Enzyme Endopeptidase Research Articles

Ontogeny of prolyl endopeptidase, pyroglutamyl peptidase I, TRH, and its metabolites in rat pancreas.

We demonstrate that two enzymes, soluble unspecific pyroglutamyl peptidase I and prolyl endopeptidase, able to degrade thyrotropin-releasing hormone (TRH) in vitro were present in pancreas at the early stage of rat development. Specific particulate pyroglutamyl peptidase II remained undetectable during ontogenesis. Pyroglutamyl peptidase I specific activity increased until day 3 and decreased after day 5. Furthermore, prolyl endopeptidase specific activity rose slightly to a peak on postnatal day 20. A good correlation between immunoreactive TRH and deaminated TRH (TRH-OH) was found in the 1st wk after birth. However, His-Pro diketopiperazine (DKP) levels were stable and low during development. We show that hot acidic extraction conditions could artefactually generate His-Pro DKP. In vivo, active site-directed inhibitors of pyroglutamyl peptidase I and prolyl endopeptidase enzymes do not show any TRH-deamidating and/or pyroglutamyl peptidase I pathways in neonatal rat pancreas. The data suggest that these two enzymes are not involved in intra- or extracellular control of TRH levels in neonatal rat pancreas and that pancreatic TRH content appears to be principally regulated by biosynthetic steps. Nevertheless, low levels of endogenous His-Pro DKP and TRH-OH identified in neonatal rat pancreas suggest that TRH or TRH-like peptides may be metabolized in this tissue in intact rats, albeit at low rates.

Capsaicin-induced release of tachykinins: effects of enzyme inhibitors

We studied the effects of neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE) inhibition on the airway responses and the recovery of endogenously released substance P- and neurokinin A-like immunoreactivities (SP-LI and NKA-LI) after tracheal injection of capsaicin in isolated guinea pig lungs superfused through the trachea. Capsaicin in doses from 10(-10) to 10(-7) mol induced a dose-dependent increase in airway opening pressure and release of SP-LI and NKA-LI. Airway opening pressure changes and the recovery of SP-LI and NKA-LI were significantly greater in lungs superfused with the NEP inhibitor SCH 32615 than in control lungs. ACE inhibition with captopril did not increase the mechanical response or the recovery of SP-LI compared with lungs not receiving captopril. In lungs from guinea pigs pretreated with high doses of capsaicin 7-10 days before study, a regimen designed to deplete endogenous tachykinins, there was a significant decrease in the content and release of NKA-LI and SP-LI. There were no detectable airway effects of acute capsaicin infusion even after doses of 10(-5) mol. Because NEP is important in modulating the airway effects of endogenously released tachykinins after tracheal infusion of capsaicin, but ACE is not, it seems likely that tracheal administration of capsaicin releases tachykinins from epithelial rather than endothelial loci.

Prodrugs of peptides. 8. In vitro study of intestinal metabolism and penetration of thyrotropin-releasing hormone (TRH) and its prodrugs

The feasibility of improving the poor oral bioavailability of the tripeptide TRH (pGlu-His-Pro-NH 2) was examined using the prodrug approach. The prodrugs studied were various N-alkoxycarbonyl derivatives formed by reacting TRH with the appropriate chloroformates at its imidazole moiety. In vitro metabolism studies with rabbit and rat intestinal homogenates showed that TRH and the prodrugs were rapidly degraded in the rabbit homogenate by virtue of prolyl endopeptidase which cleaves the C-terminal proline amide moiety. Whereas TRH showed a high stability in rat gut homogenates, the prodrug derivatives were readily degraded, partly due to prolyl endopeptidase and partly due to non-specific esterases. In vitro penetration studies using the modified Ussing chamber showed that the prodrugs did not improve the penetration of TRH across the jejunal, ileal and colonic segments of the rat. It is concluded that although the prodrugs are much more lipophilic than TRH in terms of octanol-buffer partition coefficients, the greater susceptibility of the derivatives to undergo enzymatic degradation more than the offsets the improved lipophilicity characteristics. Prodrugs suitable for improving the oral absorption of TRH should not only possess a certain lipophilicity, but should also be resistant towards the prolyl endopeptidase enzyme.

