[Calpain and alpha-crystallin in the lens of cac mouse].
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Aging↗
Biomedical subjects
T Murachi
Publications and source records attributed to T Murachi.
Changes in contents of calpain and calpastatin in rat liver during growth.
Variation of calpain I, calpain II, and calpastatin in rat liver during growth from 0 to 14 weeks was studied by chromatographic fractionation of the liver cytosol and enzyme assays on the eluted fractions. When compared in terms of units per g wet liver, high-Ca2+-requiring calpain II always exceeded low-Ca2+-requiring calpain I in male and female rats. The level of calpain II in neonatal (0 week) rat liver was 1.9-2.9 times higher than that for the adults (7 to 14 weeks). The contents of calpastatin, calpain-specific inhibitor protein, were were always higher than those of calpain II in adult rat liver, but the difference was much less, or sometimes even reversed, in neonatal and young (1 and 2 weeks) animals. In general, the variation was more pronounced in female than in male rats.
Aging↗
Direct spectrophotometry of magnesium in serum after reaction with hexokinase and glucose-6-phosphate dehydrogenase.
We describe a simple method for determining magnesium in serum by using hexokinase (EC 2.7.1.1) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49). The method is based on determination of the reaction rate of hexokinase activated by Mg2+, which participates in the hexokinase reaction as the substrate in the form of a Mg X ATP2- complex. The reaction rate is determined from the change in absorbance at 340 nm as NADPH is produced by the action of glucose-6-phosphate dehydrogenase. This simple and rapid spectrophotometric method does not require expensive instrumentation, but results correlate satisfactorily with those obtained by atomic absorption spectroscopy. Thus, the present method gives a "true" value for magnesium in serum, a value appreciably lower than that obtained by an earlier colorimetric method, the Xylidyl Blue II method (Biochem Med 7: 208-217, 1973), which lacks specificity.
Adenosine Triphosphate↗
Reconstitution of calpain I and calpain II from their subunits: interchangeability of the light subunits.
Calpain (Ca2 +-dependent cysteine proteinase) is known to be a heterodimer, composed of one heavy (called 80K) and one light (called 30K) subunit. Calpain I, a low-Ca2 +-requiring form from porcine and human erythrocytes, and calpain II, a high-Ca2 +-requiring form from porcine kidney, were separated into their respective 80K and 30K subunits by high-performance liquid chromatography on a TSK-Gel G3000SW column in 1 M NaSCN. The isolated 80K subunits from porcine calpains I and II showed Ca2 +-dependent proteolysis, though much depressed and requiring higher Ca2 + concentrations compared with the respective parent calpains. The optimal conditions were set for the reconstitution of a heterodimeric molecule from the once denatured and separated 80K and 30K subunits. In such reconstitution, the 30K subunits of calpains I and II were found to be functionally identical and interchangeable; either a homologous or heterologous 30K subunit lowered the Ca2 + requirement of an 80K subunit significantly and enhanced the proteolytic activity. Subunit interchange could also be demonstrated between porcine and human calpains.
Animals↗
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.
Amino Acid Sequence↗
Calpain and calpastatin in porcine retina. Identification and action on microtubule-associated proteins.
Two forms of Ca2+-dependent cysteine proteinase (calpain, EC 3.4.22.17) and their specific endogenous inhibitor (calpastatin) were partially purified from porcine retina: calpain I (low-Ca2+-requiring form) was half-maximally activated at 8 microM-Ca2+, and calpain II (high-Ca2+-requiring form) at 250 microM-Ca2+. Both calpain I and calpain II were inhibited by calpastatin. Calpain I from porcine retina was shown to be composed of 83 000- and 29 000-Mr subunits, and calpain II of 80 000- and 29 000-Mr subunits, by the use of monospecific antibodies. Calpains I and II were both found to hydrolyse microtubule-associated proteins 1 and 2 rapidly.
Animals↗
Molecular diversity of calpastatin in mammalian organs.
The crude homogenates of various human and porcine organs were subjected to immunoelectrophoretic blot analysis using affinity-purified anti-calpastatin antibody which specifically reacts with human erythrocyte 70 kDa calpastatin. Multiple immuno-reactive bands were revealed which ranged from 100 to 50 kDa. The results indicated the diversity of monomeric calpastatin molecules. The band patterns were different from one organ to the other. Among them, lung, heart and skeletal muscle were characterized by the predominance of 90-100 kDa calpastatin, having a common antigenicity to erythrocyte 70 kDa calpastatin. Such molecular diversity of calpastatins was also substantiated by enzymatic and chromatographic analyses.
Animals↗
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.
Animals↗
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.
Antibodies, Monoclonal↗
Degradation of actin and vimentin by calpain II, a Ca2+-dependent cysteine proteinase, in bovine lens.
