The Experts below are selected from a list of 177 Experts worldwide ranked by ideXlab platform
Ben M Dunn - One of the best experts on this subject based on the ideXlab platform.
-
ANALYSIS OF BINDING INTERACTIONS OF PEPSIN INHIBITOR-3 TO MAMMALIAN AND MALARIAL ASPARTIC PROTEASES
2015Co-Authors: Mavis Agb, Ben M DunnAbstract:The nematode Ascaris suum primarily infects pigs, but also causes disease in humans. As part of its survival mechanism in the intestinal tract of the host, the worm produces a number of protease inhibitors, including pepsin inhibitor-3 (PI3), a 17 kDa protein. Recombinant PI3 expressed in E. coli has previously been shown to be a competitive inhibitor of a sub-group of aspartic proteinases: pepsin, Gastricsin and cathepsin E. The previously determined crystal structure of the complex of PI3 with porcine pepsin (p. pepsin) showed that there are two regions of contact between PI3 and the enzyme. The first three N-terminal residues (QFL) bind into the prime side of the active site cleft and a polyproline helix (139–140) in the C-terminal domain of PI3 packs against residues 289–295 that form a loop in p. pepsin. Mutational analysis of both inhibitor regions was conducted to assess their contributions to the binding affinity for p. pepsin, human pepsin (h. pepsin) and several malarial aspartic proteases, the plasmepsins. Overall, the polyproline mutations have a limited influence on the Ki values for all the enzymes tested, with the values for p. pepsin remaining in the low nanomolar range. The largest effect was seen with a Q1L mutant, with a 200-fold decrease in Ki for plasmepsin 2 from Plasmodium falciparum (PfPM2). Thermodynamic measurements of the binding of PI3 to p
-
Detailed Analysis of Human Cathepsin E Prime Region Specificity
Advances in experimental medicine and biology, 1998Co-Authors: Marina Bukhtiyarova, John Kay, Chetana Rao-naik, Peter J. Tatnell, Paul C. White, Ben M DunnAbstract:Aspartic proteinases are produced in the human body by a variety of cells. Some of these proteins, such as pepsin, Gastricsin and renin, are secreted and exert their effects in the extracellular environment. Cathepsin D and cathepsin E, on the other hand, are intracellular enzymes. Three-dimensional structures have been determined for four of these enzymes by X-ray crystallography with the exception of cathepsin E.
-
Expression, Purification, and Characterization of the Recombinant Pepsin Inhibitor from Ascaris suum
Advances in experimental medicine and biology, 1998Co-Authors: Jeffrey J. Zalatoris, John Kay, Chetana Rao-naik, Gregory Fecho, Karen Girdwood, Ben M DunnAbstract:Only a few natural inhibitors of aspartic proteinases have been discovered to date. A small number of peptides from bacterial origins have been shown to inhibit members of this protease family.1-2 Larger proteinaceous inhibitors of aspartic proteases have been discovered in the potato3 (cathepsin D inhibitor), in yeast,4 and the intestinal nematode Ascaris.5–7 Similar genes have been discovered in other parasitic nematodes.8 The Ascaris aspartic proteinase inhibitor (PI-3) is a 16.7 kDa protein that specifically inhibits pepsin, Gastricsin, and, less strongly, cathepsin E. This inhibitor possesses six cysteine residues which form three disulfide bridges, and it contains helical and strand structural motifs.
-
Exploring the binding preferences/specificity in the active site of human cathepsin E.
