scholarly journals Human recombinant membrane-bound aminopeptidase P: production of a soluble form and characterization using novel, internally quenched fluorescent substrates

2005 ◽  
Vol 385 (2) ◽  
pp. 389-397 ◽  
Author(s):  
Giuseppe MOLINARO ◽  
Adriana K. CARMONA ◽  
Maria A. JULIANO ◽  
Luiz JULIANO ◽  
Elena MALITSKAYA ◽  
...  

APP (aminopeptidase P) has the unique ability to cleave the N-terminal amino acid residue from peptides exhibiting a proline at P1′. Despite its putative involvement in the processing of bioactive peptides, among them the kinins, little is known about the physiological roles of both human forms of APP. The purpose of the present study is first to engineer and characterize a secreted form of hmAPP (human membrane-bound APP). Our biochemical analysis has shown that the expressed glycosylated protein is fully functional, and exhibits enzymic parameters similar to those described previously for mAPP purified from porcine or bovine lungs or expressed from a porcine clone. This soluble form of hmAPP cross-reacts with a polyclonal antiserum raised against a 469-amino-acid hmAPP fragment produced in Escherichia coli. Secondly, we synthesized three internally quenched fluorescent peptide substrates that exhibit a similar affinity for the enzyme than its natural substrates, the kinins, and a higher affinity compared with the tripeptide Arg-Pro-Pro used until now for the quantification of APP in biological samples. These new substrates represent a helpful analytical tool for rapid and reliable screening of patients susceptible to adverse reactions associated with angiotensin-converting enzyme inhibitors or novel vasopeptidase (mixed angiotensin-converting enzyme/neprilysin) inhibitors.

1983 ◽  
Vol 26 (9) ◽  
pp. 1267-1277 ◽  
Author(s):  
James L. Stanton ◽  
Norbert Gruenfeld ◽  
Joseph E. Babiarz ◽  
Michael H. Ackerman ◽  
Robert C. Friedmann ◽  
...  

2011 ◽  
Vol 365 ◽  
pp. 180-186
Author(s):  
Ya Lin Ren ◽  
Yan Jun Cong ◽  
Cun She Chen

A lot of peptides were synthesized on the basis of the sequence of αs1-casein. These tri-peptides with the characteristics of low toxicity and high biological activity will be applied to screen a new type of antihypertensive drug. Angiotensin-converting enzyme inhibitors (ACEI) with the better biological activity were screened and their structure-activity relationship was studied. The method of Fmoc solid-phase synthesis had been used to synthesize tri-peptides, and the principle, species, steps of which had been introduced respectively. HPLC assay was applied to screen the activity of ACEI by detecting the concentrate of hippuric acid, which can correspond with the inhibitory activity of tri-peptides. The results showed that the tri-peptides containing hydrophobic amino acid or praline had higher inhibition activity, and the same to the one that containing an aromatic amino acid in N-terminal.


Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Koichi Sugamura ◽  
Seigo Sugiyama ◽  
Koichiro Fujisue ◽  
Yoshihiro HIrata ◽  
Hirofumi Kurokawa ◽  
...  

Background: Inhibiting Neutral Endpeptidase-24.11 (NEP) combined with angiotensin receptor blocker (ARB) has recently been employed as an effective treatment for hypertension (HT). Although soluble form of NEP (sNEP) is able to measure in human plasma, the relationship between plasma levels of sNEP and clinical condition is still unknown in HT patients. Methods: We consecutively measured plasma levels of sNEP and B-type natriuretic peptide (BNP) in 210 patients with HT (144 male, 66 female, mean age; 69.5±10.4) who were suspected cardiovascular disease with multiple risk factors from April 2013 to December 2013. The levels of plasma sNEP were measured by using commercially available ELISA kits (SEB785Hu, USCN life science inc.). Results: Plasma levels of sNEP were non-normally distributed in whole high-risk HT patients assessed by Shapiro-Wilk test (p<0.01, median; 431.7 pg/ml, interquartile range; 302.0 to 540.9 pg/ml). sNEP levels were not significantly different among the patients with or without CAD [n=176, 431.7 pg/ml, (310.7 to 532.1) vs. n=34, 437.1 pg/ml, (284.4 to 574.6), p=0.851]. Plasma levels of sNEP were significantly greater in the HT patients treated with angiotensin converting enzyme inhibitors (ACEI) compared with those treated without ACEI [n=46, 493.4 pg/ml, (395.2 to 614.0) vs. n=164, 403.4 pg/ml, (299.6 to 523.8), p=0.012]. Plasma levels of sNEP were not significantly different between the patients with or without using ARB [n=107, 397.7 pg/ml, (284.8 to 506.8) vs. n=103, 456.4 pg/ml, (335.0 to 568.0), p=0.098]. There is no significant association between the plasma levels of sNEP and log BNP (ρ=0.018, P=0.801). Multivariable logistic regression analysis revealed that the administration of ACEI was independently associated with the higher levels of sNEP above median (odds ratio=2.48, 95% confidence interval; 1.23 to 5.03, p<0.02). Conclusion: Among high risk patients with HT, patients treated with ACEI demonstrated significantly elevated levels of sNEP and the administration of ACEI was independently associated with the higher levels of sNEP. NEP could be up-regulated by ACEI and ARB did not significantly affect on NEP activity in HT patients. NEP could be effectively inhibited by a NEP-inhibitor in HT patients treated by ARB.


2010 ◽  
Vol 43 ◽  
pp. 253-256
Author(s):  
Ming Yu Huang ◽  
Jing Jing Lv ◽  
Hong Jun Ni ◽  
Yu Zhu ◽  
Fu Bao Zhang

A lot of peptides were synthesized on the basis of the sequence of αs1-casein. These tri-peptides with the characteristics of low toxicity and high biological activity will be applied to screen a new type of antihypertensive drug. Angiotensin-converting enzyme inhibitors (ACEI) with the better biological activity were screened and their structure-activity relationship was studied. The method of Fmoc solid-phase synthesis had been used to synthesize tri-peptides, and the principle, species, steps of which had been introduced respectively. HPLC assay was applied to screen the activity of ACEI by detecting the concentrate of hippuric acid, which can correspond with the inhibitory activity of tri-peptides. The results showed that the tri-peptides containing hydrophobic amino acid or praline had higher inhibition activity, and the same to the one that containing an aromatic amino acid in N-terminal.


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