Thiol Enzyme Research Articles

Urease activity and l-ascorbic acid

In this work, we studied the behaviours of urease in the presence of l-ascorbic acid (AA) and dehydroascorbic acid (DHA) in different conditions. The inactivations of urease were carried out in an unbuffered and buffered system. We show that in the unbuffered system AA inactivated urease in a biphasic manner by denaturation brought about by AA-lowered pH. Further, we show that in the buffered system neither AA nor DHA themselves are inhibitors of urease. The inhibitory action of AA and DHA was revealed in the presence of Fe3+ ions and most importantly, unlike reported in the literature, it was found to be primarily mediated by H2O2. The resulting inhibition by DHA-Fe3+ consisted of enzyme thiol oxidation and its effectiveness grew with increasing pH. The results may shed light on the roles of AA in therapies applied in ureolytic bacteria infections, notably those with Helicobacter pylori.

Mechanisms underlying the inhibition of the cytochrome P450 system by copper ions

Copper toxicity has been associated to the capacity of free copper ions to catalyze the production of superoxide anion and hydroxyl radical, reactive species that modify the structure and/or function of biomolecules. In addition, nonspecific Cu2+-binding to thiol enzymes, which modifies their catalytic activities, has been reported. Cytochrome P450 (CYP450) monooxygenase is a thiol protein that binds substrates in the first and limiting step of CYP450 system catalytic cycle, necessary for the metabolism of lipophilic xenobiotics. Therefore, copper ions have the potential to oxidize and bind to cysteinyl residues of this monooxygenase, altering the CYP450 system activity. To test this postulate, we studied the effect of Cu2+ alone and Cu2+/ascorbate in rat liver microsomes, to independently evaluate its nonspecific binding and its pro-oxidant effects, respectively. We assessed these effects on the absorbance spectrum of the monooxygenase, as a measure of structural damage, and p-nitroanisole O-demethylating activity of CYP450 system, as a marker of functional impairment. Data obtained indicate that Cu2+ could both oxidize and bind to some amino acid residues of the CYP450 monooxygenase but not to its heme group. The differences observed between the effects of Cu2+ and Cu2+/ascorbate show that both mechanisms are involved in the catalytic activity inhibition of CYP450 system by copper ions. The significance of these findings on the pharmacokinetics and pharmacodynamics of drugs is discussed.

Possible involvement of radical intermediates in the inhibition of cysteine proteases by allenyl esters and amides

In order to investigate crystallographically the mechanism of inhibition of cysteine protease by α-methyl-γ,γ-diphenylallenecarboxylic acid ethyl ester 3, a cysteine protease inhibitor having in vivo stability, we synthesized N-(α-methyl-γ,γ-diphenylallenecarbonyl)-l-phenylalanine ethyl ester 4. Reaction of 4 with thiophenol, the SH group of which has similar pKa value to that of cysteine protease, produced oxygen-mediated radical adducts 6 and 7 in ambient air but did not proceed under oxygen-free conditions. Catalytic activities of two thiol enzymes including cathepsin B were also lowered in the absence of oxygen. These results suggest that cysteine protease can act through an oxygen-dependent radical mechanism.

Covalent modification of proteins by green tea polyphenol (–)-epigallocatechin-3-gallate through autoxidation

Green tea polyphenol (–)-epigallocatechin-3-gallate (EGCG) has various beneficial properties including chemopreventive, anticarcinogenic, and antioxidant actions. The interaction with proteins known as EGCG-binding targets may be related to the anticancer effects. However, the binding mechanisms for this activity remain poorly understood. Using mass spectrometry and chemical detection methods, we found that EGCG forms covalent adducts with cysteinyl thiol residues in proteins through autoxidation. To investigate the functional modulation caused by binding of EGCG, we examined the interaction between EGCG and a thiol enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Concentration-dependent covalent binding of EGCG to GAPDH was found to be coupled to the irreversible inhibition of GAPDH activity. Mutation experiments revealed that EGCG is primarily bound to the cysteinyl thiol group of the active center, indicating that the irreversible inhibition of GAPDH is due to the covalent attachment of EGCG to the active-center cysteine. Moreover, using EGCG-treated cancer cells, we identified GAPDH as a target of EGCG covalent binding through specific interactions between catechols and aminophenyl boronate agarose resin. Based on these findings, we propose that the covalent modification of proteins by EGCG may be a novel pathway related to the biological activity of EGCG.

