. Author manuscript; available in PMC: 2015 Jun 1.

Published in final edited form as: Proteomics Clin Appl. 2014 Mar 31;8(0):317–326. doi: 10.1002/prca.201300118

Table 2.

Direct activity assays of urinary aminopeptidases

Target Aminopeptidase	Urinary protein (μg)	Substrate 0.5 mM	pH	NaCl (mM)	CaCl₂ (mM)	DTT (mM)	Inhibitors
Target Aminopeptidase	Urinary protein (μg)	Substrate 0.5 mM	pH	NaCl (mM)	CaCl₂ (mM)	DTT (mM)	PMSF	EDTA	AMA	BEST	SITAG	CYST

ANPEP	1	Ala-AMC	7.5	150			+	+
ENPEP	5	Glu-AMC	8.0	200	50		+	+
DPP4	5	Gly-Pro-AMC	7.5	150				+			Δ
DPP7	5	Lys-Pro-AMC	6.0				+		+	+	+
CTSC	0.5	Pro-Arg-AMC	6.0	50		5	+		+	+		Δ

ANPEP, alanyl aminopeptidase; ENPEP, glutamyl aminopeptidase; DPP(4, 7), dipeptidyl aminopeptidase (4, 7); CTSC, cathepsin C; AMC, 7-amino-4-methylcoumarin.

Buffers: 50 mM Tris. HCl (pH 7.5 and 8.0); 25 mM MES (pH 6.0); all reaction mixtures contained 1 mg/mL BSA.

Inhibitors: 2 mM PMSF; 2 mM EDTA; 10 μM amastatin (AMA); 10 μM bestatin (BEST); 10 μM sitagliptin(SITAG); 10 μg/mL cystatin (CYST); Δ = difference between −/+ inhibitor