Leucine p nitroanilide

A brush border membrane (BBM) preparation method by English et al [32 ] was adapted for whole neonates. Briefly, frozen WCR neonates were homogenized in ice-cold 5 mM Tris-HCl, pH 7.4, 50 mM sucrose supplemented with cytidine 5’-diphosphocholine (Sigma), protease inhibitor cocktail (Sigma), and PMSF, using three 30 s pulses of a polytron PT 2500E (Kinematica, Inc) homogenizer at 15,000 rpm. One volume of 5 mM Tris-HCl, pH 7.4, 50 mM sucrose was added to homogenates, supplemented with 10 mM CaCl₂, stirred on ice for 30 min and centrifugated at 4,500 x g for 30 min. Cleared lysates were passed through a four-layer cheese cloth and further centrifuged at 27,000 x g for 30 min. Resulting pellets were re-suspended in half the previous volume, homogenized on ice with a Dounce homogenizer (Sigma), supplemented with 10 mM CaCl₂, and further centrifuged at 27,000 x g for 30 min. BBM pellets were re-suspended in 0.32 M sucrose, aliquoted, flashed frozen in liquid nitrogen, and stored at -80°C. Total protein concentration was determined using the Bradford assay (Bio-Rad), using bovine serum albumin (BSA) as a standard. Enrichment was determined using the enzyme biomarker aminopeptidase-N (APN) and the leucine-p-nitroanilide (Sigma) as substrates [33 ]. BBM samples with 23 to 36-fold enrichment were used in binding assays.

PBS was precooled to serve as the medium to homogenize the intestine. Then the supernatant was extracted after centrifuging the homogenate at 2, 500×g for 15 min. According to the method described by Yin et al. (20 (link)), brush border membranes (BBM) were purified from the homogenate of the intestine.
The method of measuring the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), total antioxidant capacity (T-AOC), trypsin, lipase (LPS), α-amylase (α-AMS), alkaline phosphatase (AKP), acid phosphatase (ACP), and the levels of endothelin-1 (ET-1), D-lactic acid (D-Lac), reduced glutathione (GSH), malondialdehyde (MDA), complement 3 (C3) was referred to the instruction of commercial kit (Nanjing Jiancheng Bioengineering Institute, China). Besides, the kit for measuring ET-1, D-Lac, and C3, was commercial ELISA kit. The activity of leucine-aminopeptidase (LAP) was determined according to the description of Liu et al. (22 (link)). In brief, leucine-p-nitroanilide (Sigma, USA) servicing as substrate was incubated with 100 μL supernatant fraction (37°C) and phosphate buffer. The absorbance of samples was continuously monitored in Multiskan Spectrum (Thermo, USA) at 405 nm for 15 min.