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B M Dunn

Publications and source records attributed to B M Dunn.

At least 109 records · Page 6Linked to original sources
PubMed1988

Use of synthetic peptides to identify an N-terminal epitope on mouse gamma interferon that may be involved in function.

We previously have assigned N-terminal specificity to three hamster monoclonal antibodies (mAbs I, II, and III) produced to mouse recombinant gamma interferon (IFN-gamma), based on the ability of the N-terminal peptide IFN-gamma-(1-39) to block binding of 125I-labeled IFN-gamma (125I-IFN-gamma) and on the ability of these antibodies to bind 125I-IFN-gamma-(1-39). Only mAb I blocked function and binding to the IFN-gamma receptor, suggesting that it may bind to a region of the molecule involved in interaction with the receptor. To further define the epitope specificities of the antibodies, a series of N-terminal peptides were synthesized and tested for their ability to block antibody binding of 125I-IFN-gamma. Peptides IFN-gamma-(1-39), IFN-gamma-(1-20), IFN-gamma-(3-20), and IFN-gamma-(5-20) inhibited binding of 125I-IFN-gamma by mAb I in order of decreasing effectiveness, while peptide IFN-gamma-(7-20) was without effect. Peptides IFN-gamma-(1-39), IFN-gamma-(1-20), and IFN-gamma-(3-20) also inhibited binding of 125I-IFN-gamma by mAb II but were less effective when compared with their inhibition of mAb I. IFN-gamma-(5-20) and IFN-gamma-(7-20) did not inhibit binding by mAb II. Peptides IFN-gamma-(1-10), IFN-gamma-(10-30), and IFN-gamma-(21-44) did not inhibit either mAb I or mAb II. While IFN-gamma-(1-39) and IFN-gamma-(10-30) inhibited binding by mAb III, neither IFN-gamma-(1-20) nor any of its truncated forms were inhibitory. All three antibodies had similar Kd values for 125I-IFN-gamma. A prediction of the secondary structure of the molecule and the peptide inhibition data suggest that the epitope (possible receptor binding region) for mAb I involves a loop in the area containing residues 12-20, with sequences N-terminal to these residues possibly stabilizing the loop conformation. Direct evidence that the N-terminal 1-39 region of IFN-gamma is important in receptor binding was the observation that IFN-gamma-(1-39), but not the C-terminal IFN-gamma-(95-133), competed with 125I-IFN-gamma for the receptor on mouse L cells. IFN-gamma-(1-39) also specifically blocked IFN-gamma antiviral activity at concentrations that blocked binding to the receptor. The fact that IFN-gamma-(1-39) was the only peptide that blocked both IFN-gamma binding to receptor and function is consistent with the antibody competition data, where it was the most effective peptide in blocking binding of 125I-IFN-gamma by the N-terminal-specific mAbs. The combination of peptide mapping of epitope specificities and receptor competition should further help define the structural basis for IFN-gamma action.

Animals↗
PubMed1987

The pH dependence of the hydrolysis of chromogenic substrates of the type, Lys-Pro-Xaa-Yaa-Phe-(NO2)Phe-Arg-Leu, by selected aspartic proteinases: evidence for specific interactions in subsites S3 and S2.

Variation in the kinetic parameters, kcat and Km, with pH has been used to obtain evidence for significant acid-dissociation processes in the hydrolysis of octapeptide substrates by three aspartic proteinases. These substrates are all cleaved at the peptide bond between a Phe (P1) and a p-nitroPhe (P1') residue resulting in a shift in absorbance at 300 nm that facilitates kinetic measurements. The substrates differ in the amino-acid residues present in the P3 and the P2 positions. Porcine pepsin, calf chymosin, and the aspartic proteinase from Endothia parasitica all show pH dependencies that imply that favorable or unfavorable interactions can occur with the S3 or S2 areas of the enzyme-active site. Examination of the crystallographically determined structure of the E. parasitica proteinase and consideration of the amino-acid sequence differences between the three enzymes suggests that the origin of the pH effects arises from favorable interactions between Glu-13 (COO-) of pig pepsin and Thr (OH) or His (ImH+) in P3 of a substrate. Similarly, Lys-220 (NH3+) of chymosin and a Glu (COO-) in P2 of a substrate may produce a favorable interaction and Asp-77 (COO-) of E. parasitica proteinase and a Glu (COO-) in P2 of a substrate may produce an unfavorable interaction. These results lead to possible explanations for subtle specificity differences within a family of homologous enzymes, and suggest loci for study by site-directed mutagenesis.

