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Biomedical subjects

B M Dunn

Publications and source records attributed to B M Dunn.

At least 91 records · Page 5Linked to original sources
PubMed1990

Sub-site preferences of the aspartic proteinase from the human immunodeficiency virus, HIV-1.

A series of synthetic, chromogenic substrates for HIV-1 proteinase with the general structure Ala-Thr-His-Xaa-Yaa-Zaa*Nph-Val-Arg-Lys-Ala was synthesised with a variety of residues introduced into the Xaa, Yaa and Zaa positions. Kinetics parameters for hydrolysis of each peptide by HIV-1 proteinase at pH 4.7, 37 degrees C and u = 1.0 M were measured spectrophotometrically and/or by reverse phase FPLC. A variety of residues was found to be acceptable in the P3 position whilst hydrophobic/aromatic residues were preferable in P1. The nature of the residue occupying the P2 position had a strong influence on kcat (with little effect on Km); beta-branched residues Val or Ile in this position resulted in considerably faster peptide hydrolysis than when e.g. the Leu-containing analogue was present in P2.

Amino Acid Sequence↗
PubMed1990

Sensitive, soluble chromogenic substrates for HIV-1 proteinase.

By replacement of the P1' residue in a capsid/nucleocapsid cleavage site mimic with 4-NO2-phenylalanine (Nph), an excellent chromogenic substrate, Lys-Ala-Arg-Val-Leu*Nph-Glu-Ala-Met, for HIV-1 proteinase (kappa cat = 20 s-1, Km = 22 microM) has been prepared. Substitution of the Leu residue in P1 with norleucine, Met, Phe, or Tyr had minimal effects on the kinetic parameters (kappa cat and kappa cat/Km) determined at different pH values, whereas peptides containing Ile or Val in P1 were hydrolyzed extremely slowly. The spectrophotometric assay has been used to characterize the proteinase further with respect to pH dependence, ionic strength dependence, and the effect of competitive inhibitors of various types.

Amino Acid Sequence↗
PubMed1990

The selectivity of statine-based inhibitors against various human aspartic proteinases.

The interactions of five human enzymes (renin, pepsin, gastricsin, cathepsin D and cathepsin E) and the aspartic proteinase from Endothia parasitica with several series of synthetic inhibitors were examined. All of the inhibitors contained the dipeptide analogue statine or its phenylalanine or cyclohexylalanine homologues in the P1-P1' positions. The residues occupying the peripheral sub-sites (P4 to P3') were varied systematically and inhibitory constants were determined for the interactions with each of the proteinases. Inhibitors were elucidated that specifically inhibited human renin and did not affect any of the other human enzymes or the fungal proteinase. With suitable selection of residues to occupy individual sub-sites, effective inhibitors of specific human aspartic proteinases may now be designed.

Amino Acid Sequence↗
PubMed1990

Chemical synthesis of a neurotoxic polypeptide from the sea anemone Stichodactyla helianthus.

The sea anemone Stichodactyla helianthus neurotoxin I, a 48-residue polypeptide, was synthesized by automated solid phase methodology. The fully reduced polypeptide was subsequently refolded in the presence of a glutathione oxidoreduction buffer to the biologically active species containing three disulfide bonds. The overall yield after rigorous purification was 12.5%. The circular dichroism (CD), and proton nuclear magnetic resonance (1H NMR) spectra of the HPLC-purified synthetic toxin were indistinguishable from those obtained concurrently with the natural toxin. A subtilisin digest of the synthetic neurotoxin generated peptide fragments identical to that of a sample of the natural toxin subjected to the same treatment. The toxicity of the synthetic polypeptide was identical to that of the natural toxin (crab LD50, 3.1 micrograms/kg). The equilibrium dissociation constant (28 nM) for interaction of the synthetic toxin with crab axolemma vesicles was nearly identical to that of the natural toxin (25 nM).

Amino Acid Sequence↗
PubMed1989

A consensus sequence for substrate hydrolysis by rhinovirus 3C proteinase.

Kinetic constants were determined for the hydrolysis of a series of synthetic peptide substrates by recombinant rhinovirus (HRV 14) 3C proteinase. Systematic removal or replacement of individual residues indicated that the minimum sequence required for effective cleavage by the viral cysteine proteinase was P5-Val/Thr-P3-P2-Gln-Gly-Pro.

