Quantitation of tissue calpain activity after isolation by hydrophobic chromatography

A rapid and reliable method for quantitating tissue calpains (Ca2+-activated, neutral, thiol proteases) was developed using hydrophobic chromatography with phenyl-Sepharose. Calpains I and II isolated by this method are free of endogenous inhibitor(s) (calpastatin), activator(s), and nonspecific proteases. These calpains expose hydrophobic regions in the presence of Ca2+ and bind tightly to phenyl-Sepharose. Inactivation of bound calpain is prevented by the addition of leupeptin (20 microM). Calpains I and II bound initially by phenyl-Sepharose in a Ca2+-dependent manner are then eluted successively on the basis of their Ca2+-independent binding to phenyl-Sepharose. Because calpastatin may prevent binding of calpain to phenyl-Sepharose by forming a protease-inhibitor complex in the presence of Ca2+, preadsorbing the protease to a suspension of phenyl-Sepharose beads initially in the absence of Ca2+ separates most of the calpain present in tissue extracts from calpastatin. The isolated calpains obtained are assayed by casein digestion. This quantitation procedure is suitable for measuring calpain activity in various tissues and cells including erythrocytes.     

Comparative studies on heat stability and autolysis of scallop (Patinopecten yessoensis) calpain II-like proteinase and rabbit calpain II.

1. The scallop calpain-like proteinase is about five times more labile than the rabbit calpain II upon heat treatment at 35 degrees C. 2. By autolysis of the scallop proteinase of two 100 kDa subunits, 90, 45 and 30 kDa fragments were formed. Thereby the activity decreased monophasically in the presence of millimolar order of Ca2+, but did not increase in the presence of micromolar order of Ca2+ unlike the rabbit calpain II.     

Purification and characterization of a calpain activator from human platelets.

A calpain (Ca(2+)-activated neutral protease) activator was purified from human platelets by ammonium sulfate fractionation, gel-filtration, ion-exchange chromatography, followed by heat-treatment. The purified calpain activator with a Mr of 47.5 kDa was a heat-stable protein as demonstrated in other cells. The calpain activator did not change the Ca2+ sensitivity of calpain but activated calpain activity about 2-fold. This calpain activator may play an important role in the activation of the protease system leading to the Ca(2+)-mediated physiological process of platelets.     

Degradation of the proto-oncogene product p39mos is not necessary for cyclin proteolysis and exit from meiotic metaphase: requirement for a Ca(2+)-calmodulin dependent event.

Exit from M phase, which requires cyclin degradation, is prevented from occurring in unfertilized eggs of vertebrates arrested at second meiotic metaphase due to a cytostatic factor recently identified as p39mos, the product of the proto-oncogene c-mos. Calpain can destroy both p39mos and cyclin in vitro in extracts prepared from metaphase-arrested Xenopus eggs, but only when free Ca2+ concentration is raised to the millimolar range. When free Ca2+ concentration is raised for only 30 s to the micromolar range, as occurs in physiological conditions after fertilization, cyclin degradation is induced, byt p39mos is not degraded. Cyclin proteolysis at micromolar free Ca2+, is not inhibited by calpastatin, and therefore does not involve calpain. A cyclin mutant modified in the destruction box is found to be resistant at micromolar, but not millimolar free Ca2+, suggesting that the ubiquitin pathway mediates cyclin degradation at micromolar Ca2+ concentration whereas calpain is involved at the millimolar level. A synthetic peptide which binds Ca(2+)-calmodulin with high affinity suppresses cyclin degradation at micromolar but not millimolar free Ca2+, and this only when it is present in the extract during the first 30 s after raising free Ca2+ concentration. The inhibition of the cyclin degradation pathway by the Ca(2+)-calmodulin binding peptide can be overcome by adding calmodulin. These results strongly suggest that a Ca(2+)-calmodulin process is required as an early event following fertilization to release the cyclin degradation pathway from inhibition in metaphase-arrested eggs. In contrast, p39mos degradation is not required.     

