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Kenji Takahashi - One of the best experts on this subject based on the ideXlab platform.
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identification of a glutamic Acid and an aspartic Acid residue essential for catalytic activity of aspergillopepsin ii a non pepsin type Acid Proteinase
Journal of Biological Chemistry, 2000Co-Authors: Xiang-ping Huang, Hideshi Inoue, Naofumi Kagami, Masaki Kojima, Osamu Makabe, Takao Kimura, Koichi Suzuki, Kenji TakahashiAbstract:Abstract Aspergillopepsin II from Aspergillus niger var. macrosporus is a non-pepsin type or pepstatin-insensitive Acid Proteinase. To identify the catalytic residues of the enzyme, all Acidic residues that are conserved in the homologous Proteinases of family A4 were replaced with Asn, Gln, or Ala using site-directed mutagenesis. The wild-type and mutant pro-enzymes were heterologously expressed in Escherichia coli and refolded in vitro. The wild-type pro-enzyme was shown to be processed into a two-chain active enzyme under Acidic conditions. Most of the recombinant mutant pro-enzymes showed significant activity under Acidic conditions because of autocatalytic activation except for the D123N, D123A, E219Q, and E219A mutants. The D123A, E219Q, and E219A mutants showed neither enzymatic activity nor autoprocessing activity under Acidic conditions. The circular dichroism spectra of the mutant pro- and mature enzymes were essentially the same as those of the wild-type pro- and mature enzyme, respectively, indicating that the mutant pro-enzymes were correctly folded. In addition, two single and one double mutant pro-enzyme, D123E, E219D, and D123E/E219D, did not show enzymatic activity under Acidic conditions. Taken together, Glu-219 and Asp-123 are deduced to be the catalytic residues of aspergillopepsin II.
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Aspergillus niger Acid Proteinase A
Advances in experimental medicine and biology, 1998Co-Authors: Kenji Takahashi, Naofumi Kagami, Xiang-ping Huang, Masaki Kojima, Hideshi InoueAbstract:Aspergillus niger var. macrosporus produces two kinds of extracellular Acid Proteinases, i.e., Proteinase B (aspergillopepsin I or proctase B) and Proteinase A (aspergillopepsin II or proctase A).1 Proteinase B is a typical pepsin-type aspartic Proteinase, whereas Proteinase A is a non-pepsin type Acid Proteinase rather insensitive to specific inhibitors of aspartic Proteinases, including pepstatin, DAN and EPNP.2 Proteinase A has a molecular mass of 22,265 dalton, and is composed of two peptide chains, namely Lchain (39 residues) and Hchain (173 residues), which are noncovalently bound to each other.3,4 It has no similarity in amino Acid sequence with ordinary pepsin-type aspartic Proteinases and appears to have fairly different substrate specificity. The active site residues have not been identified in the previous studies, although the results of chemical modification as well as its pH-activity profile indicated that certain carboxyl groups are involved in the activity.
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Acid Proteinase from nepenthes distillatoria badura
Advances in Experimental Medicine and Biology, 1998Co-Authors: Senarath B. P. Athauda, Hideshi Inoue, Akihiro Iwamatsu, Kenji TakahashiAbstract:Plant aspartic Proteinases have so far received much less attention in contrast to the well characterized mammalian, fungal and viral aspartic Proteinases.1 They are widely distributed in the plant kingdom, and have been detected in seeds, leaves and flowers of different plants as well as in the digestive fluid of some insectivorous species.1 Aspartic Proteinases from barley, rice and cardoon flower have been well characterized and their cDNA-derived primary structures have been reported.2–4 However, only a few studies were reported on Proteinases of insectivorous plants.5–8 Insectivorous plant Nepenthes distillatoria is available in Sri Lanka in a large quantity and will be a good source of the insectivorous plant Proteinases. They are interesting not only from the view point of plant physiology, but also from the view point of structure/function relationship and molecular evolution of aspartic Proteinases.