Proteins, 1995Co-Authors: Chetana Rao-naik, John Kay, Brian L. Batley, Stephen T. Rapundalo, Kunchur Guruprasad, Jeffrey Hill, Tom L. Blundell, Ben M DunnAbstract:Aspartic proteinases are produced in the human body by a variety of cells. Some of these proteins, examples of which are pepsin, Gastricsin, and renin, are secreted and exert their effects in the extracellular spaces. Cathepsin D and cathepsin E on the other hand are intracellular enzymes. The least characterized of the human aspartic proteinases is cathepsin E. Presented here are results of studies designed to characterize the binding specificities in the active site of human cathepsin E with comparison to othermechanistically similar enzymes. A peptide series based on Lys-Pro-Ala-Lys-Phe*Nph-Arg-Leu was generatedto elucidate the specificity in the individual binding pockets with systematic substitutions in the P5− P2 and P2′-P3′ based on charge, hydrophobicity, and hydrogen bonding. Also, to explore the S2 binding preferences, asecond series of peptides based on Lys-Pro-Ile-Glu-Phe*Nph-Arg-Leu was generated with systematic replacements in the P2 position. Kinetic parameters were determined forboth sets of peptides. The results were correlated to a rule-based structural model of human cathepsin E, constructed on the known three-dimensional structures of several highly homologous aspartic proteinases; porcine pepsin, bovine chymosin, yeast proteinase A, human cathepsin D, andmouse and human renin. Important specificity-determining interactions were found in the S3 (Glu13) and S2 (Thr-222, Gln-287, Leu-289, Ile-300)subsites. © 1995 Wiley-Liss, Inc.
-
Inhibition of aspartic proteinases by synthetic peptides derived from the propart region of human prorenin.
International Journal of Biochemistry, 1992Co-Authors: Anthony D. Richards, C. M. Bessant, Ben M Dunn, P.a. CharltonAbstract:1. 1. Five synthetic peptides which together spanned the propart segment of human prorenin were tested for their ability to interact with human renin, pepsin, Gastricsin, cathepsin D, cathepsin E, calf chymosin and the aspartic proteinase from Endothia parasitica. 2. 2. While two peptides showed no significant effect with any of the enzymes, a further two were cleaved by several enzymes. 3. 3. Only one (corresponding to the 32P–43P residues in the propart sequence) acted as a weak competitive inhibitor of most of the enzymes.
Michael N. G. James - One of the best experts on this subject based on the ideXlab platform.
-
Crystallographic Studies of an Activation Intermediate of Human Gastricsin
Advances in experimental medicine and biology, 1998Co-Authors: Amir R. Khan, Nadezhda I. Tarasova, Maia M. Chernaia, Michael N. G. JamesAbstract:The human digestive enzyme proGastricsin (hPGC) is an aspartic proteinase zymogen that is synthesized as an inactive precursor, having a positively charged and inhibitory N-terminal prosegment of 43 residues (Ala1p to Leu43p; the “p” suffix refers to the prosegment). Conversion of proGastricsin to mature Gastricsin occurs upon the lowering of pH, and involves conformational changes that uncover the active site.1 The initial hydrolytic event is the uni-molecular, auto-catalytic cleavage of the peptide bond between Phe26p and Leu27p in the prosegment.1,3 Further proteolytic processing is then initiated, ultimately resulting in the removal of the entire prosegment and formation of the mature enzyme. Our lab has previously determined the crystal structure of hPGC.2 The structure of mature Gastricsin is currently undetermined, although it is expected to resemble human pepsin and the other aspartic proteinases.
-
Structural characterization of activation ‘intermediate 2’ on the pathway to human Gastricsin
Nature Structural Biology, 1997Co-Authors: Amir R. Khan, Nadezhda I. Tarasova, Maia M. Cherney, Michael N. G. JamesAbstract:The crystal structure of an activation intermediate of human Gastricsin has been determined at 2.4 Å resolution. The human digestive enzyme Gastricsin (pepsin C) is an aspartic proteinase that is synthesized as the inactive precursor (zymogen) proGastricsin (pepsinogen C or hPGC). In the zymogen, a positively-charged N-terminal prosegment of 43 residues (Ala 1p–Leu 43p; the suffix ‘p’ refers to the prosegment) sterically prevents the approach of a substrate to the active site. Zymogen conversion occurs in an autocatalytic and stepwise fashion at low pH through the formation of intermediates. The structure of the non-covalent complex of a partially-cleaved peptide of the prosegment (Ala 1p–Phe 26p) with mature Gastricsin (Ser 1–Ala 329) suggests an activation pathway that may be common to all gastric aspartic proteinases.
-
Structural characterization of activation 'intermediate 2' on the pathway to human Gastricsin.
