrate. It was found that NEM acted as a time and concentration dependent inactivator of urease. The dependence of urease residual activity on the incubation time showed that the activity decreased with time until the total loss of enzyme activity. The process followed a pseudo-first-order reaction. A monophasic loss of enzyme activity was observed at pH 7.4 and 8.4, while a biphasic reaction occurred at pH 6.4. Moreover, the alkaline pH promoted the inactivation. The presence of thiol-compounds, such as L-cysteine, glutathione or dithiothreitol (DTT), in the incubation mixture significantly slowed down the rate of inactivation. The interaction test showed that the decrease of inactivation was an effect of NEM-thiol interaction that lowered NEM concentration in the incubation mixture. The reactivation of NEM-blocked urease by DTT application and multidilution did not result in an effective activity regain. The applied DTT reacted with the remaining inactivator and could stop the progress of enzyme activity loss but did not cause the reactivation. This confirmed the irreversibility of inactivation. Similar results obtained at pH 6.4, 7.4 and 8.4 indicated that the mechanism of urease inactivation by NEM was pH-independent. However, the pH value significantly influenced the process rate.     

Lysis of virus-infected target cells by antibody-dependent cellular cytotoxicity. II. Reversibility of the heart inactivation of K cell killing and relationship of Fc receptor capping to lysis.

The heat inactivation of human blood mononuclear cells active in antibody-dependent cellular cytotoxicity (ADCC) is largely reversed after 24 hr in culture at 37 °C. The reactivation process is inhibited by actinomycin D, cycloheximide, and emetine but not by mitomycin-C, indicating that recovery requires RNA and protein synthesis but not DNA synthesis. The ability of lymphocytes to cap surface immunoglobulin (SIg) and IgG-Fc receptors (FcR) was also studied. As with ADCC effector cell activity, both SIg and FcR capping were abolished by heating, and the kinetics of inactivation was similar to that of the inactivation of ADCC effector activity. In addition, the heat inactivation of capping was reversible in culture and followed kinetics of reactivation similar to that of K cell reactivation. These results suggest the participation of heat-labile proteins at or near the surface of the effector cell, which are also apparently involved in the capping of surface receptors. Presumably these heat-labile proteins are membrane-associated enzymes, but they may also be cytoskeletal structures such as microfilaments or microtubules whose heat-sensitivity is currently unknown. The mounting of lethal hits may involve the same membrane machinery which is responsible for capping or a capping process itself.     

Reactivation of Giardia lamblia cysts after exposure to polychromatic UV light

Aims: In this study, we determined the ability of a promising alternative UV technology – a polychromatic emission from a medium‐pressure UV (MP UV) technology – to inhibit the reactivation of UV‐irradiated Giardia lamblia cysts. Methods and Results: A UV‐collimated beam apparatus was used to expose shallow suspensions of purified G. lamblia cysts in PBS (pH 7·2) or filtered drinking water to a low dose (1 mJ cm^?2) of MP UV irradiation. After UV irradiation, samples were exposed to two repair conditions (light or dark) and two temperature conditions (25°C or 37°C for 2–4 h). The inactivation of G. lamblia cysts by MP UV was very extensive, and c. 3 log₁₀ inactivation was achieved with a dose of 1 mJ cm^?2. Meanwhile, there was no apparent reactivation (neither in vivo nor in vitro) of UV‐irradiated G. lamblia under the conditions tested. Conclusion: The results of this study indicated that, unlike the traditional low‐pressure (LP) UV technology, an alternative UV technology (MP UV) could inhibit the reactivation of UV‐irradiated G. lamblia cysts even when the cysts were exposed to low UV doses. Significance and Impact of the Study: It appears that alternative UV technology has some advantages over the traditional LP UV technology in drinking water disinfection because of their high level of inactivation against G. lamblia cysts and also effective inhibition of reactivation in UV‐irradiated G. lamblia cysts.     

