Comp. Biochem. PhysioL Vol. 10211, No. 2, pp. 247-253, 1992 0305-0491/92$5.00 + 0.00 Printedin Great Britain © 1992PergamonPress Ltd DISTRIBUTION OF 5'-NUCLEOTIDASE IN MUSCLE OF SOME MARINE FISHES DJAGALW. MARSENO,KANJI HORI and KEISUKE MIYAZAWA Department of Food Science, Faculty of Applied Biological Science, Hiroshima University, Higashi-Hiroshima 724, Japan (Tel: 0824-22-7111) (Received 29 October 1991) Abstraet--A preliminary examination for the purification and characterization of 5'-nucleotidase of fish muscle was carried out and the following results were obtained: 1. The activities of 5'-nucleotidase in the muscles of marine vertebrates and invertebrates (total 11 species) were determined. The highest activity of 5'-nucleotidase was found in Blackrock fish Sebastes inermis, which was then used as a material for estimation of subcellular distribution and solubilization of the enzyme. 2. The 5'-nucleotidase of ordinary muscle of the fish Sebastes inermis was found in nuclear, microsomal and cytosolic fractions. About half of the total activity was found in the nuclear fraction, whereas the highest specific activity was observed in the mierosomal fraction. 3. Complete solubilization of the enzyme was attained by using a high concentration of detergent such as Triton X-100, CHAPS, octylglucoside,octylthioglucoside and sodium deoxycholate, suggestingthat the enzyme was tightly bound to the membrane. 4. Based on the results of solubility and stability tests, Triton X-100 seemed suitable for solubilizing 5'-nucleotidase from the membrane. 5. Microsomal 5'-nucleotidase was an Mg2+-activated enzyme, and no inactivation was observed up to 50 mM of Mg2+. INTRODUC~ON The post-mortem dephosphorylation of nucleotides by autolytic processes in marine animal skeletal muscle has been studied for many years, and the change of metabolite composition has been accepted and used as an index of fish freshness (Saito et al., 1959; Jones et al., 1964; Fatima et al., 1981; Ehira and Uchiyama, 1987). Dephosphorylation of IMP to inosine in fish muscle during storage by the activity of enzyme 5'-nucleotidase was found to be highly correlated to the lowering freshness of fishes as indicated by an increase of K-value (Tomioka, 1984a). On the other hand, IMP was found to be an "umami" substance of dried bonito "katsuoboshi" by Kodama (1913), and later, Kuninaka (1960) demonstrated that 5'-nucleotides such as IMP, in combination with glutamic acid, play an important role as a taste enhancer. Thus the role of 5'-nucleoti- dase in fish muscle, especially from the viewpoint of quality, palatability and freshness, is important. The purification of 5'-nucleotidase from some fish skeletal muscle such as cod (Yamamoto et al., 1986), Pacific cod (Tarr et al., 1969), carp (Tomioka and Endo, 1984b,c) and bonito (Hirota, 1973a,b) has been reported. These enzymes have been characterized as a membrane-bound glycoprotein, Abbreviations--IMP: Inosine 5'-monophosphate; AMP: Adenosine 5'-monophosphate; CHAPS: 3-[(3-¢holamido- propyl)-dimethylammonio]- 1-propane-sulfonate; octyl- glucoside: n-oetyl-fl-~glucoside; oetylthioglueoside: n- oetyl-fl-D-thioglucoside;EGTA: ethyleneglycol-O-O'-bis (2-aminoethyl)-N,N,N',N'-tetraaeetic acid. so far. However, little information on the intra- cellular distribution of enzyme 5'-nucleotidase, and its solubilization with detergents in fish skeletal muscle, is available. Against this background, we attempted to clarify the properties of 5'-nucleotidase and its role in re- lation to the quality of fish muscle, in order to develop better storage of fish. As a preliminary experiment for the purification and characterization of 5'-nucleotidase of fish muscle, the following examinations were carded out. The distribution of 5'-nucleotidase among some marine vertebrates and invertebrates was examined. The intracellular distribution of 5'-nucleotidase and its solubilization with detergents in skeletal muscle of fish were also investigated using Sebastes inermis as a model, because the activity of 5'-nucleotidase of this fish muscle is higher than those of other specimens tested. MATERIALS AND METHODS Materials AMP was obtained from the Oriental Yeast Co. (Osaka, Japan), IMP was purchased from Sigma (St Louis, MO). Detergent Triton X-100, octylthioglucoside,octylglucoside, CHAPS, and sodium deoxycholate were purchased from Wako Pure Chemicals (Osaka, Japan), and zwittergent 3-14 was obtained from Calbiochem Co. (San Diego, CA). All other chemicalswere of analytical grade. The distilled water used throughout the experiments was filtered by Milli-Q Laboratories (Millipore Bedford, MA). Specimens The specimens of fish, mollusks and crustaceans were purchased from a local market and used for experiments as 247