Nature structural biology, 1997Co-Authors: Amir R. Khan, Nadezhda I. Tarasova, Maia M. Cherney, Michael N. G. JamesAbstract:Structural characterization of activation ‘intermediate 2’ on the pathway to human Gastricsin
-
The Molecular Structure of Human ProGastricsin and its Comparison with that of Porcine Pepsinogen
Advances in experimental medicine and biology, 1995Co-Authors: Nadezhda I. Tarasova, Michael N. G. James, Stanley A. Moore, Anita R. Sielecki, Maia M. ChernaiaAbstract:Mammalian aspartic proteinases are synthesized as inactive precursors or zymogens. Stomach zymogens undergo a conversion to the active enzyme form autocatalytically at pH < 5.0 (1). The human gastric juice has two major groups of aspartic proteinases, the pepsins (EC3.4.23.1) and the Gastricsins (EC3.4.23.3). ProGastricsin or pepsinogen C (PGC) is converted to Gastricsin by removal of the 43 amino-terminal residues of the prosegment. The resulting mature Gastricsin has 329 amino acid residues. The sequence of human PGC has been determined independently in two laboratories by nucleotide sequencing of the gene (2) and of cDNA clones (3).
-
Separation of porcine pepsinogen A and proGastricsin. Sequencing of the first 73 amino acid residues in proGastricsin.
Biochimica et biophysica acta, 1992Co-Authors: Bent Foltmann, Helle B. Drøhse, Peter K. Nielsen, Michael N. G. JamesAbstract:Porcine pepsinogen A (EC 3.4.23.1) and proGastricsin (EC 3.4.23.3) have been separated by chromatography on DEAE-cellulose followed by chromatography on DEAE-Sepharose. Agar gel electrophoresis at pH 6.0 showed the presence of three components of pepsinogen A and two of proGastricsin. During activation at pH 2 a segment of 43 amino acid residues (the prosegment peptide) is cleaved from the N-terminus of proGastricsin. The sequence of this was determined; in addition, the first 30 residues of Gastricsin were sequenced. The sequence of the first 73 amino acid residues of proGastricsin shows an overall identity with proGastricsins from man, monkey and rat of 67%. The overall identity with other zymogens for gastric proteinases is 27%. The highly conserved Lys36p (pig pcpsinogen A numbering) is changed to Arg in porcine proGastricsin.
Bent Foltmann - One of the best experts on this subject based on the ideXlab platform.
-
Human proGastricsin. Analysis of intermediates during activation into Gastricsin and determination of the amino acid sequence of the propart.
European journal of biochemistry, 2005Co-Authors: Bent Foltmann, Arne L. JensenAbstract:Human proGastricsin was prepared from extracts of gastric mucosa by chromatography on columns of DEAE-cellulose. The amino acid compositions of proGastricsin and Gastricsin were determined and calculated on the basis of the molecular weights 38 000 and 32 000 respectively. The activation of proGastricsin at pH 2 was investigated and monitored by agarose gel electrophoresis at pH 5.4. Two intermediates were observed. Determination of the amino acid sequence showed that the propart consists of 43 amino acid residues. A pronounced homology with other gastric zymogens was found. With the proenzyme amino acid residue numbering used previously [B. Foltman (1981) Essays in Biochemistry, 17, 52-84] the activation of proGastricsin at pH 2 may be summarized as follows. The first cleavage occurs after Phe (p27). At pH 5.4 the peptide remains associated with the protein (intermediate I). Subsequent proteolysis removes the peptides from Leu (p28) to Leu (p45). At pH 5.4 the N-terminal peptide from proGastricsin (p2-p27) remains associated with Gastricsin (intermediate II) until the propart peptide is hydrolysed to smaller fragments.
