All four repeating domains of the endogenous inhibitor for calcium-dependent protease independently retain inhibitory activity. Expression of the cDNA fragments in Escherichia coli

We have already determined the primary structure of the endogenous inhibitor for calcium-dependent protease (CANP inhibitor, calpastatin) from the cDNA sequence and revealed that the CANP inhibitor contains four internally repeating units which could be responsible for its multiple reactive sites (Emori, Y., Kawasaki, H., Imajoh, S., Imahori, K., and Suzuki, K. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 3590-3594). Restriction fragments of the cDNA corresponding to each of the four domains (encoding 104-156 amino acid residues of the total 718 residues) were subcloned into the multicloning site of pUC9 or pUC18 in a direction and frame matched to the lacZ' open reading frame of the vector. Under the lac operator-promoter system, we succeeded in producing truncated fragments of the CANP inhibitor in Escherichia coli. The CANP inhibitor fragments were partially purified, and the inhibitory activities toward calcium-dependent protease (CANP) were examined. All fragments containing well conserved regions of about 30 amino acid residues (domains I-IV) located in the middle of the four units exhibited the inhibitory activity. However, their inhibitory activities varied considerably. Further truncation experiments revealed that small fragments containing 30-70 amino acid residues of the CANP inhibitor still retained inhibitory activity. From these experimental results the following conclusions can be drawn: 1) each of the four repeating units of the CANP inhibitor (about 140 amino acid residues) is a real functional unit and can inhibit CANP activity independently; and 2) domains corresponding to well conserved sequences of about 30 amino acid residues containing a consensus Thr-Ile-Pro-Pro-X-Tyr-Arg sequence are essential for the inhibitory activity, and the bordering regions are important for its modulation.     

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.     

Identification and characterization of inhibitory sequences in four repeating domains of the endogenous inhibitor for calcium-dependent protease

We reported previously the cDNA cloning of the endogenous inhibitor for calcium-dependent protease (CANP inhibitor, calpastatin) and the expression of its fragments in Escherichia coli. The CANP inhibitor has four internal repeating domains each spanning about 140 amino acid residues. The inhibitory activity arises from these domains which have a well-conserved sequence, TIPPXYR, in their central positions. The inhibitory activities of various fragments expressed in E. coli suggest the involvement of the regions around the well-conserved sequences. In this report, we describe further detailed investigation on the interaction site of the CANP inhibitor with CANP by truncating inhibitor fragments and by using chemically synthesized peptides. The results clearly indicate that the sequence around the well-conserved sequence, TIPPXYR, is an interaction site. A peptide as short as 23 amino acid residues retained inhibitory activity, but a 9-residue peptide corresponding to the conserved sequence, VTIPPKYRE had none. The inhibitory sequence is suggested as LGXKDREXTIPPXYRXLL. The analysis of the competition between an inhibitor peptide and an irreversible inhibitor, E-64 for the reaction with the active site suggests no involvement of the active site cysteine residue of CANP in the inhibitory interaction between CANP and the CANP inhibitor. The high specificity of the CANP inhibitor to CANP arises from its interaction with residues other than the active site cysteine residue, possibly the subsite for substrate-binding of CANP.     

Calcium-activated neutral protease and its endogenous inhibitor in tissues of dystrophic and normal mice

Calcium-activated neutral protease (milli-CANP) and its endogenous inhibitor are elevated in muscle tissues, primarily the skeletal muscle and heart, of dystrophic mice (C57BL/6J dy/dy) as compared to the control strain (C57BL/10J). Tissues showing relative increase of CANP also show significant loss of enzymes such as CK, LDH in comparison to plasma, where these enzymes register a significant increase. PK is lost minimally from these tissues, probably showing a "sparing effect." Absence of any significant change in CANP activity in the liver points to a specific role of CANP in the dystrophic process. In the skeletal muscle the endogenous CANP inhibitor registers a concomitant increase with CANP without altering the enzyme/inhibitor ratio.     

A protein inhibitor of the mitochondrial adenosine triphosphatase complex of rat liver. Purification and characterization.

A heat-stable protein has been purified from rat liver mitochondria which inhibits the ATP hydrolytic activity of both the soluble and membrane-bound mitochondrial F1-ATPase. The overall purification is about 2400-fold with the major purification step consisting of Sephadex "affinity" chromatography. The purified rat liver inhibitor is homogeneous as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 12,300. Amino acid analysis reveals a high content of glutamic acid, lysine, and arginine and the absence of cysteine, proline and methionine. Whether tested with the rat liver or bovine heart ATPase, the liver inhibitor is equally as potent and specific as the heart inhibitor preparation of Pullman and Monroy (Pullman, M.E., and Monroy, G.C. (1963) J. Biol. Chem. 238, 3762-3769). Although the results presented show that the rat liver ATPase inhibitor resembles closely the ATPase inhibitors from other tissues with respect to specific activity and reaction specificity, it is important to note that the rat liver inhibitor is almost 2000 daltons larger than the bovine heart inhibitor, about 5000 daltons larger than ATPase inhibitors of yeast, and contains significantly more lysine residues than both the bovine heart and yeast inhibitors.     

