For the reg ularization procedure a confidence level tech support of 0. 68 was used. The molecular mass of LAPTc in solution was also determined by size exclusion chromatography coupled to multiangle laser light scattering and refractometry. rLAPTc, purified by affinity chromatography as above, at 170 uM in 25 mM Tris HCl, pH 7. 5, 100 mM NaCl, was injected in a KW 804 column preceded by a guard column, equilibrated in the same solvent, at 20 C with a flow rate of 0. 5 ml min. Protein concentration was measured on line by refractive index measurements using an Optilab rEX and considering ?n ?c 0. 186 ml g. On line MALLS detection was performed with a miniDAWN TREOS detector using laser emitting at 658 nm. Data were analyzed and weight averaged molar masses were calculated using the ASTRA software.
Elution profiles were monitored by RI. The molecular mass distribution was determined from combined MALLS and RI data. Assay of optimal pH and temperature for activity and thermostability of LAPTc The optimal pH for activity of both endogenous and recombinant LAPTc was determined as described above in 50 mM acetic acid 50 mM MES 50 mM Tris HCl buffer adjusted to the desired pH. To assay the optimal temperature for aminopeptidase activity, reactions took place at 20, 25, 30, 37, 40, 50, 60, 70, 80 or 100 C in reaction buffer. Enzyme thermostability was assayed by incubating the purified proteins at the same tempera tures for either 15 or 240 min in reaction buffer before the aminopeptidase activity assay on Leu AMC. An 8% SDS PAGE analysis of the molecular organization of the native or recombinant LAPTc followed.
PAGE was per formed in the presence of 0. 1 or 0. 01% SDS without previous boiling of either protein. Inhibition pattern and cation dependence of LAPTc Different concentrations of tosyl lysylchloromethane, bestatin, EDTA, L trans epoxysuccinylleucyla mido butane, phenylmethylsulfonyl fluoride, 1,10 phenanthroline, leupeptin, or phosphoramidon were incubated with 50 ng of purified LAPTc in 100 ul reaction buffer for 20 min at room temperature before the substrate was added. Enzymatic reactions were monitored as described above. All inhibitors were from Sigma Aldrich. To assess the effects of cations on enzymatic activity, purified LAPTc was incubated in reaction buffer containing 10 mM EDTA or 250 uM 1,10 phenanthroline for 30 min at room temperature.
After extensive dialysis against reac tion buffer at 4 C, AV-951 20 uM Leu AMC and AlCl3, CaCl2, FeCl2, CoCl2, MgCl2, MnCl2, or ZnCl2 were added to the reaction system, followed by a 15 min incubation at 37 C. Hydrolysis of the substrate was measured as described above. Controls consisted of enzymatic reac tions carried out either without EDTA or 1,10 phenan throline treatments or in the absence of cations.