-
Comparative Investigations on Pig Gastric Proteases and Their Zymogens
Advances in experimental medicine and biology, 1995Co-Authors: Bent Foltmann, Peter K. Nielsen, Kenneth W. Harlow, Gunnar Houen, Per T. SangildAbstract:Pepsin (pepsin A EC 3.4.23.1) is the predominant proteolytic enzyme in the gastric juice of most vertebrates. It was named by Schwann (1836) long before the term enzyme was introduced, and in the early days of biochemistry pepsin A was used as a model enzyme in numerous investigations. The minor proteolytic components of the gastric juice have received much less attention. Pepsin B (EC 3.4.23.2) and Gastricsin (EC 3.4.23.3) were isolated by Ryle and Porter (1959) and first designated parapepsin I and II. The corresponding zymogens were subsequently isolated (Ryle, 1960; Ryle, 1965) but no structural data were reported. Chymosin (EC 3.4.23.4) was for many years regarded as a milk-clotting enzyme that was characteristic for young ruminants (Foltmann, 1966), but immunochemical cross reactivity between calf chymosin and extracts of stomachs from newborn piglets showed that the gastric juice of piglets also contains chymosin (Foltmann et al., 1978).
-
Separation of porcine pepsinogen A and proGastricsin. Sequencing of the first 73 amino acid residues in proGastricsin.
Biochimica et biophysica acta, 1992Co-Authors: Bent Foltmann, Helle B. Drøhse, Peter K. Nielsen, Michael N. G. JamesAbstract:Porcine pepsinogen A (EC 3.4.23.1) and proGastricsin (EC 3.4.23.3) have been separated by chromatography on DEAE-cellulose followed by chromatography on DEAE-Sepharose. Agar gel electrophoresis at pH 6.0 showed the presence of three components of pepsinogen A and two of proGastricsin. During activation at pH 2 a segment of 43 amino acid residues (the prosegment peptide) is cleaved from the N-terminus of proGastricsin. The sequence of this was determined; in addition, the first 30 residues of Gastricsin were sequenced. The sequence of the first 73 amino acid residues of proGastricsin shows an overall identity with proGastricsins from man, monkey and rat of 67%. The overall identity with other zymogens for gastric proteinases is 27%. The highly conserved Lys36p (pig pcpsinogen A numbering) is changed to Arg in porcine proGastricsin.
Hans Lilja - One of the best experts on this subject based on the ideXlab platform.
-
Gastricsin-mediated proteolytic degradation of human seminal fluid proteins at pH levels found in the human vagina.
Journal of andrology, 1993Co-Authors: Pal B. Szecsi, Hans LiljaAbstract:The proteolytic degradation of human seminal fluid proteins at acidic conditions has been investigated. Upon acidification to the pH level of the human vagina, autoproteolysis of most seminal fluid proteins occurred after 30 minute of incubation at 37 degrees C. The degradation was unaffected by inhibitors of serine, thiol, or metallo proteases, whereas pepstatin prevented any proteolysis. The proteins in seminal fluid depleted of the aspartic protease proGastricsin did not degrade upon acidification. Readdition of the proGastricsin restored the autoproteolytic ability of seminal fluid. Prostate-specific antigen, prostatic acid phosphatase, and Zn-alpha 2-glycoprotein are quickly degraded; albumin, transferrin, and lactoferrin are degraded more slowly. The low molecular weight fragments of semenogelin I and II and especially beta-microseminoprotein are somewhat resistant to proteolysis. These observations strongly suggest that the aspartic protease proGastricsin is responsible for the autoproteolysis of seminal fluid proteins under acidic conditions. This suggests that the function of the enzyme is to degrade seminal fluid proteins deposited in the vagina; this in turn may decrease the antigenic load in the vagina and prevent immuno-infertility.
Norman B. Roberts - One of the best experts on this subject based on the ideXlab platform.
