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.     

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.     

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.     

Activation of tyrosine hydroxylase by Ca2+-dependent neutral protease, calpain

Two molecular species of Ca2+-dependent neutral protease (calpains I and II) and its endogenous inhibitor (calpastatin) in cytosol fraction of bovine adrenal medulla were separated by hydrophobic interaction chromatography. Both calpains I and II, having low and high Ca2+ requirements for casein hydrolysis, respectively, were found to activate tyrosine hydroxylase(TH) that had been purified from cytosol fraction of bovine adrenal medulla. This activation of TH by calpain was inhibited by leupeptin and the endogenous inhibitor, calpastatin. The activated TH with calpain II, characterized by high-performance gel permeation chromatography, had a reduced Mr of 120,000 from the Mr of 230,000 of native enzyme.     

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.     

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.     

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.     

Comparison of tryptic peptides from the heavy and light subunits of calpain I and calpain II by high performance liquid chromatography

Two different forms of Ca2+-dependent cysteine proteinase, low-Ca2+-requiring calpain I and high-Ca2+-requiring calpain II, are known to be heterodimers, each composed of one heavy (called 80K) and one light (called 30K) subunit. The most probable identity of the 30K and the substantial difference between the 80K subunits of porcine calpains I and II were clearly demonstrated by comparing the tryptic peptide maps obtained upon running a high performance liquid chromatography which permitted parallel detection of tryptophan-containing peptides by fluorometry. Comparison of the amino acid compositions of the two 30K and 80K subunits also confirmed this conclusion. The same chromatographical analysis also revealed close structural similarity of the human calpain I 30K subunit, and even some similarity existing between the calpain I 80K subunits of human and porcine origins.     

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.     

Effects of Substance P on Carbachol-Stimulated 45Ca2+ Uptake into Cultured Adrenal Chromaffin Cells

Substance P is known to modulate acetylcholine-induced catecholamine release from adrenal chromaffin cells. To investigate the mechanisms involved in this modulation, the present study examined the effects of substance P on net 45Ca2+ fluxes in cultures of bovine adrenal chromaffin cells. Two effects of substance P were observed: (1) Substance P inhibited carbachol-induced 45Ca2+ uptake and 45Ca2+ efflux and (2) substance P protected against desensitization of carbachol-induced 45Ca2+ uptake and 45Ca2+ efflux. Thus substance P modulates two other cholinergic responses, 45Ca2+ uptake and 45Ca2+ efflux, in a manner similar to its modulation of catecholamine release. The results also indicate that substance P's inhibition of net carbachol-induced 45Ca2+ uptake is due to inhibition of 45Ca2+ uptake rather than enhancement of 45Ca2+ efflux. Substance P almost completely inhibited carbachol-induced 45Ca2+ uptake in both Na+-containing and Na+-free media, suggesting that substance P can inhibit the uptake of 45Ca2+ induced by carbachol regardless of whether 45Ca2+ is taken up through voltage-sensitive or acetylcholine receptor-linked channels. However, substance P produced only a small inhibition of K+-induced 45Ca2+ uptake, indicating that substance P does not interact directly with voltage-sensitive Ca2+ channels. In addition, substance P's inhibition of carbachol-induced 45Ca2+ uptake was noncompetitive with respect to Ca2+, were unable to overcome substance P's inhibition of [3H]-norepinephrine ( [3H]NE) release. It is concluded that substance P does not interact directly with Ca2+ channels in bovine adrenal chromaffin cells.     

Nuclear magnetic resonance studies of the internal dynamics in Apo, (Cd2+)1 and (Ca2+)2 calbindin D9k. The rates of amide proton exchange with solvent.

The backbone dynamics of the EF-hand Ca(2+)-binding protein, calbindin D9k, has been investigated in the apo, (Cd2+)1 and (Ca2+)2 states by measuring the rate constants for amide proton exchange with solvent. 15N-1H correlation spectroscopy was utilized to follow direct 1H-->2H exchange of the slowly exchanging amide protons and to follow indirect proton exchange via saturation transfer from water to the rapidly exchanging amide protons. Plots of experimental rate constants versus intrinsic rate constants have been analyzed to give qualitative insight into the opening modes of the protein that lead to exchange. These results have been interpreted within the context of a progressive unfolding model, wherein hydrophobic interactions and metal chelation serve to anchor portions of the protein, thereby damping fluctuations and retarding amide proton exchange. The addition of Ca2+ or Cd2+ was found to retard the exchange of many amide protons observed to be in hydrogen-bonding environments in the crystal structure of the (Ca2+)2 state, but not of those amide protons that were not involved in hydrogen bonds. The largest changes in rate constant occur for residues in the ion-binding loops, with substantial effects also found for the adjacent residues in helices I, II and III, but not helix IV. The results are consistent with a reorganization of the hydrogen-bonding networks in the metal ion-binding loops, accompanied by a change in the conformation of helix IV, as metal ions are chelated. Further analysis of the results obtained for the three states of metal occupancy provides insight into the nature of the changes in conformational fluctuations induced by ion binding.     

Conformational changes in the metal-binding sites of cardiac troponin C induced by calcium binding.

