Purification And Characterization Of Leucine Aminopeptidases From The Skeletal Muscle Of Freshwater Fish

Leucine aminopeptidases (LAPs), a class of cellular exoproteases preferably catalyze the hydrolysis of leucine residue from the amino-termini of proteins or peptides,play important roles physiologically and in the formation of flavor. However, till now no report on LAP from fish muscle has been documented. In the present study, LAPs from the skeletal muscle of three typical freshwater fishes, namely common carp, grass carp and silver carp, were purified to high homogeneity and their biochemical characteristics were investigated. The purification and characterization of LAPs in the present study represents a primary step in answering specific questions about the physiological function of such enzymes in vivo.Using the same methods, three LAPs could be purified from common carp, grass carp and silver carp, and they were named cmLAP, gmLAP and smLAP, respectively. The purification procedure consisted of ammonium sulfate fractionation and sequential chromatographic steps, including DEAE-Sephacel, Sephacryl S-200, hydroxyapatite, Phenyl-Sepharose 6-Fast Flow, and Econo-Pac Macro-Prep High Q column (only used for cmLAP purification).The molecular masses of the three enzymes were all estimated to be approximately 105 kDa by SDS-PAGE and gel filtration chromatography on Sephacryl S-200, and they were all monomers. The enzyme activities were optimal at 35-40℃and pH 7.0-7.5 and of poor thermal stability. Metal-chelating agents EDTA, EGTA and 1,10-phenanthroline specifically inhibited enzyme activities, while inhibitors to other proteinases did not show much effect, indicating they were metalloproteinases. Furthermore, bestatin, a specific aminopeptidase inhibitor strongly inhibited their activities. Divalent cation ions such as Mn2+ and Mg2+ enhanced the enzyme activities slightly, while Co2+, Cu2+ and Zn2+ inhibited the activities to different extents. In addition, sulfhydryl reagent was necessary to maintain enzyme activities. All of the three enzymes preferentially hydrolyzed substrate Leu-MCA followed by other substrates, while they did not reveal any endoproteinase activity toward N-terminus blocked substrates and did not exhibit any proteolytic activity toward protein substrates such as myofibrillar proteins and bovine serum albumin. The apparent Km and Vmax values of cmLAP were 4.6μmol/L and 9.62μmol·min-1·mg-1, respectively for substrate Leu-MCA. In conclusion,LAPs are quite possibly ubiquitous in freshwater fish muscle and have similar biochemical functions and characteristics.