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Aspartic Proteinase
The Experts below are selected from a list of 4434 Experts worldwide ranked by ideXlab platform
Rickey Y. Yada – 1st expert on this subject based on the ideXlab platform
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structure function characterization of the recombinant Aspartic Proteinase a1 from arabidopsis thaliana
Phytochemistry, 2010Co-Authors: Miguel A Mazorramanzano, Takuji Tanaka, Rickey Y. YadaAbstract:Abstract Aspartic Proteinases (APs) are involved in several physiological processes in plants, including protein processing, senescence, and stress response and share many structural and functional features with mammalian and microbial APs. The heterodimeric Aspartic Proteinase A1 from Arabidopsis thaliana (AtAP A1) was the first acid protease identified in this model plant, however, little information exists regarding its structure function characteristics. Circular dichroism analysis indicated that recombinant AtAP A1 contained an higher α-helical content than most APs which was attributed to the presence of a sequence known as the plant specific insert in the mature enzyme. rAtAP A1 was stable over a broad pH range (pH 3–8) with the highest stability at pH 5–6, where 70–80% of the activity was retained after 1 month at 37 °C. Using calorimetry, a melting point of 79.6 °C was observed at pH 5.3. Cleavage profiles of insulin β-chain indicated that the enzyme exhibited a higher specificity as compared to other plant APs, with a high preference for the Leu 15 –Tyr 16 peptide bond. Molecular modeling of AtAP A1 indicated that exposed histidine residues and their interaction with nearby charged groups may explain the pH stability of rAtAP A1.
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An Investigation Of Gastric-like Aspartic Proteinase Molecular Chimeras
Biophysical Journal, 2009Co-Authors: Charity L. Parr, Rickey Y. YadaAbstract:Proplasmepsin II (zPMII) represents a structurally unique member of the Aspartic Proteinase family, with a prosegment-enzyme interaction that has never been reported. It has been a generally accepted assertion that the prosegment in pepsin-like Aspartic Proteinases is critical to Aspartic Proteinase folding, and to investigate this further two chimeric proteins were generated, one with the pepsinogen prosegment fused to the mature region of plasmepsin II (PMII) (pepproPMII) and a second with the prosegment of PMII fused to pepsin (PMIIpropep). Both chimeras were expressed from E. coli, however, PMIIpropep was extremely unstable and was rapidly degraded by trypsin, suggesting protein misfolding. Since a stable enzyme could not be generated PMIIpropep was not further studied. Alternatively, pepproPMII was capable of both autoactivation and synthetic substrate cleavage. In addition, both the zymogen and mature form of the enzyme had the same predicted secondary structures, suggesting that altering the PMII prosegment did not affect this level of protein conformation although the prosegment may play a role in enzyme stability. DSC and CD measurements indicated that pepproPMII had reduced thermal stability as compared to zPMII. It is proposed that this reduction of temperature stability resulted from the loss of the ability of the prosegment in PMII to stabilize the C-terminal domain of the enzyme. The ability of PMII to fold in the presence of a completely non-homologous prosegment suggests that the prosegment in gastric-like Aspartic Proteinases is not always critical to enzyme folding but likely plays a role in protein stabilization.
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expression and characterization of the recombinant Aspartic Proteinase a1 from arabidopsis thaliana
Phytochemistry, 2008Co-Authors: Miguel A Mazorramanzano, Rickey Y. YadaAbstract:Abstract The present study reports the recombinant expression, purification, and partial characterization of a typical Aspartic Proteinase from Arabidopsis thaliana (AtAP A1). The cDNA encoding the precursor of AtAP A1 was expressed as a functional protein using the yeast Pichia pastoris . The mature form of the rAtAP A1 was found to be a heterodimeric glycosylated protein with a molecular mass of 47 kDa consisting of heavy and light chain components, approx. 32 and 16 kDa, respectively, linked by disulfide bonds. Glycosylation occurred via the plant specific insert in the light chain. The catalytic properties of the rAtAP A1 were similar to other plant Aspartic Proteinases with activity in acid pH range, maximal activity at pH 4.0, K m of 44 μM, and k cat of 55 s −1 using a synthetic substrate. The enzyme was inhibited by pepstatin A.
Jukka Kervinen – 2nd expert on this subject based on the ideXlab platform
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crystal structure of plant Aspartic Proteinase prophytepsin inactivation and vacuolar targeting
The EMBO Journal, 1999Co-Authors: Jukka Kervinen, Gregory J Tobin, Julia Costa, David S Waugh, Alexander Wlodawer, Alexander ZdanovAbstract:We determined at 2.3 A resolution the crystal structure of prophytepsin, a zymogen of a barley vacuolar Aspartic Proteinase. In addition to the classical pepsin‐like bilobal main body of phytepsin, we also traced most of the propeptide, as well as an independent plant‐specific domain, never before described in structural terms. The structure revealed that, in addition to the propeptide, 13 N‐terminal residues of the mature phytepsin are essential for inactivation of the enzyme. Comparison of the plant‐specific domain with NK‐lysin indicates that these two saposin‐like structures are closely related, suggesting that all saposins and saposin‐like domains share a common topology. Structural analysis of prophytepsin led to the identification of a putative membrane receptor‐binding site involved in Golgi‐mediated transport to vacuoles.