Modification of jack bean urease thiols by thiosulphinates contained in garlic extract: DTNB titration studies

The reactivity of the thiol groups in urease with thiosulphinate contained in garlic extract was spectroscopically characterised. The enzyme was incubated with the garlic extract and the reaction progress curves were recorded in the presence of thiol-selective reagent 5,5′-dithiobis(2-nitrobenzoic acid). Simultaneously the enzyme residual activity was also determined. The process was studied in 50 mM phosphate buffer, at pH 7.8 and ambient temperature. It was found that thiosulphinates act as time- and concentration-dependent inactivators of urease. The observed decrease of the enzyme activity corresponds to the number of the urease thiols modified by thiosulphinates. The modification of half of all the urease thiols (18 of 36) causes a slight (only 8–10%) decrease of the catalytic activity. The modification of the remaining 18 thiols results in significant disturbance of urease action until complete loss of the catalytic function occurs. This provides the evidence that Cys 592, the critical residue for urease activity, belongs to the enzyme thiols, which are less reactive and more resistant to chemical modification than the other thiols.

Regulation of thiol enzyme activity by (−)‐epigallocatechin gallate through covalent binding to cysteine

(−)‐Epigallocatechin gallate (EGCg), which has an antitumor activity, is a major component of polyphenol in green tea. Some mechanisms on the anticancer effect of EGCg have been proposed, and EGCg‐protein interaction is thought to be a significant step. However, the mechanisms on the regulation of protein function through binding are still unclear. To obtain the proof of functional modulation by EGCg, we used thiol enzymes, glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) and papain, as model proteins. Incubation of GAPDH with EGCg resulted in a dose‐dependent loss of the enzyme activity, which was accompanied by the loss of free thiol groups. Furthermore, by mass spectrometry (MS) and Western blotting with redox cycling staining, it is clarified that EGCg covalently binds to the enzyme. In addition, the results on the modification of papain were similar to that of GAPDH. To identify the binding site of GAPDH by EGCg, both the native and EGCg‐treated GAPDH were purified with catechol affinity resin and then digested with trypsin, and the resulting peptides were subjected to MS. We identified the EGCg‐binding peptides containing cysteine residues. These results suggest that modulation of GAPDH activity by EGCg is due to the selective modification of the cysteine residues in the GAPDH molecule. These findings may contribute to the understanding of anticancer mechanisms based on binding of tea catechins to proteins.

Mono- (Ag, Hg) and di- (Cu, Hg) valent metal ions effects on the activity of jack bean urease. Probing the modes of metal binding to the enzyme

The inhibition of urease by heavy metal ions has been habitually ascribed to the reaction of the ions with enzyme thiol groups, resulting in the formation of mercaptides. To probe the modes of metal binding to the enzyme, in this work the reaction of mono- (Ag, Hg) and di- (Cu, Hg) valent metal ions with jack bean urease was studied. The enzyme was reacted with different concentrations of the metal ions for different periods of times, when its residual activitiy was assayed and thiol content titrated. The titration carried out with DTNB was done to examine the involvement of urease thiol groups in metal ion binding. The binding was further probed by reactivation of the metal ion-enzyme complexes with DTT, EDTA and dilution. The results are discussed in terms of the HSAB concept. In inhibiting urease the metal ions showed a common feature in that they inhibited the enzyme within a comparable micromolar range, and also in that their inhibition was multisite. By contrast, the main distinguishing feature in their action consisted of the involvement of enzyme thiol groups in the reaction. Hg2 + and inhibition was found thoroughly governed by the reaction with the enzyme thiols, and the complete loss of enzyme activity involved all thiols available in the enzyme under non-denaturating conditions. In contrast, Ag+ and Cu2 + ions for the complete inactivation of the enzyme required 53 and 60% of thiols, respectively. Accordingly, Ag+ and Cu2 + binding to functional groups in urease other than thiols, i.e. N- and O-containing groups, cannot be excluded. Based on the reactivation experiments this seems particularly likely for Cu2 + , whose concurrent binding to thiols and other groups might distort the architecture of the active site (the mechanism of which remains to be elucidated) resulting in the observed inhibitory effects.