Purification and characterization of thiol proteinase inhibitor from rat liver

A thiol proteinase inhibitor was purified from rat liver by essentially the same procedure as reported previously (Kominami, E., Wakamatsu, N., and Katunuma, N. (1981) Biochem. Biophys. Res. Commun. 99, 568-575), but without heat treatment. The purified inhibitor appears homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate and displayed no multiple forms. The inhibitor has Mr = 12,500 and contains 50.5% of polar amino acid residues, 9.3% aromatic amino acids, and no tryptophan. The presence of 2 half-cystines/molecule and the absence of free thiol groups indicate that the inhibitor possesses one disulfide bridges. The inhibitor inhibits cathepsin H by forming an enzyme-inhibitor complex in a molar ratio of 1:1. It inhibits most thiol proteinases such as cathepsin H, L, B, and C, papain, and ficin, but not calcium-activated neutral proteinase or serine proteinases or carboxyl proteinases. The inhibitor was found in various rat tissues. Immunological diffusion analysis with anti-liver thiol proteinase inhibitor serum indicated that the rat liver inhibitor is immunologically identical with the inhibitors from other rat tissues. On subcellular fractionation of rat liver, the thiol proteinase inhibitor was recovered in the cytosol fraction.     

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 an inhibitor of calcium-activated neutral protease from rabbit skeletal muscle: purification of 50,000-dalton inhibitor

An endogenous inhibitor of calcium-activated neutral protease (CANP), which was isolated from rabbit skeletal muscle under mild conditions, comprised high- and low-molecular-weight components. The latter (LMW-inhibitor; Mr=50,000) was purified to homogeneity by means of chromatography on DEAE-cellulose and phenyl-Sepharose CL-4B and chromatofocusing. The purified inhibitor is a protein composed of two polypeptide chains with molecular weights of 26,000 and 24,000 daltons. It contains large amounts of glutamic acid, alanine, and serine, and small amounts of aromatic amino acids. It was specific for CANPs having low (m-type) and high (mu-type) Ca2+-sensitivity, had no effect on any other protease examined (trypsin, alpha-chymotrypsin, bromelain, ficin, papain, thermolysin, etc.), and inhibited rabbit mCANP more effectively than rabbit muCANP or chicken mCANP. It was demonstrated that the inhibition is due to the formation of a stoichiometric complex between two molecules of rabbit mCANP and one inhibitor molecule.     

Activation of intracellular calcium-activated neutral proteinase in erythrocytes and its inhibition by exogenously added inhibitors

Intracellular calcium-activated neutral proteinase (CANP) in rabbit erythrocytes was activated by an influx of Ca2+ into the cells. The catalytic large subunit changed from the original 79 kDa from to the 77 kDa and 76 kDa forms on activation just in the same manner as occurs in the autolytic activation of purified CANP in vitro. The activation required both extracellular Ca2+ and A23187, and was accompanied by the degradation of some membrane proteins and morphological changes in erythrocyte shape from discocytes to echinodisks, echinocytes, and spherocytes. Exogenously added Cbz-Leu-Leu-Leu-aldehyde inhibited the activation of intracellular CANP as well as the degradation of membrane proteins and the morphological changes indicating that the latter two processes are due to the action of CANP. Leupeptin and E64d were without effect on intracellular CANP.     