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the specificity of peptide bond cleavage of Acid Proteinase a from aspergillus niger var macrosporus toward oxidized ribonuclease a
Bioscience Biotechnology and Biochemistry, 1997Co-Authors: Kenji TakahashiAbstract:In order to investigate the specificity of peptide bond cleavage by Acid Proteinase A from Aspergillus niger var. macrosporus (Aspergil-lopepsin II), performic Acid-oxidized bovine pancreatic ribonuclease A was digested by the enzyme at pH 1.8 or 5.5, and the resulting peptides were separated by HPLC and analyzed. Among the total 123 peptide bonds, approximately thirty and thirteen bonds were cleaved at pH 1.8 for 2h and at pH 5.5 for 20 h, respectively. Cleavages occurred fairly specifically at Tyr-X, Phe-X, His-X, Asn-X, Asp-X, Gln-X, and Glu-X bonds.
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Expression in E.Coli of Aspergillus Niger Var. Macrosporus Proteinase A, a Non-Pepsin Type Acid Proteinase
Advances in experimental medicine and biology, 1995Co-Authors: Naofumi Kagami, Hideshi Inoue, Osamu Makabe, Takao Kimura, Kenji TakahashiAbstract:In addition to the extensively studied pepsin type aspartic Proteinases, there exists a distinct family of Acid Proteinases that differ widely in both functional and structural aspects. Functionally, they are insensitive to more than one of the specific inhibitors for the pepsin type aspartic Proteinases; i.e., pepstatin, l,2-epoxy-3-(p-nitrophenoxy)propane and diazoacetyl-DL-norleucine methyl ester. Structurally, no homology is observed in the primary structures, even in the Asp-Thr/Ser-Gly sequence that is conserved in the active sites of the aspartic Proteinases. Because of these fundamental differences, this family is discriminated by the name, “non-pepsin type Acid Proteinase”.
Hideshi Inoue - One of the best experts on this subject based on the ideXlab platform.
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identification of a glutamic Acid and an aspartic Acid residue essential for catalytic activity of aspergillopepsin ii a non pepsin type Acid Proteinase
Journal of Biological Chemistry, 2000Co-Authors: Xiang-ping Huang, Hideshi Inoue, Naofumi Kagami, Masaki Kojima, Osamu Makabe, Takao Kimura, Koichi Suzuki, Kenji TakahashiAbstract:Abstract Aspergillopepsin II from Aspergillus niger var. macrosporus is a non-pepsin type or pepstatin-insensitive Acid Proteinase. To identify the catalytic residues of the enzyme, all Acidic residues that are conserved in the homologous Proteinases of family A4 were replaced with Asn, Gln, or Ala using site-directed mutagenesis. The wild-type and mutant pro-enzymes were heterologously expressed in Escherichia coli and refolded in vitro. The wild-type pro-enzyme was shown to be processed into a two-chain active enzyme under Acidic conditions. Most of the recombinant mutant pro-enzymes showed significant activity under Acidic conditions because of autocatalytic activation except for the D123N, D123A, E219Q, and E219A mutants. The D123A, E219Q, and E219A mutants showed neither enzymatic activity nor autoprocessing activity under Acidic conditions. The circular dichroism spectra of the mutant pro- and mature enzymes were essentially the same as those of the wild-type pro- and mature enzyme, respectively, indicating that the mutant pro-enzymes were correctly folded. In addition, two single and one double mutant pro-enzyme, D123E, E219D, and D123E/E219D, did not show enzymatic activity under Acidic conditions. Taken together, Glu-219 and Asp-123 are deduced to be the catalytic residues of aspergillopepsin II.
