Dependent Proteinase Activity Research Articles

Modulation of Ca(2+)-Dependent Proteiolysis under the Action of Weak Low-Frequency Magnetic Fields

The study aimed to determine the molecular targets of magnetic fields in living objects. Time-dependent effects of weak low-frequency magnetic field tuned to the parametric resonance for calcium ions were studied on model organisms (fish, whelk). The dynamics of Ca(2+)-dependent proteinase activity under the exposure to magnetic fields with given parameters was determined and minimal time of exposure in order to achieve inactivation of these proteinases was find out as well. As hyperactivation of Ca(2+)-dependent proteinases is a basis of degenerative pathology development the therapeutic potential of weak low-frequency magnetic fields enabling to modulate Ca(2+)-dependent proteinase activity is supported.

Active recombinant rat calpain II. Bacterially produced large and small subunits associate both in vivo and in vitro.

cDNA for the C-terminal Ca(2+)-binding domain of rat calpain small subunit was cloned by means of the polymerase chain reaction. The encoded protein (21 kDa), which corresponds closely to the natural autolysis product of the small subunit, was produced in soluble form in Escherichia coli at a level of 20 mg/liter of cell culture. Rat calpain II large subunit (80 kDa) was produced from a cDNA clone in E. coli in soluble form at a level of approximately 1 mg/liter. The 80-kDa subunit alone had no proteinase activity, with or without Ca2+, but Ca(2+)-dependent proteinase activity was obtained following association of the two subunits, which was achieved either by co-expression of the two subunit cDNAs in E. coli, or by mixing the two partially purified subunits in the presence of 1 M NaSCN followed by dialysis. The heterodimeric (80 + 21 kDa) proteinase had a Ca2+ requirement for 50% activity of 0.35 mM and a specific activity at 2 mM Ca2+ of approximately 1 unit/microgram, values essentially identical to those of natural (80 + 30 kDa) calpain II. The results establish association and biological activity of the bacterially produced subunits and provide a system for studying structure-function relationships in calpain by means of mutagenesis.

Structural and functional analysis of the human neutrophil 1-15 antigen, an Mr 65,000 to 70,000 activation-associated membrane proteinase.

Previous studies with the anti-neutrophil/antichymotrypsin mAb 1-15 have identified an activation-associated, chymotrypsin-like activity within the membrane fraction of isolated human neutrophils (PMN). In the present study, the molecular and biochemical characteristics of mAb 1-15 Ag/proteinase were determined. On casein/acrylamide sizing gels, PMN membrane preparations were found to contain an Mr 58,000 to 84,000 band of Ca2(+)-dependent proteinase activity. Reducing and nonreducing SDS-PAGE of mAb 1-15-affinity-purified membrane proteins demonstrated specific recovery of an enzymatically active Mr 65,000 to 70,000 chymotrypsin-like Ag. The presence of a distinct membrane serine esterase of isoelectric point 6.3/Mr 65,000 to 70,000 was confirmed in active site-labeling experiments with the serine proteinase inhibitor [3H]diisopropylfluorophosphate (DFP). Substrate-affinity chromatography with phe-Sepharose or FMLP-Sepharose provided partial purification of enzyme activity among Mr 65,000 to 70,000 FMLP- or phe-binding proteins. Enzyme inhibition was obtained by incubation with mAb 1-15, DFP, N-carbobenzoxyl-phe-chlormethyl ketone, or PMSF, but not tosyl-amide-phenylethylchlormethyl ketone, bestatin, aprotinin, or phosphoramidon. In HPLC analysis, [3H]DFP labeled proteinase was found to comigrate with one of three FMLP-affinity-labeled membrane peaks, but unlike the FMLP surface receptor the DFP-labeling membrane proteinase was not modified by endoglycosidase F. We conclude that the mAb 1-15 Ag, which appears to play a role in PMN activation, is a distinct, active, Mr 65,000 to 70,000 serine proteinase with affinity for substrate sites containing aromatic amino acids.

The Ca 2+-dependent proteolytic system—Calpain-calpastatin—In the neural tissue of the honeybee Apis mellifera

Ca 2+-dependent proteinase activity was detected in the neural tissue of the honeybee Apis mellifera. DEAE-chromatography revealed two Ca 2+-dependent proteinase activities, with elution positions corresponding to vertebrate calpain I and II. Both activities yielded apparent M r = 80,000 , had a strict requirement for Ca 2+ and a SH-reducing agent. PMSF and trypsin inhibitor did not affect the proteinase activities. Alkylating agents and mercaptide forming compounds as well as the calpain inhibitor calpastatin purified from bovine brain inhibited the activity. The natural inhibitor protein detected in the neural tissue of the honeybee showed properties equal to calpastatin from other sources. All observed properties are in accordance with those of the Ca 2+-dependent proteolytic system calpain-calpastatin.

