Enzyme Derived Pig Livers. By Shoei ISEKI and Haruko YAMAMOTO Department of Legal Medicine, School of Medicine, Gunma University, Maebashi

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1 No. 4] Proc. Japan Acad., 44 (1968) A-Decomposing Human and Enzyme Derived Pig Livers from By Shoei ISEKI and Haruko YAMAMOTO Department of Legal Medicine, School of Medicine, Gunma University, Maebashi (Comm. by Tanemoto FURUHATA, M. J. A., April 12, 1968) It was observed by Weissmann and Friedericil~ that pig liver extracts contain a-n-acetylgalactosaminidase acting on phenyl a-nacetyl-d-galactosaminide at ph 4.3, and that the action is inhibited by N-acetyl-D-galactosamine. The extracts contained also a- and n-nacetylglucosaminidases, but the former was almost lost through partial purification. It was further observed by the same authors that N-acetylgalactosaminidase is also present in the spleen, kidney, liver, and other organs of rat, and that in this case the enzyme of the liver is found in lysosomes. Later, Weissmann et al.2~ obtained a fraction of pig liver homogenates by ammonium sulphate saturation and dialysis of the precipitate, and examined it for a- and ~3-acetylglucosaminidases, a- and 8-acetylmannosaminidases, a- and j3-glucosidases, a-monnosidase, p-galactosidase, j3-glucuronidase, a-l-f ucosidase and j3-d-xylosidase as well as a- and,9-acetylgalactosaminidases. The present report deals with the a-n-acetylgalactosaminidase as A-decomposing enzyme, which is found in human and pig liver extracts. Materials and methods. 1. Enzyme preparations. According to Weissmann and Friederici's method,' fresh pig and human livers were each homogenized, acidified with citric acid to ph 3.25, stored for one day at 4 C, adjusted to ph 4.35 by adding sodium citrate solution and centrifuged. The supernatants were saturated to 2O-6O% with ammonium sulphate, the precipitates, after dialysis, again saturated with ammonium sulphate to 4050 %, and the precipitates redialyzed and then freeze-dried. 2. Blood group substances. Water-soluble blood group substance was purified from human stomach linings by the pepsin digestion and phenol extraction method and blood group substance from red cells was prepared from the stromata by the phenol/saline procedure according to the Baranowski et al.'s method.3>'4> Formalin- This paper is dedicated to Dr. T. Furuhata, Member of Japan Academy, Professor emeritus of Tokyo University and Director of National Research Institute of Police Science, on the occasion of his 77th birthday.

2 270 S. ISEKI and H. YAMAMOTO [Vol. 44, treated red cells were prepared by combined use of the Flick's,5~ Moskowitz and Carb's,6> and McKenna's method.7> Two volumes of a 20% formalin-containing saline buffer (mixture of a 0.2 M, ph 7.3 phosphate buffer and saline at 1: 5) and one volume of washed red cells were mixed and allowed to stand for one day at 25 C. After washing with distilled water, the cell suspension in a 5 % NaHSO3 solution was placed into a seamless cellulose tubing to be dialyzed against water, and the cells were again washed with distilled water. 3. Antiserum. Anti-deacetylase treated A substance immune serum was prepared by injecting a rabbit with A substance, treated with deacetylase8) from Cl. tertium A. This is the antibody against an antigen, which contains galactosamine as the antigenic determinant. 4. Application o f enzyme. Water-soluble blood group substance, formalin-treated red cells, and blood group substance from red cells were incubated with a 1 % enzyme solution in citrate-phosphate buffer of ph 4.3, and stored for 4 to 5 days at 37 C. Untreated red cells were incubated with a 2 % enzyme solution in a mixture of citrate-phosphate buffer and saline at 1: 5, and allowed to stand for 10 hours at 37 C. In examination for chemical effects of the enzyme, HC1 was used instead of the buffer to adjust ph to 4.3. In examination for oligosaccharides, the equal volumes of a 1 % sugar solution and 1 % enzyme solution were mixed, and after incubation at 37 C for 4 days, the mixture was subjected to paper chromatography. 5. Chemical analysis. Sugar examination was performed by the usual method. Determination of galactosamine in the presence of glucosamine was performed approximately by the Slein's method.9> After incubation with the enzyme solution, the material was dialyzed, and both the dialyzable and non-dialyzable portions were hydrolyzed with 2 N HC1 for 4 hours. After removal of HC1, each was deviled into 2 equal parts. One part, 0.4 ml in amount, was incubated with 0.3 ml of the hexokinase solution, and allowed to stand for 1 hour at 30 C, for 2 hours at 20 C, and lastly in the ice box overnight, and 0.3 ml of the hexokinase solution was further added, to be incubated at 30 C for 1 hour. The 0.2 ml of a 0.15 M ZnSO4 and 0.2 ml of 0.15 M Ba (OH) 2 solution were added to the mixture, and after the centrifugation, the supernatant was quantitatively examined for galactosamine by the Blix's modification of the Elson and Morgan's method. The other part was subjected to the same procedure without adding the hexokinase, and from the difference in hexosamine amount between the two parts, the ratio of galactosamine to glucosamine was calculated. Results. 1. Serological effects. As shown in Table I, the A