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transport and activation of the vacuolar Aspartic Proteinase phytepsin in barley hordeum vulgare l
Journal of Biological Chemistry, 1998Co-Authors: Stefanie Glathe, Jukka Kervinen, Gregory J Tobin, Alexander Wlodawer, Manfred Nimtz, Grace H Li, Terry D Copeland, David A Ashford, Julia CostaAbstract:Abstract The primary translation product of barley Aspartic Proteinase, phytepsin (EC 3.4.23.40), consists of a signal sequence, a propart, and mature enzyme forms. Here, we describe post-translational processing and activation of phytepsin during its transport to the vacuole in roots, as detected by using metabolic labeling and immunoprecipitation. After removal of the signal sequence, the glycosylated precursor of 53 kDa (P53) was produced and further processed to polypeptides of 31 and 15 kDa (P31 + P15) and, subsequently, to polypeptides of 26 and 9 kDa (P26 + P9), 45 min and 24 h after synthesis, respectively. The processing occurred in a late-Golgi compartment or post-Golgi compartment, because brefeldin A inhibited the processing, and P53 acquired partial endoglycosidase H resistance 30 min after synthesis, whereas P15 was completely resistant. The N-glycosylation inhibitor tunicamycin had no effect on transport, but the absence of glycans on P53 accelerated the proteolytic processing. Phytepsin was also expressed in baculovirus-infected insect cells. The recombinant prophytepsin underwent autoproteolytic activation in vitro and showed enzymatic properties similar to the enzyme purified from grains. However, a comparison of the in vitro/in vivoprocessing sites revealed slight differences, indicating that additional proteases are needed for the completion of the maturationin vivo.
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tissue specific localization of Aspartic Proteinase in developing and germinating barley grains
Planta, 1994Co-Authors: Kirsi Tormakangas, Jukka Kervinen, Anne Ostman, Teemu H TeeriAbstract:Resting seeds of several plant species, including barley grains, have been reported to contain Aspartic Proteinase (EC 3.4.23) activity. Here, the expression of the Hordeum vulgare L. Aspartic Proteinase (HvAP) was studied in developing and germinating grains by activity measurements as well as by immunocytochemical and in-situ hybridization techniques. Southern blotting suggests the presence of one to two HvAP-encoding genes in the barley genome, while Northern analysis reveals a single 2.1-kb mRNA in grains and vegetative tissues. Western blotting with antibodies to HvAP shows the same subunit structure in different grain parts. In developing grains, HvAP is produced in the embryo, aleurone layer, testa and pericarp, but in the starchy endosperm HvAP is present only in the crushed and depleted area adjacent to the scutellum. During seed maturation, HvAP-encoding mRNA remains in the aleurone layer and in the embryo, but the enzyme disappears from the aleurone cells. The enzyme, however, remains in the degenerating tissues of the testa and pericarp as well as in resting embryo and scutellum. During the first three days of germination, the enzyme reappears in the aleurone layer cells but is not secreted into the starchy endosperm. The HvAP is also expressed in the flowers, stem, leaves, and roots of barley. The wide localization of HvAP in diverse tissues suggests that it may have several functions appropriate to the needs of different tissues.
Miguel A Mazorramanzano – 3rd expert on this subject based on the ideXlab platform
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structure function characterization of the recombinant Aspartic Proteinase a1 from arabidopsis thaliana
Phytochemistry, 2010Co-Authors: Miguel A Mazorramanzano, Takuji Tanaka, Rickey Y. YadaAbstract:Abstract Aspartic Proteinases (APs) are involved in several physiological processes in plants, including protein processing, senescence, and stress response and share many structural and functional features with mammalian and microbial APs. The heterodimeric Aspartic Proteinase A1 from Arabidopsis thaliana (AtAP A1) was the first acid protease identified in this model plant, however, little information exists regarding its structure function characteristics. Circular dichroism analysis indicated that recombinant AtAP A1 contained an higher α-helical content than most APs which was attributed to the presence of a sequence known as the plant specific insert in the mature enzyme. rAtAP A1 was stable over a broad pH range (pH 3–8) with the highest stability at pH 5–6, where 70–80% of the activity was retained after 1 month at 37 °C. Using calorimetry, a melting point of 79.6 °C was observed at pH 5.3. Cleavage profiles of insulin β-chain indicated that the enzyme exhibited a higher specificity as compared to other plant APs, with a high preference for the Leu 15 –Tyr 16 peptide bond. Molecular modeling of AtAP A1 indicated that exposed histidine residues and their interaction with nearby charged groups may explain the pH stability of rAtAP A1.
-
expression and characterization of the recombinant Aspartic Proteinase a1 from arabidopsis thaliana
Phytochemistry, 2008Co-Authors: Miguel A Mazorramanzano, Rickey Y. YadaAbstract:Abstract The present study reports the recombinant expression, purification, and partial characterization of a typical Aspartic Proteinase from Arabidopsis thaliana (AtAP A1). The cDNA encoding the precursor of AtAP A1 was expressed as a functional protein using the yeast Pichia pastoris . The mature form of the rAtAP A1 was found to be a heterodimeric glycosylated protein with a molecular mass of 47 kDa consisting of heavy and light chain components, approx. 32 and 16 kDa, respectively, linked by disulfide bonds. Glycosylation occurred via the plant specific insert in the light chain. The catalytic properties of the rAtAP A1 were similar to other plant Aspartic Proteinases with activity in acid pH range, maximal activity at pH 4.0, K m of 44 μM, and k cat of 55 s −1 using a synthetic substrate. The enzyme was inhibited by pepstatin A.