Characterization of cDNA clones encoding a novel calcium-activated neutral proteinase from Schistosoma mansoni

To identify and characterize Schistosoma mansoni proteins that are recognized by infected hosts, we have used a pool of sera from infected humans to screen cDNA libraries constructed from poly(A)+ mRNA of adult S. mansoni. The deduced amino acid sequences of the three isolated clones showed a high degree of similarity to the large subunit of calcium-activated neutral proteinase (CANP) from humans and chicken. These overlapping clones, which include a nearly full-length clone with an open reading frame of 758 amino acid residues, together encode the entire large subunit of CANP. The deduced sequence of this S. mansoni protein can be divided into four domains (I-IV) that include the two domains characteristic of other large subunits of CANP: a thiol-protease domain (II) and a calcium-binding domain (IV) containing EF hand motifs. However, the schistosome protein is unique in having only three EF hand motifs in the calcium-binding domain and in having an additional EF hand motif that is shared between domains II and III. We have shown that these EF hand motifs are capable of binding 45Ca2+. Furthermore, the large subunit is S. mansoni contains an NH2-terminal sequence of 28 residues that is absent from the mammalian CANPs and has a high degree of similarity to the presumed receptor binding sequence of colicin Ia and Ib.     

Molecular cloning of the cDNA for the large subunit of the high-Ca2+-requiring form of human Ca2+-activated neutral protease

A nearly full-length cDNA clone for the large subunit of high-Ca2+-requiring Ca2+-activated neutral protease (mCANP) from human tissues has been isolated. The deduced protein, determined for the first time as an mCANP, has essentially the same structural features as those revealed previously for the large subunits of the low-Ca2+-requiring form (muCANP) [Aoki, K., Imajoh, S., Ohno, S., Emori, Y., Koike, M., Kosaki, G., & Suzuki, K. (1986) FEBS Lett. 205, 313-317] and chicken CANP [Ohno, S., Emori, Y., Imajoh, S., Kawasaki, H., Kisaragi, M., & Suzuki, K. (1984) Nature (London) 312, 566-570]. Namely, the protein, comprising 700 amino acid residues, is characterized by four domains, containing a cysteine protease like domain and a Ca2+-binding domain. The overall amino acid sequence similarities of the mCANP large subunit with those of human muCANP and chicken CANP are 62% and 66%, respectively. These values are slightly lower than that observed between muCANP and chicken CANP (70%). Local sequence similarities vary with the domain, 73-78% in the cysteine protease like domain and 48-65% in the Ca2+-binding domain. These results suggest that CANPs with different Ca2+ sensitivities share a common evolutionary origin and that their regulatory mechanisms are similar except for the Ca2+ concentrations required for activation.     

Primary structure and evolution of calcium-activated neutral protease (CANP)

The amino acid sequences of two subunits (80K and 30K) of calcium-activated neutral protease (CANP) were examined to clarify the structure-function relationship of CANP. The 80K subunit is composed of four clear domains (I–IV from the N-terminus). Domain II is a cysteine proteinase domain homologous to cathepsins B, L, and H. Domain IV is a calcium binding domain with four consecutive EF-hand structures known as typical calcium-binding sites found in calmodulin. The 30K subunit also has a clear domain structure (two domains). The N-terminal domain, a Gly-rich hydrophobic domain, probably determines the location of CANP through association with cellular membrane. The C-terminal domain is a calmodulinlike calcium-binding domain highly homologous to IV in the 80K subunit. The protease activity ascribable to II is regulated by 2 moles of built-in calmodulins, though its precise regulation mechanism is unknown. These results are discussed together with the molecular evolution of CANP on the basis of the gene structures of the two subunits.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

E-F hand structure-domain of calcium-activated neutral protease (CANP) can bind Ca2+ ions

The cDNA fragments corresponding to the domains with four consecutive E-F hand structures in the large and small subunits of chicken and rabbit calcium-activated neutral protease (CANP) were inserted into an expression vector (pUC8 or pUC18). The resulting plasmids were used to transform E. coli, and isopropyl-1-thio-beta-D-galactoside (IPTG)-inducible expression was performed. The resulting four kinds of E-F hand structure-domains (the chicken large subunit, rabbit high- and low-calcium-requiring large subunits, and rabbit small subunit) were purified and analyzed for their calcium-binding abilities and capacities by the microscale filter assay. Most of the E-F hand structures could bind calcium and 2 or 4 mol of Ca2+ ions bound to the four consecutive E-F hand structures. The calcium-binding affinity of the E-F hand structures in the large subunit roughly corresponds to the calcium concentration required for its CANP activity.     

