A continuous fluorimetric assay for aminopeptidase P detailed analysis of product inhibition

Stöckel-Maschek, Angela; Stiebitz, B.; Koelsch, R; Neubert, Klaus

doi:10.1016/s0003-2697(03)00464-0

Cited by 26 publications

(9 citation statements)

References 22 publications

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“…The reaction was first attempted on amine 7 using equimolar amounts of cbz-proline and slight excess of both DCC and DMAP 20,21 in DCM under an argon atmosphere. This approach was partially successful, producing desired product 8, albeit in only 15% yield.…”

Section: Resultsmentioning

confidence: 99%

Inhibition of prolyl oligopeptidase with a synthetic unnatural dipeptide

Racys¹,

Rea²,

Fülöp³

et al. 2010

Bioorganic & Medicinal Chemistry

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The synthesis of a new inhibitor of prolyl oligopeptidase, containing a piperidine ring, together with an X-ray structure of its complex with the enzyme, is described. This provides evidence that covalent inhibitors of POP do not have to be limited to structures containing 5-membered N-containing heterocyclic rings. inhibitor:Leave this area blank for abstract info. Abstract-A new inhibitor, containing a linked proline-piperidine structure, for the enzyme prolyl oligopeptidase (POP) has been synthesised and demonstrated to bind covalently with the enzyme at the active site. This provides evidence that covalent inhibitors of POP do not have to be limited to structures containing 5-membered N-containing heterocyclic rings.

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Section: Resultsmentioning

confidence: 99%

Inhibition of prolyl oligopeptidase with a synthetic unnatural dipeptide

Racys¹,

Rea²,

Fülöp³

et al. 2010

Bioorganic & Medicinal Chemistry

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“…Aminopeptidase P is inhibited by a number of cleavage products [23], [24]. The NS3 proteases of hepatitis C and dengue viruses, necessary for specific cleavage of the viral polyprotein, are both inhibited by their products [25], [26].…”

Section: Discussionmentioning

confidence: 99%

Product Inhibition in Native-State Proteolysis

2014

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The proteolysis kinetics of intact proteins by nonspecific proteases provides valuable information on transient partial unfolding of proteins under native conditions. Native-state proteolysis is an approach to utilize the proteolysis kinetics to assess the energetics of partial unfolding in a quantitative manner. In native-state proteolysis, folded proteins are incubated with nonspecific proteases, and the rate of proteolysis is determined from the disappearance of the intact protein. We report here that proteolysis of intact proteins by nonspecific proteases, thermolysin and subtilisin deviates from first-order kinetics. First-order kinetics has been assumed for the analysis of native-state proteolysis. By analyzing the kinetics of proteolysis with varying concentrations of substrate proteins and also with cleavage products, we found that the deviation from first-order kinetics results from product inhibition. A kinetic model including competitive product inhibition agrees well with the proteolysis time course and allows us to determine the uninhibited rate constant for proteolysis as well as the apparent inhibition constant. Our finding suggests that the likelihood of product inhibition must be considered for quantitative assessment of proteolysis kinetics.

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“…Serum APP activity was measured using a modified version of the assay previously described by Lefebvre et al[13] Two or 4μl serum samples, plated in a 96-well format, were incubated at 37°C for 3h with 5μM L-lysyl(ε-2-aminobenzoyl)-L-prolyl-L-prolyl-4-nitroanilide [H-Lys(ε-Abz)-Pro-Pro-pNA] (Bachem, Torrance, CA), an internally quenched fluorescent substrate. [14] Fluorescence was measured at 5 to 7 time points in a Flexstation II 384 spectrofluorometer (Molecular Devices, Sunnyvale, CA). After the 3h reading, 1nmol of internal standard, Abz-Gly (Bachem, Torrance, CA), was routinely added to each well to normalize for serum and substrate quenching effects.…”

Section: Methodsmentioning

confidence: 99%

Sex-dependent and race-dependent association of XPNPEP2 C-2399A polymorphism with angiotensin-converting enzyme inhibitor-associated angioedema

Woodard‐Grice

Lucisano

Byrd³

et al. 2010

Pharmacogenetics and Genomics

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Background-Angioedema is a rare adverse effect of angiotensin converting enzyme (ACE) inhibitors that occurs more commonly in women and black Americans. Angioedema is thought to result from decreased degradation of vasoactive peptides. During ACE inhibition, bradykinin is primarily inactivated by aminopeptidase P (APP). Previous studies have provided conflicting data regarding serum APP activity in patients with a history of ACE inhibitor-associated angioedema. A single nucleotide polymorphism, −2399C>A (rs3788853, C-2399A), in XPNPEP2, the X-linked gene that encodes membranous APP, has been reported to associate with APP activity.

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A continuous fluorimetric assay for aminopeptidase P detailed analysis of product inhibition

Cited by 26 publications

References 22 publications

Inhibition of prolyl oligopeptidase with a synthetic unnatural dipeptide

Inhibition of prolyl oligopeptidase with a synthetic unnatural dipeptide

Product Inhibition in Native-State Proteolysis

Sex-dependent and race-dependent association of XPNPEP2 C-2399A polymorphism with angiotensin-converting enzyme inhibitor-associated angioedema

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