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Aspergillus niger Acid Proteinase A
Advances in experimental medicine and biology, 1998Co-Authors: Kenji Takahashi, Naofumi Kagami, Xiang-ping Huang, Masaki Kojima, Hideshi InoueAbstract:Aspergillus niger var. macrosporus produces two kinds of extracellular Acid Proteinases, i.e., Proteinase B (aspergillopepsin I or proctase B) and Proteinase A (aspergillopepsin II or proctase A).1 Proteinase B is a typical pepsin-type aspartic Proteinase, whereas Proteinase A is a non-pepsin type Acid Proteinase rather insensitive to specific inhibitors of aspartic Proteinases, including pepstatin, DAN and EPNP.2 Proteinase A has a molecular mass of 22,265 dalton, and is composed of two peptide chains, namely Lchain (39 residues) and Hchain (173 residues), which are noncovalently bound to each other.3,4 It has no similarity in amino Acid sequence with ordinary pepsin-type aspartic Proteinases and appears to have fairly different substrate specificity. The active site residues have not been identified in the previous studies, although the results of chemical modification as well as its pH-activity profile indicated that certain carboxyl groups are involved in the activity.
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Acid Proteinase from nepenthes distillatoria badura
Advances in Experimental Medicine and Biology, 1998Co-Authors: Senarath B. P. Athauda, Hideshi Inoue, Akihiro Iwamatsu, Kenji TakahashiAbstract:Plant aspartic Proteinases have so far received much less attention in contrast to the well characterized mammalian, fungal and viral aspartic Proteinases.1 They are widely distributed in the plant kingdom, and have been detected in seeds, leaves and flowers of different plants as well as in the digestive fluid of some insectivorous species.1 Aspartic Proteinases from barley, rice and cardoon flower have been well characterized and their cDNA-derived primary structures have been reported.2–4 However, only a few studies were reported on Proteinases of insectivorous plants.5–8 Insectivorous plant Nepenthes distillatoria is available in Sri Lanka in a large quantity and will be a good source of the insectivorous plant Proteinases. They are interesting not only from the view point of plant physiology, but also from the view point of structure/function relationship and molecular evolution of aspartic Proteinases.
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Expression in E.Coli of Aspergillus Niger Var. Macrosporus Proteinase A, a Non-Pepsin Type Acid Proteinase
Advances in experimental medicine and biology, 1995Co-Authors: Naofumi Kagami, Hideshi Inoue, Osamu Makabe, Takao Kimura, Kenji TakahashiAbstract:In addition to the extensively studied pepsin type aspartic Proteinases, there exists a distinct family of Acid Proteinases that differ widely in both functional and structural aspects. Functionally, they are insensitive to more than one of the specific inhibitors for the pepsin type aspartic Proteinases; i.e., pepstatin, l,2-epoxy-3-(p-nitrophenoxy)propane and diazoacetyl-DL-norleucine methyl ester. Structurally, no homology is observed in the primary structures, even in the Asp-Thr/Ser-Gly sequence that is conserved in the active sites of the aspartic Proteinases. Because of these fundamental differences, this family is discriminated by the name, “non-pepsin type Acid Proteinase”.
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The primary structure of Aspergillus niger Acid Proteinase A.
The Journal of biological chemistry, 1991Co-Authors: Kenji Takahashi, Hideshi Inoue, K. Sakai, T. Kohama, S. Kitahara, K. Takishima, M. Tanji, Senarath B. P. Athauda, Takayuki Takahashi, H. AkanumaAbstract:Abstract The complete amino Acid sequence of the Acid Proteinase A, a non-pepsin type Acid Proteinase from the fungus Aspergillus niger var. macrosporus, was determined by protein sequencing. The enzyme was first dissociated at pH 8.5 into a light (L) chain and a heavy (H) chain, and the L chain was sequenced completely. Further sequencing was performed with the reduced and pyridylethylated or aminoethylated derivative of the whole protein, using peptides obtained by digestions with Staphylococcus aureus V8 protease, trypsin, chymotrypsin, and lysylendopeptidase. The location of the two disulfide bonds was determined by analysis of cystine-containing peptides obtained from a chymotryptic digest of the unmodified protein. These results established that the protein consists of a 39-residue L chain and a 173-residue H chain that associate noncovalently to form the native enzyme of 212 residues (Mr 22,265). This is, to our knowledge, the first time that such a protein with a rather short peptide chain associated noncovalently has been found. No sequence homology is found with other Acid or aspartic Proteinases, except for Scytalidium lignicolum Acid Proteinase B, an enzyme unrelated to pepsin by sequence, which has about 50% identity with the present enzyme. These two enzymes, however, are remarkably different from each other in some structural features.
