STUDIES ON THE MUSCLE CATHEPSIN ACTIVITY OF MACKEREL BADANIA NAD AKTYWNOSCIJ\. KATEPSYN Mlf;SNIOWYCH MAK.RELi

Various biochemical processes inducing profoud changes in the basic muscle tissue components occur during fish storage, both fresh and frozen. The most significant changes are those associated with the protein system; they are influenced by proteolytic enzymes. The protein splitting enzymes are specific in their activity which depends on many factors, pH and temperature of the environment being some of them. In the present study the effects of pH and temperature on the catheptic activity during mackerel muscle autolysis were followed, the muscle cathepsin was isolated and purified and some of its properties examined.


INTRODUCTION
Various biochemical processes inducing profoud changes in the basic muscle tissue components occur during fish storage, both fresh and frozen.The most significant changes are those associated with the protein system; they are influenced by proteolytic enzymes.The protein -splitting enzymes are specific in their activity which depends on many factors, pH and temperature of the environment being some of them.
In the present study the effects of pH and temperature on the catheptic activity during mackerel muscle autolysis were followed, the muscle cathepsin was isolated and purified and some of its properties examined.

Materials
Colias mackerel,Scombercolias (Gmelin), caught in September on the North-West African shelf and appropriately preserved on board of a commercial trawler were the object of the investigations.Fishes from a 2-hour night haul were frozen.Immediately after catching the fishes were thoroughly washed and arranged on trays in a manner eliminating any possible mechanic damage on freezing.In several minutes after preparing the fishes, the freezing process was started in Danish freezers "Sabroe".The process lasted 170 mins, the internal temperature of the frozen block reaching ea -20 °C.Having completed their freezing and taken them out of trays the fish blocks were glazed, packed in cartons and placed in ship's holds at -25 °C.After 24 h the blocks were thoroughly glazed again and stored in the holds at -25 °C for 18 days.Having landed and delivered fish by a refrigerated van to the department, they were stored in a freezer at -25 °C.The investigations began immediately after the fishes had been brought to the laboratory, i.e., after 21 days of storage.

Methods
fish muscles autolysis was performed in a manner described in the previous paper (Fik, 1972), using the Sorensen method (Katzenellenbogen and Mochnacka, 1969) to determine the amino nitrogen increments and expressing them as percentages of the maximum increment.The enzyme extracts were prepared by the method reported in the previous paper (Fik, 1972)

DISCUSSION OF RESULTS
Initially the influence of pH and temperature on the mackerel muscle autolysis rate was determined, the results being presented in Figs. 1 and 2.
The autolysis proceeded with its highest intensity at pH range 4.0---4.4.After the time allowed for the autolysis had been increased from 4 to 6 hours, the course of the curve did not change and the same optimal value was maintained.resistant to denaturation changes occurring with temperature than the muscle cathepsins of horse mackerel (Fik, 1972).
Carrying on the studies, the mackerel muscle cathepsin was isolated and purified, and some of its properties examined.The enzyme was purified 345 fold with 10% yield (Table 1).The first acid treatment using 2N acetic acid at 35 °C resulted in 3.4-fold enrichment of the enzyme.The enzyme thus obtained could be stored overnight at 0 °C with no noticeable changes in its activity.Ammonium sulphate fractionation increased the enzyme activity 8-fold over the • non-purified extract.Lyophilized enzyme preparate of the 4 th fraction maintained its activity for several months at 0 °C.Acetone fractio nation yielded a 3.4-fold increase in the enzyme activity over its initial value.Pooling the highest activity fractions after the Sephadex G-200 filtration (Fig. 3), the main peak of catheptic activity was obtained.This fraction was freeze-stored for several weeks, no change in its enzymatic activity being detected.Obviously, durability of the lyophilized enzyme is unlimited.
The purified cathepsin showed a maximum activity against the denatured hemoglobin at pH 4.4 (Fig. 4).This result is close to the pH optimum for purified muscle cathepsins of cod (Siebert, 1962) and hake (Fik, 1973), differing slightly from the optimum for Miroslaw Fik purified cod spleen cathepsin (Siebert et al., 1963) and greatly deviating from the optimum for purified tuna muscle proteinase (Groning, �r, 1964).The pH optimum for the purified cathepsin is almost identical with that for the mackerel muscle autolysis (Fig. 1).The enzyme purified during the studies presented showed good hemoglobin -splitting properties but remained inactive against synthetic substrates specific for the mammalian cathepsins A, B and C. Similar results were obtained by Siebert and Schmitt (1965) and Groninger (1964) for the cod muscle cathepsin and tuna muscle proteinase, respectively.Thus the mackerel muscle cathepsin is different from the mammalian cathepsins which can be classified according to their synthetic substrates -splitting properties (Tallan et al., 1952).The classification of the cathepsin discussed could only be made basing on an appropriate standard protein decomposition.Siebert and Schmitt (1965) made an attempt to classify the cod cathepsins of muscles and spleen against insuline.However, the insuline B chain decomposition showed no different properties when compared to the cathepsins tested so far against this protein and did not allow an exact enzyme specificity classification to be made.Therefore, the intracellular occurrence and optimum activity within the acid range are still retained as classification characters of the fish proteinases catheptic nature.
The temperature .coefficients,Q 10 , of the purified mackerel muscle cathepsin were comprised within 1.6-2.3when measured at 10-40 °C.Regarding the thermal lability, usually the temperature the 30-minute action of which results in a 50% enzyme inactiva tion, is determined.According to Siebert and Schmitt (1965) this temperature is alike for various enzymes of fish and mammalian muscles and is comprised within 42-48 °C.CONCLUSIONS 1.The mackerel muscle cathepsin showed the optimal activity at pH 4-4.4 and ea 30 °C.
2. The purified mackerel muscle cathepsin did not split the synthetic substrates specific for the mammalian cathepsins A, Band C.
. Proteolytic activities fo raw extracts and partly purified preparates.were determined on urea-and NaQH -denatured hemoglobin by the Anson method (Bergmayer, 1965).The purified cathepsin was incubated with synthetic substra tes specific for cathepsins A, B and C of mammals in the conditions reported by Tallan et al. (1952).The cathepsin A activity against N-carbobenzoxy-1-glutamyl-L-tyrosine was determined by the ninhydrin method (Bailey, 1962), whereas cathepsin A and cathe psin B activities were determined with N-benzoyl-L-arginine amide and glycyl-L-tyrosine amide, respectively, by the Conway method (Johnston et al., 1950).The protein content was determined by the biuret method (Gomall et al., 1949), bovine serum crystalline albumin being used as a standard, and by the ultraviolet absorption method.The method described by Siebert and Schmitt (1965) was applied to the cathepsin purification.