Molecular cloning, expression, purification and characterization of calcineurin from bovine cardiac muscle

Calcineurin (CaN), also known as calmodulin-dependent phosphatase, was cloned from bovine cardiac muscle and the deduced amino acid sequences of CaN A revealed that it had an open reading frame of 511 amino acid residues. As compared to bovine brain CaN A, the cardiac enzyme contains a 10 amino acid (ATVEAIEADE) deletion before the autoinhibitory region. A deletion analysis of the catalytic domain revealed a 20% decrease in phosphatase activity when the N-terminal 200 amino acids were removed from CaN A as compared to the wild type enzyme. The C-terminal deletions of CaN A revealed that in addition to the autoinhibitory domain (residues 457-480), additional adjacent residues (407-456) also inhibited CaN activity. These results point to either a second autoinhibitory region within CaN A or an extension of the previously noted autoinhibitory region within the cardiac CaN A enzyme.     

Blocking gastrin and CCK-B autocrine loop affects cell proliferation and apoptosis in vitro

Since its initial discovery as Ca²⁺/calmodulin (CaM)-dependent serine/threonine protein phosphatase, calcineurin (CaN) has been extensively studied in many mammalian tissues. CaN has been shown to be involved in various biological and Ca²⁺-dependent signal transduction pathways. Over the last decade, our laboratory has been interested and has carried out numerous experiments on this specific protein phosphatase. While, a lot of research has been performed studying CaN’s involvement in ischemia, the immune system, and various mammalian tissues, not much is known about the potential role of CaN in various eye diseases. This review focuses on the studies that have been carried out in our laboratory on CaN, and specifically CaN’s involvement in the eye. We demonstrated that CaN is localized in various eye tissues (cornea, iris, ciliary body, vitreous body, retina, choroid, sclera, and optic nerve) and that both its protein expression and activity were observed in high amounts in the retina, optic nerve and cornea. Recently, we have cloned and characterized the CaN A and B subunits in the bovine retina. These initial findings suggest that CaN may play a potential role in visual transduction and various ocular diseases, including cancer.     

Divalent cation effects on calcineurin phosphatase: differential involvement of hydrophobic and metal binding domains in the regulation of the enzyme activity

Summary The effects of divalent metals, metal chelators (EDTA, EGTA) and sodium dodecyl sulfate were investigated on the phosphatase activity of isolated bovine brain calcineurin assayed in the absence (called intrinsic) and presence of calmodulin. Intrinsic phosphatase was increased by Mn²⁺, was unaffected by Mg²⁺, Ca^2–, and Ba⁺, and was markedly inhibited by Ni^2–, Fe²⁺, Zn²⁺ and Cu^2–. When assayed in the presence of calmodulin, many divalent metals (Ni^2–, Zn²⁺, Pb²⁺, Cd²⁺), besides Mn²⁺, increased modestly the phosphatase activity at low concentrations (10–100 M) and inhibited it markedly at high concentrations. Ca^2–-calmodulin stimulated phosphatase activity was antagonized by Ni²⁺, Zn²⁺, Fe²⁺, Cu²⁺, Pb²⁺, at low concentrations (50 M), and by Ba²⁺, Cd²⁺ at slightly higher concentrations (> 100 M); Mn²⁺ and Co^2– (50 M to 1 mM) in fact augmented it. EDTA and EGTA in a concentration and time dependent fashion inhibited the intrinsic phosphatase activity, particularly that of trypsinized calcineurin. SDS in low concentrations (0.005%) augmented the phosphatase activity and inhibited it at high concentrations. Mn²⁺ (± calmodulin) and Ca²⁺ only with calmodulin present increased the phosphatase activity assayed with low concentrations of SDS. The EDTA dependent inhibition of intrinsic phosphatase was almost abolished in assays containing SDS. Prior exposure of calcineurin to Mn²⁺ led to a high activity conformation state of calcineurin that was long-lived or pseudo-irreversible. Such Mn²⁺-activated state of calcineurin exhibited no discerbible change in the affinity towards myelin basic protein or its inhibition by trifluoperazine. At alkaline pH, Mg²⁺ supported the intrinsic phosphatase activity, although to a lesser degree than Mn²⁺. The latter cation, compared to Mg²⁺ and Ni²⁺, was also a more powerful stimulator of the calcineurin phosphatase assayed with histone (III-S) and myosin light chain as substrates.     