Hepatic proliferation inhibitor

Summary Although a long held tenet of biology has been that endogenous inhibitors can modulate cell proliferation, little progress was made in purifying any such inhibitor. This was largely due to the rarity of non-malignant cell cultures in which regulation of cell division was still operative, and to problems in separating cytotoxic and cytostatic effects in the complex biological extracts which were being studied. During the last decade, hepatic proliferation inhibitors of varying degrees of purity have been isolated using regenerating rat liver or hepatoma cell cultures as test systems. In these early studies, a number of inhibitors with differing molecular weights, physicochemical properties and biological responses were purified from liver cytosol and/or serum. Some of them could inhibit DNA synthesis or mitosis and thus were considered to be G1 or G2 inhibitors. However, experiments which could give precise answers about mechanisms of action could not be done until an inhibitor purified to homogeneity was available.Using well-characterized rat liver diploid epithelial cell cultures, which maintain a number of liver properties and which do not possess any transformation markers or malignant properties, we recently purified an hepatic proliferation inhibitor to a homogenous protein. It has a molecular weight of 26 000 daltons and an isoelectric point of 4.65. It specifically inhibits cell division and DNA synthesis in a number of non-malignant rat liver epithelial cell types, and has no effect on transformed liver cells, or hepatoma cells, in culture. Its effect is not mediated through destruction or sequestration of essential nutrients or calcium ions. Nor have preliminary experiments shown the hepatic proliferation inhibitor to interfere with the binding of epidermal growth factor to its receptors. The majority of the cells treated with the inhibitor are blocked in the G1 phase. Further experiments to study its mechanism of action and the inter-relationship, if any, between the cell cycle block induced by serum or nutrient deprivation, and the inhibitor-induced cycle block are in progress.     

Human plasma alpha 1- and alpha 2-thiol proteinase inhibitors strongly inhibit Ca-activated neutral protease from muscle

Human plasma alpha 1- and alpha 2-thiol proteinase inhibitors (alpha 1,2TPIs) inhibited purified Ca-activated neutral protease (CANP) most strongly among a number of thiol proteinases tested. When CANP was added to plasma, it was also inhibited by alpha 2-macroglobulin (alpha 2M). At low CANP concentrations, CANP was bound mainly to alpha 1,2TPIs; and after saturation of alpha 1,2TPIs the additional CANP was bound to alpha 2M. These data suggested that a probable role of alpha 1,2TPIs is to neutralize the proteolytic activity of the CANP derived from the tissues in collaboration with alpha 2M.     

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.     

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.     

Pectin methylesterase inhibitor

Pectin methylesterase (PME) is the first enzyme acting on pectin, a major component of plant cell wall. PME action produces pectin with different structural and functional properties, having an important role in plant physiology. Regulation of plant PME activity is obtained by the differential expression of several isoforms in different tissues and developmental stages and by subtle modifications of cell wall local pH. Inhibitory activities from various plant sources have also been reported. A proteinaceous inhibitor of PME (PMEI) has been purified from kiwi fruit. The kiwi PMEI is active against plant PMEs, forming a 1:1 non-covalent complex. The polypeptide chain comprises 152 amino acid residues and contains five Cys residues, four of which are connected by disulfide bridges, first to second and third to fourth. The sequence shows significant similarity with the N-terminal pro-peptides of plant PME, and with plant invertase inhibitors. In particular, the four Cys residues involved in disulfide bridges are conserved. On the basis of amino acid sequence similarity and Cys residues conservation, a large protein family including PMEI, invertase inhibitors and related proteins of unknown function has been identified. The presence of at least two sequences in the Arabidopsis genome having high similarity with kiwi PMEI suggests the ubiquitous presence of this inhibitor. PMEI has an interest in food industry as inhibitor of endogenous PME, responsible for phase separation and cloud loss in fruit juice manufacturing. Affinity chromatography on resin-bound PMEI can also be used to concentrate and detect residual PME activity in fruit and vegetable products.     

Calcium-activated neutral protease and its endogenous inhibitor Activation at the cell membrane and biological function

The structures of calcium-activated neutral protease (CANP) and its endogenous inhibitor elucidated recently have revealed novel features with respect to their structure-function relationship and enzyme activity regulation. The protease is regarded as a proenzyme which can be activated at the cell membrane in the presence of Ca²⁺ and phospholipid, and presumably regulates the functions of proteins, especially membrane-associated proteins, by limited proteolysis. Protein kinase C is hydrolysed and activated by CANP at the cell membrane to a cofactor-independent form. These results are reviewed and the possible involvement of CANP in signal transduction is discussed.     

Molecular cloning and functional expression of cDNA encoding the cysteine proteinase inhibitor with three cystatin domains from sunflower seeds

Two cysteine proteinase inhibitors, cystatins Sca and Scb, were previously isolated from sunflower seeds [Kouzuma et al. J. Biochem. 119 (1996) 1106-1113]. A cDNA clone encoding a novel phytocystatin with three repetitive cystatin domains was isolated from a cDNA library of sunflower seeds using the Sca cDNA fragment as a hybridization probe. The cDNA insert comprises 1,093 bp and encodes 282 amino acid residues. The deduced amino acid sequences of the domains are highly similar to each other (66-81%), sharing 65-90% identical residues with Sca. The cDNA was expressed in Escherichia coli cells, and then the recombinant sunflower multicystatin (SMC) was purified and its inhibitory activity toward papain was examined. SMC exhibited strong inhibitory activity toward papain, with a stoichiometry of 1:3, indicating that each cystatin domain independently functions as a potent cysteine proteinase inhibitor. Proteolysis of SMC with Asn-specific proteinase suggested that post-translational processing by an Asn-specific proteinase may give rise to mature Sca-like phytocystatins.     