Amino Acid Sequence↗
PubMed1986

A systematic series of synthetic chromophoric substrates for aspartic proteinases.

The hydrolysis of the chromogenic peptide Pro-Thr-Glu-Phe-Phe(4-NO2)-Arg-Leu at the Phe-Phe(4-NO2) bond by nine aspartic proteinases of animal origin and seven enzymes from micro-organisms is described [Phe(4-NO2) is p-nitro-L-phenylalanine]. A further series of six peptides was synthesized in which the residue in the P3 position was systematically varied from hydrophobic to hydrophilic. The Phe-Phe(4-NO2) bond was established as the only peptide bond cleaved, and kinetic constants were obtained for the hydrolysis of these peptide substrates by a representative selection of aspartic proteinases of animal and microbial origin. The value of these water-soluble substrates for structure-function investigations is discussed.

Animals↗
PubMed1986

Epitope and functional specificity of monoclonal antibodies to mouse interferon-gamma: the synthetic peptide approach.

Spleen cells from hamsters immunized with recombinant mouse interferon-gamma (IFN-gamma) were fused with mouse myeloma cells, resulting in the production of four anti-IFN-gamma monoclonal antibodies. Binding of 125I-IFN-gamma by these protein A-bound antibodies was specifically blocked by cold IFN-gamma. Binding by three of these antibodies was also blocked by a synthetic peptide corresponding to the N-terminal 1-39 amino acids of IFN-gamma, whereas a corresponding C-terminal (95-133) peptide had no effect on binding. The N-terminal specificity of these three antibodies was confirmed by their specific binding of 125I-N-terminal (1-39) peptide. One of the N-terminal specific monoclonal antibodies inhibited both antiviral and macrophage priming (for tumor cell killing) activities of IFN-gamma, whereas the other two had no effect on either biologic function. The selectivity of the inhibition of IFN-gamma function was not due to a differential ability of the N-terminal specific antibodies to bind IFN-gamma. Blocking experiments with cold IFN-gamma and N-terminal peptide suggest that the epitope specificities of the monoclonal antibodies could be determined by the conformational or topographic structure of IFN-gamma. An exact determination of the epitope specificity of the monoclonal antibody that inhibited IFN-gamma function could provide insight into the structural basis for the role of the N-terminal domain in the biologic function of IFN-gamma. Polyclonal antibodies to either the N-terminal or the C-terminal peptides also inhibited both the antiviral and the macrophage-priming activities of IFN-gamma. All of the antibodies that inhibited IFN-gamma function also blocked binding of IFN-gamma to membrane receptor on cells, whereas antibodies that did not block function also did not inhibit binding. The data suggest that both the N-terminal and the C-terminal domains of IFN-gamma play an important role in its antiviral and macrophage-priming functions, possibly in a cooperative manner.

Amino Acid Sequence↗
PubMed1986

Anti-VPg antibody precipitation of product RNA synthesized in vitro by the poliovirus polymerase and host factor is mediated by VPg on the poliovirion RNA template.

Antibody to the poliovirus genome-linked protein, VPg, specifically immunoprecipitated the product RNA synthesized in vitro by the poliovirus RNA polymerase and HeLa cell host factor when VPg-linked poliovirion RNA was used as a template. The largest product RNA that was immunoprecipitated was twice the size of the template RNA. The complete denaturation of the product RNA with CH3HgOH had no effect on the immunoprecipitation reaction. In contrast, CH3HgOH denaturation prevented the immunoprecipitation of the oligo(U)-primed product RNA. Immunoprecipitation of the product RNA synthesized in the host-factor-dependent reaction was prevented if VPg was removed from the template RNA by pretreatment with proteinase K or if an RNA template without VPg was used in the reaction. The results support our previous evidence that a covalent linkage exists between the labeled negative-strand product RNA and the VPg-linked template RNA and suggest that the purified polymerase and host factor initiated RNA synthesis in vitro in the absence of VPg or a VPg-precursor protein.

Antibodies, Viral↗
PubMed1985

The selectivity of action of the aspartic-proteinase inhibitor IA3 from yeast (Saccharomyces cerevisiae).