La3+ stimulate the activity of calcineurin in two different ways

It is well known that the activity of calcineurin (CaN) could be modulated by several transitional metal ions. In the present work, the effects of a calcium analog, lanthanum ion (La³⁺), on the activity of CaN were studied. It was found that La³⁺ exerted multiple effects on CaN activity. La³⁺ could stimulate CaN in the absence of calmodulin (CaM); whereas at low concentrations of La³⁺, there was a slight inhibition of activation of CaN in the presence of CaM. Competitive experiments and limited trypsin proteolysis confirmed that La³⁺ did not act on the catalytic core of CaN, but exerted its effect through direct action on the CaN regulatory domain similar to Mg²⁺. In activity titration and spot blotting studies, La³⁺-containing CaM complexes were less effective in stimulating CaN than Ca²⁺ or Mn²⁺-containing CaM; however, the binding affinity of these metal–CaM complexes to CaN was similar. These effects of La³⁺ on CaN activity are unique among metal ions and may provide clues to understand the biological effects of La³⁺.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N,and <Superscript>13</Superscript>C chemical shift assignments of the regulatory domain of human calcineurin

Calcineurin (CaN) plays an important role in T-cell activation, cardiac system development and nervous system function. Previous studies have demonstrated that the regulatory domain (RD) of CaN binds calmodulin (CaM) towards the N-terminal end. Calcium-loaded CaM activates the serine/threonine phosphatase activity of CaN by binding to the RD, although the mechanistic details of this interaction remain unclear. It is thought that CaM binding at the RD displaces the auto-inhibitory domain (AID) from the active site of CaN, activating phosphatase activity. In the absence of calcium-loaded CaM, the RD is disordered, and binding of CaM induces folding in the RD. In order to provide mechanistic detail about the CaM–CaN interaction, we have undertaken an NMR study of the RD of CaN. Complete ¹³C, ¹⁵N and ¹H assignments of the RD of CaN were obtained using solution NMR spectroscopy. The backbone of RD has been assigned using a combination of ¹³C-detected CON-IPAP experiments as well as traditional HNCO, HNCA, HNCOCA and HNCACB-based 3D NMR spectroscopy. A ¹⁵N-resolved TOCSY experiment has been used to assign Hα and Hβ chemical shifts.     

Endonuclease Activity of MutL Protein of the <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> Mismatch Repair System

We have purified the MutL protein from Rhodobacter sphaeroides mismatch repair system (rsMutL) for the first time. rsMutL demonstrated endonuclease activity in vitro, as predicted by bioinformatics analysis. Based on the alignment of 1483 sequences of bacterial MutL homologs with presumed endonuclease activity, conserved functional motifs and amino acid residues in the rsMutL sequence were identified: five motifs comprising the catalytic site responsible for DNA cleavage were found in the C–terminal domain; seven conserved motifs involved in ATP binding and hydrolysis and specific to the GHKL family of ATPases were found in the N–terminal domain. rsMutL demonstrated the highest activity in the presence of Mn²⁺. The extent of plasmid DNA hydrolysis declined in the row Mn²⁺ > Co²⁺ > Mg²⁺ > Cd²⁺; Ni²⁺ and Ca²⁺ did not activate rsMutL. Divalent zinc ions inhibited rsMutL endonuclease activity in the presence of Mn²⁺ excess. ATP also suppressed plasmid DNA hydrolysis by rsMutL. Analysis of amino acid sequences and biochemical properties of five studied bacterial MutL homologs with endonuclease activity revealed that rsMutL resembles the MutL proteins from Neisseria gonorrhoeae and Pseudomonas aeruginosa.     

