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Biomedical subjects

H U Demuth

Publications and source records attributed to H U Demuth.

At least 19 recordsLinked to original sources
PubMed1999

Improved glucose tolerance in rats treated with the dipeptidyl peptidase IV (CD26) inhibitor Ile-thiazolidide.

The incretins glucose-dependent insulinotropic polypeptide (GIP1-42) and truncated forms of glucagon-like peptide-1 (GLP-1) are hormones released from the gut in response to ingested nutrients, which act on the pancreas to potentiate glucose-induced insulin secretion. These hormones are rapidly inactivated by the circulating enzyme dipeptidyl peptidase IV ([DPIV] CD26). This study describes the effect on glucose tolerance and insulin secretion of inhibiting endogenous DPIV in the rat using Ile-thiazolidide, a specific DPIV inhibitor. High-performance liquid chromatography (HPLC) analysis of plasma following in vivo administration of 125I-labeled peptides showed that inhibition of DPIV by about 70% prevented the degradation of 90.0% of injected 125I-GLP-17-36 after 5 minutes, while only 13.4% remained unhydrolyzed in rats not treated with the DPIV-inhibiting agent after only 2 minutes. Ile-thiazolidide treatment also increased the circulating half-life of intact GLP-17-36 released in response to intraduodenal (ID) glucose (as measured by N-terminal specific radioimmunoassay [RIA]). In addition, inhibition of DPIV in vivo resulted in an earlier increase and peak of plasma insulin and a more rapid clearance of blood glucose in response to ID glucose challenge. When considered with the HPLC data, these results suggest that the altered insulin profile is an incretin-mediated response. DPIV inhibition resulting in improved glucose tolerance may have therapeutic potential for the management of type 2 diabetes mellitus.

Animals↗
PubMed1998

Intrathecal administration of p-hydroxymercuribenzoate or phosphoramidon/bestatin-combined induces antinociceptive effects through different opioid mechanisms.

The antinociceptive effect of intrathecally (i.t.) administered protease inhibitors was tested against capsaicin (800 ng) injected into the dorsal surface of a hindpaw. Both p-hydroxymercuribenzoate (2-8 nmol), a cysteine protease inhibitor, and phosphoramidon (1-4 nmol), an endopeptidase 24.11 inhibitor in the presence of bestatin (0.25 nmol) an aminopeptidase inhibitor, administered i.t. 60 min prior to the injection of capsaicin produced a dose-dependent reduction of the capsaicin-induced paw licking and biting response. p-Hydroxymercuribenzoate (4 nmol)-induced antinociception was significantly antagonized by nor-binaltorphimine, a selective kappa-opioid receptor antagonist, but not by naltrindole, a selective delta-opioid receptor antagonist. On the other hand, phosphoramidon (4 nmol) /bestatin-induced antinociception was significantly antagonized by naltrindole, but not by nor-binaltorphimine. The results indicate that the antinociceptive effect of p-hydroxymercuribenzoate may be due to the inhibition of a cysteine protease degrading endogenous dynorphins whereas phosphoramidon in the presence of bestatin blocks the degradation of enkephalins.

Animals↗
PubMed1998

CD26 (dipeptidyl peptidase i.v.) on human T lymphocytes does not mediate adhesion of these cells to endothelial cells or fibroblasts.

We have examined the role of CD26 (dipeptidyl peptidase IV) in the adhesion of resting and activated T lymphocytes to endothelial cells and fibroblasts. For this purpose, we ran a short-time adhesion assay under different strategies: Adhesion of T lymphocytes was determined in the presence of different anti-CD26 monoclonal antibodies, or in the presence of synthetic inhibitors of the enzymatic function of CD26. In addition, the expression of CD26 on T lymphocytes, which were adherent to endothelial cells or fibroblasts, was performed by flow cytometric analysis. We found that the anti-CD26 monoclonal antibodies tested here were not able to inhibit T cell adhesion to monolayers of endothelial cells or fibroblasts. Secondly, synthetic inhibitors of the enzymatic function of CD26 had no effect on the adhesion of T lymphocytes to endothelial cells or fibroblasts. Furthermore, CD26-positive T cells were not accumulated in the adherent population. These results suggest that CD26 on T lymphocytes plays no role in T cell adhesion to endothelial cells or fibroblasts.

Dipeptidyl Peptidase 4↗
PubMed1998

Improved glucose tolerance in Zucker fatty rats by oral administration of the dipeptidyl peptidase IV inhibitor isoleucine thiazolidide.

The hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide (GLP)-1 act on the pancreas to potentiate glucose-induced insulin secretion (enteroinsular axis). These hormones (incretins) are rapidly hydrolyzed by the circulating enzyme dipeptidyl peptidase IV (DP IV) into biologically inactive NH2-terminally truncated fragments. This study describes the effect of inhibiting endogenous DP IV with a specific DP IV inhibitor, isoleucine thiazolidide (Ile-thiazolidide), on glucose tolerance and insulin secretion in the obese Zucker rat. In initial studies, the specificity of Ile-thiazolidide as an inhibitor of incretin degradation was determined using matrix-assisted laser desorption/ionization-time of flight mass spectrometry. These results showed that inhibiting DP IV activity with Ile-thiazolidide blocked the formation of NH2-terminally truncated GIP and GLP-1. Oral administration of Ile-thiazolidide resulted in rapid inhibition of circulating DP IV levels by 65% in obese and lean Zucker rats. Suppression of DP IV levels enhanced insulin secretion in both phenotypes with the most dramatic effect occurring in obese animals (150% increase in integrated insulin response vs. 27% increase in lean animals). Ile-thiazolidide treatment improved glucose tolerance in both phenotypes and restored glucose tolerance to near-normal levels in obese animals. This was attributed to the glucose-lowering actions of increasing the circulating half-lives of the endogenously released incretins GIP and, particularly, GLP-1. This study suggests that drug manipulation of plasma incretin activity by inhibiting the enzyme DP IV is a valid therapeutic approach for lowering glucose levels in NIDDM and other disorders involving glucose intolerance.

Administration, Oral↗
PubMed1997

Effects of the CLIP fragment ACTH 20-24 on the duration of REM sleep episodes.

Substances acting upon rapid eye movement (REM) sleep or paradoxical sleep (PS) can affect the number and/or the duration of PS episodes. In the present study, we investigated the effects of the proopiomelanocortin-derived peptide CLIP (corticotropin-like intermediate lobe peptide, ACTH 18-39) and its N-terminal fragments ACTH 18-24 and ACTH 20-24 on the duration of PS episodes in rats. Intracerebroventricular injection of ACTH 20-24 caused a pronounced prolongation of PS episodes (up to 7 min duration, never seen under baseline conditions), whereas ACTH 18-24 acted in a similar way but without reaching statistical significance. We suggest that short N-terminal CLIP fragment(s) may represent endogenous hypnogenic factor(s) involved in the regulation of paradoxical sleep.

Adrenocorticotropic Hormone↗
PubMed1996

Investigation of glucose-dependent insulinotropic polypeptide-(1-42) and glucagon-like peptide-1-(7-36) degradation in vitro by dipeptidyl peptidase IV using matrix-assisted laser desorption/ionization-time of flight mass spectrometry. A novel kinetic approach.

The incretins glucose-dependent insulinotropic polypeptide (GIP1-42) and glucagon-like peptide-1-(7-36)-amide (GLP-17-36), hormones that potentiate glucose-induced insulin secretion from the endocrine pancreas, are substrates of the circulating exopeptidase dipeptidyl peptidase IV and are rendered biologically inactive upon cleavage of their N-terminal dipeptides. This study was designed to determine if matrix-assisted laser desorption/ionization-time of flight mass spectrometry is a useful analytical tool to study the hydrolysis of these hormones by dipeptidyl peptidase IV, including kinetic analysis. Spectra indicated that serum-incubated peptides were cleaved by this enzyme with only minor secondary degradation due to other serum protease activity. Quantification of the mass spectrometric signals allowed kinetic constants for both porcine kidney- and human serum dipeptidyl peptidase IV-catalyzed incretin hydrolysis to be calculated. The binding constants (Km) of these incretins to purified porcine kidney-derived enzyme were 1.8 +/- 0.3 and 3.8 +/- 0.3 microM, whereas the binding constants observed in human serum were 39 +/- 29 and 13 +/- 9 microM for glucose-dependent-insulinotropic polypeptide and glucagon-like peptide-1-(7-36)-amide respectively. The large range of Km values found in human serum suggests a heterogeneous pool of enzyme. The close correlation between the reported kinetic constants and those previously described validates this novel approach to kinetic analysis.

Animals↗
PubMed1996

N-peptidyl, O-acyl hydroxamates: comparison of the selective inhibition of serine and cysteine proteinases.