Masaaki Yasuda - One of the best experts on this subject based on the ideXlab platform.
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application of an Acid Proteinase from monascus purpureus to reduce antigenicity of bovine milk whey protein
Journal of Industrial Microbiology & Biotechnology, 2011Co-Authors: P Nilantha L Lakshman, Shinjiro Tachibana, Hirohide Toyama, Toki Taira, Toshihiko Suganuma, Worapot Suntornsuk, Masaaki YasudaAbstract:An Acid Proteinase from Monascus purpureus No. 3403, MpuAP, was previously purified and some characterized in our laboratory (Agric Biol Chem 48:1637–1639, 1984). However, further information about this enzyme is lacking. In this study, we investigated MpuAP’s comprehensive substrate specificity, storage stability, and prospects for reducing antigenicity of whey proteins for application in the food industry. MpuAP hydrolyzed primarily five peptide bonds, Gln4–His5, His10–Leu11, Ala14–Leu15, Gly23–Phe24 and Phe24–Phe25 in the oxidized insulin B-chain. The lyophilized form of the enzyme was well preserved at 30–40°C for 7 days without stabilizers. To investigate the possibility of reducing the antigenicity of the milk whey protein, enzymatic hydrolysates of the whey protein were evaluated by inhibition ELISA. Out of the three main components of whey protein, casein and α-lactalbumin were efficiently degraded by MpuAP. The sequential reaction of MpuAP and trypsin against the whey protein successfully degraded casein, α-lactalbumin and β-lactoglobulin with the highest degree of hydrolysis. As a result, the hydrolysates obtained by using the MpuAP–trypsin combination showed the lowest antigenicity compared with the single application of pepsin, trypsin or pepsin–trypsin combination. Therefore, the overall result suggested that the storage-stable MpuAP and trypsin combination will be a productive approach for making hypoallergic bovine milk whey protein hydrolysates.
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production of angiotensin i converting enzyme inhibitory peptides from soybean protein with monascus purpureus Acid Proteinase
Process Biochemistry, 2005Co-Authors: Megumi Kuba, C Tana, Shinkichi Tawata, Masaaki YasudaAbstract:Abstract Soybean proteins, β-conglycinin and glycinin were hydrolysed by an Acid Proteinase from Monascus purpureus . The degree of hydrolysis and inhibitory activities of angiotensin I-converting enzyme (ACE) increased with increasing proteolysis time. After 10 h of incubation, the IC 50 values of the β-conglycinin and glycinin hydrolysates were determined as 0.126 mg/ml and 0.148 mg/ml, respectively. Four ACE inhibitory peptides were isolated from the soybean protein hydrolysates and identified by protein sequencer. ACE inhibitory peptides isolated from the β-conglycinin hydrolysate were identified as LAIPVNKP (IC 50 = 70 μM) and LPHF (670 μM), and those from the glycinin hydrolysate as SPYP (850 μM) and WL (65 μM). The inhibitory activity of SPYP markedly increased after successive digestion by pepsin, chymotrypsin and trypsin in vitro.
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degradation of soybean protein by an Acid Proteinase from monascus anka
Food Science and Technology International Tokyo, 1998Co-Authors: Masaaki Yasuda, Maki SakaguchiAbstract:Degradation of soybean protein by Monascus-Proteinase was investigated in order to reveal the role of the enzyme in the process of tofuyo ripening. The ratio of trichloroacetic Acid-insoluble nitrogen of soybean protein to the total nitrogen in the reaction mixture decreased with increasing enzymatic reaction time. It was found that the digestion of soybean protein by this enzyme progressed as follows: initially, α'-, α-, and β-subunits in β-conglycinin, and then, the Acidic subunit in glycinin were degraded. However, the basic subunit of glycinin still remained, and some polypeptide bands (around 10 kDa) were formed during the enzyme reaction. The degradation rate of soybean protein by this enzyme was affected by the ethyl alcohol concentration in the reaction mixtures.