The ability of the aspartic-proteinase inhibitor IA3 from yeast (Saccharomyces cerevisiae) to affect the activities of a range of mammalian and microbial aspartic proteinases was examined. The inhibitor appeared to be completely selective in that only the aspartic proteinase A from yeast was inhibited to any significant extent. IA3 thus represents the first example of a totally specific, naturally occurring, aspartic-proteinase inhibitor.

Animals↗
PubMed1984

Cryoenzymology of porcine pepsin.

Physical and kinetic properties of porcine pepsin have been examined in aqueous methanol solvents at temperatures below ambient to seek evidence for covalent intermediates in the catalyzed hydrolysis of good substrates. It was first demonstrated that aqueous methanol cryosolvents have no significant deleterious effects upon this protein. The addition of methanol does lead to a drastic reduction in the midpoint of the thermal melting curve of pepsin. This could account for rate reductions previously observed in catalysis by this enzyme. This effect is lessened by the addition of active-site ligands including substrates and is fully reversible upon dilution into aqueous solution. Two substrates were chosen which have chromophoric groups on opposite sides of the scissile peptide bond. The UV spectral changes from hydrolysis of Pro-Thr-Glu-Phe-(NO2)Phe-Arg-Leu and the fluorescence spectral changes from hydrolysis of DNS-Ala-Ala-Phe-Phe-OP4P+-CH3 were studied at temperatures down to -60 degrees C. The resulting Arrhenius plots were linear in the region where pepsin exists in the native state with downward curvature exhibited at higher temperatures where the reversible denaturation occurs. No "burst" reactions were observed with either substrate. In addition, efforts at trapping intermediates by low-temperature denaturation and precipitation have provided no evidence for covalent intermediates on the reaction pathway. Although this evidence is negative, we cannot rule out the possibility of the formation of covalent intermediates following an initial rate-limiting step.

Animals↗
PubMed1984

Cryoenzymology of penicillopepsin; with an appendix: mechanism of action of aspartyl proteinases.

Intrinsic spectral and kinetic properties of penicillopepsin and its action on N-acetylalanylalanyllysyl-p-nitrophenylalanylalanylalanine amide have been investigated at subzero temperatures in aqueous methanol and dimethyl sulfoxide solutions in an attempt to find evidence for or against a covalent mechanism in the catalyzed hydrolysis of peptide bonds. The study of fluorescence and circular dichroism spectra as a function of solvent concentrations gave no evidence for any solvent-induced structural effects at temperatures below the thermal denaturation transition. The effect of temperature on the intrinsic fluorescence of penicillopepsin in either 60% (v/v) methanol or 50% (v/v) dimethyl sulfoxide did not indicate any temperature-induced structural changes. On the other hand, Arrhenius plots for the hydrolysis reaction over the range 0 to -50 degrees C showed downward curvature. A probable explanation for this phenomenon is that the reduction in flexibility of the enzyme due to thermal and viscosity factors leads to the stabilization of a nonproductive conformation. The pH optima of kcat/Km are shifted from 5.1 in aqueous solvents to 5.6 in 60% methanol and to 6.6 in 50% dimethyl sulfoxide. Aqueous methanol caused small decreases of Km and of Kcat; the decrease in the latter was greater than that brought about by the decrease in the water concentration. In aqueous dimethyl sulfoxide, there was no detectable change in kcat up to 15%, but Km increased by more than an order of magnitude. Above 15%, only kcat/Km could be measured. No evidence for the accumulation of either covalent amino or covalent acyl intermediates was obtained when penicillopepsin was incubated at -70 degrees C in 67% methanol with several substrates. Although negative, these experiments do not rule out conclusively the involvement of covalent intermediates in penicillopepsin-catalyzed reactions.

Aspartic Acid Endopeptidases↗
PubMed1984

The synthesis, purification, and evaluation of a chromophoric substrate for pepsin and other aspartyl proteases: design of a substrate based on subsite preferences.

