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S Imajoh

Publications and source records attributed to S Imajoh.

At least 19 recordsLinked to original sources
PubMed1992

Active-site residues of the transpeptidase domain of penicillin-binding protein 2 from Escherichia coli: similarity in catalytic mechanism to class A beta-lactamases.

By means of amino acid sequence alignment with class A beta-lactamases, the residues essential for the catalytic activity of the peptidoglycan transpeptidase of penicillin-binding protein 2 (PBP2) have been predicted to be Lys333, Asp447, and Lys544, in addition to the acylation site residue for the acyl-enzyme mechanism, Ser330. Accordingly, these residues were replaced by site-directed mutagenesis, and the resultant mutants were examined as to penicillin-binding activity and genetic complementation, which represent only the acylation step and the total reaction during transpeptidation, respectively. All the mutants at position 333 showed the complete loss of both the binding and complementation activities. Most of the mutants at position 447 retained the binding activity but lost the complementation activity, the exception being the D447E mutant, which retained both. The binding rates for various penicillins of the D447N mutant, which had lost the complementation activity, were almost identical to those of the wild type. The binding of the mutants at position 544 tended to require a higher penicillin concentration, and that of the K544H mutant required a lower pH. When the roles of the counterpart residues, Lys73, Glu166, and Lys234, in class A beta-lactamases were considered, the results suggested that Lys333 and Asp447 are essential for the acylation and acyl-transfer steps, respectively, and that Lys544 stabilizes the Michaelis complex through its side-chain positive charge.

Amino Acid Sequence↗
PubMed1992

Distribution of calcium-activated neutral protease inhibitor in the central nervous system of the rat.

The ubiquitous existence of calcium-activated neutral protease (CANP, calpain), an enzyme whose activity is regulated by calcium ions and a specific endogenous CANP inhibitor (calpastatin), is well known. Although there has been much investigation concerning the distribution and role of CANP, investigations of the distribution of the CANP inhibitor using immunohistochemical techniques are rare. We made antiserum against a 40K fragment of cDNA corresponding to two C-terminal repeats of rat liver CANP inhibitor expressed in Escherichia coli. Using this antiserum, we examined the distribution of CANP inhibitor in the rat central nervous system by the ABC technique and compared it with the distribution of CANP. Neurons and glias were stained, with the cytosol stained diffusely and the cell membranes stained clearly and strongly. Axons and myelin were stained faintly, but nuclei and vessels were not stained. The distribution of CANP inhibitor was thus found to be similar to that of CANP.

Animals↗
PubMed1989

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.

Animals↗
PubMed1989

Purification and some characteristics of a zinc metalloprotease from the venom of Bothrops jararaca (jararaca).

A metalloprotease from Bothrops jararaca venom (J protease) was purified by DEAE-Sephacel, CM-cellulose, Sephacryl S-200 and Sephadex G-75 chromatograph. The proteolytic activity was inactivated by EDTA, o-phenanthroline and DTNB. Phosphoramidon and cysteine protease inhibitors (leupeptin, E64 and its derivatives) were inactive on this enzyme. J protease was activated by calcium and the metal content analysis showed the presence of one mole each of tightly bond zinc and calcium per mole of this J protease. The amino acid composition, N-terminal amino acid sequence (29 residues) and the cleavage sites on the oxidized insulin B chain and angiotensin I were determined.

Amino Acid Sequence↗
PubMed1988

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.

Amino Acid Sequence↗
PubMed1988

Calcium-activated neutral protease (CANP), a putative processing enzyme of the neuropeptide, kyotorphin, in the brain.

Kyotorphin (Tyr-Arg) accumulation in the dialysed synaptosol from the rat brain in the presence of an inhibitor of kyotorphin-degrading enzyme, was maximal at neutral pH. This accumulation was activated by calcium ions, but was inhibited by leupeptin and SH-blocking agents, a finding which suggests the involvement of calcium-activated neutral protease (CANP or calpain). In addition, the kyotorphin-precursor protein, being processed by purified mu- or m-CANP, was detected at about 160 kDa on Sephacryl S-300 chromatography of the synaptosol. The present findings seem to be the first evidence for the role of CANP as a processing enzyme of neuropeptide-precursor in nerve terminals.