Evidence that the agonist action of dynorphin A(1–8) in the guinea‐pig myenteric‐plexus may be mediated partly through conversion to [Leu5]enkephalin

1. The agonist action of the opioid peptide dynorphin A(1-8) on the myenteric plexus-longitudinal muscle of the guinea-pig ileum has been characterized. 2. The endogenous opioid peptide dynorphin A(1-8) was rapidly degraded by slices of myenteric plexus-longitudinal muscle of the guinea-pig ileum. 3. A product of the degradation was the delta-receptor preferring [Leu5]enkephalin. Levels of [Leu5]enkephalin were markedly increased in the presence of the peptidase inhibitors bestatin, thiorphan and captopril. 4. In the myenteric plexus dynorphin A(1-8) acted as a kappa-receptor agonist. In the presence of bestatin, thiorphan and captopril a mu-receptor agonist effect was observed. This mu-agonist action was lost in the presence of N-[1-(RS)-carboxy-2-phenylethyl]Ala-Ala-Phe-p-aminobenzoate, an inhibitor of the endopeptidase enzyme EC 3.4.24.15. 5. The results suggest that formation of [Leu5]enkephalin from dynorphin A(1-8) may be an important conversion process. The enzyme responsible may be the Zn2(+)-metalloendopeptidase, EC 3.4.24.15.

Parathyroid hormone degradation by chymotrypsin-like endopeptidase in the opossum kidney cell.

Cathepsin-D has been previously reported to cleave intact PTH into PTH-(1-34) and -(35-84) in membranous fractions of rat and bovine kidney. Whether PTH degradation occurs by intact kidney cells, however, has not been examined in detail. We have, therefore, examined this possibility using an opossum kidney (OK) cell line which possesses the characteristics of proximal renal tubules and responds to PTH. PTH radioimmunoreactivity recovered in trichloroacetic acid-soluble products and in fractions eluted from reverse phase HPLC was measured using an antibody directed to the midregion and C-terminus of PTH. In this study, intact OK cells, but not extracellular enzymes, cleaved human (h) PTH-(1-84) into three discrete fragments which were released into the medium in a time- and temperature-dependent fashion. Half-maximal velocity of PTH-degrading activity (PTHDA) was observed at 9 nM hPTH-(1-84). A 1000-fold molar excess of PTH antagonists [hPTH-(3-34) and [Tyr34]hPTH-(7-34)amide] markedly inhibited PTHDA, whereas ACTH, glucagon, or big gastrin did not suppress it, suggesting an involvement of the PTH receptor in PTHDA. This PTHDA was strongly inhibited by phenylmethylsulfonylfluoride and chymostatin, but not by trypsin inhibitor, elastatinal, or inhibitors of aspartic, cysteine, or metalloproteinases, suggesting that it is due to a seryl chymotrypsin-like endopeptidase. Analysis of chymotrypsin-digested products of hPTH-(1-84) eluted from HPLC exhibited five fragments detected by UV absorbance (210 nm), three of which were measurable by PTH RIA, and each corresponded to the three PTH fragments produced by OK cells. All three fragments were predominantly suppressed in the presence of chymostatin, suggesting that chymotrypsin-like activity is solely responsible for PTHDA in intact OK cells. To further explore the cleavage sites of PTH by chymotrypsin, amino acid analysis of chymotrypsin-cleaved products was performed. The results strongly support the conclusion that a chymotrypsin-like enzyme in OK cells cleaved the hormone between residues 23-24, and 34-35 to produce, at least, hPTH-(24-84) and -(35-84). Lysosomal blockers (chloroquine, ammonium chloride, or monensin) did not affect this PTHDA. Our present study indicates that chymotrypsin-like endopeptidase, but not other endopeptidase or lysosomal enzymes, is responsible for the limited hydrolysis of PTH by intact OK cells.