3C Viral Proteases↗
PubMed1989

Inhibition of the aspartic proteinase from HIV-2.

Kinetic constants were determined for the interaction of the HIV-2 aspartic proteinase with a synthetic substrate and a number of inhibitors at several pH values. Acetyl-pepstatin was more effective towards HIV-2 proteinase than the renin inhibitor, H-261; this effect is exactly the opposite from that observed previously for the proteinase from the HIV-1 AIDS virus.

Aspartic Acid Endopeptidases↗
PubMed1989

Isolation, characterization, and amino acid sequence of a polypeptide neurotoxin occurring in the sea anemone Stichodactyla helianthus.

An aqueous exudate collected from frozen and thawed bodies of a Caribbean sea anemone, Stichodactyla (formerly Stoichactis) helianthus, contained a polypeptide neurotoxin (Sh I) selectively toxic to crustaceans. The polypeptide was purified by G-50 Sephadex, phosphocellulose, and sulfopropyl-Sephadex chromatography and shown to have a molecular size of 5200 daltons and a pI of 8.3. The amino acid sequence determined by automatic Edman degradations of whole RCM Sh I and of its clostripain, staphylococcal protease, and cyanogen bromide digest peptides is A1ACKC5DDEGP10DIRTA15PLTGT20VDLGS25CNAGW30EKCAS35YYTII40ADCCR45KKK . Only 33% of this sequence is identical with the sequence of Anemonia sulcata toxin II, a sea anemone toxin isolated from the taxonomic family Actiniidae. The six half-cystines are located in equivalent positions to those of the actiniid toxins and account for nearly half of the residues common to all of the toxins. However, 69% of the Sh I sequence is identical with that of toxin II from Heteractis paumotensis, another sea anemone belonging to the family Stichodactylidae. Stichodactylid toxins lack the initial N-terminal residue of actiniid toxins and possess three consecutive acidic residues at positions 6-8, a single tryptophan at position 30, and four consecutive basic residues at positions 45-48 (C-terminus). A rabbit IgG prepared by Sh I immunization bound Sh I with a K0.5 of 4.7 nM but failed to bind homologous actiniid (Anemonia sulcata II, Condylactis gigantea III) or bolocerid (Bolocera tuedae II) polypeptide neurotoxins.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1989

Effective blocking of HIV-1 proteinase activity by characteristic inhibitors of aspartic proteinases.

Inhibitory constants (Ki) between 5 and 35 nM were derived (under different conditions of pH and ionic strength) for the interaction of HIV-1 proteinase with acetyl-pepstatin and H-261, two characteristic inhibitors of aspartic proteinases. Thus this enzyme, essential for replication of the AIDS virus, may be classified unequivocally as belonging to this proteinase family.

Aspartic Acid Endopeptidases↗
PubMed1989

Stabilisation of cathepsin E by ATP.

The hydrolysis of 3 distinct substrates by cathepsin E from human red blood cells and gastric mucosa was measured in the presence and absence of physiologically relevant concentrations of ATP. At pH values below about 5.0, the nucleotide was without effect. However, at pH 5.8, whereas cathepsin E was virtually inactive by itself, it was restored to full activity (kcat) by ATP and the non-hydrolysable methylene-ATP analogue. At still higher pH values, kcat progressively diminished but significant levels of cathepsin E activity were readily detectable at pH 7.0. The specificity of this stabilisation effect was examined.

Adenosine Triphosphate↗
PubMed1989

Peptide histidine isoleucine and vasoactive intestinal polypeptide cause relaxation of opossum internal anal sphincter via two distinct receptors.

The purpose of this investigation was to characterize the nature of peptide histidine isoleucine (PHI) and vasoactive intestinal polypeptide (VIP) receptors, and to examine the role of PHI in internal anal sphincter (IAS) relaxation. The studies were performed on opossums anesthetized with alpha-chloralose. The pressures in the IAS were recorded using continuously perfused catheters. The IAS responses to PHI analogues, PHI-27, PHM-27, PHI-(14-27)-NH2, PHI-(1-13), to VIP, to rectal balloon distention, sacral nerve stimulation, and local intramural stimulation were evaluated before and after PHI-(14-27)-NH2, PHI tachyphylaxis, and the VIP antagonists [4 Cl-D-Phe6, Leu17] VIP (VIP analogue) and (N-Ac-Tyr1, D-Phe2)-GRF(1-29)-NH2 (growth hormone releasing factor analogue). The inhibitory responses by all of the PHI analogues and VIP were not modified by tetrodotoxin. PHI-(14-27)-NH2 and PHI tachyphylaxis caused significant antagonism of the fall in internal anal sphincter pressure by PHI-27 and PHM-27 without modifying the IAS responses to VIP and rectal balloon distention, sacral nerve stimulation, and local intramural stimulation. On the other hand, VIP and growth hormone releasing factor analogues caused significant antagonism of VIP responses without modifying the responses to PHI-27. We conclude that distinct PHI and VIP receptors are present in the IAS smooth muscle and that PHI may not play a significant role in the IAS relaxation via the rectoanal reflex.