α-Crystallin Assisted Refolding of Enzyme Substrates: Optimization of External Parameters

α-Crystallin is known to act as a molecular chaperone by preventing the aggregation of partially unfolded substrate proteins. It is also known to assist the refolding of a number of denatured enzymes, but the activity yield is often less than 20%. In this paper, we have tried to tune the refolding ability of α-crystallin in vitro by optimizing various external parameters. We wanted to find out the best possible condition under which it can exhibit maximum refolding capacity. We found that under suitable condition in vitro α-crystallin can refold denatured malate dehydrogenase, carbonic anhydrase and lactate dehydrogenase to recover more than 40% activity. We also measured the effect of several external factors such as nucleotides, osmolytes, electrolytes, temperature etc. on the in vitro α-crystallin mediated reactivation of above stated enzymes. We found that nucleotides and electrolytes had little effect on the refolding ability of α-crystallin. However, in presence of different osmolytes, we found that its ability to reactivate denatured substrate proteins enhanced significantly. Refolding in presence of pre-incubated α-crystallin reveals that hydrophobicity had stronger influence on the refolding capacity of α-crystallin than its oligomeric size.     

In vitro oxidative inactivation of glutathione S-transferase from a freeze tolerant reptile

We have previously reported that when garter snakesThamnophis sirtalis parietalis, a freeze tolerant species, were exposed to 5 h freezing at –2.5° C organs showed increases in the activities of anti-oxidant enzymes, especially catalase in skeletal muscle. This was interpreted to be an adaptation to deal with the potentially injurious postischemic situation of thawing. The present work analyzesin vitro oxidative inactivation of a possible target of postischemic-induced free radical damage, the secondary anti-oxidant defense glutathione-S transferase, and the protective role of endogenous catalase. Approximately 50% of GST activity from snake muscle homogenates was lost within 2 min after addition of H₂O₂ plus Fe(II) (0.4–2 mM) in media containing azide whereas addition of iron alone resulted in no damaging effects. The opposing effects of dimethyl sulfoxide and EDTA in modifying this process strongly suggested the involvement of ·OH radicals in the GST inactivation. A partial recovery of the activity was promoted by mercaptoethanol, indicating that sulphydryl groups oxidation participate in the mechanism of GST inactivation. Pre-incubation of the reaction media containing H₂O₂ caused protection of the GST activity only in the absence of azide, indicating that endogenous catalase modulates the extent of oxyradical damage. The protective pre-incubation effect was more efficacious when employing homogenates from lung and liver, organs that have higher catalase activities, as well as homogenates from freezing-exposed muscle (that show an 80% increase in catalase activity, compared with control). The protection against GST inactivation observed in muscle from frozen snakes demonstrates that increased anti-oxidant defenses during freezing exposure can be a key factor in controllingin vitro oxyradical damage. The implications for natural freeze tolerance are discussed.     

Thermoplasma acidophilum Cdc6 protein stimulates MCM helicase activity by regulating its ATPase activity

The minichromosome maintenance (MCM) proteins are thought to function as the replicative helicases in archaea. In most archaeal species studied, the interaction between MCM and the initiator protein, Cdc6, inhibits helicase activity. To date, the only exception is the helicase and Cdc6 proteins from the archaeon Thermoplasma acidophilum. It was previously shown that when the Cdc6 protein interacts with MCM it substantially stimulates helicase activity. It is shown here that the mechanism by which the Cdc6 protein stimulates helicase activity is by stimulating the ATPase activity of MCM. Also, through the use of site-specific substitutions, and truncated and chimeric proteins, it was shown that an intact Cdc6 protein is required for this stimulation. ATP binding and hydrolysis by the Cdc6 protein is not needed for the stimulation. The data suggest that binding of Cdc6 protein to MCM protein changes the structure of the helicase, enhancing the catalytic hydrolysis of ATP and helicase activity.     