Ca 2+-dependent proteolysis of the Ah receptor

We previously reported ( J. Biol. Chem. (1986) 261, 6352–6465) that the photoaffinity ligand for the Ah receptor, [ 125I]-2-azido-3-iodo-7,8-dibromodibenzo- p-dioxin, upon incubation with the liver cytosol fraction from C57BL/6 mice, labeled in a 1:1 ratio two peptides that had apparent molecular masses of 95 and 70 kDa and similar proteolytic fragmentation patterns. In the cytosolic fraction of Hepa 1 cells, a cloned murine hepatoma cell line, the product of photoaffinity labeling is almost exclusively a 95-kDa peptide which is rapidly hydrolyzed by a Ca 2+-dependent proteinase to a 70-kDa peptide as well as other fragments. Thus, the ligand binding unit of the Ah receptor in C57BL/6 mouse liver and Hepa 1 cell is a 95-kDa peptide, and the 70-kDa fragment is a proteolytic artifact. The Ca 2+-dependent proteinase which hydrolyzes the 95-kDa peptide has the properties of calpain II: (i) an absolute requirement for Ca 2+, with maximal activity at 0.5 to 1.0 m m Ca 2+; (ii) a pH optimum of 7.5 to 8.0; (iii) inhibition by EDTA, iodoacetamide, leupeptin and l- trans-epoxysuccinylleucylamido(4-guanidino)butane, but not by soybean trypsin inhibitor, aprotinin, or phenylmethanesufonyl fluoride. Upon Chromatographic separation of the liver cytosol of C57BL/6 mice on DEAE-Sephacel, Ca 2+-dependent proteinase activity (using casein or the labeled 95-kDa peptide as substrates) elutes with 0.25 m NaCl, and a specific proteinase inhibitor elutes with 0.15 m NaCl. Ca 2+-dependent proteinase activity that hydrolyzes the 95-kDa peptide is found in the liver cytosols of several mammalian species.

The reversible activation by Mn 2+ ions of the Ca 2+-requiring neutral proteinase of human erythrocytes

Mn 2+ (50 μ m) satisfies the requirement for activity of the purified Ca 2+-dependent neutral proteinase from human erythrocytes. Unlike the activation by Ca 2+ [ E. Melloni et al. (1984) Biochem. Int. 8, 477–489 ], the effect of Mn 2+ is fully reversible and does not involve autodigestion of the native 80-kDa catalytic subunit. However, the native dimeric proenzyme (procalpain), which contains both the 80-kDa subunit and a smaller 30-kDa subunit, is not activated by Mn 2+ alone but also requires the presence of micromolar concentrations of Ca 2+. Under these conditions, 40% of the maximum activity is expressed without dissociation of the 80- and 30-kDa subunits. Mn 2+, but not micromolar Ca 2+, can also partially satisfy the metal requirement of the native 80-kDa subunit isolated after dissociation of the heterodimer. This activity is further enhanced by the addition of 5 μ m Ca 2+, which is ineffective in the absence of Mn 2+. After procalpain is converted to active calpain by incubation with Ca 2+ and substrate [ S. Pontremoli et al. (1984) Biochem. Biophys. Res. Commun. 123, 331–337 ] full activity is observed with 5 μ m Mn 2+, which now substitutes completely for Ca 2+. Activation of procalpain by Mn 2+ represents a new mechanism for modulation of the Ca 2+-dependent proteinase activity.

Ca2+-dependent proteolytic activity in crab claw muscle. Effects of inhibitors and specificity for myofibrillar proteins.

The claw closer muscle of the Bermuda land crab, Gecarcinus lateralis, undergoes a sequential atrophy and restoration during each molting cycle. We describe here the role of Ca2+-dependent proteinases in the turnover of myofibrillar protein in normal anecdysial (intermolt) claw muscle. Crab Ca2+-dependent proteinase degrades the myofibrillar proteins actin, myosin heavy and light chains, paramyosin, tropomyosin, and troponin-T and -I. Ca2+-dependent proteinase activity in whole homogenates and 90,000 X g supernatant fractions from muscle homogenates has been characterized with respect to Ca2+ requirement, substrate specificity, and effects of proteinase inhibitors. The enzyme is inhibited by antipain, leupeptin, E-64, and iodoacetamide; it is insensitive to pepstatin A. The Ca2+-dependent proteinase is a sarcoplasmic cysteine proteinase that shows maximal activation at 1 mM Ca2+ at neutral pH. Since approximately 28% of the activity remains at 1.5 microM Ca2+, the enzyme is partially active at physiological Ca2+ concentrations. The specificity of crab Ca2+-dependent proteinase was examined with native myosin with normal ATPase activity as well as with radioiodinated myosin and radioiodinated hemolymph proteins. Hydrolysis of 125I-myosin occurs in two phases, both Ca2+-dependent: 1) heavy chain (Mr = 200,000) is cleaved into four large fragments (Mr = 160,000, 110,000, 73,000, 60,000) and numerous smaller fragments; light chain (Mr = 18,000) is cleaved to a 15,000-Da fragment; 2) the fragments produced in the first phase are hydrolyzed to acid-soluble material. Although radioiodinated native hemolymph proteins are not susceptible to the Ca2+-dependent proteinase, those denatured by carboxymethylation are degraded. These data suggest that crab Ca2+-dependent proteinase is involved in turnover of myofibrillar protein in normal muscle and muscle undergoing proecdysial atrophy.