Purification and characterization of 210,000-dalton inhibitor of calcium-activated neutral protease from rabbit skeletal muscle and its relation to 50,000-dalton inhibitor

An endogenous inhibitor of calcium-activated neutral protease (CANP), which was isolated from rabbit skeletal muscle with chemically drastic pretreatments, comprised major (high-molecular-weight form, HMW-inhibitor) and minor (low-molecular-weight form, LMW-inhibitor) components. HMW-inhibitor was purified to homogeneity using FPLC and preparative electrophoresis. The purified inhibitor appeared as a single protein with a molecular weight of 110,000 on SDS-polyacrylamide gel electrophoresis, and a molecular weight of 210,000 on gel filtration. It was therefore presumed that the inhibitor is a dimer protein under native conditions. It contained large amounts of glutamic acid, alanine, and proline, and small amounts of aromatic amino acids, showing an amino acid composition similar to that of LMW-inhibitor. HMW-inhibitor inhibited CANPs with both low (m-type) and high (mu-type) Ca2+-sensitivity but had no effect on any other proteases examined. It was demonstrated that the inhibition was due to the formation of a stoichiometric complex between rabbit mCANP and inhibitor subunit in the ratio of five to one. These results suggest that HMW-inhibitor might have several reactive sites per molecule and that LMW-inhibitor subunit might be a proteolytic fragment of HMW-inhibitor containing an active site.     

Purification and characterization of an inhibitor of calcium-activated neutral protease from rabbit skeletal muscle

An endogenous inhibitor of calcium-activated neutral protease was purified to homogeneity from rabbit skeletal muscle using ion-exchange chromatography on DEAE-cellulose and QAE-Sephadex A-50 columns, chromatofocusing, and hydrophobic interaction chromatography on a phenyl-Sepharose CL-4B column. The purified inhibitor was shown to be a dimer of identical subunits and each subunit has a molecular weight of about 34,000. This inhibitor was remarkably thermo- and acid-stable. It was specific for calcium-activated neutral protease and had no effect on any other protease examined (trypsin, papain, alpha-chymotrypsin, bromelain, etc.). It is demonstrated that the inhibition is due to the formation of stoichiometric complex between two enzyme molecules and one inhibitor molecule.     

Identity of calcium-activated neutral proteases from rabbit cardiac and skeletal muscle

1. The low-calcium-requiring form (μCANP) and the high-calcium-requiring form (mCANP) of the calcium-activated neutral proteases were purified to near homogeneity from rabbit cardiac muscle.2. Each of them was compared with the counterpart of skeletal muscle in respect to subunit composition, calcium sensitivity, pH dependency of the activity and peptide map of the fragments produced byS. aureus V8 protease digestion.3. All results suggested that mCANP and μCANP from cardiac muscle were almost indistinguishable in various properties with mCANP and μCANP of skeletal muscle, respectively, showing the lack of tissue-specificity of CANPs among these two tissues. However, the total and the relative contents of mCANP and μCANP were different among them.     

Comparison of calcium-activated neutral proteases from skeletal muscle of rabbit and chicken

Calcium-activated neutral proteases (CANPs) were purified from rabbit skeletal muscle and chicken skeletal muscle, and compared as to their electrophoretic properties, metal requirements, subunit amino acid compositions and immunological cross-reactivities. Two kinds of CANPs (mu CANP and mCANP) were isolated from rabbit but the chicken tissue lacked one corresponding to mu CANP. They were acidic in the order of chicken mCANP, rabbit mCANP, and rabbit mu CANP but the difference between the former two was very small. All of them were composed of two subunits, so-called 80K and 30K subunits. The molecular weight of the 30K subunit was the same for these CANPs (28K) but those of the 80K subunit were different (79K for rabbit mu CANP, 75K for rabbit mCANP and 81K for chicken mCANP). The calcium-sensitivity of chicken mCANP was very high when compared with that of rabbit mCANP and close to that of rabbit mu CANP. Antisera against chicken CANP and those against rabbit CANP cross-reacted with rabbit CANP and chicken CANP, respectively, when examined by immunoelectrotransfer blot techniques.     

