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Membrane peptidases on human osteoblast-like cells in culture: hydrolysis of calcitonin and hormonal regulation of endopeptidase-24.11.

ABSTRACT: Five membrane peptidase activities have been identified on cultured human osteoblast-like cells. These consisted of the four exopeptidases aminopeptidase-A, aminopeptidase-N, aminopeptidase-W and carboxypeptidase-M, and the endopeptidase, endopeptidase-24.11. The presence of endopeptidase-24.11 was confirmed immunochemically by immunofluorescent staining and by enzyme-linked immunosorbent assay. The inclusion of phosphoramidon partially inhibited the hydrolysis of human calcitonin by a membrane fraction prepared from osteoblast-like cell membranes, thus implicating endopeptidase-24.11 in its inactivation. Another metallopeptidase also contributed substantially to calcitonin hydrolysis. Purified porcine endopeptidase-24.11 (1 microgram) was shown to hydrolyse calcitonin with a half-life of 23 min, which compared to a half-life of 0.5 min for substance P under similar conditions. Sequence data revealed that the initial site of hydrolysis of calcitonin was between residues Lys18 and Phe19. The expression of endopeptidase-24.11 by cultured osteoblast-like cells was shown to be modified by various agents: expression was decreased by phorbol 12-myristate-13-acetate (160 nM for 48 h) and increased in the presence of calcitonin (1.5 nM for 48 h) and 1,25-dihydroxyvitamin D3 (0.01-1 microM for 72 h).

SUBMITTER: Howell S

PROVIDER: S-EPMC1132396 | biostudies-other | 1993 Feb

REPOSITORIES: biostudies-other

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Hydrolysis of rat melanin-concentrating hormone by endopeptidase 24.11 (neutral endopeptidase).

Project description:Melanin-concentrating hormone (MCH) is a cyclic peptide which behaves as an antagonist of the pituitary melanotropic hormone alpha-melanocyte-stimulating hormone in fishes. Cloning of the rat MCH cDNA precursor recently revealed the presence of an additional putative peptide named NEI. The present work examined the susceptibility of these novel peptides to hydrolysis by various purified exo- and endo-peptidases including endopeptidases 24.11 (NEP), 24.15, 24.16, angiotensin-converting enzyme, leucine aminopeptidase and carboxypeptidase A. NEP attacked MCH at three sites of the molecule with an apparent affinity of about 12 microM and a kcat. of 4 min-1. The first site of cleavage was at Cys-7-Met-8, i.e. within the peptide loop formed by the internal disulphide bridge. NEP could therefore be considered as an MCH-inactivating peptidase since the degradation products generated are probably devoid of biological activity. In contrast, NEI neither inhibited the degradation of the NEP chromogenic substrate glutaryl-Phe-Ala-Phe-p-aminobenzoate nor was susceptible to proteolysis by NEP. Unlike NEP, angiotensin-converting enzyme, endopeptidase 24.15 and endopeptidase 24.16 appeared totally unable to cleave MCH, whereas the peptide was readily degraded by aminopeptidase M and carboxypeptidase A.

| S-EPMC1133042 | biostudies-other

The metabolism of neuropeptides. The hydrolysis of peptides, including enkephalins, tachykinins and their analogues, by endopeptidase-24.11.