Derivatives of the purple phosphatase from red kidney bean: Replacement of zinc with other divalent metal ions

Apoenzyme, containing ⩽0.1 zinc atoms and ⩽0.2 Fe atoms per subunit and with ⩽3% of the phosphatase activity, has been prepared from native red kidney bean purple phosphatase. Treatment of this apoenzyme with Fe³⁺ or Zn²⁺ separately gave very little recovery of activity, whereas treatment with both Fe³⁺ and Zn²⁺ resulted in complete restoration of activity, indicating that both metal ions are essential. ZnFe enzyme with close to one iron and one zinc atom per subunit has been reconstituted by this procedure. Essentially full reactivation was also achieved by addition of Fe³⁺ together with Fe²⁺ or Co²⁺ to the apoenzyme; Fe³⁺ and Cd²⁺ gave 27% restoration of activity, whereas Fe³⁺ with Mn²⁺, Cu²⁺, Ni²⁺ or Hg²⁺ gave little or no increase in activity. Kinetic parameters for the hydrolysis of p-nitrophenyl phosphate and ATP by the FeFe derivative are reported.     

The catalytically active domain in the A subunit of calcineurin

Calcineurin (CaN) is a heterodimer composed of a catalytic subunit A (CaNA) and a regulatory subunit B (CaNB). We report here an active truncated mutation of the rat CaNAdelta that contains only the catalytic domain (residues 1-347, also known as a/CaNA). The p-nitrophenyl phosphatase activity and protein phosphatase activity of a/CaNA were higher than that of CaNA. Both p-nitrophenyl phosphatase activity and protein phosphatase activity of a/CaNA were unaffected by CaM and the B-subunit; the B-subunit and CaM have relatively little effect on p-nitrophenyl phosphatase activity and a crucial effect on protein phosphatase activity of CaNA. Mn2+ and Ni2+ ions effeciently activated CaNA. The Km of a/CaNA was about 16 mM, and the k(cat) of a/CaNA was 10.03 s(-1) using pNPP as substrate. With RII peptide as a substrate, the Km of a/CaNA was about 21 microM and the k(cat) of a/CaNA was 0.51 s(-1). The optimum reaction temperature was about 45 degrees C, and the optimum reaction pH was about 7.2. Our results indicate that a/CaNA is the catalytic core of CaNA, and CaN and the B-subunit binding domain itself might play roles in the negative regulation of the phosphatase activity of CaN. The results provide the basis for future studies on the catalytic domain of CaN.     

Inhibition of excitatory neuronal cell death by cell-permeable calcineurin autoinhibitory peptide

In glutamate-mediated excitatory neuronal cell death, immunosuppressants (FK506, Cys-A) are powerful agents that protect neurons from apoptosis. Immunosuppressants inhibit two types of enzyme, calcium/calmodulin-dependent protein phosphatase (calcineurin: CaN), and peptidyl-prolyl cis-trans-isomerase (PPIase) activity such as the FKBP family. In this study, we used a protein transduction approach to determine the functional role of CaN and to produce a potential therapeutic agent for glutamate-mediated neuronal cell death. We created a novel cell-permeable CaN autoinhibitory peptide using the 11 arginine protein transduction domain. This peptide was highly efficient at transducing into primary culture neurons, potently inhibited CaN phosphatase activities, and inhibited glutamate-mediated neuronal cell death. These results showed that CaN plays an important role in excitatory neuronal cell death and cell-permeable CaN autoinhibitory peptide could be a new drug to protect neurons from excitatory neuronal death.     