Comparative specificity and kinetic studies on porcine calpain I and calpain II with naturally occurring peptides and synthetic fluorogenic substrates

Homogeneous porcine calpain (Ca2+-dependent cysteine proteinase) was found to hydrolyze a variety of peptides and synthetic substrates. Leu-Trp-Met-Arg-Phe-Ala, eledoisin-related peptide, alpha-neoendorphin, angiotensin I, luteinizing hormone-releasing hormone, neurotensin, dynorphin, glucagon, and oxidized insulin B chain were cleaved with a general preference for a Tyr, Met, or Arg residue in the P1 position preceded by a Leu or Val residue in the P2 position. No great difference in specificity was found between low-Ca2+-requiring calpain I and high-Ca2+-requiring calpain II. 4-Methylcoumaryl-7-amide (MCA) derivatives having a Leu(or Val)-Met(or Tyr)-MCA or a Leu-Lys-MCA sequence were also cleaved by either calpain I or calpain II with preference for Leu over Val by a factor of 9 to 16. Calpains I and II showed similar but not identical kinetic behavior for individual substrates. The Km and kcat values ranged from 0.23 to 7.08 mM and 0.062 to 0.805 s-1 for the calpains, while kcat/Km values for the calpains were only 1/433 to 1/5 of those for papain with a given substrate. With succinyl-Leu-Met(or Tyr)-MCA, calpains I and II were half-maximally activated at 12 and 260 microM Ca2+, respectively, and competitively inhibited by leupeptin (Ki = 0.32 microM for I and 0.43 microM for II) or antipain (Ki = 1.41 microM for I and 1.45 microM for II). Thus, this is the first report describing the specificity and kinetics of calpains I and II.     

Detection and some properties of calpain II (high-Ca2+-requiring form of calpain) in carp (Cyprinus carpio) erythrocytes

In order to examine the existence of calpain I, a low (micromolar)-Ca2+-requiring form of calpain, in fish tissues, carp erythrocytes were chosen as the experimental material, since only calpain I is known to exist in mammalian erythrocytes. By DEAE-cellulose chromatography, calpain and calpastatin (specific inhibitor for calpain) were separated from carp erythrocyte hemolysate. Carp erythrocyte calpain is classified as calpain II, a high (millimolar)-Ca2+-requiring form of calpain, from the result of Ca2+-requirement for the activity.     

Purification and characterization of a Ca(2+)-activated thiol protease from Drosophila melanogaster.

A Ca(2+)-activated thiol protease was purified from Drosophila melanogaster. The procedure involves Phenyl-Sepharose, Reactive Red-Agarose and Q-Sepharose fast flow (or MonoQ) chromatographic steps. The enzyme eluting from Q-Sepharose fast flow seems to be homogeneous as judged by silver staining on SDS-PAGE: it consists of a single polypeptide chain of M(r),app = 94K and pI = 5.46. The proteolytic activity of the purified enzyme is absolutely Ca(2+)-dependent, characterized by 0.6 mM free Ca2+ at half-maximal activity. Ca2+ ions cannot be replaced effectively by the divalent cations Mg2+, Mn2+, Zn2+, Ba2+, and Cd2+. The enzyme shows the inhibitor pattern of thiol proteases. Human recombinant calpastatin (domain I) completely inhibits the enzyme at a nearly 1:1 molar ratio. Several of these properties resemble those of vertebrate calpain II. However, various attempts to detect a small subunit of M(r) approximately 30K, common with vertebrate calpains, remained unsuccessful. We suggest that the Drosophila enzyme is a novel calpain II-like protease.     

Elucidation of calpain dependent phosphorylation of myosin light chain in human platelets

Newly synthetized calpain inhibitors (CI-I approximately III) were used to prove potential participation of calpain in protein phosphorylation. CIs were about 1,000 times more potent against platelet calpain I than N-ethyl-maleimide (NEM) and an epoxy succinate derivative (E-64). CI-II inhibited 20K (myosin light chain) and 47K phosphorylation of Ca2+-stimulated lysed platelets as well as protein degradation (actin binding protein, P235). Both myosin light chain kinase (MLCK) and C-kinase dependent phosphorylation of 20K were inhibited by CI-II as demonstrated in phosphopeptides mapping. Electropermeabilized platelets (EP) were employed to examine the effects of CI-II on Ca2+ mediated reactions in non-lysed platelets. Phosphorylation of 20K and 47K induced by Ca2+ addition to EP was inhibited by CI-II, though secretory response was not modified. Only MLCK dependent phosphorylation of 20K was observed in Ca2+-activated EP, which was inhibited by CI-II. Collectively, the data indicated that calpain may activate both MLCK and C-kinase to phosphorylate 20K by partial proteolysis.     