Calpain II, a high Ca2+-requiring form of Ca2+-dependent cysteine proteinase (EC 3.4.22.17), isolated from bovine lens was found to cleave actin and vimentin, two major cytoskeletal elements of the lens. Polyacrylamide gel electrophoresis revealed that actin (Mr 43 000) was broken down through intermediary products of approximate Mr 42 000 and 40 000, while vimentin (Mr 57 000) was rapidly cleaved into several fragments ranging from Mr 44 000 to 20 000. The cleavage was dependent on Ca2+ and could be blocked by calpastatin , a calpain-specific inhibitor. These findings suggest that calpain might play a role in age-related degradation of the lens cytoskeleton.
Actins↗
Limited proteolysis of bovine lens alpha-crystallin by calpain, a Ca2+-dependent cysteine proteinase, isolated from the same tissue.
A Ca2+-dependent cysteine proteinase (calpain, EC 3.4.22.17) was found in the cystosolic fraction of bovine lens and purified to apparent homogeneity. The purified enzyme required 1 mM Ca2+ for its full activation and was composed of two subunits of Mr 80 000 and 29 000 as demonstrated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). This enzyme, when activated by Ca2+, degraded both A- and B-chains of alpha-crystallin, which were isolated also from bovine lens. SDS-gel electrophoresis of the digest revealed that the A-chain (Mr 19 500) was broken down to produce an 18-kDa polypeptide fragment and the B-chain (Mr 22 500) to produce a 19.5-kDa polypeptide fragment. No further cleavage occurred even upon prolonged incubation or after the second addition of the enzyme, indicating the uniquely limited proteolysis of each chain protein. The existence of calpastatin, an endogenous inhibitor protein specific for calpain, was also demonstrated in bovine lens cytosol.
Animals↗
Action of calpain on the basic estrogen receptor molecule of porcine uterus.
Basic estrogen receptor (ER) molecule (vero-ER) of the cytosol of porcine uterus was purified 1,200-fold after successive chromatographies on phenyl-Sepharose, hydroxylapatite, and DEAE-cellulose, followed by Sephadex G-150 gel filtration. The purified vero-ER was completely free from endogenous protease and ER-binding factor. The action of Ca2+-dependent cysteine proteinase (calpain) on vero-ER was studied by utilizing the purified receptor and calpains from porcine uterus (endogenous calpain), porcine kidney, and human erythrocytes. Proteolysis of vero-ER was followed by monitoring the disappearance of the binding capability of vero-ER with "8S" ER-forming factor. Vero-ER was proteolyzed by both the endogenous and the exogenous calpains in the presence of Ca2+. The calpains did not attack vero-ER in the absence of Ca2+. The results indicated the absolute requirement by calpain for Ca2+ for the limited hydrolysis of vero-ER. Uterine cytosol was shown to contain, in parallel with calpain, a protease which does not require Ca2+ for the limited proteolysis of vero-ER. The strongly hydrophobic domain of vero-ER, recently shown to be indispensable for the nuclear translocation of vero-ER (Murayama, A. & Fukai, F. (1983) FEBS Lett. 158, 255), was preferentially destroyed by both the Ca2+-requiring and -nonrequiring enzymes. It was assumed that calpain might intervene in the estrogen action by diminishing irreversibly the amount of the cytoplasmic ER capable of translocating into the nucleus.
Animals↗
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.
Amino Acids↗
Large-scale purification of porcine calpain I and calpain II and comparison of proteolytic fragments of their subunits.
Large-scale purification of calpain [Ca2+-dependent cysteine proteinase; EC 3.4.22.17] from porcine tissues is described. The methods used included chromatographies on DEAE-cellulose, Ultrogel AcA 34, Blue Sepharose CL-6B, and DEAE Bio-Gel A which yielded homogeneous enzyme proteins: 27.0 mg of calpain I (low Ca2+-requiring form) from 5 liters of blood with 17,900-fold purification and 57.6 mg of calpain II (high Ca2+-requiring form) from 1.5 kg of kidneys with 5,800-fold purification. Porcine calpains I and II are half-maximally activated at 2.8 microM and 150 microM Ca2+, respectively. They are composed of large and small subunits: Mr 83,000 and 29,000 for calpain I and Mr 80,000 and 29,000 for calpain II. Gel-electrophoretic analysis of the digest with a-chymotrypsin or Staphylococcus aureus V8 protease revealed that the large subunits of calpains I and II are markedly different in structure whereas the small subunits are most likely identical. Mono-specific antibodies directed toward the respective large and small subunits were used for immunoblotting experiments which established not only the identity among several porcine tissues of calpain I but also that of calpain II. several porcine tissues of calpain I but also that of calpain II.
Animals↗
Enzyme immunoassay of calpain I and calpastatin and its application to the analysis of human erythrocyte hemolysate.
