The aminopeptidase activity in EVs was measured by the hydrolysis of l -leucine-7-amido-4-methylcoumarin hydrochloride (Leu-AMC) and l -arginine-AMC (Arg-AMC) (Sigma-Aldrich) as previously described [15 (link)]. Briefly, the EV solution was added to assay buffer (50 mM Tris-HCl, pH 8.0) containing 10 μM Leu-AMC or Arg-AMC, followed by incubation at 37 °C for 30 min. The PBS buffer served as the control for this assay. The release of fluorescence was detected at an excitation wavelength of 370 nm and an emission wavelength of 440 nm using a FlexStation 3 Multi-Mode microplate reader (Molecular Devices, Sunnyvale, CA, USA) [38 (link)].
Leu amc
Manufactured by Merck Group
Leu-AMC is a fluorogenic substrate used for the detection and quantification of leucine aminopeptidase (LAP) activity. LAP is an enzyme involved in the hydrolysis of peptide bonds. Leu-AMC serves as a substrate for LAP, releasing a fluorescent product upon enzymatic cleavage, allowing for the measurement of LAP activity in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Leu pna, by Merck Group (2 mentions)
Llkhhafsfk, by Genecust (2 mentions)
Peptide llriqrgpgrafvti, by JPT Peptide Technologies (2 mentions)
Flexstation3 multi mode, by Molecular Devices (1 mentions)
Spark 10m, by Tecan (1 mentions)
Flexstation 3 multi mode microplate reader, by Molecular Devices (1 mentions)
Np 40, by Merck Group (1 mentions)
Superose 6 hr 10 30 column, by Cytiva (1 mentions)
Formic acid, by Merck Group (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Z phe arg amc, by Bachem (1 mentions)
Ca 074, by MedChemExpress (1 mentions)
Pepstatin a, by Merck Group (1 mentions)
Z phe leu oh, by Bachem (1 mentions)
Human hb, by Merck Group (1 mentions)
Fluorescamine, by Merck Group (1 mentions)
Bestatin, by Merck Group (1 mentions)
Bestatin, by Santa Cruz Biotechnology (1 mentions)
Amastatin, by Santa Cruz Biotechnology (1 mentions)
9 protocols using leu amc
1
Quantification of Aminopeptidase Activity in EVs
2
ERAP1 Inhibitor IC50 Determination
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ERAP1 enzymatic activity and inhibitor IC50 values were calculated as previously reported24 (link). Briefly, hydrolysis of the fluorescent substrate leucine-aminomethylcoumarin (Leu-AMC, Sigma–Aldrich, L2145) was followed at 460 nm (excitation at 380 nm) using a Spark 10 M (TECAN) multimode microplate reader. The IC50 of DG080 was calculated by using the equation log (inhibitor) versus response-variable slope of GraphPad Prism 8.0™.
3
Peptide-based Enzyme Inhibition Protocol
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Leu-pNA and Leu-AMC were purchased from Sigma–Aldrich. Peptide with the sequence LLKHHAFSFK was purchased from Genecust. Peptide LLRIQRGPGRAFVTI was purchased from JPT Peptide Technologies. Peptide YTAFTIPSI was purchased from BioPeptide Inc, and ovalbumin peptides were from CRB Discovery. All peptides were HPLC purified to >95% purity and confirmed by mass spectrometry to have the correct mass. Inhibitor DG013A was synthesized as described previously (38 (link)), and inhibitor GSK849 was obtained, purified, and characterized as described previously (16 ).
4
Isolation and Characterization of Secreted Proteases
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Following the steps described in Section 4.4 Isolation of secreted proteins and using PBS buffer as the control the activity of Asp was assayed by the hydrolysis of l -leucine-7-amido-4-methylcoumarin hydrochloride (Leu-AMC) and l -arginine-AMC (Arg-AMC, Sigma-Aldrich). Five μL of Asp solution was added to 195 μL of assay buffer (50 mM Tris-HCl, pH 8.0) containing 10 μM Leu-AMC or Arg-AMC and incubated for 30 min at 37 °C. The release of fluorescence was measured at an excitation wavelength of 370 nm and an emission wavelength of 440 nm using a FlexStation 3 Multi-Mode microplate reader (Molecular Devices, Sunnyvale, CA, USA) [23 (link)].