Comparison of calpain I and calpain II from carp muscle

1. The content of calpain II is 3.4 times more than that of calpain I when estimated by the elution profiles from a column of DEAE-cellulose. 2. Calpain I required 1 mM Ca2+ and calpain II required 5 mM Ca2+ to show the full activities. These data demonstrated that Ca2+-sensitivities of both calpains were lower than those of mammalian calpains, respectively. 3. The optimum caseinolytic activity was pH 7.2 for calpain I and pH 7.5 for calpain II. 4. The molecular weight of calpain I was estimated to be 110 k and that of calpain II to be 120 k by gel filtration. 5. Calpain I was much more heat-stable than calpain II around 50-60 degrees C. 6. Both calpains were sensitive to calpastatin, an endogenous inhibitor for calpain.     

Evidence for membrane-associated calpain I in human erythrocytes. Detection by an immunoelectrophoretic blotting method using monospecific antibody

Low and high Ca2+-requiring forms of Ca2+-dependent cysteine proteinase are known as calpain I and calpain II, respectively. We have obtained, for the first time, monospecific antibodies for calpain I and for calpain II. Using these antibodies and an electrophoretic blotting method, we have found that a small, but reproducible, amount of calpain I was associated with human erythrocyte membranes while the bulk of the protease was contained in the cytosol. Most of membrane-associated calpain I was extractable with 1% Triton X-100, but not with 0.1% detergent. In the presence of 0.1 mM Ca2+ and 5 mM cysteine, membrane-associated calpain I degraded the membrane protein band 4.1 preferentially and band 3 protein only slowly. The Ca2+-induced autodigestion of the membrane preparation was inhibited by leupeptin but not by a cytosolic calpain inhibitor, calpastatin, added to the incubation medium. No calpain II was detected in either erythrocyte cytosol or membranes when anti-calpain II antibody was used under the same conditions as those for the detection of calpain I.     

Isovalerylcarnitine is a specific activator of the high calcium requiring calpain forms

Isovalerylcarnitine, a product of the catabolism of L-leucine, is a potent activator of rat calpains isolated from erythrocytes, kidney, liver, skeletal and heart muscle. Only calpains II, but not calpains I, are activated by IVC, with the only exception of rat erythrocyte calpain I, the only species present in these cells which has a Ca2+ requirement higher than that of most calpain I isoenzymes. Activation by IVC involves a dual effect: 1) a ten fold increase in the affinity of calpain for Ca2+, and 2) an increase in the Vmax 1.3-1.6 fold above the values observed with the native enzymes at saturating [Ca2+] as well as with the autolyzed fully active calpain form at 5 microM Ca2+. The increased affinity for calcium results in an increased rate of autoproteolysis of calpain II. Activation by IVC is additive to that promoted by interaction (or association) to phospholipids vesicles. Together these results suggest that IVC may operate as a selective activator of calpain both in the cytosol and at the membrane level; in the latter case in synergism with the activation induced by association of the proteinase to the cell membrane.     

Truncations of amphiphysin I by calpain inhibit vesicle endocytosis during neural hyperexcitation

Under normal physiological conditions, synaptic vesicle endocytosis is regulated by phosphorylation and Ca(2+)-dependent dephosphorylation of endocytic proteins such as amphiphysin and dynamin. To investigate the regulatory mechanisms that may occur under the conditions of excessive presynaptic Ca(2+) influx observed preceding neural hyperexcitation, we examined hippocampal slices following high-potassium or high-frequency electrical stimulation (HFS). In both cases, three truncated forms of amphiphysin I resulted from cleavage by the protease calpain. In vitro, the binding of truncated amphiphysin I to dynamin I and copolymerization into rings with dynamin I were inhibited, but its interaction with liposomes was not affected. Moreover, overexpression of the truncated form of amphiphysin I inhibited endocytosis of transferrin and synaptic vesicles. Inhibiting calpain prevented HFS-induced depression of presynaptic transmission. Finally, calpain-dependent amphiphysin I cleavage attenuated kainate-induced seizures. These results suggest that calpain-dependent cleavage of amphiphysin I inhibits synaptic vesicle endocytosis during neural hyperexcitation and demonstrate a novel post-translational regulation of endocytosis.     

Peptide-biphenyl hybrids as calpain inhibitors

Calpain is a cysteine protease that is activated by Ca2+. The over-activation of calpain, which occurs on increasing Ca2+ concentration, causes a variety of diseases. This paper reports experimental results on the inhibition of calpain I (mu-calpain) by peptide-biphenyl hybrids. We have found that some peptide-biphenyl hybrids, with aromatic amino acids in the peptide chains, inhibit calpain with IC50 values in the nanomolar range. Since the peptide-biphenyl hybrids reported in the present paper do not possess a reactive electrophilic functionality, we hypothesize that they interfere with the activation of calpain by Ca2+, and present experimental and computational results on the binding of peptide-biphenyl hybrids to Ca2+.     