Animals↗
PubMed1988

A novel phorbol ester receptor/protein kinase, nPKC, distantly related to the protein kinase C family.

Protein kinase C (PKC)-related cDNA clones encode an 84 kd protein, nPKC. nPKC contains a cysteine-rich repeat sequence homologous to that seen in conventional PKCs (alpha, beta I, beta II, and gamma), which make up a family of 77-78 kd proteins with closely related sequences. nPKC, when expressed in COS cells, confers increased high-affinity phorbol ester receptor activity to intact cells. Antibodies raised against nPKC identified a 90 kd protein in rabbit brain extract as well as in extracts from COS cells transfected with the cDNA construct. nPKC shows protein kinase activity that is regulated by phospholipid, diacylglycerol, and phorbol ester but is independent of Ca2+. The structural and enzymological characteristics of nPKC clearly distinguish it from conventional PKCs, which until now have been the only substances believed to mediate the various effects of diacylglycerol and phorbol esters. These results suggest an additional signaling pathway involving nPKC.

Amino Acid Sequence↗
PubMed1988

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.

Amino Acid Sequence↗
PubMed1988

Regulation of activity of calcium activated neutral protease.

Various lines of evidence suggest that calcium dependent protease (CANP, calpain) exists in the cytosol as an inactive proenzyme which is converted to an active form by autolysis. During autolysis only the N-terminal regions of both subunits of proCANP are modified. About 20 and 90 residues are removed from the large and small subunits, respectively. The N-terminal region (domain I) of the large subunit modified during autolysis precedes the protease domain and corresponds to the propeptides of various cysteine proteinases. Analyses of the autocatalytic activation of CANP in the presence of plasma membranes reveal that proCANP translocates to the membrane in the presence of microM Ca2+ and is activated at the membrane. The CANP inhibitor and Ca2+ are the most important factors for the regulation of CANP activity. The primary translation product of the mRNA for rabbit liver CANP inhibitor contains four internal repeats. Structural analyses of the liver and erythrocyte inhibitors reveal that they contain four and three repeats, respectively. The repeating unit was identified as the functional unit of the inhibitor and each unit inhibits one mole of CANP. On the basis of these results, an activation mechanism for proCANP at the membrane was proposed. The native enzyme, which has been called CANP or calpain, should now be called proCANP or calpainogen. CANP and calpain should be used for the autolyzed active form.

Animals↗
PubMed1987

Molecular cloning and sequencing of cDNA for rat cathepsin H. Homology in pro-peptide regions of cysteine proteinases.

A cDNA for rat cathepsin H was isolated and sequenced. The deduced protein comprising 333 amino acid residues is composed of a typical signal sequence (21 residues), a pro-peptide region (92 residues) and a mature enzyme region (220 residues). The amino acid sequence in the pro-peptide region, in particular, residues Phe-(-41) to Ser-(-29) of cathepsin H, is highly homologous to the pro-peptide regions of other cysteine proteinases. This homologous region may play a role in the processing of cysteine proteinases.

Amino Acid Sequence↗
PubMed1987

Molecular cloning and sequencing of cDNA for rat cathepsin L.

A near full-length cDNA for rat cathepsin L was isolated. The deduced protein comprises 334 amino acid residues (Mr 37,685) containing a typical signal sequence (N-terminal 17 residues), pro-peptide (96 residues), and the sequence for mature cathepsin L (221 residues). Rat cathepsin L shows 94% amino acid identity with mouse cysteine proteinase. Amino acid sequence homologies of rat cathepsin L with rat cathepsins H and B are 45 and 25%, respectively. These facts indicate that mouse cysteine proteinase is probably mouse cathepsin L and that cathepsin L is more closely related to cathepsin H than cathepsin B.