Isotope labeling of recombinant normal cardiac troponin C (cTnC3) with 15N-enriched amino acids and multidimensional NMR were used to assign the downfield-shifted amide protons of Gly residues at position 6 in Ca(2+)-binding loops II, III, and IV, as well as tightly hydrogen-bonded amides within the short antiparallel beta-sheets between pairs of Ca(2+)-binding loops. The amide protons of Gly70, Gly110, and Gly146 were found to be shifted significantly downfield from the remaining amide proton resonances in Ca(2+)-saturated cTnC3. No downfield-shifted Gly resonance was observed from the naturally inactive site I. Comparison of downfield-shifted amide protons in the Ca(2+)-saturated forms of cTnC3 and CBM-IIA, a mutant having Asp65 replaced by Ala, demonstrated that Gly70 is hydrogen bonded to the carboxylate side chain of Asp65. Thus, the hydrogen bond between Gly and Asp in positions 6 and 1, respectively, of the Ca(2+)-binding loop appears crucial for maintaining the integrity of the helix-loop-helix Ca(2+)-binding sites. In the apo- form of cTnC3, only Gly70 was found to be shifted significantly downfield with respect to the remaining amide proton resonances. Thus, even in the absence of Ca2+ at binding site II, the amide proton of Gly70 is strongly hydrogen bonded to the side-chain carboxylate of Asp65. The amide protons of Ile112 and Ile148 in the C-terminal domain and Ile36 in the N-terminal domain data-sheets exhibit chemical shifts consistent with hydrogen-bond formation between the pair of Ca(2+)-binding loops in each domain of Ca(2+)-saturated cTnC3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of substance P and its N- and C-terminal fragments with Ca2+: implications for hormone action.

In an attempt to understand the role of Ca2+ on the bioactive conformation of peptide hormones, we have examined the interaction between Ca2+ and the neuropeptide substance P. Using CD spectroscopy to monitor conformational changes caused by Ca2+ binding, we found no significant binding of the cation by substance P in water. However, a substantial conformational change occurred in the hormone on Ca2+ addition in trifluoroethanol or an 80:20 (v/v) mixture of acetonitrile and trifluoroethanol. A biphasic binding of Ca2+ was observed in these solvents with saturation at 2 cations per hormone molecule. Mg2+ caused a relatively smaller conformational change in the hormone. A peptide corresponding to residues 1-7 at the N-terminal fragment of substance P showed a weak nonsaturating binding of Ca2+ in the nonpolar solvents whereas the 7-11 C-terminal fragment peptide displayed a binding indicative of an 1:1 Ca2+/peptide complex. Ca2+ binding by the hormone and the 7-11 fragment was also monitored by changes in fluorescence of the phenylalanyl residues. The results support the conclusion drawn from the CD data about a distinct Ca2+ binding site in the C-terminal part of substance P. The Kd values obtained from fluorescence data were 160 microM for Ca2+ and 1 mM for Mg2+ binding by substance P. The hormone and the two peptide fragments were also tested for their effect on the stability of dimyristoyl lecithin vesicles. Substance P and the N-terminal fragment caused no significant leakage of either fluorescent dyes or K+ trapped in the vesicles. Nor did they cause membrane fusion as monitored by the fluorescence quenching method.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional and physical characteristics of rat Leydig cell populations isolated by metrizamide and Percoll gradient centrifugation

The physical and functional properties of Leydig cell populations obtained by centrifugation of testicular cells in two different density gradient media, Percoll and Metrizamide, were compared. Percoll-gradient centrifugation yielded two Leydig cell bands (Peak I and Peak II) that were comparable, as to their density and testosterone-producing capacity, to the respective Leydig cell bands, Population I and Population II, isolated in a Metrizamide gradient. The denser Leydig cell band (II) had a greater capacity for testosterone production than the less dense band (I), regardless of the type of gradient used for its isolation. Metrizamide gradient centrifugation separated the majority of germ cells from the "light" (Population I) Leydig cells, whereas in the Percoll gradient, germ cells comigrated with Peak I Leydig cells. Leydig cell separation by Percoll gradients was highly dependent on the presence of Ca2+ and Mg2+ in the medium, while these cations had no effect on the separation of Leydig cells by Metrizamide. In conclusion, Metrizamide gradient centrifugation yielded two Leydig cell populations of similar functional and physical properties to the respective populations isolated in Percoll gradients.     

Possible role of calpain I and calpain II in differentiating muscle

The variable distribution of the 80-kD subunit of two calcium-activated proteases, calpain I and calpain II, has been examined in L8 and L6 myoblasts, and their non-fusing variants, fu-1 and M3A using non-cross-reacting monoclonal antibodies to both subunits. Immunofluorescence results have shown that while the 80-kD subunit of calpain I is localized in the cytoplasm of all the myoblasts, the 80-kD subunit of calpain II appears to be predominantly associated with the plasma membranes of L8 and L6 myoblasts. The distribution of the 80-kD subunit of calpain II in non-fusing myoblasts, fu-1 and M3A, is generally cytoplasmic and diffuse. Immunoblot analysis of membrane and cytosol fractions of all the myoblasts using the monoclonal antibodies described above essentially confirms the immunofluorescence findings. Because calpain II exhibits a peripheral distribution in cells which are fusion-competent, L6 and L8 myoblasts, but not in fu-1 and M3A myoblasts, we suggest that calpain II may play a role in the Ca2+-mediated fusion events of differentiating (prefusion) myoblasts.     