Ca2+-protease--an enzyme of the metabolism of cytoskeletal proteins in the olfactory membrane of vertebrates

Two forms of Ca(++)-activated protease (calpain I and calpain II) associated with an endogenous inhibitor (calpastatin) were detected in a cytosolic fraction of the olfactory tissue of vertebrates (pig, rat). Using ion exchange chromatography on DEAE-cellulose column, calpain I is divided into 2 peaks (eluting by 0.07-0.15 and 0.22-0.25 M NaCl), and calpain II is eluted by 0.35-0.40 M NaCl. The calpain activity was detected in fractions eluted by 0.1-0.17 M NaCl. The Ca(++)-activated protease was demonstrated also in a fraction of cytoskeleton of olfactory tissue insoluble in a 1% solution of Triton X-100. The activity can be detected by Ca(++)-dependent destruction of exogenous substrate (casein), and by Ca(++)-dependent degradation of cytoskeletal endogenous proteins (16, 18 and 20 kDa), of which one may be calmodulin.     

Binding of calpain fragments to calpastatin

Their cDNA-derived amino acid sequences predict that the 80-kDa subunits of the micromolar and millimolar Ca(2+)-requiring forms of the Ca(2+)-dependent proteinase (mu- and m-calpain, respectively) each consist of four domains and that the 28-kDa subunit common to both mu- and m-calpain consists of two domains. The calpains were allowed to autolyze to completion, and the autolysis products were separated and were characterized by using gel permeation chromatography, calpastatin affinity chromatography, and sequence analysis. Three major fragments were obtained after autolysis of either calpain. The largest fragment (34 kDa for mu-calpain, 35 kDa for m-calpain) contains all of domain II, the catalytic domain, plus part of domain I of the 80-kDa subunit of mu- or m-calpain. This fragment does not bind to calpastatin, a competitive inhibitor of the calpains, and has no proteolytic activity in either the absence or presence of Ca2+. The second major fragment (21 kDa for mu-calpain and 22 kDa for m-calpain) contains domain IV, the calmodulin-like domain, plus approximately 50 amino acids from domain III of the 80-kDa subunit of mu- or m-calpain. The third major fragment (18 kDa) contains domain VI, the calmodulin-like domain of the 28-kDa subunit. The second and third major fragments bind to a calpastatin affinity column in the presence of Ca2+ and are eluted with EDTA. The second and third fragments are noncovalently bound, so the 80- and 28-kDa subunits of the intact calpain molecules are noncovalently bound at domains IV and VI. After separation in 1 M NaSCN, the 28-kDa subunit binds completely to calpastatin, approximately 30-40% of the 80-kDa subunit of mu-calpain binds to calpastatin, and the 80-kDa subunit of m-calpain does not bind to calpastatin in the presence of 1 mM Ca2+.     

Intracellular localization of two distinct Ca2+-proteases (calpain I and calpain II) as demonstrated by using discriminative antibodies

Intracellular localization of two molecular species of calpain (Ca2+-dependent cysteine proteinase) was studied by immunocyto- and histochemical methods employing antibodies strictly monospecific for the respective antigens. Apparent immunological cross-reactivity between the larger subunits of calpain I (low Ca2+-requiring form) and calpain II (high Ca2+-requiring form) was calculated to be 15-17%, and two steps of affinity chromatography were needed to obtain antibodies which can discriminate between the two proteases. Indirect immunofluorescent staining of cultured PK 15 cells revealed diffuse staining of the cytoplasm with both antibodies against calpain I and calpain II. Preincubation with Ca2+-ionophore had no effect on the staining patterns. Sections of porcine kidney were stained by the avidin-biotinylated peroxidase complex method. The proximal and distal tubules and collecting duct were stained, but the glomerulus, macula densa, and vascular vessels were not stained by either anti-calpain I or anti-calpain II antibodies.     