Amino Acid Sequence↗
PubMed1987

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.

Animals↗
PubMed1987

Calcium-activated neutral protease inhibitor from rabbit erythrocytes lacks the N-terminal region of the liver inhibitor but retains three inhibitory units.

Endogenous inhibitors for calcium-activated neutral protease (CANP) were purified from rabbit erythrocytes and liver. The purified inhibitors showed single bands but with significantly different mobilities on sodium dodecylsulfate-polyacrylamide gel electrophoresis. Peptide mapping and sequencing analyses have revealed that the erythrocyte inhibitor (429 residues) retains the C-terminal three repetitive units of the liver inhibitor (639 residues), which contains four potential repetitive units for inhibition of CANP. The erythrocyte and liver inhibitors inhibited 3 and 4 moles of CANP on the basis of the molecular weights of 46,000 and 68,000, respectively.

Amino Acid Sequence↗
PubMed1987

A fragment of an endogenous inhibitor produced in Escherichia coli for calcium-activated neutral protease (CANP) retains an inhibitory activity.

A C-terminal fragment of an endogenous rabbit liver inhibitor for calcium-activated neutral protease (CANP) was produced in Escherichia coli and its inhibitory activity was examined after purification. The truncated inhibitor (373 amino acid residues), which contains two internal repeat structures, inhibits 2 mol CANP whereas the native liver inhibitor (639 residues), containing four internal repeat structures, inhibits 4 mol CANP. This supports the hypothesis that the repeating unit is the functional unit of inhibition. The results also indicate that post-translational modification of the inhibitor is not essential for inhibition.

Amino Acid Sequence↗
PubMed1987

Endogenous inhibitor for calcium-dependent cysteine protease contains four internal repeats that could be responsible for its multiple reactive sites.

A cDNA encoding an endogenous inhibitor, termed calpastatin, for calcium-dependent cysteine protease (calpain, EC 3.4.22.17) was cloned by screening rabbit cDNA libraries with a synthetic oligodeoxynucleotide probe based on the partial amino acid sequence of the purified protein. The deduced amino acid sequence contains 718 amino acid residues (Mr, 76,964), and the mature protein corresponds to the deduced sequence from the 80th residue of the primary translation product (resultant Mr, 68,113). This deduced molecular weight is significantly lower than that determined by NaDodSO4/polyacrylamide gel electrophoresis, suggesting the possibility that the inhibitor is post-translationally modified. The sequence of the mature inhibitor contains four consecutive internal repeats approximately 140 amino acid residues long, each of which might be responsible for the inhibitory activity. Calpastatin is apparently different from a typical cysteine protease inhibitor (cystatin), suggesting that the mechanism of inhibition of calcium-dependent cysteine protease by the inhibitor might be different from that of other cysteine proteases by cystatin.

Amino Acid Sequence↗
PubMed1987

The COOH-terminal E-F hand structure of calcium-activated neutral protease (CANP) is important for the association of subunits and resulting proteolytic activity.

High-Ca2+-requiring calcium-activated neutral protease (mCANP), a dimeric enzyme composed of large (Mr = 80,000) and small (Mr = 28,000) subunits, is resistant to carboxypeptidase Y (CPase Y) in the absence of NaSCN. In the presence of 0.2 M NaSCN, CPase Y digested mCANP, one or two amino acids being released from the COOH-termini of the large and small subunits, but no change occurred in the activity of the digested mCANP. In the presence of 1 M NaSCN, 8-10 amino acids were released from the subunits by CPase Y, and the COOH-terminal potential Ca2+-binding sites of both subunits were destroyed. On digestion under these conditions, mCANP lost the ability to form a complex, and the proteolytic activity was not recovered even when the digested subunits were mixed with native subunits. These results suggest that the COOH-terminal regions of the two subunits of mCANP, which constitute the helical portions of the COOH-terminal E-F hand structures in both subunits, are essential for the subunit association and resulting proteolytic activity.

Animals↗