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.     

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.     

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.     

Purification and characterization of a calcium-activated neutral protease from monkey cardiac muscle

A calcium-activated neutral protease (CANP) was purified from monkey cardiac muscle by a method involving column chromatography on DEAE-cellulose, Sepharose CL-6B, DEAE-Sephacel, organomercurial-Sepharose 4B, and Sephadex G-150 in succession. This protease required both millimolar concentration of Ca2+ and the SH-group for activation, and it was maximally active around pH 8.0. It was strongly inhibited by thiol protease inhibitors such as iodoacetic acid, antipain, leupeptin, and epoxysuccinic acid derivatives. The molecular weight of this protease was estimated to be 110,000 by gel filtration. Upon nondenaturing electrophoresis the purified protease gave two bands, both of which were active at millimolar concentration of Ca2+, indicating the existence of two forms of the protease. The less acidic band (form I CANP) contained two components with molecular weights of 74,000 and 28,000 and the more acidic one (form II CANP) contained components with molecular weights of 74,000 and 26,000. The protease was synergistically activated by Mn2+ and Ca2+ at a concentration where Mn2+ or Ca2+ alone was not effective. In the presence of millimolar level of Ca2+, limited autolysis reduced the Ca2+-requirement of this protease. The proteolysis of myofibrils by this protease resulted in the production of a component with a molecular weight of 30,000 as well as various other higher and lower molecular weight peptide fragments.     

Studies of a calcium-activated neutral protease from chicken skeletal muscle. I. Purification and characterization.

A calcium-activated neutral protease was purified 2,700-fold over the crude extract from chicken skeletal muscle. The purified protease migrated as a single band on polyacrylamide gel electrophoresis with or without SDS. Its molecular weight was 80,000 and pH optimum for activity was 7.7. The activity required strictly the presence of calcium (optimum concentration: 1.8 mM) or strontium (optimum concentration: 10 mM) ions. The protease was inhibited by leupeptin, which is known to be a strong inhibitor of papain, cathepsin B, trypsin, and plasmin.     

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.     

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.     

Purification and characterization of a calcium-activated neutral protease from monkey brain and its action on neuropeptides

A calcium-activated neutral protease was purified from Japanese monkey brain by ammonium sulfate fractionation and sequential column chromatographies monitored by assay of caseinolytic activity. The purified enzyme gave a single protein band on non-denaturing polyacrylamide gel electrophoresis, and consisted of two subunits with molecular weights of 74,000 and 20,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme required millimolar order calcium ions for activation, and was optimally active at pH 7.5-8.0. Upon incubation with various neuropeptides as substrates, the enzyme preferentially cleaved the peptide bonds with Arg, Lys, or Tyr at the P1 position and an amino acid residue with a bulky aliphatic side chain, such as Leu, Val, or Ile, at the P2 position. The hydrolytic activity toward neuropeptides as well as casein was strongly inhibited by various thiol protease inhibitors. These results suggested that the brain calcium-activated neutral protease may participate in the degradation of neuropeptides in vivo.     

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.     

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.     

Purification and characterization of dihydropyridine receptor from rabbit skeletal muscle

Digitonin and 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propane sulfonate (Chapso) were used to solubilize the receptor of dihydropyridine calcium antagonists from the transverse tubule membranes of rabbit skeletal muscle. The receptor retained the ability for selective adsorption from either detergent extract by dihydropyridine-Sepharose. Incubation of the affinity resin with nitrendipine resulted in the elution of the receptor protein composed of two main polypeptides with molecular masses of 160 kDa and 53 kDa, as shown by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Only these two subunits were found in the receptor preparation purified to a specific dihydropyridine-binding activity of 2500-2800 pmol/mg protein (60-70% purity) from digitonin-solubilized membranes by a combination of wheat-germ-agglutinin--Sepharose, anion-exchange and dihydropyridine-Sepharose chromatography steps. The individual subunits were isolated in dodecyl-sulfate-denatured form from the preparation of the receptor, enriched by a two-step large-scale procedure applied to Chapso-solubilized membranes. The 160-kDa subunit slowly changed its apparent molecular mass to 125 kDa upon disulfide bond reduction without formation of novel peptides. This finding implies that 160-kDa subunit is cross-linked by intramolecular S-S bridge(s). Chemical deglycosylation with trifluoromethanesulfonic acid showed that the carbohydrate content of large and small subunits accounted for 7.5% and 6.6% by mass, respectively. The dihydropyridine receptor subunits are glycosylated through N-glycoside bonds only. In their ratio of polar to hydrophobic amino acid residues in the amino acid composition of the receptor subunits, these polypeptides behave rather as peripheral proteins. It is suggested that the main portion of polypeptide chains is located outside the membrane in contact with solvent.     