On the interaction between glyceraldehyde-3-phosphate dehydrogenase and airborne particles: Evidence for electrophilic species

Many of the adverse health effects of airborne particulate matter (PM) have been attributed to the chemical properties of some of the large number of chemical species present in PM. Some PM component chemicals are capable of generating reactive oxygen species and eliciting a state of oxidative stress. In addition, however, PM can contain chemical species that elicit their effects through covalent bond formation with nucleophilic functions in the cell. In this manuscript, we report the presence of constituents with electrophilic properties in ambient and diesel exhaust particles, demonstrated by their ability to inhibit the thiol enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). GAPDH is irreversibly inactivated by electrophiles under anaerobic conditions by covalent bond formation. This inactivation can be blocked by the prior addition of a high concentration of dithiothreitol (DTT) as an alternate nucleophile. Addition of DTT after the reaction between the electrophile and GAPDH, however, does not reverse the inactivation. This property has been utilized to develop a procedure that provides a quantitative measure of electrophiles present in samples of ambient particles collected in the Los Angeles Basin and in diesel exhaust particles. The toxicity of electrophiles is the result of irreversible changes in biological molecules; recovery is dependent on resynthesis. If the resynthesis is slow, the irreversible effects can be cumulative and manifest themselves after chronic exposure to low levels of electrophiles.

Monomethylarsonous Acid Induced Cytotoxicity and Endothelial Nitric Oxide Synthase Phosphorylation in Endothelial Cells

Chronic arsenic poisoning is reported to be associated with peripheral and cardiovascular disease, arteriosclerosis, Raynaud's syndrome, hypertension, and Blackfoot disease. Monomethylarsonous acid (MMA(III)) is a reactive metabolite of inorganic arsenic and a potent inhibitor of endothelial nitric oxide synthase (eNOS). Arsenic is also reported to phosphorylate eNOS in cultured keratinocyte and Human T cell leukemia Jurkat cells, respectively. In the present study, we examined the cytotoxicity and eNOS phosphorylation by MMA(III) exposure in cultured bovine aortic endothelial cells (BAEC). Results showed that MMA(III) is more toxic than arsenite in BAEC cells. The IC(50) values for MMA(III) and arsenite were determined to be approximately 1.7 and 24.1 micromol/L, respectively. Exposure of BAEC to MMA(III) (0.75 micromol/L) caused a significant eNOS phosphorylation 15 min after MMA(III) exposure. However, a complex of MMA(III) with dithiothreitol (DTT) that lacks the reactivity with vicinal thiols unaffected eNOS phosphorylation. The present study shows that MMA(III )generated during biomethylation of arsenic is highly toxic in BAEC. Our study also suggests that MMA(III) could induce the eNOS phosphorylation through modification to cellular thiols of the eNOS enzyme. And the initial up-regulation of eNOS phosphorylation by MMA(III )seems to be an adaptive response against disruption of eNOS bioactivity during arsenic exposure.

Jack bean urease: The effect of active-site binding inhibitors on the reactivity of enzyme thiol groups

In view of the complexity of the role of the active site flap cysteine in the urease catalysis, in this work we studied how the presence of typical active-site binding inhibitors of urease, phenylphosphorodiamidate (PPD), acetohydroxamic acid (AHA), boric acid and fluoride, affects the reactivity of enzyme thiol groups, the active site flap thiol in particular. For that the inhibitor–urease complexes were prepared with excess inhibitors and had their thiol groups titrated with DTNB. The effects observed were analyzed in terms of the structures of the inhibitor–urease complexes reported in the literature. We found that the effectiveness in preventing the active site cysteine from the modification by disulfides, varied among the inhibitors studied, even though they all bind to the active site. The variations were accounted for by different extents of geometrical distortion in the active site that the inhibitors introduced upon binding, leaving the flap either open in AHA-, boric acid- and fluoride-inhibited urease, like in the native enzyme or closed in PPD-inhibited urease. Among the inhibitors, only PPD was found to be able to thoroughly protect the flap cysteines from the further reaction with disulfides, this apparently resulting from the closed conformation of the flap. Accordingly, in practical terms PPD may be regarded as the most suitable inhibitor for active-site protection experiments in inhibition studies of urease.

