Leucine Aminopeptidase from Red Sea Bream (Pagrus major) Skeletal Muscle: Purification, Characterization, Cellular Location, and Tissue Distribution

Abstract

A leucine aminopeptidase was purified for the first time from marine fish red sea bream ( Pagrus major) skeletal muscle to homogeneity with 4850-fold and a yield of 7.4%. The purification procedure consisted of ammonium sulfate fractionation and chromatographies including DEAE-Sephacel, Sephacryl S-200, hydroxyapatite, and phenyl-Sepharose. The enzyme was approximately 96 kDa as estimated by SDS-PAGE and gel filtration and preferentially hydrolyzed substrate Leu-MCA. The enzymatic activity was optimal at 45 degrees C and pH 7.5. The K m and k cat values of the enzyme for Leu-MCA were 1.55 microM and 26.4 S (-1) at 37 degrees C, respectively. Activation energy ( E a) of the enzyme was 59.6 kJ M (-1). The enzyme was specifically inhibited by metal-chelating agents, and Zn (2+) and (or) Mn (2+) seemed to be its metal cofactor(s). In addition, bestatin strongly inhibited its activity, and K i was 1.44 microM. Using a highly specific polyclonal antibody, the location of enzyme was demonstrated intracellularly and distributed in different tissues.

Facile electrochemical detection of botulinum neurotoxin type E using a two-step proteolytic cleavage

“…This clearly indicated successful cleavage of the N-terminal isoleucine residue by LAP. Since it is known that Zn 2 þ decreases LAP activity but increases BoNT/E activity (Tang et al, 2007;Wu et al, 2008), cleavage was also investigated in a solution containing ZnCl 2 . LAP did not cleave isoleucine residue at all or when it did, cleavage was very slow in the presence of Zn 2 þ (Fig.…”

Identification of an aminopeptidase from the skeletal muscle of grass carp (Ctenopharyngodon idellus)

“…The whole purification procedure resulted in a 1,652-fold increase in specific activity with a 5.4% recovery. The specific activity increase was relative higher than that of leucine aminopeptidase from common carp (1160) while lower than that of red sea bream (4850.5) (Wu et al 2008). After ammonium sulfate fractionation, a single sharp enzyme activity peak was obtained when eluted from DEAESephacel anion exchange column by NaCl gradient (Fig.…”

“…To understand the functions of aminopeptidase in fish muscle, it is necessary to purify and characterize such enzymes from fish. More recently, we described the purification of a leucine aminopeptidase from the skeletal muscle of common carp (Cyprinus carpio) ) and red sea bream (Pagrus major) (Wu et al 2008). The objective of the present study was to purify and characterize an aminopeptidase from the skeletal muscle of freshwater fish grass carp (Ctenopharyngodon idellus), the possible physiological function and potential application in fish processing of this enzyme were also discussed.…”

Purification and characterisation of an alanine aminopeptidase from bovine skeletal muscle

“…The tissues from which aminopeptidase were isolated originated from a number of animals including human (McClennan & Garner, 1980), pig (Flores, Aristoy, & Toldrá, 1998), rabbit (Matsuishi, Saito, Okitani, & Kato, 2003), chicken (Hui et al, 1993;Jamadar, Jamadar, Dandekar, & Harikumar 2006;Skrtic & Vitale, 1994), carp (Hara, Sakai, & Ishihara, 1988;Liu et al, 2008), red sea bream (Wu, Cao, Chen, Liu, & Su, 2008), and mullet (Chiou, Matsui, & Konosu, 1988). Many proteins are present in muscle, including cathepsins, calpains, dipeptidyl peptidases and aminopeptidases.…”

“…Aminopeptidases are involved in the production of free amino acids in meat and meat products and this contributes to an improvement of meat flavour (Flores, Marina, & Toldrá , 2000). Different kinds of muscle aminopeptidases comprising human pyroglutamyl and leucine aminopeptidases, bovine aminopeptidase D and H, porcine methionyl aminopeptidases and chicken aminopeptidase H (Flores et al, 2000;Mantle, Lauart, & Gibson, 1991;Nishimura, Rhyu, Kato, & Arai, 1994;Rhyu, Nishimura, Kato, Okitani, & Kato, 1992), red sea bream (Wu et al, 2008) and carp leucine aminopeptidases have been reported. The aim of the present investigation was to isolate an aminopeptidase from bovine skeletal muscle and to compare its characteristics with other aminopeptidases reported in the literature.…”