Isolation and sequence analysis of cDNA clones for the small subunit of rabbit calcium-dependent protease

We have isolated and sequenced cDNA clones for the small subunit (30-kDa subunit) of rabbit calcium-dependent protease (Ca2+-protease) using synthesized oligodeoxynucleotide probes based on the partial amino acid sequence of the protein. A nearly full-length cDNA clone containing the total amino acid coding sequence was obtained. From the deduced sequence, the following conclusions about possible functions of the protein are presented. The kDa subunit comprises 266 residues (Mr = 28,238). The N-terminal region (64 residues) is mainly composed of glycine (37 residues) and hydrophobic amino acids and may interact with the cell membrane or an organelle. The sequence of the C-terminal 168 residues is highly homologous to the corresponding C-terminal region of the large subunit (80-kDa subunit) which has been identified as the calcium-binding domain. This region of the 30-kDa subunit contains four E-F hand structures and presumably binds Ca2+, as in the case of the 80-kDa subunit. Thus, the 30-kDa subunit may play important roles in regulating enzyme activity and/or possibly in determining the location of the Ca2+-protease. The marked sequence homology of the C-terminal regions of the two subunits may indicate that the calcium-binding domains have evolved from the same ancestral gene.     

Binding of calpain fragments to calpastatin

Their cDNA-derived amino acid sequences predict that the 80-kDa subunits of the micromolar and millimolar Ca(2+)-requiring forms of the Ca(2+)-dependent proteinase (mu- and m-calpain, respectively) each consist of four domains and that the 28-kDa subunit common to both mu- and m-calpain consists of two domains. The calpains were allowed to autolyze to completion, and the autolysis products were separated and were characterized by using gel permeation chromatography, calpastatin affinity chromatography, and sequence analysis. Three major fragments were obtained after autolysis of either calpain. The largest fragment (34 kDa for mu-calpain, 35 kDa for m-calpain) contains all of domain II, the catalytic domain, plus part of domain I of the 80-kDa subunit of mu- or m-calpain. This fragment does not bind to calpastatin, a competitive inhibitor of the calpains, and has no proteolytic activity in either the absence or presence of Ca2+. The second major fragment (21 kDa for mu-calpain and 22 kDa for m-calpain) contains domain IV, the calmodulin-like domain, plus approximately 50 amino acids from domain III of the 80-kDa subunit of mu- or m-calpain. The third major fragment (18 kDa) contains domain VI, the calmodulin-like domain of the 28-kDa subunit. The second and third major fragments bind to a calpastatin affinity column in the presence of Ca2+ and are eluted with EDTA. The second and third fragments are noncovalently bound, so the 80- and 28-kDa subunits of the intact calpain molecules are noncovalently bound at domains IV and VI. After separation in 1 M NaSCN, the 28-kDa subunit binds completely to calpastatin, approximately 30-40% of the 80-kDa subunit of mu-calpain binds to calpastatin, and the 80-kDa subunit of m-calpain does not bind to calpastatin in the presence of 1 mM Ca2+.     

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.     

Isolation and sequence analyses of cDNA clones for the large subunits of two isozymes of rabbit calcium-dependent protease

Two sets of cDNA clones were isolated from cDNA libraries prepared from poly(A+) RNA of rabbit lung and spleen by screening with the cDNA probe for the large subunit (80-kDa subunit) of chicken calcium-dependent protease (Ca2+-protease; Ohno, S., Emori, Y., Imajoh, S., Kawasaki, H., Kisaragi, M., and Suzuki, K. (1984) Nature 312, 566-570). The two sets of clones were identified as cDNA clones for two Ca2+-protease isozymes with high (mu-type) and low (m-type) calcium sensitivities from a comparison of the primary structures deduced from the nucleotide sequences with partial amino acid sequences from the two isozymes. The cDNA clones for the 80-kDa subunits of the mu- and m-type Ca2+-proteases contained, in total, about 1.5- and 2.2-kilobase cDNA inserts, respectively, which correspond roughly to the C-terminal halves of the coding regions and the entire 3'-noncoding regions. The two isozymes are encoded by two distinct mRNA species present in all the tissues examined, although the amount of mRNA significantly differs among the various tissues. Four E-F hand structures, typical calcium-binding structures in various calcium-binding proteins such as calmodulin, were detected in the C-terminal regions of both isozymes, as in the case of chicken Ca2+-protease. Comparison of the amino acid sequences of the two rabbit isozymes and the corresponding region of the chicken enzyme revealed marked homology, which indicates that these three enzymes have the same evolutionary origin. Furthermore, we suggest that the mu-type rabbit Ca2+-protease, rather than the m-type, is similar to chicken Ca2+-protease, which is regarded as an m-type enzyme in the C-terminal region. The evolution and molecular basis of the differences in calcium sensitivities of the Ca2+-proteases are discussed.     