Project description:Endopeptidase-24.11 (EC 3.4.24.11), purified to homogeneity from pig kidney, was shown to hydrolyse a wide range of neuropeptides, including enkephalins, tachykinins, bradykinin, neurotensin, luliberin and cholecystokinin. The sites of hydrolysis of peptides were identified, indicating that the primary specificity is consistent with hydrolysis occurring at bonds involving the amino group of hydrophobic amino acid residues. Of the substrates tested, the amidated peptide substance P is hydrolysed the most efficiently (Km = 31.9 microM; kcat. = 5062 min-1). A free alpha-carboxy group at the C-terminus of a peptide substrate is therefore not essential for efficient hydrolysis by the endopeptidase. A large variation in kcat./Km values was observed among the peptide substrates studied, a finding that reflects a significant influence of amino acid residues, remote from the scissile bond, on the efficiency of hydrolysis. These subsite interactions between peptide substrate and enzyme thus confer some degree of functional specificity on the endopeptidase. The inhibition of endopeptidase-24.11 by several compounds was compared with that of pig kidney peptidyldipeptidase A (EC 3.4.15.1). Of the inhibitors examined, only N-[1(R,S)-carboxy-2-phenylethyl]-Phe-p-aminobenzoate inhibited endopeptidase-24.11 but not peptidyldipeptidase. Captopril (D-3-mercapto-2-methylpropanoyl-L-proline), Teprotide (pGlu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro) and MK422 [N-[(S)-1-carboxy-3-phenylpropyl]-L-Ala-L-Pro] were highly selective as inhibitors of peptidyldipeptidase. Although not wholly specific, phosphoramidon was a more potent inhibitor of endopeptidase-24.11 than were any of the synthetic compounds tested.

| S-EPMC1144315 | biostudies-other

The hydrolysis of brain and atrial natriuretic peptides by porcine choroid plexus is attributable to endopeptidase-24.11.

Project description:The hydrolysis of the porcine 26-residue brain natriuretic peptide (BNP-26) and its counterpart human 28-residue atrial natriuretic peptide (alpha-hANP) by pig membrane preparations and purified membrane peptidases was studied. When the two peptides were incubated with choroid plexus membranes, the products being analysed by h.p.l.c., alpha-hANP was degraded twice as fast as BNP. The h.p.l.c. profiles of alpha-hANP hydrolysis, in short incubations with choroid plexus membranes, yielded alpha hANP' as the main product, this having been previously shown to be the result of hydrolysis at the Cys7-Phe8 bond. In short incubations this cleavage was inhibited 84% by 1 microM-phosphoramidon, a specific inhibitor of endopeptidase-24.11. BNP-26 was hydrolysed by choroid plexus membranes, kidney microvillar membranes and purified endopeptidase-24.11 in a manner that yielded identical h.p.l.c. profiles. In the presence of phosphoramidon, hydrolysis by the choroid plexus membranes was 94% inhibited. Captopril had no effect and, indeed, no hydrolysis of BNP-26 by peptidyl dipeptidase A (angiotensin-converting enzyme) was observed even after prolonged incubation with the purified enzyme. The stepwise hydrolysis of BNP-26 by endopeptidase-24.11 was investigated by sequencing the peptides produced during incubation. The initial product resulted from hydrolysis at Ser14-Leu15, thereby opening the ring. This product (BNP') was short-lived; further degradation involved hydrolysis at Ile12-Gly13, Arg8-Leu9, Gly17-Leu18, Val22-Leu23, Arg11-Ile12 and Cys4-Phe5. Thus endopeptidase-24.11 is the principal enzyme in renal microvillar and choroid plexus membranes hydrolysing BNP-26 and alpha-hANP.

| S-EPMC1149565 | biostudies-other

Hydrolysis of Antimicrobial Peptides by Extracellular Peptidases in Wastewater.

Project description:Several antimicrobial peptides (AMPs) are emerging as promising novel antibiotics. When released into wastewater streams after use, AMPs might be hydrolyzed and inactivated by wastewater peptidases─resulting in a reduced release of active antimicrobials into wastewater-receiving environments. A key step towards a better understanding of the fate of AMPs in wastewater systems is to investigate the activity and specificity of wastewater peptidases. Here, we quantified peptidase activity in extracellular extracts from different stages throughout the wastewater treatment process. For all four tested municipal wastewater treatment plants, we detected highest activity in raw wastewater. Complementarily, we assessed the potential of enzymes in raw wastewater extracts to biotransform 10 selected AMPs. We found large variations in the susceptibility of AMPs to enzymatic transformation, indicating substantial substrate specificity of extracted enzymes. To obtain insights into peptidase specificities, we searched for hydrolysis products of rapidly biotransformed AMPs and quantified selected products using synthetic standards. We found that hydrolysis occurred at specific sites and that these sites were remarkably conserved across the four tested wastewaters. Together, these findings provide insights into the fate of AMPs in wastewater systems and can inform the selection and design of peptide-based antibiotics that are hydrolyzable by wastewater peptidases.

