Role Of Aminopeptidase Research Articles

Low fluences of ultraviolet irradiation stimulate HeLa cell surface aminopeptidase and candidate "TGF alpha ase" activity.

Several forms of perturbation result in the release of bioactive molecules into the microenvironment of injured cells to mediate the inflammatory or reparative reactions which restore normal tissue structure and function. Amongst other products, ultraviolet irradiation (UV) causes the release of the growth factor TGF alpha from a variety of epithelial cell sources, apparently by a post-translational mechanism. Here we have explored the hypothesis that UV results in the activation of cell surface proteases which may then be capable of excising mature TGF alpha from its plasma membrane-bound precursor. Using a recently described, sensitive assay of peptidase activity tailored to the substrate requirements for cleavage of the scissile bonds in proTGF alpha, we have found that nonlethal fluences of UV (< 12 Jm-2) to HeLa cell cultures are followed by large increases in cell surface proteolytic activities. Amongst these, endopeptidase activity produces a similar product profile from the nonapeptide substrate to that of human leukocyte elastase, an enzyme previously shown to be capable of releasing a bioactive, mature form of TGF alpha from its cell-bound precursor. However, in addition to this candidate "TGFase" activity, cell surface aminopeptidase activity was also very significantly increased. The increase in the two classes of peptidase function differed in the timing of their responses. Aminopeptidase activation occurred immediately following UV, peaking after some 15-20 h, whereas the increase in endopeptidase activity lagged 6 h behind, cresting after 20-24 h. No evidence for a role for aminopeptidase in the activation of the endopeptidase could be found. Also, there was no increase in the total proteolytic activity demonstratable in cell extracts following UV. Attempts to interrupt the UV peptidase activation by inhibiting protein synthesis with cycloheximide were unsuccessful; rather, the inhibitor itself caused an increase in both classes of peptidase activity during the first 20 h. Unlike the UV response, both the aminopeptidase and endopeptidase ectoactivities increased simultaneously within a few hours of introducing cycloheximide into the medium of unirradiated cultures. The cycloheximide induced activity peaked after 20 h. Interestingly, cycloheximide alone has previously been shown to potentiate TGF alpha release from a cell line producing its precursor constitutively. These data suggest that both UV and cycloheximide can initiate reactions in HeLa cells which result in ectopeptidase activation of a global nature. Since both agents result in rapid interruption of DNA synthesis, it is possible that this cell surface proteolytic response may be analogous to, or part of, the "mammalian genetic stress response".(ABSTRACT TRUNCATED AT 400 WORDS)

Proteolytic conversion of oxytocin by brain synaptic membranes: Role of aminopeptidases and endopeptidases

The proteolytic conversion of oxytocin and vasopressin by purified rat brain synaptic membranes was studied at 37°C and physiological pH 7.4. The formed peptide fragments were isolated by high performance liquid chromatography and characterized by amino acid analysis. When oxytocin was incubated with synaptic membranes, either C- or N-terminal fragments were found. The most abundant were [Cyt 6]oxytocin(4–9), [Cyt 6]oxytocin(3–9), [Cyt 6]oxytocin(2–9), oxytocin(1–8) and oxytocin(1–7). In contrast, only C-terminal fragments, [Cyt 6-Arg 8]vasopressin(4–9), [Cyt 6-Arg 8]vasopressin(3–9), were found by incubating [Arg 8]vasopressin. The formation of C-terminal oxytocin and vasopressin fragments was inhibited by the aminopeptidase inhibitors amastatin and bestatin, while the formation of oxytocin(1–7) and (1–8) was inhibited by the divalent cations Hg 2+ and Zn 2+. The formation of oxytocin(1–7) was also partially prevented by the endopeptidase inhibitor phosphoramidon. The formation of both C- and N-terminal fragments was inhibited by o-phenanthroline. The results suggest that, while [Arg 8]vasopressin is metabolized only by membrane-bound aminopeptidases, oxytocin is also metabolized by membrane-bound endopeptidases.

Photoaffinity labeling of membrane-bound porcine aminopeptidase n

To investigate the possible role of aminopeptidase N (α-aminoacyl-peptide hydrolase (microsomal), EC 3.4.11.2) in the transport of amino acids from oligopeptides, the modified amino acids Phe(N 3) and Phe(N 3,I) and the tetrapeptides Phe(N 3) or Phe(N 3, I)- l-or- dAla-Gly-Gly have been synthesized. The azido-amino acids were radioactively labeled by tritium or 125I before their coupling with the tripeptides. Their utilization as photoaffinity labels for aminopeptidase N has been studied. The modification imposed at the N-terminal residue of the tetrapeptides has not impaired their hydrolysis by porcine aminopeptidase N (same kinetic parameters as unmodified peptides). In addition, evidence is presented for a specific and reversible interaction in the dark of the azido-derivatives at the substrate recognition site of the enzyme. Upon photolysis, irreversible inactivation of aminopeptidase N and covalent attachment of Phe(N 3, I) have been demonstrated. Soluble and membrane-bound aminopeptidases are both labeled to the same extent, indicating that the free azido-amino acid preferentially reacts with the external part of the enzyme. Although the linkage of the azido-derivative is not strictly restricted to the region of the active site, the values obtained strongly suggest that 1 mol probe has been covalently attached per mol monomer of inhibited aminopeptidase.

The role of aminopeptidases in inflammatory and neoplastic tissues

The role of leucine andalanine aminopeptidases is stidued in three different biologic systems: experimental wound healing in the rat, experimental carrageenan induced intraderman granulomas in the rat, and human laryngeal carcinomas. The wound healing experiments indicate that the proliferating granulation tissue has high quantities of aminopeptidases activity which is residing primarily intracellularly in granulocytes, macrophages, mast cells fibroblasts, and new budding vessels. The quantititave levels of tissue aminopeptidases correlate positively with the degree of cellularity of the wound and fibroblastic activity. Some aminopeptidases (isoenzymes) are secreted or released by the fibroblasts in be blood serum. Starch gel electrophoretic analysis demonstrates thses with different concentrations and migration rates. Two are probably released or secreted into the serum, and th third is membranous bound in the cytoplasm (lysosomal). The carrageenan intradermal granuloma demonstrates a different inflammatory reaction which is rich in macrophages and produces a different pattern of aminopeptidases activity. Macrophages produce a high tissue level of aminopeptidase activity which is intracellular bound and not readily leached out into the serum. Gel electrophoresis studies of aminopeptidases produced by the granuloma demonstrate that the tissue bound enzyme is the main component. In addition, there appears to be a mechanism, which is not understood, for specific induction or activation of lysosomal proteolytic and carbohydrase enzymes. This mechanism is dependent on the nature of composition of the injuring agent. Laryngeal carcinomas demonstrate high tissue homogenate levels and normal serum levels of aminopeptidase activities. These enzymes are located in the tumor stroma. They are not directly related to tumor invasiveness but to the degree of stromal proliferation. The tumor stroma behaves as though it were a non-healing wound constantly secreted proteolytic enzymes (aminopeptidases). A major problem that needs to be resolved is to find the operating mechanism by which malignant cells interact with their constantly proliferating fibroblastic stroma.