A highly sensitive sandwich enzyme immunoassay for a Ca2+-dependent cysteine proteinase (calpain I) and its specific endogenous inhibitor protein (calpastatin) was developed. The calpain I and calpastatin used as immunogens were purified from human erythrocytes. Anti-calpastatin antisera having sufficiently high titer were obtained only when the immunogen was purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The assay method was principally based on the report by M. Imagawa et al. (1982, J. Appl. Biochem. 4, 41-57), using a specific antibody-coated polystyrene ball and horseradish peroxidase-conjugated Fab' fragment of the antibody. The sensitivity was 0.1 ng of calpain I or calpastatin per assay tube. Starting with 50 microliter of the hemolysate from human erythrocytes, the method permitted direct and simultaneous determination of calpain I and calpastatin, without prior separation of these two enzymatically counteracting components by chromatography. The present method as applied to the erythrocytes from 14 healthy adults gave 120-170 micrograms for calpain I and 164-211 micrograms for calpastatin per gram of hemoglobin, respectively.
Calcium-Binding Proteins↗
Fragmentation of a 70000-dalton calpastatin molecule upon its complex formation with calpain.
Homogenously purified porcine calpain I (Mr 112000), a low-Ca2+-requiring form of Ca2+-dependent cysteine proteinase [EC 3.4.22.17], was coupled to Sepharose 4B gel as an active form. It was used as a ligand to calpastatin (Mr 70000), calpain-specific inhibitor protein, for an affinity chromatography. Only in the presence of Ca2+, calpastatin bound to calpain-Sepharose, but the interaction resulted in rather extensive fragmentation of a calpastatin molecule into several peptides of Mr 14000 to 70000, which still retain inhibitory activities against calpain. Fragmentation was demonstrated both by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and by high-performance liquid chromatography in the presence of 6 M guanidine-HCl.
Animals↗
Calcium-dependent proteinases and specific inhibitors: calpain and calpastatin.
There are two forms of Ca2+-dependent cysteine proteinase: low Ca2+ (microM concentration)-requiring calpain I and high Ca2+ (mM concentration)-requiring calpain II. We have recently succeeded in establishing an efficient method for isolating calpains I and II from several tissues and also a method for separating the heavy (80 kDa) and light (30 kDa) subunits from a calpain molecule. These methods enabled us to obtain a variety of calpains I and II and their subunit proteins each in a highly pure state so that these proteins can be compared physically, chemically and immunologically. Calpastatin, a calpain-specific endogenous inhibitor protein, was also purified and characterized. Although the intercellular localization of calpains I and II is being elucidated by use of the respective monospecific antibodies, the true biological functions of calpain and calpastatin still remain rather obscure.
Animals↗
A mitogenic factor, released by stimulated human mononuclear cells and distinct from interleukin 2 (IL 2), B cell growth factor (BCGF), and interleukin 1 (IL 1).
Two lymphocyte mitogenic factors, interleukin 2 (IL 2) and blastogenic factor (BF), are generated concomitantly in human mixed lymphocyte cultures (MLC). The latter mitogenic factor is directly mitogenic for unstimulated lymphocytes, whereas the former mitogenic factor acts only on previously activated lymphocytes. Both factors had a m.w. range, as determined by gel filtration, of 18,000 to 30,000. Thus, these two factors were inseparable on the basis of m.w. size. However, BF and IL 2 were separable during ion exchange chromatography on the DEAE cellulose and phenyl-Sepharose chromatography. In addition, BF activity in the supernatants of MLC reached a maximum after day 5, whereas IL 2 activity peaked at day 3, thus distinguishing BF from IL 2 kinetically. These results clearly indicate that BF activity is mediated by molecules distinct from IL 2. The biochemical relationship between B cell growth factor (BCGF) and BF was also examined. Because BF was readily separable from BCGF by Con A-Sepharose chromatography, BF is distinguishable from BCGF. No augmentation of PHA-stimulated C3H mouse thymocyte proliferation was associated with the preparation of partially purified BF, demonstrating that BF and IL 1 are distinct molecules. Taken together, these results indicate that BF is clearly distinct from IL 2, BCGF, and IL 1. BF-containing MLC supernatants have direct mitogenic activity on both T and B cells. Both T and B cell blastogenic activities copurified during ammonium sulfate precipitation, gel filtration, DEAE cellulose ion exchange chromatography, and hydrophobic chromatography. Thus, these two activities appear to be biochemically inseparable. Monoclonal anti-Tac, that has been suggested to recognize the receptor for human IL 2, was highly inhibitory to the T cell response to the phenyl-Sepharose preparations of BF (IL 2-free). In contrast, this antibody had minimal or no effect on BF-induced B cell proliferation. However, when MLC supernatants were absorbed with a cloned IL 2-dependent T cell line, only IL 2 activity, but not BF activity, was removed, demonstrating that BF and IL 2 have different binding specificities. The precise mechanism(s) by which anti-Tac inhibits BF-induced proliferation of T cells is unknown at present. Additionally, during the course of these experiments, we observed that Con A-Sepharose chromatography could be used as a simple one-step method of separating BCGF from IL 2.
Animals↗