Biomedical subjects

Senarath B P Athauda

Publications and source records attributed to Senarath B P Athauda.

7 recordsLinked to original sources

PubMed2006

Isolation and characterization of recombinant Drosophila Copia aspartic proteinase.

The wild type Copia Gag precursor protein of Drosophila melanogaster expressed in Escherichia coli was shown to be processed autocatalytically to generate two daughter proteins with molecular masses of 33 and 23 kDa on SDS/PAGE. The active-site motif of aspartic proteinases, Asp-Ser-Gly, was present in the 23 kDa protein corresponding to the C-terminal half of the precursor protein. The coding region of this daughter protein (152 residues) in the copia gag gene was expressed in E. coli to produce the recombinant enzyme protein as inclusion bodies, which was then purified and refolded to create the active enzyme. Using the peptide substrate His-Gly-Ile-Ala-Phe-Met-Val-Lys-Glu-Val-Asn (cleavage site: Phe-Met) designed on the basis of the sequence of the cleavage-site region of the precursor protein, the enzymatic properties of the proteinase were investigated. The optimum pH and temperature of the proteinase toward the synthetic peptide were 4.0 and 70 degrees C respectively. The proteolytic activity was increased with increasing NaCl concentration in the reaction mixture, the optimum concentration being 2 M. Pepstatin A strongly inhibited the enzyme, with a Ki value of 15 nM at pH 4.0. On the other hand, the active-site residue mutant, in which the putative catalytic aspartic acid residue was mutated to an alanine residue, had no activity. These results show that the Copia proteinase belongs to the family of aspartic proteinases including HIV proteinase. The B-chain of oxidized bovine insulin was hydrolysed at the Leu15-Tyr16 bond fairly selectively. Thus the recombinant Copia proteinase partially resembles HIV proteinase, but is significantly different from it in certain aspects.

Amino Acid Sequence↗

PubMed2006

Identification and reverse genetic analysis of mitochondrial processing peptidase and the core protein of the cytochrome bc1 complex of Caenorhabditis elegans, a model parasitic nematode.

Mitochondria could be a good target for anti-parasitic drugs. The alpha and beta subunits of mitochondrial processing peptidase (MPP) and the core subunits of the cytochrome bc1 complex, UCR-1 and UCR-2, are homologous to one another and are important for mitochondrial functions. However, our knowledge of these proteins in nematodes is very limited. Caenorhabditis elegans, a free-living nematode, has six genes coding for proteins homologous to these subunits. On primary structure comparison, and immunochemical and enzymological analyses, the gene products were assigned as follows: Y71G12B.24, alpha-MPP; ZC410.2, beta-MPP; F56D2.1, UCR-1; VW06B3R.1, T10B10.2; and T24C4.1, UCR-2. The primary structures of beta-MPP and UCR-1 from Brugia malayi, a parasitic nematode causing human filariasis, were deduced from their cDNA structures. Phylogenetic analysis showed that the UCR-1s from both C. elegans and B. malayi were less related to mammalian UCR-1s than to MPPs from various organisms. MPP and the bc1 complex are essential for the life cycle of C. elegans, because their reverse genetic inhibition is lethal. This suggests the possibility that these proteins are also essential for the viability of B. malayi and other parasitic nematodes, and are potential targets for anti-parasitic agents.

Amino Acid Motifs↗

PubMed2005

Decrease of transthyretin synthesis at the blood-cerebrospinal fluid barrier of old sheep.

Transthyretin (TTR), synthesized by the choroid plexus (CP) and secreted into cerebrospinal fluid (CSF), is involved in thyroxine (T4) transport and chelation of beta-amyloid peptide, attenuating neurotoxicity. To characterize age-related changes in TTR synthesis, CSF and CPs were collected from young adult (1-2 years) and old (>8 years) sheep anesthetized with thiopentone sodium. TTR in old sheep CSF was low compared to young (n = 4 each); however, CP messenger RNA (mRNA) for TTR did not change. CPs were perfused with Ringer containing 14C-leucine to assess de novo protein synthesis, or with 125I-T4 to assess T4 transport. Protein synthesis, including TTR, was reduced in old sheep CP and in newly secreted CSF. 125I-T4 Vmax and Kd (but not Km) were reduced in old sheep CP. These age-related changes suggest reduced capacity of CP to maintain CSF T4 homeostasis and could also reduce chelation of beta-amyloid and be an added risk for Alzheimer's disease.

Aging↗

PubMed2005

Nepenthesin, a unique member of a novel subfamily of aspartic proteinases: enzymatic and structural characteristics.