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.     

A novel plant ferritin subunit from soybean that is related to a mechanism in iron release

Ferritin is a multimeric iron storage protein composed of 24 subunits. Ferritin purified from dried soybean seed resolves into two peptides of 26.5 and 28 kDa. To date, the 26.5-kDa subunit has been supposed to be generated from the 28-kDa subunit by cleavage of the N-terminal region. We performed amino acid sequence analysis of the 28-kDa subunit and found that it had a different sequence from the 26.5-kDa subunit, thus rendering it novel among known soybean ferritins. We cloned a cDNA encoding this novel subunit from 10-day-old seedlings, each of which contained developed bifoliates, an epicotyl and a terminal bud. The 26.5-kDa subunit was found to be identical to that identified previously lacking the C-terminal 16 residues that correspond to the E helix of mammalian ferritin. However, the corresponding region in the 28-kDa soybean ferritin subunit identified in this study was not susceptible to cleavage. We present evidence that the two different ferritin subunits in soybean dry seeds show differential sensitivity to protease digestions and that the novel, uncleaved 28-kDa ferritin subunit appears to stabilize the ferritin shell by co-existing with the cleaved 26.5-kDa subunit. These data demonstrate that soybean ferritin is composed of at least two different subunits, which have cooperative functional roles in soybean seeds.     

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.     

The 30-kilodalton subunit of bovine mitochondrial complex I is homologous to a protein coded in chloroplast DNA

In cattle, 7 of the 30 or more subunits of the respiratory enzyme NADH:ubiquinone reductase (complex I) are encoded in mitochondrial DNA, and potential genes (open reading frames, orfs) for related proteins are found in the chloroplast genomes of Marchantia polymorpha and Nicotiana tabacum. Homologues of the nuclear-coded 49- and 23-kDa subunits are also coded in chloroplast DNA, and these orfs are clustered with four of the homologues of the mammalian mitochondrial genes. These findings have been taken to indicate that chloroplasts contain a relative of complex I. The present work provides further support. The 30-kDa subunit of the bovine enzyme is a component of the iron-sulfur protein fraction. Partial protein sequences have been determined, and synthetic oligonucleotide mixtures based on them have been employed as hybridization probes to identify cognate cDNA clones from a bovine library. Their sequences encode the mitochondrial import precursor of the 30-kDa subunit. The mature protein of 228 amino acids contains a segment of 57 amino acids which is closely related to parts of proteins encoded in orfs 169 and 158 in the chloroplast genomes of M. polymorpha and N. tabacum. Moreover, the chloroplast orfs are found near homologues of the mammalian mitochondrial genes for subunit ND3. Therefore, the plant chloroplast genomes have at least two separate clusters of potential genes encoding homologues of subunits of mitochondrial complex I. The bovine 30-kDa subunit has no extensive sequences of hydrophobic amino acids that could be folded into membrane-spanning alpha-helices, and although it contains two cysteine residues, there is no clear evidence in the sequence that it is an iron-sulfur protein.     

Proteolytic modification of calcium-dependent protease 1 in erythrocytes treated with ionomycin and calcium

In vitro, limited proteolytic cleavage of the subunits of the purified calcium-dependent proteases [also known as calpains (EC 3.4.22.17) or calcium-activated neutral proteinases (CANPs)] appears to be required for enzyme activity. It has not yet been demonstrated if similar processing of the protease subunits occurs in vivo. To directly assess proteolytic modification of these proteases in cells, we have measured the loss of the proenzyme form of the regulatory subunit (a 26-kDa protein) and/or the appearance of the modified regulatory subunit (a 17-kDa protein) by densitometric analysis of immunoblots. In rat erythrocytes, proteolytic modification of the endogenous calcium-dependent protease (calcium-dependent protease 1, mu CANP) occurs in vivo in response to ionomycin and calcium. The extent of enzyme modification was dependent on time, ionomycin concentration, and calcium concentration, suggesting that in this cellular model Ca2+ regulates proteolytic modification of the enzyme.     

Structure of the 116-kDa polypeptide of the clathrin-coated vesicle/synaptic vesicle proton pump.