| S-EPMC10785756 | biostudies-literature

The metabolism of neuropeptides. Endopeptidase-24.11 in human synaptic membrane preparations hydrolyses substance P.

Project description:Synaptic membrane preparations from human striatum and human diencephalon were shown to contain a phosphoramidon-sensitive metalloendopeptidase that appeared identical with endopeptidase-24.11. The activity of endopeptidase-24.11 was determined with an enzymic assay employing [D-Ala2,Leu5]enkephalin as substrate, and its distribution in human brain was similar to that in pig brain, with the striatum containing the highest levels. The choroid plexus and pons also contained substantial activity. A good correlation (r = 0.97) was obtained for the distribution of the endopeptidase in pig brain and pituitary by the enzymic assay and by an immunoradiometric assay specific for pig endopeptidase-24.11. Synaptic membrane preparations from human striatum and diencephalon hydrolysed substance P at the same sites as did preparations of pig striatal synaptic membranes, and hydrolysis was substantially abolished by phosphoramidon. These results suggest that endopeptidase-24.11 is the principal enzyme hydrolysing substance P in human synaptic membrane preparations.

| S-EPMC1145007 | biostudies-other

The hydrolysis of alpha-human atrial natriuretic peptide by pig kidney microvillar membranes is initiated by endopeptidase-24.11.

Project description:alpha-Human atrial natriuretic peptide, a 28-amino-acid-residue peptide, was rapidly hydrolysed by pig kidney microvillar membranes in vitro, with a t1/2 of 8 min, comparable with the rate observed with angiotensins II and III. The products of hydrolysis were analysed by h.p.l.c., the pattern obtained with membranes being similar to that with purified endopeptidase-24.11 (EC 3.4.24.11). No hydrolysis by peptidyl dipeptidase A (angiotensin I converting enzyme, EC 3.4.15.1) was observed. The contribution of the various microvillar membrane peptidases was assessed by including specific inhibitors. Phosphoramidon, an inhibitor of endopeptidase-24.11, caused 80-100% suppression of the products. Captopril and amastatin (inhibitors of peptidyl dipeptidase A and aminopeptidases respectively) had no significant effect. Hydrolysis at an undefined site within the disulphide-linked ring occurred rapidly, followed by hydrolysis at other sites, including the Ser25--Phe26 bond.

| S-EPMC1147830 | biostudies-other

Simple Culture Methods and Treatment to Study Hormonal Regulation of Ovule Development.

Project description:Ovule development is one of the most important processes in the reproductive development of higher plants and is a determinant of seed quality and quantity. Phytohormones play key roles in this process since loss-of-function mutants in hormone signaling show defective ovule phenotypes and reduced fertility. However, it is difficult to distinguish the direct effects of hormones on ovule development because it is parts of reproductive development and the defective phenotypes would be the indirect effects following the defective vegetative development. The treatment of hormones is a direct method to investigate the hormonal regulation of ovule development, but ovule is embedded inside several layers of floral organs, and traditional methods for hormone (or inhibitor) treatments have various limitations. We have developed simple methods to apply treatments to the flowers in a living plant, where an inflorescence apex is immersed into a solution in an inverted tube. We have also developed a specific system to culture and treat excised flowers/pistils. These procedures will be useful for research on the hormonal regulation of ovule development. We provide examples of how treatments with brassinosteroids (BR) and BR biosynthesis inhibitor. We cultured and treated plant materials using our newly developed methods, and observed the morphology of wild type ovules and fluorescence signals in a marker line to monitor the progress of ovule development. The results demonstrate BR promotes ovule development and our new methods are efficient and repeatable.