A convenient chromophoric assay for porcine pepsin has been developed using a new synthetic substrate. The sequence of this substrate was chosen based on the known subsite preferences for this enzyme. The peptide contains a phenylalanyl-p-nitrophenylalanine sequence at the reactive site. Cleavage of this bond yields a change in absorbance at 310 nm of between 1700 and 2000 per mole. This allows kinetic data to be obtained readily and accurately. The products of cleavage have been identified by isolation of a peptide fragment by high-performance liquid chromatography. Values of kcat, Km, and kcat/Km of 94 +/- 6 s-1, 0.13 +/- .04 mM, and 815 +/- 210 s-1/mM-1 were obtained at pH 3.0 and 37 degrees C. The peptide is soluble over the pH range from 2 to 7, thus facilitating determination of the pH dependence of the kinetic parameters. The substrate is also valuable in studying the inhibition of pepsin.

Amino Acid Sequence↗
PubMed1983

Inhibition of pepsin by analogues of pepsinogen-(1-12)-peptide with substitutions in the 4-7 sequence region.

Derivatives of the 1-12 sequence of pig pepsinogen were prepared by solid-phase peptide synthesis. The three derivatives contain substitutions in the 4-7 region of the 1-12 sequence. Glycine was used to replace the hydrophobic residues -Val-Pro-Leu-Val- in pairs. After cleavage and purification, the synthetic peptides were compared with a synthetic peptide of the native sequence, prepared at the same time, with respect to their ability to inhibit the pepsin-catalysed clotting of milk. Inhibitory potency, determined from plots of percentage inhibition versus concentration of synthetic peptide, is inversely correlated with the substitution of glycine residues for the hydrophobic residues. Therefore the equilibrium inhibition of pepsin by these peptides is dominated by the hydrophobic nature of the 4-7 sequence region.

Amino Acids↗
PubMed1983

A new substrate for porcine pepsin possessing cryptic fluorescence properties.

A fluorescent substrate for porcine pepsin, 50-dimethylaminonaphthalene-1-sulfonyl (Dns)-Ala-Ala-Phe-Phe-3-[4-(N-CH3)-pyridyl]propyl-1-oxy ester has been synthesized. It is stable, soluble from pH 1 to 7, and is readily hydrolyzed by pepsin with values of 288 (+/- 40) s-1 for kcat, 0.039 mM (+/- 0.005) for Km, and 7510 s-1 mM-1 (+/- 500) for kcat/Km in sodium formate, pH 3.1. Kinetic studies were carried out by following the increased fluorescence (300-nm excitation, 525-nm emission) as hydrolysis occurred. The products of hydrolysis were identified and established that the peptide bond between the phenylalanine residues is cleaved by pepsin. The inhibition of pepsin catalysis by pepsinogen (1-12) activation peptide was studied in order to compare the inhibition of the reaction of pepsin with Dns-Ala-Ala-Phe-Phe-OP4P-CH3+ with that obtained by the standard milk-clotting assay. The inhibition results were comparable. Dns-Ala-Ala-Phe-Phe-OP4P-CH3+ should be a valuable tool for studies of pepsin because of its solubility over an extended pH range, its excellent turnover rate, and the ease with which the hydrolysis can be followed.

Animals↗
PubMed1982

The effects of lactoyl-pepstatin and the pepsin inhibitor peptide on pig cathepsin D.

Lactoyl-pepstatin (an acylated tetrapeptide) is much more readily soluble in aqueous media than the more common isovaleryl- and acetyl-pepstatins (acylated pentapeptides). However, the K1 value for inhibition of cathepsin D by lactoyl-pepstatin at pH 3.5 is approx. 10(-7) M, some two to three orders of magnitude weaker than has been obtained previously for isovaleryl- or acetyl-pepstatins. One of the peptides released during activation of pig pepsinogen is known to be an effective inhibitor of pig pepsin, but it does not alter the activity of the similar aspartic proteinase, pig cathepsin D.

Animals↗
PubMed1981

Interaction of alpha-dansylated peptide inhibitors with porcine pepsin: detection of complex formation by fluorescence energy transfer and chromatography and evidence for a two-step binding scheme.

Peptide inhibitors, specifically labeled at the alpha-amino terminus by dansylation, have been prepared by utilizing solid-phase peptide synthesis. Changes in fluorescence have been observed upon mixing these peptides with porcine pepsin that can be attributed to the formation of at least two complexes. Energy transfer between tryptophan residues of the protein and the dansyl group of the inhibitors has been detected by the unique excitation spectra generated. The kinetics of formation of the second complex can be correlated with inhibition of the catalytic activity of pepsin. Evidence for complex formation has also been obtained from gel filtration experiments using the fluorescent peptides.

Animals↗