A 116-kDa polypeptide has recently been found to be a common component of vacuolar proton pumps isolated from a variety of sources. The 116-kDa subunit of the proton pump was purified from clathrin-coated vesicles of bovine brain, and internal sequences were obtained from proteolytic peptides. Oligonucleotide probes designed from these peptide sequences were utilized in polymerase chain reactions to isolate partial bovine cDNA clones for the protein. Sequences from these were then utilized to isolate rat brain cDNA clones containing the full-length coding region. RNA blots indicate the presence of an abundant 3.9-kilobase message for the 116-kDa subunit in brain, and primer extension analysis demonstrates that the cloned sequence is full-length. The rat cDNA sequences predict synthesis of a protein of 96,267 Da. Analysis of the deduced amino acid sequence of the 116-kDa subunit suggests that it consists of two fundamental domains: a hydrophilic amino-terminal half that is composed of greater than 30% charged residues, and a hydrophobic carboxyl-terminal half that contains at least six transmembrane regions. The structural properties of the 116-kDa proton pump polypeptide agree well with its proposed function in coupling ATP hydrolysis by the cytoplasmic subunits to proton translocation by the intramembranous components of the pump.     

Sequence comparison among muscle-specific calpain, p94, and calpain subunits.

While conventional calpains, m- and mu-calpains named according to their calcium-dependence, are expressed in almost every tissues, mRNA of newly identified p94, which has a significant sequence similarity to the conventional calpain large subunits, is abundantly expressed only in skeletal muscle. In addition to this specific expression, p94 is distinct from conventional calpains in that it contains three unique regions showing no similarity to conventional calpain subunits. When rat and human p94 are compared, overall sequence similarity is 94.0%, which is close to those for m- and mu-calpain large subunits; 93.1% and 95.4% between human and rabbit, respectively, suggesting the evolutionary importance of p94. These calpain large subunit proteins, p94, m- and mu-types, can be considered to constitute a super family, whose p94, m- and mu-types represent the three major types. Sequences of the calpain large-subunit family members, including the recently reported Schistosoma calpain, are compared. Their evolutionary correlation and function are discussed on the basis of the results thus far obtained.     

Molecular cloning and expression of cDNAs coding for soluble guanylate cyclase from rat lung.

Complementary DNA clones corresponding to the 70- and 82-kDa subunits of soluble guanylate cyclase of rat lung have been isolated. Blot hybridization of total poly(A)+ RNA from rat tissues detected mRNA of about 3.4 kilobases for the 70-kDa subunit and about 5.5 kilobases for the 82-kDa subunit. Messenger RNA levels of both subunits were abundant in lung and cerebrum, moderate in cerebellum, heart, and kidney, and low in liver and muscle, consistent with previously described enzyme activities in these tissues. Southern blot analysis of high molecular weight genomic DNA from rat liver indicated that the genes for the 70- and 82-kDa subunits are different. The carboxyl-terminal region of the 70- and 82-kDa subunits showed a high degree of homology and also had a partial homology with the putative catalytic domain of particulate guanylate cyclase and adenylate cyclase, indicating that both the 70- and 82-kDa subunits have catalytic domains. The cDNAs were subcloned to an expression vector and transfected to L cells. The cells transfected with cDNA of the 70-kDa subunit or the 82-kDa subunit showed no guanylate cyclase activity, whereas the cells transfected with both the 70- and 82-kDa subunit cDNAs showed significant guanylate cyclase activity that was activated markedly by sodium nitroprusside. These data suggest that both subunits are required for both the basal catalytic and regulatory activity of soluble guanylate cyclase. Presumably both catalytic subunits must be present and interactive to permit synthesis of cyclic GMP and nitrovasodilator activation.     

Calmodulin-dependent multifunctional protein kinase. Evidence for isoenzyme forms in mammalian tissues