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Characterization of neutral endopeptidase 24.11 in dog glomeruli.

Project description:Neutral endopeptidase (NEP; also known as neprilysin and enkephalinase; EC 3.4.24.11) is a cell-surface metallopeptidase that is present in many mammalian tissues. It is particularly abundant on the brush-border membranes of the kidney proximal tubule. In this paper, the presence of NEP in purified glomeruli from dog kidney was assessed by measuring phosphoramidon- and thiorphan-sensitive [D-Ala2,Leu5]enkephalin-degrading activity. Using this assay, the Km and kcat. of the glomerular enzyme were found to be identical to those of the tubular enzyme. By Western blotting the apparent M(r) of the glomerular enzyme was found to be 104,000, compared with 94,000 for the tubular enzyme. This might be due to a different glycosylation pattern, since endoglycosidase F treatment of NEP obtained from both tissues yielded deglycosylated enzymes with similar electrophoretic mobilities. The glomerular enzyme also appears to be membrane-bound, since it was retained in the detergent-rich phase after phase separation with Triton X-114. Autoradiography experiments performed with RB104, a new highly selective and potent NEP inhibitor, showed that NEP was expressed in both glomeruli and proximal tubules. The presence in glomeruli of NEP and some other brush-border peptidases (dipeptidyl-dipeptidase IV, aminopeptidase N and angiotensin I-converting enzyme) suggests that cell-surface peptidases might play an important role as regulators of plasma-derived peptides in this part of the nephron.

| S-EPMC1132435 | biostudies-other

Hormonal regulation of epithelial organization in a three-dimensional breast tissue culture model.

Project description:The establishment of hormone target breast cells in the 1970's resulted in suitable models for the study of hormone control of cell proliferation and gene expression using two-dimensional (2D) cultures. However, to study mammogenesis and breast tumor development in vitro, cells must be able to organize in three-dimensional (3D) structures like in the tissue. We now report the development of a hormone-sensitive 3D culture model for the study of mammogenesis and neoplastic development. Hormone-sensitive T47D breast cancer cells respond to estradiol in a dose-dependent manner by forming complex epithelial structures. Treatment with the synthetic progestagen promegestone, in the presence of estradiol, results in flat epithelial structures that display cytoplasmic projections, a phenomenon reported to precede side-branching. Additionally, as in the mammary gland, treatment with prolactin in the presence of estradiol induces budding structures. These changes in epithelial organization are accompanied by collagen remodeling. Collagen is the major acellular component of the breast stroma and an important player in tumor development and progression. Quantitative analysis of second harmonic generation of collagen fibers revealed that collagen density was more variable surrounding budding and irregularly shaped structures when compared to more regular structures; suggesting that fiber organization in the former is more anisotropic than in the latter. In sum, this new 3D model recapitulates morphogenetic events modulated by mammogenic hormones in the breast, and is suitable for the evaluation of therapeutic agents.

| S-EPMC3870574 | biostudies-literature

A Chemical-Intervention Strategy To Circumvent Peptide Hydrolysis by d-Stereoselective Peptidases.

Project description:d-Stereoselective peptidases that degrade nonribosomal peptides (NRPs) were recently discovered and could have serious implications for the future of NRPs as antibiotics. Herein, we report chemical modifications that can be used to impart resistance to the d-peptidases BogQ and TriF. New tridecaptin A analogues were synthesized that retain strong antimicrobial activity and have significantly enhanced d-peptidase stability. In vitro assays confirmed that synthetic analogues retain the ability to bind to their cellular receptor, peptidoglycan intermediate lipid II.

| S-EPMC6887851 | biostudies-literature

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