Role of Calcineurin-Mediated Dephosphorylation in Modulation of an Inwardly Rectifying K+ Channel in Human Proximal Tubule Cells

Activity of an inwardly rectifying K⁺ channel with inward conductance of about 40 pS in cultured human renal proximal tubule epithelial cells (RPTECs) is regulated at least in part by protein phosphorylation and dephosphorylation. In this study, we examined involvement of calcineurin (CaN), a Ca²⁺/calmodulin (CaM)–dependent phosphatase, in modulating K⁺ channel activity. In cell-attached mode of the patch-clamp technique, application of a CaN inhibitor, cyclosporin A (CsA, 5 μM) or FK520 (5 μM), significantly suppressed channel activity. Intracellular Ca²⁺ concentration ([Ca²⁺] _i ) estimated by fura-2 imaging was elevated by these inhibitors. Since inhibition of CaN attenuates some dephosphorylation with increase in [Ca²⁺] _i , we speculated that inhibiting CaN enhances Ca²⁺-dependent phosphorylation, which might result in channel suppression. To verify this hypothesis, we examined effects of inhibitors of PKC and Ca²⁺/CaM-dependent protein kinase-II (CaMKII) on CsA-induced channel suppression. Although the PKC inhibitor GF109203X (500 nM) did not influence the CsA-induced channel suppression, the CaMKII inhibitor KN62 (20 μM) prevented channel suppression, suggesting that the channel suppression resulted from CaMKII-dependent processes. Indeed, Western blot analysis showed that CsA increased phospho-CaMKII (Thr286), an activated CaMKII in inside–out patches, application of CaM (0.6 μM) and CaMKII (0.15 U/ml) to the bath at 10^?6 M Ca²⁺ significantly suppressed channel activity, which was reactivated by subsequent application of CaN (800 U/ml). These results suggest that CaN plays an important role in supporting K⁺ channel activity in RPTECs by preventing CaMKII-dependent phosphorylation.     

Regulation of calcineurin phosphatase activity by its autoinhibitory domain

The Ca(2+)-dependent activation of calcineurin phosphatase activity is regulated by an autoinhibitory element (residues 457-482) located 43 residues COOH-terminal of the calmodulin-binding domain (residues 390-414). Removal of residues 457-482 does not result in full Ca(2+)/calmodulin-independent activity. Full activity in the absence of Ca(2+) requires the removal of residues 420-457. In the present study the presence of additional autoinhibitory elements within residues 420-457 was tested using two calcineurin A subunit COOH-terminal region constructs containing residues 420-511 (AI(420-511)) or 328-511 (AI(328-511)). Using recombinant, Ca(2+)/calmodulin-independent calcineurin, AI(420-511) and AI(328-511) were three- to fourfold more potent inhibitors of calcineurin phosphatase activity than the synthetic calcineurin autoinhibitory peptide(457-482). Calmodulin reversed the inhibition of calcineurin phosphatase activity by AI(328-511) but not AI(420-511). Kinetic studies indicated that AI(420-511) exhibited mixed-type inhibition and that the enzyme/substrate/inhibitor complex is partially active. These results indicate that (i) additional autoinhibitory elements are present within residues 420-457, (ii) calmodulin-binding to the autoinhibitory domain neutralizes the inhibitory function of the 420-457 autoinhibitory segment, (iii) the full-length autoinhibitory domain is a mixed-type inhibitor of calcineurin phosphatase activity, and (iv) the enzyme/substrate/inhibitor complex is partially catalytically active.     

Activation of the ATP.Mg-dependent type 1 protein phosphatase by the Fe2+/ascorbate system

The ATP.Mg-dependent type 1 protein phosphatase is inactive as isolated but can be activated in several different ways. In this report, we show that the phosphatase can also be activated by the Fe²⁺/ascorbate system. Activation of the phosphatase requires both Fe²⁺ ion and ascorbate and the level of activation is dependent on the concentrations of Fe²⁺ ion and ascorbate. In the presence of 20 mM ascorbate, the Fe²⁺ ion concentrations required for half-maximal and maximal activation are about 0.3 and 3mM, respectively. Several common divalent metal ions, including Co²⁺, Ni²⁺, Cu²⁺, Mg²⁺, and Ca²⁺ ions, cannot cooperate with ascorbate to activate the phosphatase, and SH-containing reducing agents such as 2-mercaptoethanol and dithiothreitol cannot cooperate with Fe²⁺ ion to activate the phosphatase, indicating that activation of the phosphatase by the Fe²⁺/ascorbate system is a specific process. Moreover, H₂O₂, a strong oxidizer, could significantly diminish the phosphatase activation by the Fe²⁺/ascorbate system, suggesting that reduction mechanism other than SH-SS interchange is a prerequisite for the Fe²⁺/ascorbate-mediated phosphatase activation. Taken together, the present study provides initial evidence for a new mode of type 1 protein phosphatase activation mechanism.     