Calcium/calmodulin-dependent multifunctional protein kinases, extensively purified from rat brain (with apparent molecular mass 640 kDa), rabbit liver (300 kDa) and rabbit skeletal muscle (700 kDa), were analysed for their structural, immunological, and enzymatic properties. The immunological cross-reactivity with affinity-purified polyclonal antibodies to the 50-kDa catalytic subunit of the brain calmodulin-dependent protein kinase confirmed the presence of common antigenic determinants in all subunits of the protein kinases. One-dimensional phosphopeptide patterns, obtained by digestion of the autophosphorylated protein kinases with S. aureus V8 protease, and two-dimensional fingerprints of the 125I-labelled proteins digested with a combination of trypsin and chymotrypsin, revealed a close similarity between the two subunits (51 kDa and 53 kDa) of the liver enzyme. Similar identity was observed between the 56-kDa and/or 58-kDa polypeptides of the skeletal muscle calmodulin-dependent protein kinase. The data suggest that the subunits of the liver and muscle protein kinases may be derived by partial proteolysis or by autophosphorylation. The peptide patterns for the 50-kDa and 60-kDa subunits of the brain enzyme confirmed that the two catalytic subunits represented distinct protein products. The comparison of the phosphopeptide maps and the two-dimensional peptide fingerprints, indicated considerable structural homology among the 50-kDa and 60-kDa subunits of the brain calmodulin-dependent protein kinase and the liver and muscle polypeptides. However, a significant number of unique peptides in the liver 51-kDa subunit, skeletal muscle 56-kDa, and the brain 50-kDa and 60-kDa polypeptides were observed and suggest the existence of isoenzyme forms. All calmodulin-dependent protein kinases rapidly phosphorylated synapsin I with a stoichiometry of 3-5 mol phosphate/mol protein. The two-dimensional separation of phosphopeptides obtained by tryptic/chymotryptic digestion of 32P-labelled synapsin I indicated that the same peptides were phosphorylated by all the calmodulin-dependent protein kinases. Such data represent the first structural and immunological comparison of the liver calmodulin-dependent protein kinase with the enzymes isolated from brain and skeletal muscle. The findings indicate the presence of a family of highly conserved calmodulin-dependent multifunctional protein kinases, with similar structural, immunological and enzymatic properties. The individual catalytic subunits appear to represent the expression of distinct protein products or isoenzymes which are selectively expressed in mammalian tissues.     

The protease core of the muscle-specific calpain,p94, undergoes Ca2+-dependent intramolecular autolysis

Limb girdle muscular dystrophy type 2A is linked to a skeletal muscle-specific calpain isoform known as p94. Isolation of the intact 94-kDa enzyme has been difficult to achieve due to its rapid autolysis, and uncertainty has arisen over its Ca2+-dependence for activity. We have expressed a C-terminally truncated form of the enzyme that comprises the protease core (domains I and II) along with its insertion sequence, IS1, and N-terminal leader sequence, NS. This 47-kDa p94I-II mini-calpain was stable during purification. In the presence of Ca2+, p94I-II cleaved itself within the NS and IS1 sequences. Mapping of the autolysis sites showed that NS and IS1 have the potential to be removed without damage to the protease core. Ca2+-dependent autolysis must be an intramolecular event because the inactive p94I-II C129S mutant was not cleaved by incubation with wild-type p94I-II. In addition, the rate of autolysis of p94I-II was independent of the concentration of the enzyme.     

Calcium-Activated Neutral Proteinase of Human Brain: Subunit Structure and Enzymatic Properties of Multiple Molecular Forms

Abstract Calcium-activated neutral proteinase (CANP) was purified 2,625-fold from postmortem human cerebral cortex by a procedure involving chromatography on diethylaminoethyl (DEAE)-cellulose, phenyl-Sepharose, Ultrogel AcA-44, and DEAE-Biogel A. The major active form of CANP exhibited a molecular weight of 94–100 kilodaltons (Kd) by gel filtration on Sephacryl 300 and consisted of 78-Kd and 27-Kd subunits. Two-dimensional gel electrophoresis resolved the small subunit into two molecular species with different isoelectric points. CANP degraded most human cytoskeletal proteins but was particularly active toward fodrin and the neurofilament protein subunits (145 Kd > 200 Kd > 70 Kd). The enzyme required 175 μMCa²⁺ for half-maximal activation and 2 mM Ca²⁺ for optimal activity toward [methl-¹⁴C]azocasein. Other divalent metal ions were poor activators of the enzyme, and some, including copper, lead, and zinc, strongly inhibited the enzyme. Aluminum, a neurotoxic ion that induces neurofilament accumulations in mammalian brain, inhibited the enzyme 47% at 1 mM and 100% at 5 mM A second CANP form lacking the 27-Kd subunit was partially resolved from the 100-Kd heterodimer during DEAE-Biogel A chromatography. The 78-Kd monomer exhibited the same specific activity, calcium ion requirement, pH optimum, and specificity for cytoskeletal proteins as the 100-Kd heterodimer, suggesting that the 27-Kd subunit is not essential for the major catalytic properties of the enzyme. The rapid autolysis of the 27-Kd subunit to a 18-Kd intermediate when CANP is exposed to calcium may explain differences between our results and previous reports, which describe brain mCANP in other species as a 76-80-Kd monomer or a heterodimer containing 76-80-Kd and 17-20-Kd subunits. The similarity of the 100-Kd human brain CANP to CANPs in nonneural tissues indicates that the heterodimeric form is relatively conserved among various tissues and species.     

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.