Localization of angiotensin-converting enzyme, prolyl endopeptidase and other peptidases in cultured neuronal or glial cells

To determine the cellular localization of nervous tissue peptidases, 7 peptidases and 2 lysosomal marker enzyme activities were measured in cultured mouse and rat cells. Neuronal cells of both species exhibited higher activities of angiotensin-converting enzyme (ACE) and prolyl endopeptidase (Pro-EP) than glial cells did. In contrast, arginyl endopeptidase and lysosomal enzymes (acid phosphatase, β-glucuronidase) in the neuronal cell lines were lower than those in the glial cell lines. Other peptidases (alanyl aminopeptidase, arginyl aminopeptidase, leucyl aminopeptidase, dipeptidyl aminopeptidase) activities were not specifically localized in either cell lines. The effects of cellular differentiation on these peptidase activities in the PC 12h cell line and rat glioblasts were also examined using nerve growth factor (NGF) and glia maturation factor (GMF), respectively. Neuron specific peptidase (ACE and Pro-EP) activities were decreased in PC12h cells cultured with NGF, and Pro-EP activity was increased in the glioblast cells cultured with GMF. These results support the idea that some of the peptidases are differentially localized in neuronal or glial cells, and play physiological roles in central or peripheral neural tissues.

Histochemical observations on the localization of some enzymes associated with digestion in four species of Brazilian nemerteans.

The processes of digestion have been investigated histochemically for several nemertean species (Jennings, 1962a ; Gibson and Jennings, 1969 ; Jennings and Gibson, 1969 ; Gibson, 1970) , and results so far obtained suggest that although the fundamental digestive sequence is essentially similar for all the species, differences in details can be related either to their systematic position or mode of life. Irrespective of the nature of the food utilized, digestion occurs in two distinct phases. Initial extracellular digestion is accomplished in the intestinal lumen at an acidic pH and involves mainly proteolytic enzymes secreted by the gastrodermis. Food particles are subsequently engulfed by lamellar outgrowths of the distal ciliated cell walls (Jennings, 1969) and food vacuoles passed back into the gastro dermis for the second, intracellular, stage in digestion. This involves exopeptidases (arylamidases), acid and alkaline phosphatases and, in some species at least, carbohydrases and lipases. The most uniform patterns of digestion are found in anoplan neinerteans (Jennings, 1962a ; Jennings and Gibson, 1969). Acidophilic gland cells in the foregut, rich in carbonic anhydrase, discharge their contents during ingestion which serve both to kill prey taken alive and provide the correct lumenar pH for extracellular digestion . Mucoid secretions, di scharged from other foregut glands, lubricate the food as it is passed into the intestine. Food entering the intestinal lumen stimulates the gastrodernial gland cells to discharge endopeptidases, which are responsible for early proteolysis and function optimally under acidic conditions. Intracellular digestion initially involves endopeptidases phagocytosed along with food particles and is marked by a sharp increase in acid phosphatase activity in and around food vacuoles. Acid phosphatases may be concerned in some way with the maintenance of the correct pH for intralumenar endopeptic activity (Jennings and Gibson, 1969) or with food vacuole formation (Rosenbaum and Rolon, 1960). Slinger and Gibson (1974) demonstrating biochemically that these enzymes func tion optimally in the range pH 4.1—5.0,depending upon the species. A few hours after the commencement of phagocytosis acid phosphatase and endopeptidase activity in the food vacuoles and surrounding cytoplasm declines, being replaced by alkaline phosphatases and exopeptidases. This final, alkaline, phase in digestion operates within the pH range 8.7—10.1 (Slinger and Gibson, 1974), and continues until digestion has been completed. Endopeptidase enzymes can be demonstrated in the gastrodermal gland cells at all times, irrespective of the nutritive state, but exopeptidase activity can only be visualized histochemically in gut cells at the appropriate stage in digestion. 352