Ayse Kalkanci - One of the best experts on this subject based on the ideXlab platform.
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fluorometric determination of Acid Proteinase activity in candida albicans strains from diabetic patients with vulvovaginal candidiasis
Mycoses, 2011Co-Authors: Zuhal Yildirim, Nedret Kilic, Ayse KalkanciAbstract:Summary Vulvovaginal candidiasis is one of the most frequent disorders in obstetrics and gynaecology. Approximately three-quarters of all adult women experience at least one episode of vulvovaginal candidiasis during their life span. Diabetes mellitus (DM) increases the rate of vaginal colonisation and infection with Candida species. The secreted Acid Proteinase might be especially relevant in the pathogenesis of vulvovaginal candidiasis. The aim of this study was to determine the Acid Proteinase activity in the samples of Candida albicans from diabetic patients with vulvovaginal candidiasis by a fluorometric method. Vaginal swabs were taken from 33 women (aged between 22 and 57 years) having symptoms of vaginitis. Patients were divided into three groups: control group, controlled diabetic group and uncontrolled diabetic group. The Proteinase activity in the culture supernatants was determined by a modified fluorometric method. Acid Proteinase activities were significantly increased in the uncontrolled diabetic group in comparison with both the control group and the controlled diabetic group (P < 0.05). Acid Proteinase may play an important role in C. albicans pathogenesis in diabetic patients. Improving glucose control may reduce the risk of Candida colonisation and potentially symptomatic infection, among women with diabetes and hence may be useful even for weaker enzyme activity measurements.
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Fluorometric determination of Acid Proteinase activity in Candida albicans strains from diabetic patients with vulvovaginal candidiasis
Mycoses, 2010Co-Authors: Zuhal Yildirim, Nedret Kilic, Ayse KalkanciAbstract:Summary Vulvovaginal candidiasis is one of the most frequent disorders in obstetrics and gynaecology. Approximately three-quarters of all adult women experience at least one episode of vulvovaginal candidiasis during their life span. Diabetes mellitus (DM) increases the rate of vaginal colonisation and infection with Candida species. The secreted Acid Proteinase might be especially relevant in the pathogenesis of vulvovaginal candidiasis. The aim of this study was to determine the Acid Proteinase activity in the samples of Candida albicans from diabetic patients with vulvovaginal candidiasis by a fluorometric method. Vaginal swabs were taken from 33 women (aged between 22 and 57 years) having symptoms of vaginitis. Patients were divided into three groups: control group, controlled diabetic group and uncontrolled diabetic group. The Proteinase activity in the culture supernatants was determined by a modified fluorometric method. Acid Proteinase activities were significantly increased in the uncontrolled diabetic group in comparison with both the control group and the controlled diabetic group (P
Masaki Kojima - One of the best experts on this subject based on the ideXlab platform.
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identification of a glutamic Acid and an aspartic Acid residue essential for catalytic activity of aspergillopepsin ii a non pepsin type Acid Proteinase
Journal of Biological Chemistry, 2000Co-Authors: Xiang-ping Huang, Hideshi Inoue, Naofumi Kagami, Masaki Kojima, Osamu Makabe, Takao Kimura, Koichi Suzuki, Kenji TakahashiAbstract:Abstract Aspergillopepsin II from Aspergillus niger var. macrosporus is a non-pepsin type or pepstatin-insensitive Acid Proteinase. To identify the catalytic residues of the enzyme, all Acidic residues that are conserved in the homologous Proteinases of family A4 were replaced with Asn, Gln, or Ala using site-directed mutagenesis. The wild-type and mutant pro-enzymes were heterologously expressed in Escherichia coli and refolded in vitro. The wild-type pro-enzyme was shown to be processed into a two-chain active enzyme under Acidic conditions. Most of the recombinant mutant pro-enzymes showed significant activity under Acidic conditions because of autocatalytic activation except for the D123N, D123A, E219Q, and E219A mutants. The D123A, E219Q, and E219A mutants showed neither enzymatic activity nor autoprocessing activity under Acidic conditions. The circular dichroism spectra of the mutant pro- and mature enzymes were essentially the same as those of the wild-type pro- and mature enzyme, respectively, indicating that the mutant pro-enzymes were correctly folded. In addition, two single and one double mutant pro-enzyme, D123E, E219D, and D123E/E219D, did not show enzymatic activity under Acidic conditions. Taken together, Glu-219 and Asp-123 are deduced to be the catalytic residues of aspergillopepsin II.