Two series of N-aminoacyl, O-benzoyl hydroxamates were designed to investigate the influence of the substituted benzoyl residue on the hydrolytic stability and the reactivity of these potential inhibitors towards selected cysteine and serine proteinases. The inactivators react more rapidly with cysteine proteinases than with the serine enzymes tested. While Z-Phe-Gly-NHO-Nbz is the most reactive inhibitor of cathepsin L, inhibiting the target protein by a second order rate constant of 932.000 M-1 s-1, the bacterial serine proteinase thermitase is inhibited best by Z-Gly-Phe-NHO-Nbz, exhibiting a second-order rate constant of 1.170 M-1 s-1. Thiolsubtilisin, having the thiol-group as the reactive nucleophile instead of serine, exhibits specificity constants of the inactivation two orders of magnitude smaller than subtilisin. The degree of selectivity of the inhibitors relative to cathepsin B, cathepsin L, cathepsin S and papain varies up to two orders of magnitude with respect to their second order rate constant of inactivation. The inhibitory reactivity of these compounds varies only up to sixfold depending on the benzoyl substituent. Similarly, the rate constants for the hydrolytic decomposition of the compounds vary by a factor of about 6, suggesting that the structural and mechanistic features of the compounds which are responsible for decomposition as well as for the enzyme inhibition are the same. Comparing both reactions, the data allow the calculation of an acceleration factor of 2.4 x 10(10) for the inhibition of cathepsin L by its most effective inhibitor, clearly characterizing this enzyme inhibition reaction as enzyme-activated.

Cathepsins↗
PubMed1996

Substrates containing phosphorylated residues adjacent to proline decrease the cleavage by proline-specific peptidases.

Thirteen dipeptide rho-nitroanilides of the common structure H-Xaa-Pro-4-NA (Xaa = serine, threonine and tyrosine) and seven tripeptide rho-nitroanilides of the common structure H-Gly-Xaa-Pro-4-NA (Xaa = serine or threonine) were prepared and analyzed as substrates of the proline-specific peptidases dipeptidyl peptidase IV and prolyl endopeptidase, respectively. The side chains of the hydroxy amino acids were synthetically modified by various acyl-, benzyl- and phosphate residues. The presence of aliphatic or aromatic residues attached to the side chain of the P2-hydroxy amino acids resulted in no significant change of the specificity constants of the enzyme-catalyzed substrate hydrolysis. In some cases, however, substrate inhibition was observed. In contrast, the reactivity of dipeptidyl peptidase IV and prolyl endopeptidase decreases more than two orders of magnitude towards the phosphorylated di- and tripeptide substrates compared to the hydrolysis of unmodified substrates. The kinetic data obtained with the model compounds suggest that side-chain modification of proline-containing peptide substrates may influence their resistance towards the hydrolytic activity of proline-specific hydrolases. Additionally, the results support that structural changes of the substrate during enzyme-hydrolysis may be involved in the mechanism of action of proline-specific serine peptidases. From this result we speculate that posttranslational phosphorylation of peptide sequences found in protein kinase recognition motifs such as -Xaa-Ser/Thr-Pro-Yaa- and -Xaa-Pro-Ser/Thr-Yaa- may serve as structural determinants that modulate their proteolytic stability.

Amino Acid Sequence↗
PubMed1996

Cyclic morphiceptin analogs: cyclization studies and opioid activities in vitro.

Attempts were undertaken to develop cyclic beta-casomorphin-5 analogs with improved opioid activity profiles by deletion of the glycine residue in position 5, leading to analogs structurally related to the opioid peptide morphiceptin (H-Tyr-Pro-Phe-Pro-NH2). The tetrapeptide sequence Boc-Tyr(tBu)-D-Xaa-Phe-Yaa-OH (Xaa = Lys, Orn, A2bu; Yaa = Pro in L- or D-configuration) was used to study the influence of ring size and chirality on the yield of cyclization between the omega-amino group of Xaa and the C-terminal carboxyl group. In all cases the cyclization reaction was performed under identical experimental conditions to allow a direct comparison with regard to yield and homogeneity. The reaction products were purified by crystallization and liquid chromatography, and were characterized by HPLC, TLC, electrospray mass spectrometry and 1H-NMR spectroscopy. In none of the reactions performed with the cyclization precursors containing proline in the L-configuration could a cyclic monomer be detected, and the cyclodimer (7-9) was the exclusive product in each case. Cyclodimerization was also the favored reaction in the attempted formation of the 11-membered ring of the cyclic [D-A2bu2, D-Pro4]-morphiceptin analog 12, since only traces of the monomer were found. In the case of both the [D-Lys2, D-Pro4]-analog 10 and the [D-Orn2, D-Pro4]-analog 11, the cyclomonomer/cyclodimer ratio was about 80:20. The cyclic monomers 10 and 11 showed high opioid activity in the mu-receptor-representative guinea pig ileum assay (IC50 = 2-5 nM) and in the delta-receptor representative mouse vas deferens assay (IC50 = 50-60 nM), whereas the potency of the cyclodimers was 2-3 orders of magnitude lower in both in vitro bioassays.