3 No. 4] A-Decomposing Enzyme Derived from Livers 271 activity was diminished while the H activity was enhanced in all of the water-soluble A substance, A substance from red cells, intact A red cells and formalin-treated A red cells after application of the enzyme preparation from the human or pig liver. After incubation with the enzyme, these materials did not inhibit the agglutination of deacetylase-treated red cells by anti-deacetylase treated A substance rabbit serum, nor was restored the A activity of these enzyme-treated materials by N-acetylation. Table I. Serological effects of from pig and human A-decomposing livers enzymes The agglutinability of enzyme-treated A red cells with anti-a serum was reduced, while that with anti-h serum was enhanced. The f ormalin-treated A red cells usually lost the agglutinability with anti- A serum, though retaining the capacity to absorb it, so that it was difficult to know change in the agglutinability elicited by the enzyme action. When the enzyme was applied to B, H and deacetylase-treated A substances, scarcely any change was observed in their inhibitory effects on agglutination of red cells by respective antibodies. A-decomposing activity was demonstrated either by the pig or by human liver extracts irrespective of blood group of the original material. These A-decomposing enzymes are assumed to exert no action in the human or animal body, since ph, required for this action, ranges 3.5' Characteristics o f the enzyme. Investigations with citratephosphate buffer (ph 3.ON6.5) and phosphate buffer (ph 6.0'8.0) disclosed that the optimal ph for the A-decomposing enzyme from liver ranged 4.0'4.5, and that no enzyme action was observed below

4 272 S. ISEKI and H. YAMAMOTO [Vol. 44, 3.5 and above 5.0 of ph. The optimal temperature was 37 C and it was ineffective below 20 C. The enzyme action was inhibited strongly by N-acetylgalactosamine, and rather less by galactosamine and glucosamine. It was also inhibited weakly by such metal ions as 10.3 M Hg++ and Pb Enzymic activity o f the enzyme preparation. The enzyme preparations from the human and pig livers decomposed such oligosaccharides as maltose, lactose, melibiose, 0-a- and 0-~3-D-mannosyl- (1-*4)-mannose, and lacto-n-triose II(O-Q-D-N-acetylglucosaminyl- (1-*3)-O-Q-D-galactosyl-(1-*4)-D-glucose], and such glycosides as p- nitrophenyl R-N-acetyl-D-glucosaminide, O-nitrophenyl j3-d-galactoside, phenyl a-maltoside, phenyl R-glucoside, phenyl a-d-galactoside, phenyl a- and phenyl p-n-acetyl-d-galactosaminides and also human H-active oligosaccharide. Therefore the enzyme preparations are considered to contain a- and j3-glucosidases, a- and j3-galactosidases, a- and p-mannosidases, a- and p-n-acetylgalactosaminidases, p-nacetylglucosaminidase and a-l-fucosidase. But the enzyme preparation from human liver showed very weak p-glucosidase activity. 4. Chemical effects. N-acetylgalactosamine, N-acetylglucosamine, galactose, and a small amount of f ucose were demonstrated in dialysate of A substance which had lost the A activity as the result of incubation with the enzyme preparation from the human or pig liver. The dialysate after incubation with the pig enzyme contained about 14.4% of the original quantity of hexosamine in A substance, and galactosamine accounted for about 3/5 of the amount of hexosamine in the dialysate or about 20% of the original galactosamine in A substance. After incubation with the human liver enzyme, about 9% of the original hexosamine was recovered in the dialysate, and galactosamine represented about 3/4 of the amount of hexosamine in the dialysate or about 16% of the original galactosamine in A substance. These enzymes liberated, from H substance, N-acetylglucosamine, galactose and a small amount of f ucose, but not any N-acetylgalactosamine. It is therefore considered that the N-acetylgalactosamine which was liberated from A substance by the enzyme action must be the determinant sugar of the A-specificity (Table II). When the formalin-treated A red cells were incubated with the enzyme preparation from the human or pig liver, N-acetylgalactosamine, which amounted in hexosamine value 270 µg per 50 ml of cells, and galactose were liberated. However, these enzyme preparations liberated from f ormalin-treated 0 red cells smaller amount of Q N- acetylgalactosamine than from A red cells and galactose. It is therefore considered that p-n-acetylgalactosamine, which is unrelated with the A-specificity, may be liberated from A red cells together with that