Purification and characterization of peptidylarginine deiminase from rabbit skeletal muscle

The preceding paper described the identification and some properties of peptidylarginine deiminase, which catalyzes the deimination of arginyl residues in protein, from rabbit skeletal muscle, kidney, brain, and lung. In the present work we purified peptidylarginine deiminase from rabbit skeletal muscle with a 16% yield by 7 steps. The purification involved ion-exchange chromatography on DEAE-Sephacel, gel filtration on Bio-Gel A-0.5 m, and affinity chromatography on soybean trypsin inhibitor-Sepharose 4B and aminohexyl-Sepharose 4B. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate. The molecular weight of the enzyme was estimated to be about 83,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 130,000-140,000 by gel filtration on Sephadex G-200. The isoelectric point was 5.3 and the amino acid composition was also determined. The enzyme preferably catalyzed the formation of citrulline derivatives from arginine derivatives in which both the amino and carboxyl groups were substituted and showed the highest activity towards Bz-L-Arg-O-Et among the arginine derivatives tested. The Km value for Bz-L-Arg-O-Et was found to be 0.50 X 10(-3) M. The enzyme also showed marked activities towards native protein substrates, such as protamine sulfate, soybean trypsin inhibitor, histone and bovine serum albumin.     

Studies of a calcium-activated neutral protease from chicken skeletal muscle. II. Substrate specificity.

Calcium-activated neutral protease purified from chicken skeletal muscle hydrolyzed myofibrillar proteins, tubulin, spectrin, and oxidized insulin B chain, but hardly hydrolyzed synthetic or natural peptides.     

Purification and partial characterization of a myofibril-bound serine protease from ostrich skeletal muscle

A myofibril-bound serine protease (MBSP) was partially purified from ostrich (Struthio camelus) skeletal muscle. MBSP was dissociated from the myofibrillar fraction by ethylene glycol treatment at pH 8.5, followed by partial purification via Toyopearl Super Q 650 S and p-aminobenzamidine column chromatographies. Ostrich MBSP revealed a major protein band of approximately 21 kDa on SDS-PAGE, showing proteolytic activity after casein zymography. Optima pH and temperature of ostrich MBSP were 8 and 40 °C, respectively. Substrate specificity analysis revealed that the enzyme cleaved synthetic fluorogenic substrates at the carboxyl side of arginine residues. Kinetic parameters (K_m and V_max values) were calculated from Lineweaver–Burk plots. The kinetic characteristics of ostrich MBSP were compared to values obtained for commercial bovine trypsin in this study, as well as those obtained for MBSP from mouse and various fish species. The results suggest that ostrich MBSP is a tryptic-like serine protease. Ostrich MBSP exhibited low sequence identity to commercial bovine trypsin (44%), MBSP from lizard fish skeletal muscle (33%) and trypsinogen from ostrich pancreas (22%).     

Purification to homogeneity and partial characterization of a 56,000-dalton protein phosphatase from rabbit reticulocytes

A 56,000-Da peptide with inherent protein phosphatase activity was isolated from the postribosomal supernatant fraction of rabbit reticulocytes. The peptide appears to form complexes with other proteins that are present in crude fractions. It exhibits atypical retention on steric exclusion columns during high performance liquid chromatography, an unusual characteristic that facilitated its isolation. The protein phosphatase activity of the 56,000-Da peptide is dependent on Mn2+ ions, but is not activated by either the FA, ATP/Mg2+ protein phosphatase activator system or by proteolysis. The protein phosphatase activity of the peptide is increased 3-fold or more by the antigen peptides described in the accompanying paper (Fullilove, S., Wollny, E., Stearns, G., Chen, S.C., Kramer, G., and Hardesty, B. (1984) J. Biol. Chem. 259, 2493-2500).     

Immunocytochemical localization of a calcium-activated protease in skeletal muscle cells

The 80 000-D subunit of a calcium-activated protease from skeletal muscle was purified to homogeneity using preparative sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and was used to elicit antibody production in rabbits. Antiserum was purified using affinity chromatography to yield a monospecific antibody fraction (anti-80K) directed against the 80 000-D subunit. Localization studies showed that the 80 000-D subunit is present in or near the sarcolemma of cultured myoblasts and sectioned muscle tissue, in discrete areas of the cytoplasm of myoblasts, and in the Z disks of the myofibril. The location of the calcium-activated protease in the cell suggests that the enzyme may be involved in myofibril degradation and in membrane alterations in developing and mature muscle cells.     

Specificity of neuropeptide degradation by two calcium-activated neutral proteases from human skeletal muscle

Two calcium-activated neutral proteases (CAPI & II) were purified from human skeletal muscle by anion exchange, gel filtration and affinity (antipain-Sepharose and Blue Ultrogel A4R) chromatography. The enzymes were homogenous as judged by polyacrylamide gel electrophoresis, and have similar properties with the exception of the Ca2+ concentration required for optimum activity (CAP I = 0.1 mM; CAP II = 1 mM). Both enzymes hydrolysed a wide variety of neuropeptides. In six cases, the products were separated and identified by hplc and amino acid analysis. Neurotensin was hydrolysed at Tyr3-Glu4; dynorphin1-13 at Arg8-Arg9; LH-RH at Gly6-Leu7; CCK-8 at Phe8-NH2, substance-P at Met10-NH2; somatostatin at Thr10-Phe11. Although differences in the rates of neuropeptide degradation were noted for the two CAP's the specificity was the same for these six peptides. It is suggested that conformational requirements may be more important than side chains adjacent to the cleavage site in directing the specificity of CAP.