Fragmentation of an endogenous inhibitor upon complex formation with high- and low-Ca2+-requiring forms of calcium-activated neutral proteases

The interaction of an endogenous inhibitor for the calcium-activated neutral protease (CANP or calpain EC 3.4.22.17) with CANP was examined by SDS-polyacrylamide gel electrophoresis, immunoblot analysis, and gel filtration. Fragmentation of the inhibitor (Mr 110K) by mCANP, a high-Ca2+-requiring form, was shown only in the presence of Ca2+ ions of millimolar order, with decreased inhibitor activity recovered from gel extracts in the 110-kDa area. This fragmentation took place even when the inhibitor could completely inhibit the caseinolytic activity of mCANP. The fragmented inhibitor retained considerable inhibitor activity after the CANP-inhibitor complex was dissociated by the addition of EDTA, and 69% of the initial activity was recovered from the mixture reacted with excess mCANP lacking the 110-kDa band. A C-terminal fragment of CANP inhibitor produced in Escherichia coli (Mr 40K) was also hydrolyzed by mCANP in the presence of Ca2+. The interaction of both forms of the inhibitor with mu CANP, a low-Ca2+-requiring form, led to the same phenomena in the presence of micromolar levels of Ca2+. CANP inhibitor could not completely inhibit the autolysis of mCANP and mu CANP, indicating that these were intramolecular events. Gel filtration analysis revealed that the mass of the smallest fragment with inhibitor activity was about 15,000 daltons. These results suggest that CANP inhibitor may act in the manner of a suicide substrate.     

Purification and characterization of a calcium-activated neutral protease from rabbit skeletal muscle which requires calcium ions of microM order concentration

One form of calcium-activated neutral protease (CANP) highly sensitive to calcium ions was purified by column chromatographic procedures to homogeneity. The purified enzyme required microM order Ca2+ (mu CANP), and the half-maximum activity was attained at 50 microM Ca2+. The electrophoretic mobility in a non-denaturing buffer showed that this enzyme is less acidic than another CANP which required mM order Ca2+ (mCANP). On SDS-polyacrylamide gel electrophoresis, the enzyme separated into two components with molecular weights of 79,000 and 28,000, respectively. Of these, the former was slightly larger than the counterpart of mCANP (Mr 76,000). Thus, mu CANP cannot be derived from mCANP by limited autolysis.     

Comparison of low and high calcium requiring forms of the calcium-activated neutral protease (CANP) from rabbit skeletal muscle

Two distinct calcium-dependent neutral proteases (CANPs) with different sensitivities to calcium ions were purified concurrently by almost the same procedures from rabbit skeletal muscle and their enzymatic properties were compared (sensitivity to various divalent metal ions, the pH dependency and heat-stability of the activity, and the hydrolytic activity towards various substrates). They were further compared chemically in terms of the state of thiol groups, the amino acid compositions of subunits and the peptide fragments by digestion with S. aureus V8 protease. The low calcium requiring form of CANP (microCANP) was more sensitive to other divalent metal ions such as Sr2+ and Ba2+ than the high calcium requiring form of CANP (mCANP). The comparison of the pH dependency of these CANP activities showed that microCANP was active in a broader pH range than mCANP and the former was more heat-stable than the latter. Both CANPs had similar affinity to various substrates, but the hydrolytic velocity was several times higher with microCANP than with mCANP. Although they were inhibited by thiol protease inhibitors to the same extent, the states of thiol groups in them were quite different. The thiol group involved in the catalytic activity of the enzyme was exposed without adding Ca2+ in microCANP, whereas the group in mCANP became exposed only when sufficient Ca2+ was added. The large subunits of these two CANPs were different in their amino acid compositions and in the peptide fragment patterns produced by S. aureus V8 protease but the small subunits were indistinguishable from each other. These results led us to conclude that these two CANPs are quite different in nature and are not in a simple relationship, i.e., one of them is not derived from the other by autolysis or modification.     

Lack of tissue-specificity of calcium-activated neutral proteases from skeletal muscle and lung of rabbit

Calcium-activated neutral proteases (CANPs) with high sensitivity (microCANP) and low sensitivity (mCANP) to calcium ions were purified individually from rabbit skeletal muscle and rabbit lung and compared as to their electrophoretic properties, calcium requirements and peptide mapping of fragments produced by S. aureus V8 protease digestion of separated subunits. All of the results suggested that there is no difference between the microCANPs as well as between the mCANPs obtained from the two tissues, with respect to the chemical and enzymatic properties. However, the contents of CANPs in these tissues were different.     