5 No. 4] A-Decomposing Enzyme Derived from Livers 273 Table II, Chemical effects of the enzyme preparation from liver which is the determinant of the A-specificity. When these enzymes were applied to O-a-N-acetyl-D-galactosaminyl-(1- >3)-[O-a-L-fucosyl- (1--e2)-]-0-t3-D-galactosyl-(1---4)-N-acetyl-D-glucosamine, which has the A activity, and to O-a-L-f ucosyl-(1--2)-o-p-d-galactosyl-(1- >4)-Nacetyl-D-glucosamine, which has the H activity, the A or H activity was lost, both liberating monosaccharides. It is therefore considered that these enzymes contain also a-l-fucosidase. Discussion. Enzyme preparations from pig and human livers contain a-n-acetylgalactosaminidase, and when applied to A substance or A red cells, they reduce or destroy their A activity while enhancing their H activity. Their actions on A substance are similar to that of a-n-acetylgalactosaminidase derived from Trichomonas f oetus10) and Helix pomatia,11 but different from those of deacetylase8> and galactosaminidase from Cl. tertium and of acylasel2~ from the pig kidney. The A-decomposing action of the enzymes from pig and human livers are demonstrated without any regard to the blood groups of the original materials. Since the ph for the action of these enzymes ranges , they are not considered to exert, in the living bodies, their specific actions on A substance which is naturally present in livers of blood group A. These enzyme preparations contain also a-galactosidase and a small amount of a-l-fucosidase, but they hardly destroy the specific activities of intact B and H substances. When, however, applied' to H-active oligosaccharide, they destroy its H activity by splitting it into simple sugars.

6 274 S. ISEKI and H. YAMAMOTO [Vol. 44, Summary. Extracts from pig and human livers of all blood groups, contain a-n-acetylgalactosaminidase as A-decomposing enzyme, which destroys A activity of A substance or A red cells with enhancement of H activity. They also contain a-galactosidase and a - fucosidase, which hardly destroy the blood group specific activities of intact B and H substances, but the a-f ucosidase destroys H activity of oligosaccharide. References 1) Weissmann, B., and Friederici, D.: Occurrence of a mammalian a-n-acetyl- D-galactosaminidase. Biochim. Biophys. Acta, 117(2), (1966). 2) Weissmann, B., Rowin, G., Marshall, J., and Friederici, D.: Mammalian a-acetylglucosaminidase. Enzymic properties, tissue distribution, and intracellular localization. Biochemistry, 6(1), (1967). 3) Baranowski, T., Lisowska, E., i Romanowska, E.: Badania nad antigenami grupowymi M i N. Doniesienie. I. Proby wydzielenia antygenow grupowych M i N z krwinek czerwonych. Arch. Immunol. i Terapii Dow., 4(1), (1956). 4) Baranowski, T., Lisowska, E., Morawiecki, A., Romanowska, E., and Strozecka, K.: Studies on blood group antigens M and N. III. Chemical composition of purified antigens. Arch. Immunol. i Terapii Dow., 7(1), (1959). 5) Flick, J. A.: Use of f ormalin-treated red cells for the study of influenza A virus hemagglutinating activity. Proc. Soc. Exp. Biol. Med., 68(3), (1948). 6) Moskowitz, M., and Carb, S.: Surface alteration and the agglutinability of red cells. Nature, 180(4594), (1957). 7) McKenna, J. M.: A stable preparation of antigen-sensitized erythrocytes. Proc. Soc. Exp. Biol. Med., 95(3), (1957). 8) Marcus, D. M., Kabat, E. A., and Schiffman, G.: Immunochemical studies on blood groups. XXXI. Destruction of blood group A activity by an enzyme from Clostridium tertium which deacetylates N-acetylgalactosamine in intact blood group substances. Biochemistry, 3(3), (1964). 9) Slein, M. W.: A rapid method for distinguishing D-glucosamine from galactosamine in biological preparations. Proc. Sac. Exp. Biol. Med., 80(4), (1952). 10) Harrap, G. J., and Watkins, W. M.: Characterization of the enzyme from Trichomonas foetus that destroys the serological specificity of blood-group A substance. Biochem. J., 93(3), 9p-10p (1964). 11) Tuppy, H., and Staudenbauer, W. L.: The action on soluble blood group A substances of an a-n-acetylgalactosaminidase from Helix pomatia. Biochemistry, 5(5), (1966). 12) Chattoraj, A.: Effect of acylase on the agglutinability of human erythrocytes. Nature, 212(5062), 628 (1966).