In vitro Proteolytic Degradation of Bovine Brain Calcineurin by m-Calpain

A major cause of neuronal dysfunction is due to altered Ca2+ regulation. An increase in Ca2+ influx can activate Ca2+-dependent enzymes including calpains, causing the proteolysis of its specific substrates. In the present study, calcineurin (CaN) was found to be proteolysed by a Ca2+-dependent cysteine protease, m-calpain. In the presence of Ca2+, the 60 kDa subunit (CaN A) was degraded to a 46 kDa immunoreactive fragment, whereas in the presence of Ca2+ /calmodulin (CaM) immunoreactive fragments of 48 and 54 kDa were observed. The beta-subunit (CaN B) was not proteolysed in either condition. The proteolysis of CaN A increased its phosphatase activity and rendered it totally CaM-independent after 10 min of proteolysis. The molecular weight of the proteolytic fragments suggested that the m-calpain cleaved CaN A in the CaN B binding domain. A CaM-overlay experiment revealed that the CaM-binding site was present only in the 54 kDa fragment produced by CaN A proteolysis in the presence of Ca2+ /CaM. Thus, the increase in CaN A phosphatase activity observed in many neuronal disorders, may be due to the action of calpain.     

Activation of the ATP.Mg-dependent type 1 protein phosphatase by the Fe2+/ascorbate system

The ATP.Mg-dependent type 1 protein phosphatase is inactive as isolated but can be activated in several different ways. In this report, we show that the phosphatase can also be activated by the Fe²⁺/ascorbate system. Activation of the phosphatase requires both Fe²⁺ ion and ascorbate and the level of activation is dependent on the concentrations of Fe²⁺ ion and ascorbate. In the presence of 20 mM ascorbate, the Fe²⁺ ion concentrations required for half-maximal and maximal activation are about 0.3 and 3mM, respectively. Several common divalent metal ions, including Co²⁺, Ni²⁺, Cu²⁺, Mg²⁺, and Ca²⁺ ions, cannot cooperate with ascorbate to activate the phosphatase, and SH-containing reducing agents such as 2-mercaptoethanol and dithiothreitol cannot cooperate with Fe²⁺ ion to activate the phosphatase, indicating that activation of the phosphatase by the Fe²⁺/ascorbate system is a specific process. Moreover, H₂O₂, a strong oxidizer, could significantly diminish the phosphatase activation by the Fe²⁺/ascorbate system, suggesting that reduction mechanism other than SH-SS interchange is a prerequisite for the Fe²⁺/ascorbate-mediated phosphatase activation. Taken together, the present study provides initial evidence for a new mode of type 1 protein phosphatase activation mechanism.Abbreviations MAPK mitogen-activated protein kinase - MCO metal ion-catalyzed oxidation - kinase F_A the activating factor of ATP.Mg-dependent protein phosphatase - I₂ inhibitor-2 - EDTA ethylenediaminetetraacetic acid - MBP myelin basic protein     

Identification of an autoinhibitory domain in the insulin receptor tyrosine kinase