Glucose-induced inactivation of isocitrate lyase in Aspergillus nidulans

     The existence of a second mechanism of catabolite control of isocitrate lyase of Aspergillus nidulans, in addition to the carbon catabolite repression phenomenon recently reported was analysed. Isocitrate lyase was rapidly and specifically inactivated by glucose. The inactivation was irreversible at all stages in the presence of cycloheximide, showing that reactivation depends on de novo protein synthesis. In addition, analysis of glucose-induced inactivation of isocitrate lyase in a creA ^d -30 strain showed that the creA gene is not involved in this process. Received: 13 May 1994 / Accepted 12 August 1994     

Controlled X‐chromosome dynamics defines meiotic potential of female mouse in vitro germ cells

The mammalian germline is characterized by extensive epigenetic reprogramming during its development into functional eggs and sperm. Specifically, the epigenome requires resetting before parental marks can be established and transmitted to the next generation. In the female germline, X‐chromosome inactivation and reactivation are among the most prominent epigenetic reprogramming events, yet very little is known about their kinetics and biological function. Here, we investigate X‐inactivation and reactivation dynamics using a tailor‐made in vitro system of primordial germ cell‐like cell (PGCLC) differentiation from mouse embryonic stem cells. We find that X‐inactivation in PGCLCs in vitro and in germ cell‐competent epiblast cells in vivo is moderate compared to somatic cells, and frequently characterized by escaping genes. X‐inactivation is followed by step‐wise X‐reactivation, which is mostly completed during meiotic prophase I. Furthermore, we find that PGCLCs which fail to undergo X‐inactivation or reactivate too rapidly display impaired meiotic potential. Thus, our data reveal fine‐tuned X‐chromosome remodelling as a critical feature of female germ cell development towards meiosis and oogenesis.     

The GINS complex from the thermophilic archaeon, <Emphasis Type="Italic">Thermoplasma acidophilum</Emphasis> may function as a homotetramer in DNA replication

The eukaryotic GINS heterotetramer, consisting of Sld5, Psf1, Psf2, and Psf3, participates in “CMG complex” formation with mini-chromosome maintenance (MCM) and Cdc45 as a key component of a replicative helicase. There are only two homologs of the GINS proteins in Archaea, and these proteins, Gins51 and Gins23, form a heterotetrameric GINS with a 2:2 molar ratio. The Pyrococcus furiosus GINS stimulates the ATPase and helicase activities of its cognate MCM, whereas the Sulfolobus solfataricus GINS does not affect those activities of its cognate MCM, although the proteins bind each other. Intriguingly, Thermoplasma acidophilum, as well as many euryarchaea, have only one gene encoding the sequence homologous to that of archaeal Gins protein (Gins51) on the genome. In this study, we investigated the biochemical properties of the gene product (TaGins51). A gel filtration and electron microscopy revealed that TaGins51 forms a homotetramer. A physical interaction between TaGins51 and TaMcm was detected by a surface plasmon resonance analysis. Unexpectedly, TaGins51 inhibited the ATPase activity, but did not affect the helicase activity of its cognate MCM. These results suggest that another factor is required to form a stable helicase complex with MCM and GINS at the replication fork in T. acidophilum cells.     

Redox interconversion of Escherichia coli glutathione reductase. A study with permeabilized and intact cells