Limited autolysis of calcium-activated neutral protease (CANP): reduction of the Ca2+-requirement is due to the NH2-terminal processing of the large subunit

Calcium-activated neutral protease (rabbit mCANP), composed of large and small subunits, was converted to a lower-Ca2+-requiring form (derived microCANP) by limited autolysis in the presence of Ca2+. The NH2-terminal regions of the two subunits of mCANP were cleaved by autolysis, but the COOH-termini remained intact after autolysis. When native mCANP or derived microCANP was dissociated into subunits, the proteolytic activity of the large subunit was reduced to 2-5% of that of the native dimeric enzyme. The Ca2+-sensitivity of one hybrid CANP reconstituted from the large subunit of derived microCANP and the small subunit of native mCANP was similar to that of derived microCANP. However, the other hybrid molecule composed of the large subunit of native mCANP and the small subunit of derived microCANP required a high concentration of Ca2+ for activity, like native mCANP. These results indicate that the Ca2+-sensitivity of derived microCANP is determined by the structural change of the large subunit resulting from loss of its NH2-terminal region. The autolysis of the small subunit apparently has no effect on the reduction of the Ca2+-requirement.     

Hydrolytic and autolytic behavior of two forms of calcium-activated neutral protease (CANP)

Some endogenous substrates were incubated with two forms of calcium-activated neutral protease (CANP) with high (muCANP) and low (mCANP) sensitivities to calcium ions. In addition to analyses of the processes of their degradation, changes in the molecular properties of these CANPs were also examined. Among the tested substrate proteins, the myosin heavy chain of rabbit skeletal muscle myofibrils and spectrin or band 3 protein of human erythrocyte membranes were degraded relatively rapidly. So far as these proteins were concerned, a higher degradation velocity was observed for muCANP than for mCANP. Vimentin from ascites tumor cells was degraded most rapidly and no difference was observed in degradation velocity between muCANP and mCANP. In all cases, muCANP and mCANP produced different proteolytic peptide fragments, suggesting the different substrate-specificities of these CANPs. The degradation of substrates always accompanied the autodigestion of CANPs, and the small subunits of both CANPs were degraded in the early stage of the autodigestion. The large subunit of muCANP (79K) was converted to a 76K polypeptide via a 77K polypeptide as an intermediate. The autodigested muCANP with 76K polypeptide retained sufficient protease activity and, moreover, its calcium-sensitivity was higher than that of intact muCANP. The possibility is thus proposed that restricted autodigestion is a necessary activation step for the appearance of activity of muCANP. No such transition was observed for mCANP.     

Isolation and characterization of monoclonal antibodies against calcium-activated neutral protease with low calcium sensitivity

Fifteen hybridomas secreting antibodies against calcium-activated neutral protease (CANP), especially those for rabbit muscle mCANP with low calcium sensitivity, have been produced by the cell fusion technique. Eight of the monoclonal antibodies belong to the class IgG1, one to the class IgG2a, and six to the class IgG2b. The antibodies from these clones were characterized with regard to their relative binding affinities to the large subunits (80K) and the small subunits (30K) of mCANP as well as mu CANP, which is another type of CANP with high calcium sensitivity. Fourteen antibodies bound only to the 80K subunit of mCANP and one antibody bound to the 80K subunit of both mCANP and mu CANP. These antibodies recognized rat mCANP but not chicken CANP, with the exception of one antibody. Examination of the effects of these antibodies on the enzyme activity of mCANP showed that six antibodies partially inhibited the enzyme activity and the others were noninhibitory. These monoclonal antibodies should be useful for analyzing the fine structure of CANPs and the mechanism of the activation of mCANP, and also for determining the intracellular localization of mCANP.     

Isolation and characterization of a proteinase inhibitor from marama beans

A protease inhibitor was purified from the African marama bean (Tylosema esculenturm). The inhibitor is present in large amounts, representing about 10.5% of the total protein. The molecular weight is slightly larger than soybean trypsin inhibitor and was estimated at 23,000 by SDS-gel electrophoresis or 24,500 by amino acid analysis. The amino acid composition was atypical of most other plant inhibitors with a cysteine content of only one or possibly two residues/mole and a blocked amino terminus. Inhibition studies indicated virtually no inhibition of chymotrypsin activity. Elastase, however, was inhibited to the same extent as trypsin, requiring about 2 moles of inhibitor for complete inhibition of the enzyme.     