Analgesics↗
PubMed1995

Nature of the inactivation of elastase by N-peptidyl-O-aroyl hydroxylamine as a function of pH.

The mechanism of inactivation of porcine pancreatic elastase (PPE) by N-peptidyl-O-aroylhydroxylamine was studied by X-ray crystallography. The inactivator forms a stable complex with the enzyme by means of a covalent attachment to the active site Ser 203(195) O gamma. The nature of the complex is, however, different depending on the pH at which the inactivation reaction occurs. At pH 5, the complex formed is a hydroxylamine derivative of Ser 203(195) in which the O gamma of serine is the oxygen of the hydroxylamine derivative. At pH 7.5, the complex formed is a carbamate derivative at Ser 203(195) O gamma. In both types of complexes, the inactivator binds in the S' subsites of the enzyme instead of forming the usual antiparallel beta-sheet with the S subsites. The implication for the mechanism of inactivation at different pHs is discussed.

Animals↗
PubMed1994

Inactivation of subtilisin Carlsberg by N-((tert-butoxycarbonyl)alanylprolylphenylalanyl)-O-benzoylhydroxyl- amine: formation of a covalent enzyme-inhibitor linkage in the form of a carbamate derivative.

The mechanism of inactivation of serine proteases by N-peptidyl-O-aroylhydroxylamines was studied by X-ray crystallography. Cocrystals of subtilisin Carlsberg inactivated with N-((tert-butoxycarbonyl)alanylprolylphenylalanyl)-O-nitrobenzoy lhydroxylamine were grown, and diffraction data to 1.8-A resolution were obtained. The resulting electron density maps clearly reveal that the gamma-oxygen of the catalytic serine forms a carbamate derivative with the inhibitor. The peptide part of the inhibitor does not form the usual antiparallel beta-sheet in the P binding cleft but protrudes out of the active site and is stabilized by a network of water molecules. These results, combined with kinetic characterization reported previously [Demuth, H.-U., Schoenlein, C., & Barth, A. (1989b) Biochim. Biophys. Acta 996, 19-22; Schmidt, C., Schmidt, R., & Demuth, H.-U. (1990) Peptides (Giralt, E., & Andreu, D., Eds.) ESCOM Science Publishers B.V., Amsterdam] support the existence of at least one intermediate between the formation of the Michaelis complex and the final product. We suggest a mechanism for the inactivation of subtilisin Carlsberg by N-((tert-butoxycarbonyl)alanylprolylphenylalanyl)-O-benzoylhydr oxylamine whereby a negatively charged Michaelis complex undergoes a Lossen rearrangement giving rise to an isocyanate intermediate that reacts with the side chain of the active site serine.

Amino Acid Sequence↗
PubMed1993

Inhibition of aminopeptidases by N-aminoacyl-O-4-nitrobenzoyl hydroxamates.

10 N-aminoacyl-O-4-nitrobenzoyl hydroxamates were investigated as potential inhibitors of aminopeptidases. While the metal-depending enzymes aminopeptidase M, aminopeptidase P and leucine aminopeptidase were inhibited reversibly by the compounds, the thiol enzyme cathepsin H was inhibited efficiently in time-dependent reactions according to its substrate specificity. N-phenylalanyl-O-4-nitrobenzoyl hydroxamate was shown to be most effective, exhibiting a second-order-rate constant of inhibition of 31,766 M-1 s-1.

Amino Acid Sequence↗
PubMed1993

Novel N-peptidyl-O-acyl hydroxamates: selective inhibitors of cysteine proteinases.