Summary The redox interconversion of Escherichia coli glutathione reductase has been studied both in situ, with permeabilized cells treated with different reductants, and in vivo, with intact cells incubated with compounds known to alter their intracellular redox state.The enzyme from toulene-permeabilized cells was inactivated in situ by NADPH, NADH, dithionite, dithiothreitol, or GSH. The enzyme remained, however, fully active upon incubation with the oxidized forms of such compounds. The inactivation was time-, temperature-, and concentration-dependent; a 50% inactivation was promoted by just 2 M NADPH, while 700 M NADH was required for a similar effect. The enzyme from permeabilized cells was completely protected against redox inactivation by GSSG, and to a lesser extent by dithiothreitol, GSH, and NAD(P)⁺. The inactive enzyme was efficiently reactivated in situ by physiological GSSG concentrations. A significant reactivation was promoted also by GSH, although at concentrations two orders of magnitude below its physiological concentrations. The glutathione reductase from intact E. coli cells was inactivated in vivo by incubation with DL-malate, DL-isocitrate, or higher L-lactate concentrations. The enzyme was protected against redox inactivation and fully reactivated by diamide in a concentration-dependent fashion. Diamide reactivation was not dependent on the synthesis of new protein, thus suggesting that the effect was really a true reactivation and not due to de novo synthesis of active enzyme. The glutathione reductase activity increased significantly after incubation of intact cells with tert-butyl or cumene hydroperoxides, suggesting that the enzyme was partially inactive within such cells. In conclusion, the above results show that both in situ and in vivo the glutathione reductase of Escherichia coli is subjected to a redox interconversion mechanism probably controlled by the intracellular NADPH and GSSG concentrations.     

Reversible inactivation of nitrate reductase in Chlorella vulgaris in vivo

Summary The NADH-nitrate oxidoreductase of Chlorella vulgaris has an inactive form which has previously been shown to be a cyanide complex of the reduced enzyme. This inactive enzyme can be reactivated by treatment with ferricyanide in vitro. In the present study, the activation state of the enzyme was determined after different prior in vivo programs involving environmental variations. Oxygen, nitrate, light and CO₂ all affect the in vivo inactivation of the enzyme in an interdependent manner. In general, the inactivation is stimulated by O₂ and inhibited by nitrate and CO₂. Light may stimulate or inhibit, depending on conditions. Thus, the effects of CO₂ and nitrate (inhibition of reversible inactivation) are clearly manifested only in the light. In contrast, light stimulates the inactivation in the presence of oxygen and the absence of CO₂ and nitrate. Since the inactivation of the enzyme requires HCN and NADH, and it is improbable that O₂ stimulates NADH formation, it is reasonable to conclude that HCN is formed as the result of an oxidation reaction (which is stimulated by light). The formation of HCN is probably stimulated by Mn²⁺, since the formation of reversibly-inactivated enzyme is impaired in Mn²⁺-deficient cells. The prevention of enzyme inactivation by nitrate in vivo is in keeping with previous in vitro results showing that nitrate prevents inactivation by maintaining the enzyme in the oxidized form. A stimulation of nitrate uptake by CO₂ and light could account for the effect of CO₂ (prevention of inactivation) which is seen mainly in the presence of nitrate and light. Ammonia added in the presence of nitrate has the same effect on the enzyme as removing nitrate (promotion of reversible inactivation). Ammonia added in the absence of nitrate has little extra effect. It is therefore likely that ammonia acts by preventing nitrate uptake. The uncoupler, carbonylcyanide-m-chloro-phenylhydrazone, causes enzyme inactivation because it acts as a good HCN precursor, particularly in the light. Nitrite, arsenate and dinitrophenol cause an enzyme inactivation which can not be reversed by ferricyanide in crude extracts. This suggests that there are at least two different ways in which the enzyme can be inactivated rather rapidly in vivo.     

Modulation of nitrate reductase in vivo and in vitro: Effects of phosphoprotein phosphatase inhibitors,free Mg2+ and 5′-AMP

