Purification and immunological characterisation of two calcium-activated neutral proteinases from rabbit skeletal muscle

Two Ca2+-activated neutral proteinases have been prepared to a high degree of purity from rabbit skeletal muscle. One, calpain I, is optimally activated by 100 microM Ca2+ and the other, calpain II, by 1 to 2 mM Ca2+. Both enzymes have two subunits of molecular weight 80 000 and 28 000. Antibodies have been raised against the native forms of both enzyme. It was found that the antibody to native calpain I reacted only with calpain I and not with calpain II, and similarly the antibody to native calpain II reacted only to calpain II. This suggested that the epitopes in the two enzymes are located in regions that are structurally different. However, immunoblotting of the denatured calpains after SDS-polyacrylamide-gel electrophoresis revealed cross-reaction between the two subunits for both enzymes. Therefore, although the denatured enzymes have common antigenic sites it would appear that these are not exposed equally in the native proteins.     

Regulation of muscle phosphorylase activity by carnosine and anserine

Carnosine (β-alanyl-L-histidine) activates rabbit muscle phosphorylase $\text{a}$ in the presence and absence of AMP and phosphorylase $\text{b}$ in the presence of AMP in a biphasic manner with a maximal activation at about 50mM carnosine and with phosphorylase $\text{b}$ showing a greater degree of activation than phosphorylase $\text{a}$. Anserine (β-alanyl-L-N^π-methyl-histidine) activates phosphorylase $\text{a}$ to a lesser extent than carnosine up to a concentration of 90mM, whereas with phosphorylase $\text{b}$ a weak activation below 30mM and a concentration-dependent inhibition above this concentration occurs. These effects are specific for the dipeptides and are not shown by their constituent amino acids. Carnosine and anserine activate phosphorylase $\text{a}$ in the presence of the allosteric inhibitors ATP, D-glucose and caffeine, and the inhibition of phosphorylase $\text{b}$ by anserine is also observed in the presence of these inhibitors.     

Identification of an endogenous activator of calpain in rat skeletal muscle

An additional component of the regulatory system of rat skeletal muscle calpain has been identified. It exerts a potent activating effect on calpain activity and is a heat stable small molecular weight protein. Of the two calpain isozymes present in muscle, the activator is specific for calpain II, being uneffective with calpain I. It promotes activation of the proteinase by reducing 50 fold, from 1 mM to of 20 microM, the requirement of Ca2+ for maximum catalytic activity of the proteinase. However in the presence of the activator calpain II expresses a consistent fraction of the maximum activity even at significantly lower concentrations of Ca2+ (below 5 microM Ca2+). The activator effect follows kinetics that are consistent with the presence of specific binding sites on the calpain molecules. The activator not only removes in a dose dependent fashion the inhibition of calpain by calpastatin, but also prevents inhibition of the proteinase upon the addition of calpastatin. Competition experiments revealed that the proteinase contains distinct sites for the activator and the inhibitor, and that both ligands can bind to calpain with the formation of an almost fully active ternary complex.     

Differential effects of aluminum ion on smooth muscle calpain I and calpain II activities.

1. In millimolar Ca2+, smooth muscle calpains I and II were inhibited by aluminum ion. 2. At sub-millimolar Ca2+, calpain II, but not calpain I, was activated by low millimolar aluminum ion. 3. Calpastatin inhibited aluminum ion-activated calpain II. 4. Aluminum ion-activated and Ca(2+)-activated calpain II gave almost identical patterns of desmin cleavage. 5. Aluminum-activated calpain II, unlike the Ca(2+)-activated enzyme, did not autolyze and retained its proteolytic activity over extended periods of time.     

Identification of two calpastatin forms in rat skeletal muscle and their susceptibility to digestion by homologous calpains

Two forms of calpastatin, differing in their specificity for the homologous calpain isozymes I and II, have been separated from rat skeletal muscle extracts and purified to homogeneity. Calpastatin I, the first form to elute in chromatography on DE32, is more effective against calpain I, while calpastatin II is more effective as an inhibitor of calpain II. Based on their molecular mass (approximately 105 kDa) both calpastatin forms belong to the high molecular mass class found in muscles of other animal species (Murachi, T., 1989, Biochem. Int. 18, 263-294). For calpain I, which is active with low (mu-M) concentrations of Ca2+, maximum inhibition with either calpastatin form was observed over a wide range of Ca2+ concentrations. With calpain II, which requires high (mM) concentrations of Ca2+ for activity, maximum inhibition required Ca2+ concentrations above 1 mM. Both calpastatin forms were found to be highly sensitive to degradation by calpain II, but almost completely resistant to degradation by calpain I. Degradation of calpastatin by calpain II is competitively inhibited by the addition of a calpain substrate. Isovaleryl carnitine (IVC), an intermediate product of L-leucine catabolism, previously demonstrated to be a potent and specific activator of rat skeletal muscle calpain II (Pontremoli, S., Melloni, E., Viotti, P. L., Michetti, M., Di Lisa, F., and Siliprandi, N., 1990. Biochem. Biophys. Res. Commun. 167, 373-380) greatly enhances the rate of degradation of calpastatins by calpain II. IVC, which decreases the Ca2+ requirement for maximal calpain II activity, also decreases the concentration of Ca2+ required for digestion of the inhibitor. For calpain II, regulation by either calpastatins may occur only in the presence of high [Ca2+].     