Theromin, a novel leech thrombin inhibitor

We purified the most potent thrombin inhibitor described to date from the rhynchobdellid leech Theromyzon tessulatum. Designated theromin, it was purified to apparent homogeneity by gel permeation and anion exchange chromatography followed by two reverse-phase steps of high performance liquid chromatography. The primary sequence of theromin (a homodimer of 67 amino acid residues including 16 cysteine residues) was determined by a combination of reduction and s-beta-pyridylethylation, Edman degradation, trypsin enzymatic digestion, and matrix-assisted laser desorption mass spectrometry measurement. Theromin exhibits no sequence homology with any other thrombin inhibitors. Furthermore, theromin significantly diminishes, in a dose-dependent manner, the level of human granulocyte and monocyte activation induced by lipopolysaccharides. In summary, this potent thrombin inhibitor promises to have high biomedical significance.     

A protein modulator of erythrocyte membrane (Ca2+ + Mg2+)-ATPase inhibitor protein

A protein modulator of erythrocyte membrane (Ca²⁺ + Mg²⁺)-ATPase inhibitor protein was purified to apparent homogeneity from pig membrane-free hemolysate by a combination of carboxymethyl-Sephadex chromatography, gel filtration, chromatofocusing (pH 7-4) and subsequent removal of trace inhibitor protein by salt treatment. Gel filtration gave a molecular weight of 57 500 for the purified protein modulator, while SDS-polyacrylamide gel electrophoresis of dithiothreitol-treated modulator revealed one single band with a molecular weight of 29 000. Isoelectric focusing of the dithiothreitol-treated protein revealed one band (isoelectric pH 4.85), while untreated modulator gave an extra band (isoelectric pH 4.96). It contains no methionine and has an acidic content 73% higher than that of its basic residues. Freshly prepared or dithiothreitol-treated modulator suppressed both pig and human erythrocyte (Ca²⁺ + Mg²⁺)-ATPase inhibitor protein activity, but did not affect ATPase and calmodulin activities. Modulator-coupled Affi-Gel 15 could be employed for purification of the protein inhibitor.     

Trypsin inhibitor from the seeds of Acacia confusa.

A trypsin inhibitor (ACTI) was isolated and purified from the seeds of Acacia confusa by gel filtration, and trypsin-Sepharose 4B column affinity chromatography. The molecular weight of ACTI was found to be 21,000 +/- 1,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid composition analysis. ACTI contained four half-cystine and no methionine residues, and was rich in aspartic acid, glutamic acid, glycine, leucine, and lysine residues. The native trypsin inhibitor was composed of two polypeptide chains, and it inhibited trypsin and alpha-chymotrypsin stoichiometrically at the molar ratio of 1:1 and 2:1, respectively. The amino-terminal sequence analysis of the A. confusa trypsin inhibitor A and B chains revealed a more extensive homology with Acacia elata and silk tree trypsin inhibitors, and a less extensive homology with Kunitz soybean trypsin inhibitor.     

Trypsin reactivity site of the Vicia angustifolia proteinase inhibitor

Trypsin [EC 3.4.21.4] modified (reactive site cleaved) Vicia angustifolia proteinase inhibitor was prepared at pH 3 with a catalytic amount of trypsin and purified using columns of Sephadex G-50 and DEAE-Sephadex A-25. The modified inhibitor, which still retained antitryptic activity, lost its activity upon treatment with carboxypeptidase B or citraconic anhydride. End-group analyses revealed that the carboxyl-terminal Arg and the amino-terminal Ser residues were newly exposed end-groups in the modified inhibitor. It takes a much longer incubation time (about 1 h) to exhibit the maximal inhibitory activity against trypsin. Reduction and carboxymethylation of the modified inhibitor produced two fragments on Sephadex G-50 chromatography. The smaller fragment consisted of about 32 amino acid residues and possessed a new carboxyl-terminal Arg residue. The larger fragment consisted of about 80 residues and possessed a Ser residue at its amino-terminus. These results indicate that the small fragment was derived from the amino-terminal portion of the modified inhibitor and the large fragment from the carboxyl-terminal. It is also concluded that an Arg-Ser bond is the reactive site as well as the inhibitory site of the V. angustifolia inhibitor against trypsin. The sequence around the antitryptic site exhibits high degrees of homology with other double-headed inhibitors of legume origin, such as the Bowman-Birk inhibitor, lima beam inhibitor, and the major inhibitor in chick-peas.