5
Biochemical Properties of Recombinant Protein M28AP
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The biochemical properties of recombinant protein M28AP, while using PBS buffer as the control and the activity of Asp, was assayed by the hydrolysis of L-leucine-7-amido-4-methylcoumarin hydrochloride (Leu-AMC) and l -arginine-AMC (Arg-AMC, Sigma-Aldrich, st. Louis, MO, USA). We added approximately 1 μg/mL the recombinant protein M28AP solution to 195 μL of assay buffer (50 mM Tris-HCl, pH 8.0) containing 10 μM Leu-AMC or Arg-AMC up to 200 μL, which was then incubated for 30 min at 37 °C. The release of fluorescence was measured at an excitation wavelength of 370 nm and an emission wavelength of 440 nm while using a FlexStation 3 Multi-Mode microplate reader (Molecular Devices, Sunnyvale, CA, USA) [21 (link)].
6
Peptide Separation and Characterization
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Superose 6 HR10/30 column was from Amersham Biosciences (Buckinghamshire, UK). DMSO, formic acid, NP-40, trifluoracetic acid (TFA), acetonitrile, and Leu-AMC were obtained from Sigma-Aldrich (St. Louis, MO). The LSTVIVR (786.953 Da) and STVIVR* (683.794 Da) peptides were supplied by Cambridge Research Biochemicals (Billingham, UK) and dissolved in water.
7
Protease Activity Assay Reagents
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The following reagents, substrates, and protease inhibitors were utilized in this research: Bz-Arg-Gly-Phe-Phe-Pro-4M2NA, Leu-AMC, pepstatin A, bestatin, RKLLW-NH2, human Hb, and fluorescamine from Sigma-Aldrich. Z-Phe-Arg-AMC and Z-Phe-Leu-OH from Bachem, Z-Ala-Ala-Asn-AMC, CA-074, and legumain inhibitor1 from MedChemExpress. E-64 and EDTA from Merck. PMSF and EDTA-free protease inhibitor cocktail from Roche (Merck). The stock solutions of these products were prepared, following the instructions provided by the respective providers.
8
Ld Leucine Aminopeptidase Kinetics
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The amidolytic activity of the LdLAP was determined in vitro in a metal cofactor-supplemented
biochemical assay
in 50 mM Tris-HCl (pH 8.0) buffer by measuring the liberation ofl -leucine from the fluorogenic peptide substrate, Leu-AMC (Sigma-Aldrich).
Release of AMC was monitored at 37 °C on a TECAN Infinite M200
Pro spectrofluorometer with a λex and λem pair of 355 and 460 nm, respectively. For steady-state kinetic
parameters, fluorescence was employed to deduce product formation
(AMC) by utilizing an AMC standard curve. Data was plotted, and all
parameters were determined in GraphPad Prism. To study the inhibitory
efficacy of peptidomimetics like bestatin, amastatin, and actinonin
(Santa Cruz Biotechnology) on the amidolytic activity, the LdLAP was preincubated in assay buffer with inhibitors (0
to 20 nM) for 30 min at 37 °C before the addition of a substrate
to measure the residual amidolytic activity. The mode of inhibition
was deduced with double-reciprocal plots.
biochemical assay
in 50 mM Tris-HCl (pH 8.0) buffer by measuring the liberation of
Release of AMC was monitored at 37 °C on a TECAN Infinite M200
Pro spectrofluorometer with a λex and λem pair of 355 and 460 nm, respectively. For steady-state kinetic
parameters, fluorescence was employed to deduce product formation
(AMC) by utilizing an AMC standard curve. Data was plotted, and all
parameters were determined in GraphPad Prism. To study the inhibitory
efficacy of peptidomimetics like bestatin, amastatin, and actinonin
(Santa Cruz Biotechnology) on the amidolytic activity, the LdLAP was preincubated in assay buffer with inhibitors (0
to 20 nM) for 30 min at 37 °C before the addition of a substrate
to measure the residual amidolytic activity. The mode of inhibition
was deduced with double-reciprocal plots.
9
Peptide Reagents for Protease Activity Assays
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Leu-pNA and Leu-AMC were purchased from Sigma-Aldrich. Peptide with the sequence LLKHHAFSFK was purchased from Genecust. Peptide LLRIQRGPGRAFVTI was purchased from JPT peptide technologies. Peptide YTAFTIPSI was purchased from BioPeptide Inc. and ovalbumin peptides were from CRB Discovery. All peptides were HPLC-purified to >95% purity and confirmed by mass spectrometry to have the correct mass. Inhibitor DG013A was synthesized as described previously [26] and inhibitor GSK849 was obtained, purified and characterized as described previously [16] .
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