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.     

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.     

Mechanism of action of the calpain activator protein in rat skeletal muscle.

Rat skeletal muscle contains a calpain activator protein characterized by a high specificity for calpain II, the high Ca(2+)-requiring isoform of this class of proteinases. The activator protein increases the rate of intramolecular conversion of the native 80-kDa catalytic subunit of calpain into the autolysed 75-kDa forms with maximal rate at concentrations of calcium approximately 25 times lower than those required by the native proteinase. The activator protein interacts with native calpain II forming a 1:1 complex; interaction does not occur with the fully activated form, produced by autoproteolysis. Even after immobilization to membranes, the activator binds to calpain, which then undergoes sequential activation and release from its bound form. The activator is itself resistant to digestion by calpain II, whereas it increases the rate at which homologous calpastatin is degraded by the proteinase. Taken together, these results are indicative of the existence in rat skeletal muscle of an activating system specific for calpain II which is potentially involved in the regulation of the inhibitory efficiency of calpastatin, through modulation of its intracellular level.     

Comparative enzymatic properties of avian liver and skeletal muscle D-fructose-1,6-bisphosphate 1-phosphohydrolase.

The enzymatic properties of purified preparations of chicken liver and chicken skeletal muscle fructose bisphosphatases (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) were compared. Both enzymes have an absolute requirement for Mg2+ or Mn2+. The apparent Km for MgCl2 at pH 7.5 was 0.5 mM for the muscle enzyme and 5 mM for the liver enzyme. Fructose bisphosphate inhibited both enzymes. At pH 7.5, the inhibitor constants (Ki) were 0.18 and 1.3 mM for muscle and liver fructose bisphosphatases, respectively. The muscle enzyme was considerably more sensitive to AMP inhibition than the liver enzyme. At pH 7.5 and in the presence of 1 mM MgCl2, 50% inhibition of muscle and liver fructose bisphosphatases occurred at AMP concentrations of 7 X 10(-9) and 1 X 10(-6) M, respectively. EDTA activated both enzymes. The degree of activation was time and concentration dependent. The degree of EDTA activation of both enzymes decreased with increasing MgCl2 concentration. Ca2+ was a potent inhibitor of both liver (Ki, 1 X 10(-4) M) and muscle (Ki, 1 X 10(-5) M) fructose bisphosphatase. This inhibition was reversed by the presence of EDTA. Ca2+ appears to be a competitive inhibitor with regard to Mg2+. There is, however, a positive homeotropic interaction among Mg2+ sites of both enzymes in the presence of Ca2+.     

Calpain and calpastatin in porcine retina. Identification and action on microtubule-associated proteins.

Two forms of Ca2+-dependent cysteine proteinase (calpain, EC 3.4.22.17) and their specific endogenous inhibitor (calpastatin) were partially purified from porcine retina: calpain I (low-Ca2+-requiring form) was half-maximally activated at 8 microM-Ca2+, and calpain II (high-Ca2+-requiring form) at 250 microM-Ca2+. Both calpain I and calpain II were inhibited by calpastatin. Calpain I from porcine retina was shown to be composed of 83 000- and 29 000-Mr subunits, and calpain II of 80 000- and 29 000-Mr subunits, by the use of monospecific antibodies. Calpains I and II were both found to hydrolyse microtubule-associated proteins 1 and 2 rapidly.     

Calpain II-like proteinase of scallop (Patinopecten yessoensis) striated adductor muscle.

1. A Ca(2+)-dependent cysteine proteinase was purified from scallop striated adductor muscle by ammonium sulfate fractionation and column chromatography on DEAE-cellulose and Sephacryl S-300. 2. The enzyme is of Mr approximately 200,000, composed of two Mr 100,000 subunits. 3. The enzyme is a cysteine proteinase with optimum activity at pH 6.8 and about 18 degrees C. In addition, it requires 1.7 mM Ca2+ for half-maximal activity and more than 10 mM Ca2+ for maximal activity. Thus the enzyme can be classified as calpain II.     