-
Molecular Mass Determination by Electrospray Mass Spectrometry of Human Pepsins, Gastricsin, and Porcine Pepsin A Variants
Advances in experimental medicine and biology, 1995Co-Authors: Arwyn Tomos Jones, Brian N. Green, Steve P. Wood, Norman B. RobertsAbstract:Proteins are normally analysed by Electrospray Mass Spectrometry (ESMS) as their multiply charged positive ions, when their molecular masses are typically measured to within 0.01% of the values calculated from the primary sequences. The aspartic proteinases obtained from gastric secretions, however, have an insufficient number of basic amino acids to be analysed in this way, but should be well suited to negative ion analysis because they contain a large number of acidic residues. The amount of available data on the analysis of acidic proteins by ESMS is sparse and in order to assess the efficacy of negative ion ESMS, a commercial sample of porcine pepsin A and several proteinases isolated from human gastric juice were analysed and their measured molecular masses compared with values calculated from cDNA sequences (1–3). Attempts were made from mass data, to characterise purified human pepsin A variants with the published cDNA sequences(2); and particular emphasis was placed on obtaining a true molecular mass for human pepsin 1 (fast moving pepsin) for which there is evidence of carbohydrate attachment(4).
-
Five human gastric aspartic proteinases: N-terminal amino acid sequences and amino acid composition.
The international journal of biochemistry & cell biology, 1995Co-Authors: Norman B. Roberts, Keith Peek, Jeff N. Keen, William H. TaylorAbstract:Human pepsin A consists of 4 or more isoenzymes (designated 1, 3a, 3b and 3c) one of which, pepsin 1, contains up to 50% carbohydrate moieties. The amino-acid composition and N-terminal sequence of pepsin 1 and the other isoforms have been determined and compared with data obtained for pepsin 3b and Gastricsin (pepsin C or pepsin 5). Pepsins were isolated from penta-gastrin stimulated gastric juice using repetitive chromatography on DEAE-cellulose, or high performance ion-exchange chromatography. Sequencing was performed using automated solid-phase Edman degradation with a microsequence facility. The amino-acid compositions were similar for pepsins 1, 3a, b and c and the N-terminal sequences of pepsins 1, 3a and c, reported for the first time, were shown to be identical with that for pepsin 3b (the main component of pepsin A) although residue 28 was unassigned in pepsin 1. Residue 30 in all four isoenzymes is valine and we cannot confirm reports of major pepsins with leucine in this position. For Gastricsin the sequence differed from the pepsin isoenzymes and in position 24 we find pro rather than ala as was first described. These observations suggest that pepsin 1 is identical to 3b or a mixture of 3a, 3b and 3c but not Gastricsin. This data supports the hypotheses that the four pepsin isoenzymes are products of the same gene(s) but have undergone varying levels of post translational modification.
-
Assay of Gastricsin and individual pepsins in human gastric juice.
Journal of clinical pathology, 1993Co-Authors: Arwyn Tomos Jones, K. K. Balan, S. A. Jenkins, R. Sutton, M. Critchley, Norman B. RobertsAbstract:AIMS: To develop and validate an analytical procedure for the quantitation of pepsins and Gastricsin in human gastric juice and to assess its potential in a controlled gastric secretory study. METHODS: High performance ion-exchange chromatography was used to separate human pepsin 1, 3a, 3b, 3c and Gastricsin from gastric juice. Computed chromatographic areas for each enzyme were quantified by relation to a known amount of a secondary standard porcine pepsin. The assay procedure was validated by recovery and analytical precision studies. Gastric secretions after pentagastrin and insulin stimulation from 10 patients with portal hypertension were used to assess the potential of the analytical procedure. RESULTS: The assay precision varied from 1.5 to 9.0% within batch and 7.5 to 18.1% between batch, with about 100% recoveries of porcine pepsin A from human gastric juice over the assay range 0.025-0.5 mg/ml. A fourfold increase in combined pepsin and Gastricsin concentration was observed following pentagastrin and insulin stimulation. The mean percentage content of pepsins 3a, 3b, 3c, and 1 in non-stimulated gastric juice were 4%, 72%, 12% and 1.4%, respectively, and did not change significantly after gastric stimulation. An approximate doubling of the percentage of Gastricsin (10% to 20%) relative to the pepsins was observed, however, after both insulin and pentagastrin stimulation. CONCLUSIONS: This procedure for quantifying individual human pepsins and Gastricsin in gastric juice is simple and reliable. It may be of considerable importance in determining the mechanisms involved in the control and secretion of these digestive enzymes in man, including the effect of anti-ulcer drugs and our understanding of the pathophysiology of peptic ulcer disease.