Nitrate reductase in spinach (Spinacia oleracea L.) leaves was rapidly inactivated in the dark and reactivated by light, whereas in pea (Pisum sativum L.), roots, hyperoxic conditions caused inactivation, and anoxia caused reactivation. Reactivation in vivo, both in leaves and roots, was prohibited by high concentrations (10–30 M) of the serine/threonine-protein phosphatase inhibitors okadaic acid or calyculin, consistent with the notion that protein dephosphorylation catalyzed by type-1 or type-2A phosphatases was the mechanism for the reactivation of NADH-nitrate reductase (NR). Following inactivation of leaf NR in vivo, spontaneous reactivation in vitro (in desalted extracts) was slow, but was drastically accelerated by removal of Mg²⁺ with excess ethylenediaminetetraacetic acid (EDTA), or by desalting in a buffer devoid of Mg²⁺. Subsequent addition of either Mg²⁺, Mn²⁺ or Ca²⁺ inhibited the activation of NR in vitro. Reactivation of NR (at pH 7.5) in vitro in the presence of Mg²⁺ was also accelerated by millimolar concentrations of AMP or other nucleoside monophosphates. The EDTA-mediated reactivation in desalted crude extracts was completely prevented by protein-phosphatase inhibitors whereas the AMP-mediated reaction was largely unaffected by these toxins. The Mg²⁺-response profile of the AMP-accelerated reactivation suggested that okadaic acid, calyculin and microcystin-LR were rather ineffective inhibitors in the presence of divalent cations. However, with partially purified enzyme preparations (5–15% polyethyleneglycol fraction) the AMPmediated reactivation was also inhibited (65–80%) by microcystin-LR. Thus, the dephosphorylation (activation) of NR in vitro is inhibited by divalent cations, and protein phosphatases of the PP1 or PP2A type are involved in both the EDTA and AMP-stimulated reactions. Evidence was also obtained that divalent cations may regulate NR-protein phosphatase activity in vivo. When spinach leaf slices were incubated in Mg²⁺ -and Ca²⁺-free buffer solutions in the dark, extracted NR was inactive. After addition of the Ca²⁺ /Mg²⁺-ionophore A 23187 plus EDTA to the leaf slices, NR was activated in the dark. It was again inactivated upon addition of divalent cations (Mg²⁺ or Ca²⁺). It is tentatively suggested that Mg²⁺ fulfills several roles in the regulatory system of NR: it is required for active NR-protein kinase, it inactivates the protein phosphatase and is, at the same time, necessary to keep phospho-NR in the inactive state. The EDTA- and AMP-mediated reactivation of NR in vitro had different pH optima, suggesting that two different protein phosphatases may be involved. At pH 6.5, the activation of NR was relatively slow and the addition or removal of Mg²⁺ had no effect. However, 5-AMP was a potent activator of the reaction with an apparent K _m of 0.5 mM. There was also considerable specificity for 5AMP relative to 3- or 2-AMP or other nucleoside monophoposphates. We conclude that, depending upon conditions, the signals triggering NR modulation in vivo could be either metabolic (e.g. 5-AMP) or physical (e.g. cytosolic [Mg²⁺]) in nature.Abbreviations DTT dithiothreitol - Mops 3-(N-morpholino)propanesulfonic acid - NR NADH-nitrate reductase - NRA nitrate-reductase activity - PP protein phosphatase This paper is dedicated to Prof. O.K. Volk on the occasion of his 90th birthdayThe skilled technical assistance of Elke Brendle-Behnisch is gratefully acknowledged. The investigations were cooperatively supported by the Deutsche Forschungsgemeinschaft (SFB 251), the U.S. Department of Agriculture, Agricultural Research Services, Raleigh, NC. This work was also supported in part by a grant from the U.S. Department of Energy (Grant DE-A I05-91 ER 20031 to S.C.H.).     

Evaluation of the Protective Activities of a Late Embryogenesis Abundant (LEA) Related Protein,Cor15am,during Various Stresses in Vitro

Arabidopsis Cor15am is a late embryogenesis abundant (LEA) related protein that has been shown to exhibit cryoprotective activity in vitro. In this study, we further investigated the mechanisms by which Cor15am protects substrates from inactivation. Although Cor15am did not exhibit refolding activity, it showed protective activity against various stresses in vitro. This might be attributable to the activity of Cor15am in attenuating the aggregation of the substrates. Our data indicate that Cor15am functions as a protectant against various stresses by preventing protein aggregation.     