Reversible activation of human neutrophil calpain promoted by interaction with plasma membranes

Human neutrophil calpain is a monomer of 85 kDa molecular weight. The proteinase shows an absolute requirement for Ca2+ with maximal catalytic activity at 0.1-0.2 mM Ca2+ and negligible activity at 1-5 microM Ca2+. At this concentration of Ca2+ neutrophil calpain becomes active and reaches 65% of its maximal catalytic activity following interaction with plasma membranes. The activation is fully reversible since the enzyme returns to its native, high Ca2+ requiring form following removal of the membranes. Membrane phospholipids appear to be the physiological compounds responsible for the promotion of such reversible activation. Unlike other Ca2+ dependent proteinases, neutrophil calpain does not undergo conversion to a low Ca2+ requiring form by limited autoproteolysis.     

In vitro and in vivo inhibition of muscle lipid and protein oxidation by carnosine.

Carnosine, a beta-alanyl-L-histidine dipeptide with antioxidant properties is present at high concentrations in skeletal muscle tissue. In this study, we report on the antioxidant activity of carnosine on muscle lipid and protein stability from both in vitro and in vivo experiments. Carnosine inhibited lipid peroxidation and oxidative modification of protein in muscle tissue prepared from rat hind limb homogenates exposed to in vitro Fenton reactant (Fe2+, H2O2)-generated free radicals. The minimum effective concentrations of carnosine for lipid and protein oxidation were 2.5 and 1 mM, respectively. Histidine and beta-alanine, active components of carnosine, showed no individual effect towards inhibiting either lipid or protein oxidation. Skeletal muscle of rats fed a histidine supplemented diet for 13 days exhibited a marked increase in carnosine content with a concomitant reduction in muscle lipid peroxidation and protein carbonyl content in skeletal muscle caused by subjecting rats to a Fe-nitrilotriacetate administration treatment. This significant in vitro result confirms the in vivo antioxidant activity of carnosine for both lipid and protein constituents of muscle under physiological conditions.     

Purification and properties of carp (Cyprinus carpio) muscle calpain II (high-Ca2+-requiring form of calpain)

Calpain (Ca2+-dependent cysteine proteinase) was purified to apparent homogeneity from carp muscle by the method of DEAE-cellulose, hydroxylapatite and Ultrogel AcA 34 column chromatographies. The purified enzyme is classified as calpain II (high-Ca2+-requiring form of calpain) from the effects of Ca2+ concentration, pH and the antibiotics on the activity. Carp muscle calpain II was inhibited by rat liver calpastatin, the specific inhibitor for calpain. It is probable that the calpain-calpastatin system may play a biologically fundamental and common role in various cells, since the inhibitory effect of calpastatin on calpain from different tissues of different species is well conserved.     

Protection by carnosine-related dipeptides against hydrogen peroxide-mediated ceruloplasmin modification

Carnosine, homocarnosine, and anserine are present in high concentrations in the muscle and brain of many animals and humans. Previous studies showed that these compounds have an antioxidant function. We investigated the protective effects of carnosine and related compounds on the modification of human ceruloplasmin that is induced by H2O2. Carnosine, homocarnosine, and anserine significantly inhibited the fragmentation and inactivation of ceruloplasmin that is induced by H2O2. All three compounds also inhibited the release of copper ion from protein, and the formation of hydroxyl radicals in the ceruloplasmin/H2O2 system. These compounds inhibited the fragmentation of human serum albumin that is induced by the copper-catalyzed oxidation system, as well as by the iron-catalyzed oxidation system. These results suggest that carnosine, homocarnosine, and anserine might protect ceruloplasmin against H2O2-mediated oxidative damage through a combination of copper chelation and free radical scavenging.     

Mg(Ca)-ATPase of arterial muscle cells]

We could show an ATPase in mitochondrial and microsomal fractions of sheep arteria carotis communis and arteria coronaria of cattle which can be stimulated by Ca2+ of Mg2+, respectively. The enzyme has a higher affinity for Ca2+ than for Mg2+. The maximum activity of the Mg(Ca)-ATPase was found at 2-4 mM Ca2+ or Mg2+, respectively. Higher concentrations of these ions inhibit the enzyme. Mn2+, Sr2+ and Co2+ can substitute Ca2+ in splitting of ATP by the ATPase of both fractions of ateria coronaria of cattle. The ions K+ and Na+, variation of temperature and pH and a variety of pharmacological active compounds has the same effect on the ATPase stimulated by Ca2+ or Mg2+. These findings prove that Ca2+ and Mg2+ act at the same site of the ATPase of the mitochondrial and microsomal fraction of vascular smooth muscle.     