We have tested the hypothesis that activation of the insulin receptor tyrosine kinase is due to autophosphorylation of tyrosines 1146, 1150 and 1151 within a putative autoinhibitory domain. A synthetic peptide corresponding to residues 1134–1162, with tyrosines substituted by alanine or phenylalanine, of the insulin receptor subunit was tested for its inhibitory potency and specificity towards the tyrosine kinase activity. This synthetic peptide gave inhibition of the insulin receptor tyrosine kinase autophosphorylation and phosphorylation of the exogenous substrate poly(Glu, Tyr) with an approximate IC₅₀ of 100 M. Inhibition appeared to be independent of the concentrations of insulin or the substrate poly(Glu, Tyr) but was decreased by increasing concentrations of ATP. This same peptide also inhibited the EGF receptor tyrosine kinase but not a serine/threonine protein kinase. These results are consistent with the hypothesis that this autophosphorylation domain contains an autoinhibitory sequence. (Mol Cell Biochem120: 103–110, 1993)Abbreviations IR Insulin Receptor - SDS/PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis - CaM Calmodulin - HEPES 4-(2-Hydroxyethyl)-Piperazineethane-Sulfonic Acid - DMEM Dulbecco's Modified Eagle' Medium - PMSF Phenylmethyl-Sulfonyl Fluoride - HPLC High Performance Liquid Chromatography - PKC Protein Kinase C - PKI Inhibitory Peptide for cAMP-Kinase - CaMK II Ca²⁺/Calmodulin-Dependent Protein Kinase II - CaN A A Subunit of Calcineurin     

The low-molecular-weight acid phosphatase from bovine liver: Isolation,amino acid composition,and chemical modification studies

The smallest of the three molecular weight forms of acid phosphatase from bovine liver was purified to a specific activity of 100 μmol min^?1 mg^?1 (measured at pH 5.5 and 37 °C with p-nitrophenyl phosphate). Using several chromatographie and electrophoretic methods, no evidence of heterogeneity was detected. The enzyme was characterized with respect to its stability as a function of pH, molecular weight, amino acid composition, steady-state kinetic parameters in the pH range 4–7 and inhibition by common acid phosphatase inhibitors at pH 5.5. The amino acid composition differed somewhat from a previous literature report. The enzyme was stoichiometrically inactivated upon incubation with Hg²⁺, Ag⁺, and iodoacetate. Inactivation also occurred upon photoinactivation in the presence of Rose Bengal but no inactivation occurred with diethyl pyrocarbonate. The alkylation of one of five cysteine residues by iodoacetate was shown to cause complete inactivation of the enzyme. This alkylation was prevented by the presence of phosphate ion. A tryptic dipeptide containing this essential cysteine was isolated following inactivation with iodo[2-¹⁴C]acetate.     

C-Terminal Membrane Association of Bestrophin 3 and Its Activation as a Chloride Channel

Bestrophin 3 (Best3), a member of the bestrophin Cl^? channel family, is a candidate of cGMP-sensitive, Ca²⁺-activated Cl^? channel in vascular smooth muscle cells. The Best3 channel was recently found to play an important role in vasomotion. However, the mechanism for its activation has not been clarified. In previous studies, we found that a Best3 C-terminal sequence (amino acids 353–404) was associated with the cellular membrane. The sequence includes an autoinhibitory domain (³⁵⁶IPSFLGS³⁶²) and a downstream basic residue domain (amino acids 384–397). In this study, we found that the sequence (368–383) between the two domains is actually a determinant for Best3 C-terminal membrane associability. Deletion of the sequence almost abolished the membrane association but did not activate the Best3 channel. Treatment of Best3-expressing HEK293 cells with the PI3Kα inhibitor IV (a Best3 activator) could not abolish but weakened the Best3 membrane association. The result supports the assumption that the positively charged basic residues in the Best3 C terminus are likely associated with the membranous negatively charged phospholipids, which plays a role in the regulation of Best3 activation. But the relationship between membrane associability and Best3 activation seems more complicated than expected.     