Modulation in vitro of 3-hydroxy-3-methylglutaryl coenzyme A reductase in brain microsomes: Evidence for the phosphorylation and dephosphorylation associated with inactivation and activation of the enzyme

The activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in brain microsomes was modified in vitro. The inactivation of the enzyme required Mg²⁺ and ATP or ADP, and an inactivator present both in S₁₀₅ and microsomes. Inactivation was dependent on inactivator concentration and time of preincubation. The inactive reductase in brain microsomes could be completely reactivated by a factor present in brain S₁₀₅. Reactivation of the enzyme also depended on incubation time and the activator concentration. Activator activity was inhibited by NaF, a phosphatase inhibitor. Both the inactivator and the activator appear to be proteins. Our data thus suggest that the inactivation and the reactivation of the reductase in brain microsomes occurs via protein-mediated interconversion to phosphorylated and dephosphorylated forms of the enzyme with differing catalytic activity. The HMG-CoA reductase activity increases almost two-fold during isolation of the brain microsomes. This increase in activity is blocked when brain tissue is homogenized in the medium containing NaF. In rat brain about 50% of the reductase exists in an inactive form in both young and adult rats. The low reductase activity in brain of adult animals does not appear to be related to an increase in the proportion of an inactive phosphorylated form of the enzyme. This suggests that developmental change in the reductase activity is not associated with the change in the proportion of phosphorylated and dephosphorylated forms of the enzyme.     

Regulation of steady state pyruvate dehydrogenase complex activity in plant mitochondria : reactivation constraints

The requirements for reactivation (dephosphorylation) of the pea (Pisum sativum L.) leaf mitochondrial pyruvate dehydrogenase complex (PDC) were studied in terms of magnesium and ATP effects with intact and permeabilized mitochondria. The requirement for high concentrations of magnesium for reactivation previously reported with partially purified PDC is shown to affect inactivation rather than reactivation. The observed rate of inactivation catalyzed by pyruvate dehydrogenase (PDH) kinase is always greater than the reactivation rate catalyzed by PDH-P phosphatase. Thus, reactivation would only occur if ATP becomes limiting. However, pyruvate which is a potent inhibitor of inactivation in the presence of thiamine pyrophosphate, results in increased PDC activity. Analysis of the dynamics of the phosphorylation-dephosphorylation cycle indicated that the covalent modification was under steady state control. The steady state activity of PDC was increased by addition of pyruvate. PDH kinase activity increased threefold during storage of mitochondria suggesting that there may be an unknown level of regulation exerted on the enzyme complex.     

Comparison of the conformation and stability of the native dimeric,monomelic, tetrameric and the desensitized forms of the nucleotide pyrophosphatase from mung bean (Phaseolus aureus) seedlings

A homogenous and crystalline form of nucleotide pyrophosphatase (EC 3.6.1.9) fromPhaseolus aureus (mung bean) seedlings was used for the study of the regulation of enzyme activity by adenine nucleotides. The native dimeric form of the enzyme had a helical content of about 65% which was reduced to almost zero values by the addition of AMP. In addition to this change in the helical content, AMP converted the native dimer to a tetramer. Desensitization of AMP regulation, without an alteration of the molecular weight, was achieved either by reversible denaturation with 6 M urea or by passage through a column of Blue Sepharose but additionofp-hydroxymercuribenzoate desensitized the enzyme by dissociating the native dimer to a monomer. The changes in the quaternary structure and conformation of the enzyme consequent to AMP interaction or desensitization were monitored by measuring the helical content, EDTA inactivation and Zn²⁺ reactivation, stability towards heat denaturation, profiles of urea denaturation and susceptibility towards proteolytic digestion. Based on these results and our earlier work on this enzyme, we propose a model for the regulation of the mung bean nucleotide pyrophosphatase by association-dissociation and conformational changes. The model emphasizes that multiple mechanisms are operative in the desensitization of regulatory proteins.