Biochemical and Physiological Evidence that Carnosine Is an Endogenous Neuroprotector Against Free Radicals

1. Carnosine, anserine, and homocarnosine are endogenous dipeptides concentrated in brain and muscle whose biological functions remain in doubt.2. We have tested the hypothesis that these compounds function as endogenous protective substances against molecular and cellular damage from free radicals, using two isolated enzyme systems and two models of ischemic brain injury. Carnosine and homocarnosine are both effective in activating brain Na, K-ATPase measured under optimal conditions and in reducing the loss of its activity caused by incubation with hydrogen peroxide.3. In contrast, all three endogenous dipeptides cause a reduction in the activity of brain tyrosine hydroxylase, an enzyme activated by free radicals. In hippocampal brain slices subjected to ischemia, carnosine increased the time to loss of excitability.4. In in vivo experiments on rats under experimental hypobaric hypoxia, carnosine increased the time to loss of ability to stand and breath and decreased the time to recovery.5. These actions are explicable by effects of carnosine and related compounds which neutralize free radicals, particularly hydroxyl radicals. In all experiments the effective concentration of carnosine was comparable to or lower than those found in brain. These observations provide further support for the conclusion that protection against free radical damage is a major role of carnosine, anserine, and homocarnosine.     

Identification of both calpains I and II in nucleated chicken erythrocytes

Chicken erythrocytes were found to contain two species of calpains which differ in elution profile from DEAE-cellulose and in Ca2+ requirement. After partial purification, one of them was half-maximally activated by 10 microM Ca2+ and the other by 180 microM Ca2+. The low- and high-Ca2+-requiring proteases cross-reacted only with the respective monospecific antibodies for mammalian calpain I and calpain II, respectively. Approximately 5 times more calpain I than calpain II is present in chicken erythrocytes. By immunoelectrophoretic blot analysis, both calpains I and II from chicken erythrocytes were proved to be heterodimers composed of 76 and 28 kDa, and 80 and 28 kDa subunits, respectively. Our present finding that the heavy subunit of calpain I is smaller than that of calpain II is noteworthy, since the opposite is known to be true of various mammalian calpains. An immunological study has revealed that the calpain I newly found in chicken erythrocytes is not derived from calpain II. Thus, the co-existence of calpains I and II in one animal species also holds in chickens, contrary to the previously advocated notion that chickens have only one type of calpain.     

Carnosine and related compounds protect the double-chain DNA from oxidative damages

Carnosine and its derivatives in the concentrations corresponding to their level in excitable tissues have been shown to protect DNA from oxidative damages. Their efficiency (5 mM) was of the following order: ophidine > carnosine ≈ anserine > homocarnosine > N-acetylcarnosine. β-Alanine and gamma-aminobutyric acid (GABA) did not have any capability for protection. The revealed effect can be one of the causes of oxidative stability of the brain and muscle tissue in vertebrate animals.     

Phosphorylation modulates the function of the calcium release channel of sarcoplasmic reticulum from skeletal muscle.

The modulation of the calcium release channel (CRC) by protein kinases and phosphatases was studied. For this purpose, we have developed a microsyringe applicator to achieve sequential and multiple treatments with highly purified kinases and phosphatases applied directly at the bilayer surface. Terminal cisternae vesicles of sarcoplasmic reticulum from rabbit fast twitch skeletal muscle were fused to planar lipid bilayers, and single-channel currents were measured at zero holding potential, at 0.15 microM free Ca2+, +/- 0.5 mM ATP and +/- 2.6 mM free Mg2+. Sequential dephosphorylation and rephosphorylation rendered the CRC sensitive and insensitive to block by Mg2+, respectively. Channel recovery from Mg2+ block was obtained by exogenous protein kinase A (PKA) or by Ca2+/calmodulin-dependent protein kinase II (CalPK II). Somewhat different characteristics were observed with the two kinases, suggesting two different states of phosphorylation. Channel block by Mg2+ was restored by dephosphorylation using protein phosphatase 1 (PPT1). Before application of protein kinases or phosphatases, channels were found to be "dephosphorylated" (inactive) in 60% and "phosphorylated" (active) in 40% of 51 single-channel experiments based on the criterion of sensitivity to block by Mg2+. Thus, these two states were interconvertable by treatment with exogenously added protein kinases and phosphatases. Endogenous Ca2+/calmodulin-dependent protein kinase (end CalPK) had an opposite action to exogenous CalPK II. Previously, dephosphorylated channels using PPT (Mg2+ absent) were blocked in the closed state by action of endogenous CalPK. This block was removed to normal activity by the action of either PPT or by exogenous CalPK II. Our findings are consistent with a physiological role for phosphorylation/dephosphorylation in the modulation of the calcium release channel of sarcoplasmic reticulum from skeletal muscle. A corollary of our studies is that only the phosphorylated channel is active under physiological conditions (mM Mg2+). Our studies suggest that phosphorylation can be at more than one site and, depending on the site, can have different functional consequences on the CRC.