SNARE protein degradation upon platelet activation: calpain cleaves SNAP-23

In order to better understand the molecular mechanisms of platelet granule secretion, we evaluated the effect of activation-induced degranulation on three functional platelet SNARE proteins, SNAP-23, VAMP-3, and syntaxin 4. Initial studies showed that SNAP-23 is lost upon SFLLRN-induced platelet activation. Experiments with permeabilized platelets demonstrated that proteolysis of SNAP-23 was Ca(2+)-dependent. Ca(2+)-dependent proteolysis of SNAP-23 was inhibited by the cell-permeable calpain inhibitors, calpeptin and E-64d, as well as by the naturally occurring calpain inhibitor, calpastatin. In addition, purified calpain cleaved SNAP-23 in permeabilized platelets in a dose-dependent manner. In intact platelets, calpeptin prevented SFLLRN-induced degradation of SNAP-23. In contrast, calpeptin did not prevent SFLLRN-induced degradation of VAMP-3 and syntaxin 4 did not undergo substantial proteolysis following platelet activation. Calpain-induced cleavage of SNAP-23 was a late event occurring between 2.5 and 5 min following exposure of permeabilized platelets to Ca(2+). Experiments evaluating platelet alpha-granule secretion demonstrated that incubation of permeabilized platelets with 10 microM Ca(2+) prior to exposure to ATP inhibited ATP-dependent alpha-granule secretion from permeabilized platelets. SNAP-23 was cleaved under these conditions. Incubation of permeabilized platelets with either calpeptin or calpastatin prevented Ca(2+)-mediated degradation of SNAP-23 and reversed Ca(2+)-mediated inhibition of ATP-dependent alpha-granule secretion. Thus, activation of calpain prior to secretion results in loss of SNAP-23 and inhibits alpha-granule secretion. These studies suggest a mechanism whereby calpain activation serves to localize platelet secretion to areas of thrombus formation.     

The evaluation of the role of endogenous Ca-dependent regulators and protein kinases in activating and inhibiting ion-transport ATPases]

The data on hormonal regulation of ATP-driving ion pumps are contradictory depending on the object used: whether native cells or isolated membranes. To eliminate this contrariety, we studied the ion transporting ATPases in saponin-permeabilized cells in the presence of all endogenous regulators. In permeabilized erythrocytes we obtained the presence of Ca(2+)-dependent activation of Ca(2+)-ATPase by factor(s) not affected by calmodulin antagonist R24571. We obtained also Ca(2+)-dependent activation and inhibition of Na+,K(+)-ATPase. At a concentration of Mg(2+)-ions corresponding to the intracellular level (370 microM), the 0.5-0.7 microM Ca(2+)-activated Na+,K(+)-ATPase (up to 3-fold), whereas the 1-5 microM Ca2+ inhibited it. The cyclic AMP (10(-5) M) inhibited or eliminated Ca(2+)-dependent activation. The decrease in Mg(2+)-ion concentration to 50 microM eliminated the activation and strengthened the inhibition, which reached 100% at the 1-2 microM Ca2+ concentration. The washing of membranes with EGTA eliminated Ca2+ effects on Na+,K(+)-ATPase. These data suggest that the ion-transporting ATPases are activated or inhibited by Ca(2+)-dependent regulators whose activities may be changed by protein kinase catalysed phosphorylation.     

Ischemia-reperfusion-induced calpain activation and SERCA2a degradation are attenuated by exercise training and calpain inhibition

The Ca2+-activated protease calpain has been shown to play a deleterious role in the heart during ischemia-reperfusion (I/R). We tested the hypothesis that exercise training would minimize I/R-induced calpain activation and provide cardioprotection against I/R-induced injury. Hearts from adult male rats were isolated in a working heart preparation, and myocardial injury was induced with 25 min of global ischemia followed by 45 min of reperfusion. In sedentary control rats, I/R significantly increased calpain activity and impaired cardiac performance (cardiac work during reperfusion = 24% of baseline). Compared with sedentary animals, exercise training prevented the I/R-induced rise in calpain activity and improved cardiac work (recovery = 80% of baseline). Similar to exercise, pharmacological inhibition of calpain activity resulted in comparable cardioprotection against I/R injury (recovery = 86% of baseline). The exercise-induced protection against I/R-induced calpain activation was not due to altered myocardial protein levels of calpain or calpastatin. However, exercise training was associated with increased myocardial antioxidant enzyme activity (Mn-SOD, catalase) and a reduction in oxidative stress. Importantly, exercise training also prevented the I/R-induced degradation of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a. These findings suggest that increases in endogenous antioxidants may diminish the free radical-mediated damage and/or degradation of Ca2+ handling proteins (such as SERCA2a) typically observed after I/R. In conclusion, these results support the concept that calpain activation is an important component of I/R-induced injury and that exercise training provides cardioprotection against I/R injury, at least in part, by attenuating I/R-induced calpain activation.