Conformational changes in concanavalin A associated with demetallization and alpha-methylmannose binding studied by Fourier transform infrared spectroscopy

Infrared spectra of concanavalin A have been obtained both in the absence and in the presence of the metal ions, Mn2+ and Ca2+, and the saccharide, alpha-methylmannose. Second derivative calculations have been used to determine the frequencies of the different amide I and II components. In the demetallized protein dissolved in H2O buffer, absorptions in the amide I, II and III regions at 1695 and 1634, 1532 and 1237 cm-1, respectively, are assigned to beta-structure, while absorptions at 1563 and both 1318 and 1343 cm-1 are assigned to turns and bends. After deuterium exchange, the residual amide II maximum in the difference spectrum shifts from 1538 to 1563 cm-1, indicating that exchange is faster in the beta-structure than in the turns. In the presence of Mn2+ and Ca2+, the amide II band component at 1532 cm-1 shifts 4-6 cm-1 to higher wavenumbers, and the amide I band component at 1634 shifts 1 cm-1 in the same direction, both in H2O and 2H2O buffers, suggesting changes in the hydrogen-bonding network of a large portion of the protein, particularly in the beta-sheet regions. The addition of alpha-methylmannose increases the magnitude of exchange from 55% to above 90%. Comparison with existing X-ray crystallographic data has been made, and the usefulness of FT-IR to complement this technique is discussed.     

Evidence for heterodimeric nature of calpain molecules as studied by cross-linking modification

Purified calpain I and calpain II from porcine erythrocytes and kidney were cross-linked with a bifunctional reagent, disuccinimidyl suberate, and the cross-linked products were analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The major product had a molecular mass of 105 kDa, while the starting materials were resolved into 80-kDa and 30-kDa subunits. The cross-linking in the presence of 2 mM Ca2+ yielded several higher-molecular-weight species. The cross-linked products were shown to contain both the 80-kDa and 30-kDa proteins by means of immunoblotting with antibodies monospecific for the respective subunits, suggesting that the original calpain molecule existed in solution as an 80-kDa plus 30-kDa heterodimer and that Ca2+ induced closer association of these heterodimeric molecules.     

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+].     

Free calcium and calpain I activity

Activation of purified calpain I proceeds through a Ca(2+)-induced autolysis from the 80 kDa catalytic subunit to a 76 kDa form via an intermediate 78 kDa form, and from a 30 kDa form to a 18 kDa form as the result of two autocatalytic processes (intra and intermolecular). The minimum Ca2+ requirements for autolysis and proteolysis have been determined by physico-chemical and electrophoretic methods in the presence or absence of a digestible substrate. According to our results the activation process needs less free Ca2+ than the proteolysis of a digestible substrate, which means that proteolysis is really subsequent to activation. For very low Ca2+ levels, a digestible substrate does not initiate the calpain I activation process. In the presence of phospholipid vesicles, such as PI, PS or a mixture of PI (20%), PS (20%) and PC (60%), the apparent kinetic constants of activation are greatly increased without any change in the initial velocity of the substrate proteolysis. Thus, enzyme activation and substrate proteolysis are observed as independent phenomena. These results obtained from experiments using low free Ca2+ concentrations enable us to propose a hypothesis for the mechanism of regulation by which the enzyme could be activated in the living cell.     

The effects of substance P and carbachol on inositol tris- and tetrakisphosphate formation and cytosolic free calcium in rat parotid acinar cells. A correlation between inositol phosphate levels and calcium entry

Both substance P and carbachol produced increases in inositol tris- and tetrakisphosphate and increased cytosolic free [Ca2+] in dispersed parotid acinar cells loaded with fura-2. The increase in [Ca2+]i in response to each agonist was due to a combination of mobilization of internal Ca2+ and entry of extracellular Ca2+. Kinetic studies of the initial response to substance P, and measurement of peak [Ca2+]i, demonstrated that the initial rapid rise in [Ca2+]i was due to both internal release and entry of Ca2+. Substance P could evoke a greater initial increase in [Ca2+]i and inositol trisphosphate than could carbachol. However, after 1 min in the presence of external Ca2+, the maintained [Ca2+]i level in response to substance P was considerably smaller than that seen with carbachol, an effect apparently due to homologous desensitization of the substance P receptor. The two agonists each produced a similar 4-5-fold increase in inositol tetrakisphosphate levels within 30 s; this level was maintained in the presence of carbachol, but decreased with substance P. Similarly, the level of inositol (1,4,5)-trisphosphate decreased after prolonged incubation with substance P. Thus, the maintained level of [Ca2+]i, and by deduction Ca2+ entry, correlated with the levels of inositol (1,4,5)-trisphosphate and inositol tetrakisphosphate; a result consistent with a possible role for these inositol phosphates in the control of receptor-mediated Ca2+ channels.