Double mode of inhibition-inducing interactions of 1,4-naphthoquinone with urease: Arylation versus oxidation of enzyme thiols

In their inhibition-inducing interactions with enzymes, quinones primarily utilize two mechanisms, arylation and oxidation of enzyme thiol groups. In this work, we investigated the interactions of 1,4-naphthoquinone with urease in an effort to estimate the contribution of the two mechanisms in the enzyme inhibition. Jack bean urease, a homohexamer, contains 15 thiols per enzyme subunit, six accessible under non-denaturing conditions, of which Cys592 proximal to the active site indirectly participates in the enzyme catalysis. Unlike by 1,4-benzoquinone, a thiol arylator, the inactivation of urease by 1,4-naphthoquinone under aerobic conditions was found to be biphasic, time- and concentration-dependent with a non-linear residual activity-modified thiols dependence. DTT protection studies and thiol titration with DTNB suggest that thiols are the sites of enzyme interactions with the quinone. The inactivated enzyme had ∼40% of its activity restored by excess DTT supporting the presence of sulfenic acid resulting from the oxidation of enzyme thiols by ROS. Furthermore, the aerobic inactivation was prevented in ∼30% by catalase, proving the involvement of hydrogen peroxide in the process. When H 2O 2 was directly applied to urease, the enzyme showed susceptibility to this inactivation in a time- and concentration-dependent manner with the inhibition constant of H 2O 2 K i = 3.24 mM. Additionally, anaerobic inactivation of urease was performed and was found to be weaker than aerobic. The results obtained are consistent with a double mode of 1,4-naphthoquinone inhibitory action on urease, namely through the arylation of the enzyme thiol groups and ROS generation, notably H 2O 2, resulting in the oxidation of the groups.

Chemical thiolation strategy: A determining factor in the properties of thiol-bound biocatalysts

Immobilization of enzymes on thiolsulphinate-agarose, a thiol-reactive support, is a unique method which allows reversible covalent immobilization under mild conditions, so excellent immobilization and activity yields are obtained. It allows both the formation of stable bonds as well as enzyme desorption and matrix regeneration. The impact of the source of the enzyme's thiol group involved in the immobilization (native, reduced disulphide or chemically introduced) on the properties of the resulting biocatalysts was studied using three β-galactosidases from Escherichia coli, Kluyveromices lactis and Aspergillus oryzae as a model. Chemical thiolation, which generates changes at surface exposed lysines, produced derivatives similar to their soluble counterparts. However, the reduction of native disulphide bonds prior to immobilization lead to very variable activity and stability of the derivatives depending on the accessibility and location of the disulphide bonds in the enzyme structure.

Cystine Inhibits Creatine Kinase Activity in Pig Retina

Cystinosis is an autosomal recessive disorder associated with lysosomal cystine accumulation caused by defective cystine efflux. Visual deficit is a possible consequence of cystine accumulation in cornea and retina. Fibroblasts from cystinotic patients present ATP deficit with intact mitochondrial energy-generating capacity by an unknown mechanism. Considering that creatine kinase is a thiol enzyme crucial for energy homeostasis in retina, and disulfides like cystine may alter thiol enzymes, the main objective of the present study was to investigate the effect of cystine and cysteamine, the drug used for treatment of cystinotic patients, on creatine kinase activity in cytosolic and mitochondrial fractions of the retina from adult pigs. Retina was isolated from 6-month-old Landrace pigs, homogenized and mitochondrial and cytosolic fractions separated by centrifugation. Cytosolic and mitochondrial creatine kinase activities were determined in the presence of different concentrations of cystine and/or cysteamine. Cystine inhibited the enzyme activity in a dose- and time-dependent manner and cysteamine prevented and reversed the inhibition caused by cystine, suggesting that cystine inhibits creatine kinase activity by oxidation of the sulfhydryl groups of the enzyme. Considering that creatine kinase is a crucial enzyme for retina energy homeostasis, in case cystine leaves lysosome these results provide a possible mechanism for cystine toxicity and also another beneficial effect for the use of cysteamine in patients with cystinosis.