A series of N-peptidyl-O-acyl hydroxamates with a lysine in P1 was synthesized and tested as inactivators of lysosomal cysteine proteinases (cathepsins S, L, B and H) and trypsin-like serine proteinases (trypsin, thrombin, plasmin, t-PA). N-peptidyl-O-acyl hydroxamates were shown to be selective inhibitors of cysteine proteinases. With the exception of cathepsin H, the lysosomal cysteine proteinases were inactivated 2-5 orders of magnitude more rapidly than serine proteinases with a comparable primary substrate specificity. The highest second-order rate constants of inactivation for the cysteine proteinases are in the range of 10(5)-10(6) M-1 s-1. The order of inhibitor specificity for the cysteine proteinases is comparable to the enzyme's substrate specificity.

Amino Acid Sequence↗
PubMed1993

Design of (omega-N-(O-acyl)hydroxy amid) aminodicarboxylic acid pyrrolidides as potent inhibitors of proline-specific peptidases.

A novel class of competitive, acylating inhibitors for the proline-specific peptidases: dipeptidyl peptidase IV, dipeptidyl peptidase II and prolyl endopeptidase, has been developed. The inhibitor molecules combine the efficacy of aminoacyl pyrrolidides and the potential transacylating capability of diacyl hydroxyl amines. The N-terminal deblocked inhibitors are potent reversible inhibitors of porcine kidney dipeptidyl peptidase IV, human placenta dipeptidyl peptidase II exhibiting Ki values in the microM range. Boc-protected (omega-N-hydroxy acyl amid) aminodiacarboxylic acid pyrrolidides inhibit substrate hydrolysis by prolyl endopeptidases from different sources competitively reaching Ki values of 30 nM to 60 microM. Additionally, alpha-N-BOC-(omega-N-hydroxy acetyl) glutaminyl pyrrolidide modifies human placenta prolyl endopeptidase in a time-dependent reaction.

Amino Acid Sequence↗
PubMed1993

Enzymatic activity of CD26 (dipeptidylpeptidase IV) is not required for its signalling function in T cells.

CD26 is a proteolytic enzyme (dipeptidylpeptidase IV) expressed on the T cell surface that defines an alternative activation signal for human T lymphocytes. Crosslinking of CD26 via monoclonal antibodies triggers proliferation and cytotoxicity in preactivated T cells. In this study, we used highly specific competitive and irreversible inhibitors of dipeptidylpeptidase IV to study the role of the enzymatic activity in activation of CD26-transfected T cells as well as of CD26-expressing normal human T cell clones. These inhibitors at concentrations that blocked up to 95% of the enzymatic activity, did not specifically inhibit T cell activation neither via TCR/CD3 nor via CD26 itself. This demonstrates that the enzymatic activity of CD26 is not required for its T cell activating properties.

Animals↗
PubMed1993

Inhibition of dynorphin converting enzymes from human spinal cord by N-peptidyl-O-acyl hydroxylamines.

Two cysteine proteinases, cleaving dynorphins A and B to enkephalins, were isolated from the human spinal cord. These enzymes were found to be competitively inhibited by a new class of synthetic inhibitors: N-peptidyl-O-acyl hydroxylamines. The most potent (Ki < 20 microM) were the N-terminally protected peptides Z-Phe-Phe-NHO-Ma and Boc-Phe-Gly-NHO-Bz, both containing hydrophobic amino acids at the P2 position. N-Peptidyl-O-acyl hydroxylamines were converted in water solution to the corresponding hydroxamic acids and no cleavage of the peptide bond within the inhibitor sequence was observed after prolonged incubation with the enzymes. It is anticipated that these synthetic compounds may serve as potential pharmacological tools for in vitro studies on dynorphin processing.

Amino Acid Sequence↗
PubMed1993

Peptidyl ammonium methyl ketones as substrate analog inhibitors of proline-specific peptidases.

Prolyl endopeptidase (PEP) and dipeptidyl peptidase IV (DP IV) are serine enzymes cleaving highly specific prolyl peptide bonds. Both enzymes were found to be inhibited by newly designed peptidyl ammonium and pyridinium methyl ketones acting as slow binding inhibitors. The most potent inhibitor of PEP is Z-Pro-Pro-CH2N+C5H5 exhibiting a Ki* value of 1.8 nM with a first-order rate constant of Kon 0.0022 s-1 for the formation of the tight enzyme-inhibitor complex. DP IV and H-Pro-Pro-CH2N+ (CH3)3 form an enzyme-inhibitor-complex with an apparent second order rate constant of 2713 M-1 s-1. In contrast to the very stable N-terminal protected Z-Pro-Pro-CH2N+ (CH3)3, the deblocked derivative decomposes rapidly in aqueous solution.

Dipeptidyl Peptidase 4↗