Anal Canal↗
PubMed1989

Synthetic peptides of the amino-terminus of fibronectin inhibit endothelial cell growth.

Amino-terminal and carboxyl-terminal fragments of human plasma fibronectin, at nM concentrations, reversibly inhibit the growth of cultured bovine aortic endothelial cells. To define smaller active peptides, synthetic peptides corresponding to a carboxyl-terminal disulfide bonded loop segment of one of the fragments were tested for activity and found to be active at microM concentrations. The data suggest that the entire loop sequence is required for full expression of endothelial cell growth inhibitory activity in vitro.

Amino Acid Sequence↗
PubMed1989

Hydrolysis of a series of synthetic peptide substrates by the human rhinovirus 14 3C proteinase, cloned and expressed in Escherichia coli.

The 3C proteins of several picornaviruses, including poliovirus, foot-and-mouth disease virus (FMDV) and encephalomyocarditis virus (EMCV), have been demonstrated to be cysteine-type proteinases, involved in the processing of the respective polyproteins expressed by the monocistronic RNA genome. Nucleotide sequencing data have indicated that the human rhinovirus 14 (HRV-14) RNA genome encodes a homologous 3C protein. The HRV-14 3C protein was purified to homogeneity from Escherichia coli expressing the cloned 3C genomic fragment. The enzyme was assayed against peptides corresponding to those residues, predicted (by nucleotide sequencing data) to occur at authentic cleavage sites within the polyprotein. The peptides representing the 1B/1C, 2A/2B, 2C/3A, 3A/3B, 3B/3C and 3C/3D cleavage sites, where proteolysis was predicted to occur at a Gln-Gly junction, were all cleaved by the 3C proteinase. The hydrolysis was shown (by reverse phase fast protein liquid chromatography and amino acid analysis) to occur specifically at the Gln-Gly bond in each of the peptides. The ready availability of such convenient substrates facilitated the further characterization of the 3C proteinase. By contrast, peptides corresponding to the predicted 2B/2C and 1C/1D cleavage sites, where the processing was presumed to occur at a Gln-Ala or Glu-Gly bond respectively, were not cleaved by the 3C proteinase. The ability of the HRV-14 3C proteinase to hydrolyse the synthetic peptides was inhibited if a Cys----Ser(146) mutation was introduced into the protein. Studies with known proteinase inhibitors substantiated the conclusion that the HRV-14 3C protein appears to be a cysteine proteinase and that the Cys residue at position 146 may be the active site nucleophile. The HRV-14 3C proteinase probably plays an important role, analogous to that implied for the poliovirus 3C proteinase, in the replication of the virus and thus represents a potential target for antiviral chemotherapy.

3C Viral Proteases↗
PubMed1989

Inhibition of antral gastrin cells by peptide histidine isoleucine.

The present studies were directed to determine whether peptide histidine isoleucine (PHI), like its structural analogues secretin and gastric inhibitory peptide, inhibits antral gastrin. In separate experiments, the effects of PHI on medium gastrin concentrations, the incorporation of [35S]methionine into newly synthesized gastrin, and steady-state gastrin mRNA were determined. The inclusion of PHI in the incubation medium decreased medium gastrin levels at all concentrations examined, an effect that was not altered by the addition of 10(-6) M tetrodotoxin to the medium. PHI also inhibited the incorporation of [35S]methionine into newly synthesized gastrin in a concentration-dependent manner. Steady-state levels of gastrin mRNA were determined by dot-blot hybridization, using a 32P-labeled gastrin cRNA probe. PHI inhibited gastrin mRNA levels in a concentration-dependent manner; in contrast, no effect on the levels of actin and ubiquitin mRNA could be detected, indicating specificity of PHI on gastrin mRNA. The results of these studies indicate that PHI may exert a physiological inhibitory effect on antral gastrin cells and that this inhibition may occur at several steps along the biosynthetic pathway.