Cadmium and mercury cause an oxidative stress-induced endothelial dysfunction

We investigated the ability of cadmium and mercury ions to cause endothelial dysfunction in bovine pulmonary artery endothelial cell monolayers. Exposure of monolayers for 48 h to metal concentrations greater than 3-5 microM produced profound cytotoxicity (increased lactate dehydrogenase leakage), a permeability barrier failure, depletion of glutathione and ATP and almost complete inhibition of the activity of key thiol enzymes, glucose-6-phosphate dehydrogenase (G6PDH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In contrast, metal concentrations less than 1-2 microM induced increases in glutathione and thiol-enzyme activities with minimal changes in LDH leakage, barrier function and ATP content. At shorter incubation times (24 h or less), high concentrations of cadmium caused glutathione induction rather than depletion. Thus, oxidative stress and cytotoxicity induced by lower concentrations of the metal ions stimulate compensatory responses, including increased synthesis of glutathione, which presumably preserved the activity of key thiol enzymes, however these responses were not sustainable at higher metal ion concentrations. We conclude, while high concentrations of heavy metals are cytotoxic, lower concentration induce a compensatory protective response, which may explain threshold effects in metal-ion toxicity.

Membrane Topology of Mouse Stearoyl-CoA Desaturase 1

Stearoyl-CoA desaturase (SCD) is an integral membrane protein anchored in the endoplasmic reticulum. It catalyzes the biosynthesis of monounsaturated fatty acids that are required for the synthesis of triglycerides, cholesteryl esters, and phospholipids. Four mouse isoforms of SCD (SCD1-4) and two human isoforms have been characterized. In the current study, we characterize the topology of the mouse SCD1 isoform. Hydropathy analysis of the 355-amino acid mouse SCD1 protein predicts that the protein contains four transmembrane domains (TMDs) and three loops connecting the membrane-spanning domains. To define the topology of the protein, recombinant SCD1 constructs containing epitope tags were transiently expressed in HeLa cells and analyzed by indirect immunofluorescence and cysteine derivatization. Our data provide evidence that the N and C termini of SCD1 are oriented toward the cytosol with four transmembrane domains separated by two very short hydrophilic loops in the ER lumen and one large hydrophilic loop in the cytosol. In addition, based on the previous observation that SCD is a thiol enzyme, we sought to investigate whether the cysteine residues were essential for enzyme activity through mutagenesis studies, and our data suggest that the cysteines in SCD are not catalytically essential.

Taurine chloramine is more selective than hypochlorous acid at targeting critical cysteines and inactivating creatine kinase and glyceraldehyde-3-phosphate dehydrogenase

Hypochlorous acid (HOCl) and chloramines are produced by the neutrophil enzyme, myeloperoxidase. Both react readily with thiols, although chloramines differ from HOCl in discriminating between low molecular weight thiols on the basis of their p K a. Here, we have compared the reactivity of HOCl and taurine chloramine with thiol proteins by examining inactivation of creatine kinase (CK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). With both enzymes, loss of activity paralleled thiol loss. For CK both were complete at a 1:1 taurine chloramine:thiol mole ratio. For GAPDH each chloramine oxidized two thiols. Three times more HOCl than taurine chloramine was required for inactivation, indicating that HOCl is less thiol specific. Competition studies showed that thiols of CK were 4 times more reactive with taurine chloramine than thiols of GAPDH (rate constants of 1200 and 300 M −1s −1 respectively). These compare with 205 M −1s −1 for cysteine and are consistent with their lower p K a’s. Both enzymes were equally susceptible to HOCl. GSH competed directly with the enzyme thiols for taurine chloramine and protected against oxidative inactivation. At lower GSH concentrations, mixed disulfides were formed. We propose that chloramines should preferentially attack proteins with low p K a thiols and this could be important in regulatory processes.

The effects of the interactions between amino acids on pyruvate kinase activity from the brain cortex of young rats

Considering that pyruvate kinase activity, a crucial enzyme for glucose metabolism and energy liberation in brain, may be regulated by some amino acids, it is possible that diminution of this enzyme activity may contribute to the brain damage caused by amino acids accumulated in metabolic diseases, such as phenylalanine, tryptophan and cystine. Therefore, the present study was undertaken to investigate the effect of these amino acids on pyruvate kinase activity in the brain cortex of rats. We also investigated the effect of serine and alanine on pyruvate kinase activity in the same tissue. The results suggested that phenylalanine, tryptophan, cystine, alanine, and serine act at the same site on the enzyme, phenylalanine, tryptophan, and cystine causing inhibition, and alanine and serine preventing this effect. Cystine also inhibited the enzyme activity through a different mechanism, possibly acting on the enzyme thiol groups. Considering that this enzyme is a target for amino acids accumulated in some metabolic diseases of amino acid metabolism, it is possible that its inhibition may contribute to the brain damage found in these diseases.