A Bacterial Iron Exporter for Maintenance of Iron Homeostasis

Nutritional iron acquisition by bacteria is well described, but almost nothing is known about bacterial iron export even though it is likely to be an important homeostatic mechanism. Here, we show that Bradyrhizobium japonicum MbfA (Blr7895) is an inner membrane protein expressed in cells specifically under high iron conditions. MbfA contains an N-terminal ferritin-like domain (FLD) and a C-terminal domain homologous to the eukaryotic vacuolar membrane Fe²⁺/Mn²⁺ transporter CCC1. An mbfA deletion mutant is severely defective in iron export activity, contains >2-fold more intracellular iron than the parent strain, and displays an aberrant iron-dependent gene expression phenotype. B. japonicum is highly resistant to iron and H₂O₂ stresses, and MbfA contributes substantially to this as determined by phenotypes of the mbfA mutant strain. The N-terminal FLD was localized to the cytoplasmic side of the inner membrane. Substitution mutations in the putative iron-binding amino acid residues E20A and E107A within the N-terminal FLD abrogate iron export activity and stress response function. Purified soluble FLD oxidizes ferrous iron (Fe²⁺) to incorporate ferric iron (Fe³⁺) in a 2:1 iron:protein ratio, which does not occur in the E20A/E107A mutant. The FLD fragment is a dimer in solution, implying that the MbfA exporter functions as a dimer. MbfA belongs to a protein family found in numerous prokaryotic genera. The findings strongly suggest that iron export plays an important role in bacterial iron homeostasis.     

Mouse aminoacylase 3: A metalloenzyme activated by cobalt and nickel

Aminoacylase 3 (AA3) deacetylates N-acetyl-aromatic amino acids and mercapturic acids including N-acetyl-1,2-dichlorovinyl-L-cysteine (Ac-DCVC), a metabolite of a xenobiotic trichloroethylene. Previous studies did not demonstrate metal-dependence of AA3 despite a high homology with a Zn²⁺-metalloenzyme aminoacylase 2 (AA2). A 3D model of mouse AA3 was created based on homology with AA2. The model showed a putative metal binding site formed by His21, Glu24 and His116, and Arg63, Asp68, Asn70, Arg71, Glu177 and Tyr287 potentially involved in catalysis/substrate binding. The mutation of each of these residues to alanine inactivated AA3 except Asn70 and Arg71, therefore the corrected 3D model of mouse AA3 was created. Wild type (wt) mouse AA3 expressed in E. coli contained ∼ 0.35 zinc atoms per monomer. Incubation with Co²⁺ and Ni²⁺ activated wt-AA3. In the cobalt-activated AA3 zinc was replaced with cobalt. Metal removal completely inactivated wt-AA3, whereas addition of Zn²⁺, Mn²⁺ or Fe²⁺ restored initial activity. Co²⁺ and to a lesser extent Ni²⁺ increased activity several times in comparison with intact wt-AA3. Co²⁺ drastically increased the rate of deacetylation of Ac-DCVC and significantly increased the toxicity of Ac-DCVC in the HEK293T cells expressing wt-AA3. The results indicate that AA3 is a metalloenzyme significantly activated by Co²⁺ and Ni²⁺.     

Purification and Characterization of a Fibrinolytic Protease from a Culture Supernatant of Flammulina velutipes Mycelia

In this study we purified a fibrinolytic enzyme from the culture supernatant of Flammulina velutipes mycelia by ion exchange and gel filtration chromatographies, it was designated as F. velutipes protease (FVP-I). This purification protocol resulted in 18.52-fold purification of the enzyme at a final yield of 0.69%. The molecular mass of the purified enzyme was estimated to be 37 kDa by SDS–PAGE, fibrin-zymography and size exclusion by FPLC. This protease effectively hydrolyzed fibrin, preferentially digesting α-chain over β-and γ–γ chain. Optimal protease activity was found to occur at a pH of 6.0 and a temperature of 20 to 30 °C. The protease activity was inhibited by Cu²⁺, Fe²⁺ and Fe³⁺ ions, but was found to be enhanced by Mn²⁺ and Mg²⁺ ions. Furthermore, FVP-I activity was potently inhibited by EDTA and EGTA, and it was found to exhibit a higher specificity for chromogenic substrate S-2586 for chymotrypsin, indicating that the enzyme is a chymotrypsin-like metalloprotease. The first 20 amino acid residues of the N-terminal sequence of FVP-I were LTYRVIPITKQAVTEGTELL. They had a high degree of homology with hypothetical protein CC1G_11771, GeneBank Accession no. EAU86463.