Calcium-activated neutral protease inhibitor from rabbit erythrocytes lacks the N-terminal region of the liver inhibitor but retains three inhibitory units

Endogenous inhibitors for calcium-activated neutral protease (CANP) were purified from rabbit erythrocytes and liver. The purified inhibitors showed single bands but with significantly different mobilities on sodium dodecylsulfate-polyacrylamide gel electrophoresis. Peptide mapping and sequencing analyses have revealed that the erythrocyte inhibitor (429 residues) retains the C-terminal three repetitive units of the liver inhibitor (639 residues), which contains four potential repetitive units for inhibition of CANP. The erythrocyte and liver inhibitors inhibited 3 and 4 moles of CANP on the basis of the molecular weights of 46,000 and 68,000, respectively.     

Identification and some properties of a new fast-reacting plasmin inhibitor in human plasma.

Fresh plasma was seeded with trace amounts of highly purified biologically intact iodine-labelled plasminogen and the plasmin-inhibitor complexes formed after activation with streptokinase or urokinase separated by gel filtration. Two radioactive peaks were observed, the first one eluted in the void volume and the second one just before the 7-S globulin peak. In incompletely activated samples, the second peak was always predominant over the first one. Both components were purified with high yield by a combination of affinity chromatography on lysine-agarose and gel filtration, and investigated by dodecylsulphate-polyacrylamide gel electrophoresis and immunoelectrophoresis. Neither component reacted with antisera against alpha1-antitrypsin, antithrombin III, C1-esterase inhibitor, inter-alpha-trypsin inhibitor or alpha1-antichymotrypsin. The component of the first peak appeared to be a complex between plasmin and alpha2-macroglobulin which reacted with antisera against human plasminogen and against alpha2-macroglobulin. The component of the second peak had a molecular weight (Mr) of 120000-140000 by dodecyl-sulphate-polyacrylamide gel electrophoresis and lpon reduction displayed a doublet band with an Mr of 65000-70000 and a band with Mr 11000. It reacted with antisera against plasminogen and with antisera raised against this complex and absorbed with purified plasminogen. The latter antisera reacted with a single component in plasma which is different from the above-mentioned plasma protease inhibitors. Specific removal of this component from plasma by immuno-absorption resulted in disappearance of the fast-reacting antiplasmin activity whereas alpha2-macroglobulin was found to represent the slower-reacting plasmin-neutralizing activity. In the presence of normal plasma levels of these proteins, the specific removal or absence of alpha1-antitrypsin, antithrombin III or C1-esterase inhibitor did not alter the inactivation rate of plasmin when added to plasma in quimolar amounts to that of plasminogen. It is concluded that only two plasma proteins are important in the binding of plasmin generated by activation of the plasma plasminogen, namely a fast-reacting inhibitor which is different from the known plasma protease inhibitors and which we have provisionally named antiplasmin, and alpha2-macroglobulin, which reacts more slowly.     

Tissue distribution of an endogenous inhibitor of calcium-activated neutral protease and age-related changes in its activity in rats

1. The distribution of an endogenous inhibitor of calcium-activated neutral protease (CANP) and age-related changes in its activity were studied in male and female rats of different ages by a fluorometric assay on tissue extracts after heat treatment. 2. Ubiquitous distribution of CANP inhibitor in brain, cardiac muscle, lung, spleen, liver, skeletal muscle, kidney and testis and its abundance in spleen, liver and kidney was demonstrated. 3. Comparison in terms of units/ml of crude extracts showed that the level of CANP inhibitor exceeded that of CANP in most tissues and that the relative content of CANP inhibitor to mCANP and microCANP differed greatly among tissues. 4. Sex and species differences in CANP inhibitor activity in each tissue were of little significance. 5. Changes in CANP inhibitor during aging from 6 to 12 months was not obvious but senescent rats showed a tendency toward increased inhibitor activity. This increase was especially evident in the testis.     