Carnivorous plants are known to secrete acid proteinases to digest prey, mainly insects, for nitrogen uptake. In our recent study, we have purified, for the first time, to homogeneity two acid proteinases, nepenthesin I (Nep I) and nepenthesin II (Nep II) from the pitcher fluid of Nepenthes distillatoria and investigated their enzymatic and structural characteristics. Both enzymes were optimally active at pH approx. 2.6 toward acid-denatured hemoglobin; the specificity of Nep I toward oxidized insulin B chain appears to be similar, but slightly wider than those of other aspartic proteinases (APs). At or below 50 degrees C both enzymes were remarkably stable; especially Nep I was extremely stable over a wide range of pH from 3 to 10 for over 30 days. This suggests an evolutionary adaptation of the enzymes to their specific habitat. We have also cloned the cDNAs and deduced the complete amino acid sequences of the precursors of Nep I and Nep II from the pitcher tissue of Nepenthes gracilis. Although the corresponding mature enzymes are homologous with ordinary pepsin-type APs, both enzymes had a high content of cysteine residues (12 residues per molecule), which are assumed to form six unique disulfide bonds as suggested by computer modeling and are thought to contribute toward the remarkable stability of Neps. Moreover, the amino acid sequence identity of Neps with ordinary APs, including plant vacuolar APs, are remarkably low (approx. 20%), and phylogenetic comparison shows that Neps are distantly related to them to form a novel subfamily of APs with a high content of cysteine residues and a characteristic insertion, named 'the Nep-type AP (NAP)-specific insertion', including a large number of novel, orthologous plant APs emerging in the gene/protein databases.

Amino Acid Sequence↗

PubMed2004

Enzymic and structural characterization of nepenthesin, a unique member of a novel subfamily of aspartic proteinases.

Carnivorous plants are known to secrete acid proteinases to digest prey, mainly insects, for nitrogen uptake. In the present study, we have purified, for the first time, to homogeneity two acid proteinases (nepenthesins I and II) from the pitcher fluid of Nepenthes distillatoria (a pitcher-plant known locally as badura) and investigated their enzymic and structural characteristics. Both enzymes were optimally active at pH approx. 2.6 towards acid-denatured haemoglobin; the specificity of nepenthesin I towards oxidized insulin B chain appears to be similar, but slightly wider than those of other APs (aspartic proteinases). Among the enzymic properties, however, the most notable is their unusual stability: both enzymes were remarkably stable at or below 50 degrees C, especially nepenthesin I was extremely stable over a wide range of pH from 3 to 10 for over 30 days. This suggests an evolutionary adaptation of the enzymes to their specific habitat. We have also cloned the cDNAs and deduced the complete amino acid sequences of the precursors of nepenthesins I and II (437 and 438 residues respectively) from the pitcher tissue of N. gracilis. Although the corresponding mature enzymes (each 359 residues) are homologous with ordinary pepsin-type APs, both enzymes had a high content of cysteine residues (12 residues/molecule), which are assumed to form six unique disulphide bonds as suggested by computer modelling and are supposed to contribute towards the remarkable stability of nepenthesins. Moreover, the amino acid sequence identity of nepenthesins with ordinary APs, including plant vacuolar APs, is remarkably low (approx. 20%), and phylogenetic comparison shows that nepenthesins are distantly related to them to form a novel subfamily of APs with a high content of cysteine residues and a characteristic insertion, named 'the nepenthesin-type AP-specific insertion', that includes a large number of novel, orthologous plant APs emerging in the gene/protein databases.

Amino Acid Sequence↗

PubMed2003

Inhibition of human pepsin and gastricsin by alpha2-macroglobulin.

The inhibitory effects of human alpha2-macroglobulin (alpha2-M), a major plasma proteinase inhibitor, on human pepsin and gastricsin were investigated. The activities of pepsin and gastricsin towards a protein substrate (reduced and carboxymethylated ribonuclease A) were significantly inhibited by alpha2-M at pH 5.5, whereas those towards a peptide substrate (oxidized insulin B-chain) were scarcely inhibited. Under these conditions at pH 5.5, pepsin and gastricsin cleaved alpha2-M mainly at the His694-Ala695 bond and Leu697-Val698 bond, respectively, in the bait regions sequence of alpha2-M. The conformation of alpha2-M was also shown to be markedly altered upon inhibition of these enzymes as examined by native polyacrylamide gel electrophoresis and electron microscopy. These results show the entrapment and concomitant inhibition of those proteinases by alpha2-M.

Alanine↗

PubMed2002

Distinct cleavage specificity of human cathepsin E at neutral pH with special preference for Arg-Arg bonds.

In order to clarify the potential role of cathepsin E at neutral pH, the cleavage specificity of human cathepsin E was examined at pH 7.4 toward reduced-carboxymethylated(RCm-)ribonuclease A and various bioactive and related peptides. The specificity of the enzyme at pH 7.4 was found to be considerably different from that at acidic pH; preferential cleavages were observed with Arg-X and Glu-X bonds, which are not the major cleavage sites at acidic pH. Moreover, the Arg-Arg bond was found to be the most preferential site of cleavage. This unique specificity observed at pH 7.4 suggests the possibility that cathepsin E might be involved in processing and/or degradation of certain proteins and/or peptides at or near neutral pH in vivo.

Amino Acid Sequence↗