Animals↗
PubMed1988

Identification of the aspartic proteinases from human erythrocyte membranes and gastric mucosa (slow-moving proteinase) as catalytically equivalent to cathepsin E.

Three aspartic proteinases with similar Mr values (approx. 80,000) but from distinct sources (human gastric mucosa, human erythrocyte membranes and rat spleen) were shown to have immunological cross-reactivity and comparable mobilities when subjected to polyacrylamide-gel electrophoresis under non-denaturing conditions. Kinetic parameters (kcat, Km and Ki) were determined for the interactions of the three enzymes with two synthetic chromogenic substrates and five inhibitors (naturally occurring and synthetic). On this basis it would appear that all of the enzymes should be considered equivalent to cathepsin E. pH-activity measurements indicated that the aspartic proteinase that originated from the erythrocyte membranes retained activity at a higher pH value than either of its readily soluble counterparts.

Animals↗
PubMed1988

Secondary enzyme-substrate interactions: kinetic evidence for ionic interactions between substrate side chains and the pepsin active site.

The possibility that pig pepsin has a cation binding specificity in its secondary binding subsites has been examined by the pepsin-catalyzed hydrolysis of a series of synthetic octa- to undecapeptide substrates. These chromophoric substrates are cleaved by pepsin in the phenylalanyl-p-nitrophenylalanyl (Phe-Nph) bond. Lys and Arg residues were placed into seven different positions in the substrates, and their effect on kcat and Km was examined between pH 2.8 and pH 5.8 (I = 0.1 M, 37 degrees C). Kinetic evidence indicates the existence in the enzyme binding subsites S4, S3, S2, S3', S4', and S5' of a group(s) which become(s) negatively charged at higher pH. For most substrates, the magnitude as well as the pH dependence of kcat was unaffected by the presence of Lys or Arg in these peptides. In contrast, changes up to 5 orders of magnitude were observed for Km, depending on the number of basic residues and on their positions in the sequence. Km for a group of substrates at pH greater than 5.5 was lower than 50 nM. Values for kcat/Km for some substrates exceed the level of 10(8) M-1 s-1. Therefore, the free energy derived from ionic interactions in secondary binding sites influences mostly the binding step on the reaction pathway. This result is in contrast to the previous observations that the length and the hydrophobic character of the substrate residues in some positions influence kcat with little effect on Km toward shorter substrates of pepsin [Fruton, J. (1976) Adv. Enzymol. Relat. Areas Mol. Biol. 44, 1-36].

Amino Acid Sequence↗
PubMed1988

Separation and characterization of four different amino acid sequence variants of a sea anemone (Stichodactyla helianthus) protein cytolysin.

A basic protein cytolysin previously isolated from the Caribbean sea anemone Stichodactyla helianthus was shown by CM cellulose chromatography to consist of four isotoxins possessing different N-terminal amino acid sequences. These are designated as toxins I-IV in order of increasing isoelectric point. The estimated molecular sizes (17,400-18,200) of toxins I-III were very similar; toxins I and II posses one additional amino acid at their amino terminus relative to toxin III. Under denaturing conditions, toxin IV behaved as a significantly larger (19,600) polypeptide; Edman sequencing established that it possesses a seven residue extension at the N-terminal end relative to toxin III. None of the variants contained half-cystines or reducing sugars. Toxin III contributed 83% of the total purified cytolytic (hemolytic) activity, toxin II 14%, and the relatively insoluble toxins I and IV together only contributed about 3% of the total cytolytic activity. Cytolysin III lysed Ehrlich ascitic tumour cells, but when administered intraperitoneally in nonlethal doses to mice already inoculated with this tumour, it failed to protect the mice against the tumour. Comparison of the partial amino acid sequence of equinatoxin, another sea anemone protein cytolysin, with that of Stichodactyla cytolysin III indicates they are highly homologous. Many other cytolytic proteins isolated from sea anemones share these properties with Stichodactyla cytolysins: (1) selective inhibition of hemolytic activity by preincubation with sphingomyelin, (2) a molecular size of 10,000-20,000, and (3) an isoelectric point of 9 or above.

Amino Acid Sequence↗