Purification of a trypsin inhibitor secreted by embryogenic carrot cells

A protease inhibitor with a molecular weight of about 12,800 was purified to electrophoretic homogeneity from Daucus carota cells. The protease inhibitor was heat stable and inhibited trypsin but had no activity toward chymotrypsin or subtilisin. Nonembryogenic as well as embryogenic strains contained the inhibitor in similar amounts, but in the embryogenic strains the trypsin inhibitor was released from the cells and as a result accumulated in high concentrations in the culture medium, whereas no release of the trypsin inhibitor was found during cultivation of the nonembryogenic strains. Very low amounts of acid phosphatase or α-mannosidase activity were found in the culture filtrate of both embryogenic and nonembryogenic strains, which suggest that the release of the inhibitor from embryogenic strains was not due to cell lysis.     

Characterization of a protein inhibitor of extracellular proteases produced by Erwinia chrysanthemi

Erwinia chrysanthemi, a phytopathogenic bacterium, produces a protease inhibitor which is a low-molecular-weight, heat-stable protein. In addition to its action on the three E. chrysanthemi extracellular proteases A, B and C, it also strongly inhibits the 50 kD extracellular protease of Serratia marcescens. Its structural gene (inh) was subcloned and expressed in Escherichia coli, in which it encodes an active inhibitor which was purified. The nucleotide sequence of the inh gene shows an open reading frame of 114 condons. The N-terminal amino acid sequence of the purified inhibitor was also determined. It indicated the existence of an amino-terminal signal peptide absent from the mature protein. The inhibitor is entirely periplasmic in E. chrysanthemi and partially periplasmic in E. coli.     

A low-calcium-requiring calcium-activated neutral proteinase from human placenta

A low-calcium-requiring calcium-activated neutral proteinase (mu CANP) has been purified to homogeneity from human placenta. The purification procedure includes chromatography on DEAE-cellulose, Ultrogel AcA-22 and DEAE-Sephadex in succession. The purified mu CANP is a thiol proteinase and requires calcium for activity. Half-maximal activation occurs at 40 microM calcium. It is a heterodimer with subunits of 74 kDa and 32 kDa. (The placental mCANP has subunits of 70 kDa and 32 kDa.) Mn2+ or Sr2+, in combination with Ca2+, activates the enzyme synergistically. The presence of both mCANP and mu CANP in equal proportion in human placenta is reported for the first time. This will facilitate a comparative study of these two forms of human calcium-activated neutral proteinase, especially their physiological structural and functional interrelationship. Maximal activation of the autolysed mCANP occurs at a calcium concentration much higher than that for mu CANP; and this autolysed mCANP does not cross-react with antiserum against mu CANP, suggesting that the two forms of proteinase are independent species.     

Identification of a protease inhibitor from rat peritoneal macrophages as poly(ADP-ribose)

Rat peritoneal macrophages contain a chymotrypsin-like protease and its specific inhibitor, both being associated with chromatin of the cells. The inhibitor was separated from the protease by gel filtration through a Sephadex G-75 column, further treated with trypsin, DNase and RNase, and then purified successively on Sephadex G-75, Sephadex G-25, and dihydroxyboryl Bio-Gel P-60 columns. The purified inhibitor had a molecular weight in the range from 2,000 to 3,500 and an absorption maximum at 260 nm at pH 7.0. When the inhibitor was digested by snake venom phosphodiesterase, the inhibitory potency was lost, yielding 5'-AMP and 2'-(5'-phosphoribosyl)-5'-AMP as the digestion products which were identified by high pressure liquid chromatography. The inhibitory potency was neutralized specifically by anti-poly(ADP-ribose) antiserum. The profile of inhibition by the isolated inhibitor was nearly identical with that produced by authentic poly(ADP-ribose). It was therefore concluded that the inhibitor isolated was identical with poly(ADP-ribose), whose chain length ranged from 4 to 7 ADP-ribosyl units. This is the first demonstration that a intracellular protease inhibitor can be endogenous poly(ADP-ribose).     

A small trypsin inhibitor from the frog of Odorrana grahami

A novel peptide inhibitor (OGTI) of serine protease with a molecular weight of 1949.8, was purified from the skin secretion of the frog, Odorrana grahami. Of the tested serine proteases, OGTI only inhibited the hydrolysis activity of trypsin on synthetic chromogenic substrate. This precursor deduced from the cDNA sequence is composed of 70 amino acid residues. The mature OGTI contains 17 amino acid residues including a six-residue loop disulfided by two half-cysteines (AVNIPFKVHFRCKAAFC). In addition to its unique six-residue loop, the overall structure and precursor of OGTI are different from those of other serine protease inhibitors. It is also one of the smallest serine protease inhibitors ever found.