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Aspergillus niger Acid Proteinase A
Advances in experimental medicine and biology, 1998Co-Authors: Kenji Takahashi, Naofumi Kagami, Xiang-ping Huang, Masaki Kojima, Hideshi InoueAbstract:Aspergillus niger var. macrosporus produces two kinds of extracellular Acid Proteinases, i.e., Proteinase B (aspergillopepsin I or proctase B) and Proteinase A (aspergillopepsin II or proctase A).1 Proteinase B is a typical pepsin-type aspartic Proteinase, whereas Proteinase A is a non-pepsin type Acid Proteinase rather insensitive to specific inhibitors of aspartic Proteinases, including pepstatin, DAN and EPNP.2 Proteinase A has a molecular mass of 22,265 dalton, and is composed of two peptide chains, namely Lchain (39 residues) and Hchain (173 residues), which are noncovalently bound to each other.3,4 It has no similarity in amino Acid sequence with ordinary pepsin-type aspartic Proteinases and appears to have fairly different substrate specificity. The active site residues have not been identified in the previous studies, although the results of chemical modification as well as its pH-activity profile indicated that certain carboxyl groups are involved in the activity.
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differential scanning calorimetric studies of the thermal unfolding of Acid Proteinase a from aspergillus niger at various phs
Thermochimica Acta, 1995Co-Authors: Harumi Fukada, Masaki Kojima, Masaru Tanokura, Katsutada Takahashi, Michio Sorai, Kenji TakashashiAbstract:The thermal unfolding of Acid Proteinase A, isolated from Aspergillus niger, was studied by differential adiabatic scanning calorimetry at various pHs ranging from 2 to 9. The temperature of the maximal excess heat capacity was strongly dependent on pH and highest at a pH around 3, whereas the enthalpy change of the unfolding was maximal at a pH around 5. The excess heat capacity curve at a pH below 6 showed a single asymmetric peak. In contrast, the unfolding at a pH above 6 exhibited an excess heat capacity curve which is characterized by two sharp and broad peaks. The broad peak observed at about 40°C at a pH above 6 was found to be unchanged irrespective of the pH of the solution. Curve resolution revealed that the unfolding at a pH range between 5 and 6 is characterized by a simple two-state transition, with dissociation to the light and heavy peptide chains.
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Structure and function of a pepstatin-insensitive Acid Proteinase from Aspergillus niger var. Macrosporus.
Advances in experimental medicine and biology, 1991Co-Authors: Kenji Takahashi, Hideshi Inoue, Masaki Kojima, Masaru Tanokura, Yutaka Muto, Makoto Yamasaki, Osamu Makabe, Takao Kimura, Toshio Takizawa, Toru HamayaAbstract:The fungus Aspergillus niger var. macrosporus produces two extracellular Acid Proteinases, Proteinases A and B.1–3 The Acid Proteinase B (Mr about 35 kDa) is sensitive to pepstatin, diazoacetyl-DL-norleucine methyl ester (DAN) and l,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) and thus belongs to the ordinary aspartic Proteinase family.4 On the other hand, the Proteinase A (Mr about 22 kDa) is insensitive to pepstatin and also almost insensitive to DAN and EPNP,4 and shows substrate specificity fairly different from that of pepsin-type aspartic Proteinases.5,6 These results indicate that the Proteinase A belongs to a different Acid Proteinase family. This Proteinase, therefore, seems to be an interesting object to investigate its structure/function relationships. Further, the study will contribute to a deeper understanding of the structure and function of the aspartic or Acid Proteinases in general.