Chemical modification of intracellular cytosine deaminase fromChromobacterium violaceum YK 391

Cytosine deaminase (cytosine aminohydrolase, EC 3.5.4.1) stoichiometrically catalyzes the hydrolytic deamination of cytosine and 5-fluorocytosine to uracil and 5-fluorouracil, respectively. Amino acid residues located in or near the active sites of the intracellular cytosine deaminase fromChromobacterium violaceum YK 391 were identified by chemical modification studies. The enzymic activity was completely inhibited by chemical modifiers, such as 1 mM NBS, chloramine-T, ρ-CMB, ρ-HMB and iodine, and was strongly inhibited by 1 mM PMSF and pyridoxal 5′-phosphate. This chemical deactivation of the enzymic activity was reversed by a high concentration of cytosine. Furthermore, the deactivation of the enzymic activity by ρ-CMB was also reversed by 1 mM cysteine-HCl, DTT and 2-mercaptoethanol. These results suggested that cysteine, tryptophan and methionine residues might be located in or near the active sites of the enzyme, while serine and lysine were indirectly involved in the enzymic activity. The intracellular cytosine deaminase fromC. violaceum YK 391 was assumed to be a thiol enzyme.

Oxidation of .DELTA.-ALA-D and DTT Mediated by Ascorbic Acid: Modulation by Buffers Depends on Free Iron

Ascorbic acid (AA) is one of the most important endogenous reducing agents and can participate in a variety of cellular events. In vitro, AA can act as a potent oxidant agent in the presence of free metals, promote modifications in protein structures and form reactive oxygen species during its oxidation. We have observed that AA (above 6 mmol/l) inactivates delta-aminolevulinate dehidratase (delta-ALA-D), a sulfhydryl-containing enzyme and that the inhibitory action was considerably decreased when 3-morpholinepropanesulfonic acid buffer (MOPS - pH: 6.8; 100 mmol/l) was used in the delta-ALA-D activity assay instead of potassium phosphate buffer (PB - pH: 6.8; 100 mmol/l). delta-ALA-D inhibition, probably, is mediated by the oxidation of -SH groups caused by the auto-oxidation of AA promoted by metals or another oxidizing system present in liver supernatants. This hypothesis was confirmed by studying dithiothreitol (DTT - 400 micromol/l) oxidation, as a model of enzyme thiols, where we observed that the mechanism underlying DTT and delta-ALA-D oxidation caused by ascorbate is the same. The difference observed between different buffers may be related to the oxidation of Fe(II) to Fe(III) that was more accentuated in PB than in MOPS buffer. The presence of ethilenediamintetraacetic acid (EDTA - 100 micromol/l) and Fe(III) (5 micromol/l) stimulated DTT oxidation more in PB than in MOPS buffer. Deferroxamine (DF - 100 micromol/l) considerably decreased DTT oxidation. Catalase (0.4 mg/ml) and Superoxide dismutase (SOD - 300 U/ml) had only a modest effect on DTT oxidation. The present results suggest that delta-ALA-D inhibition by AA is mediated primarily by the oxidized form of AA and reactive oxygen species play only a modest role in the process.

Activation of transglutaminase in μ-calpain null erythrocytes

Intracellular transglutaminases (protein–glutamine: amine γ-glutamyltransferase, EC 2.3.2.13) are calcium-dependent thiol enzymes that catalyze the covalent cross-linking of proteins, including those in the erythrocyte membrane. Several studies suggest that the activation of some transglutaminases is positively regulated by the calcium-dependent cysteine protease, μ-calpain. Using μ-calpain null ( Capn1 −/−) mouse erythrocytes, we demonstrate that the activation of soluble as well as membrane-bound forms of transglutaminase (TG2) in mouse erythrocytes was independent of μ-calpain. Also, the absence of μ-calpain or any detectable cysteine protease did not affect the transglutaminase activity in the erythrocyte lysate. Our studies also identify physiological substrates of μ-calpain in the erythrocyte membrane and show that their cleavage has no discernible effect on the transglutaminase mediated cross-linking of membrane proteins. Taken together, these data suggest the existence of a calpain-independent mechanism for the activation of transglutaminase 2 by calcium ions in the mouse erythrocytes and presumably also in non-erythroid cells.