Blood Group Activity of Human Sucrase from Intestinal Metaplasia”

Sucrases were purified from human small intestine and from areas of intestinal metaplasia of the stomach mucosa surrounding stomach cancers. The kinetic constants and pH activity profiles of enzyme preparations from the two sources were similar. No blood group of sucrase was in preparations from cases of intestinal metaplasia, preparations other showed activity like that of the intestine. These results indi-cate that sucrase from areas of intestinal metaplasia has similar enzymatic properties to those of enzyme from the small intestine, but that the antigenic sugar moiety of the enzyme associated with blood group activity varies. abnormal differentiation the appearance Enzymological

Blood Group Activity of Human Sucrase from Intestinal Metaplasia" (Received for publication, October 26, 1976) MARIKO KURISU, NORIKO NUMANYU, TAKASHI KAWACHI, AND TAKASHI SUGIMURA From the Biochemistry Division, National Cancer Center Research Institute, Tsukiji, Chuo-Ku, Tokyo, Japan Sucrases were purified from human small intestine and from areas of intestinal metaplasia of the stomach mucosa surrounding stomach cancers. The kinetic constants and pH activity profiles of enzyme preparations from the two sources were similar. No blood group activity of sucrase was detectable in preparations from three cases of intestinal metaplasia, but preparations from two other cases showed activity like that of the small intestine. These results indicate that sucrase from areas of intestinal metaplasia has similar enzymatic properties to those of enzyme from the small intestine, but that the antigenic sugar moiety of the enzyme associated with blood group activity varies. Gel Electrophoresis -Polyacrylamide gel electrophoresis was carried out as described by Ornstein (17) and Davis (18) using 5% acrylamide as separating gel. The buffers for the separating gel, stacking gel, and reservoir were of pH 8.9, 6.7, and 8.3, respectively.
After electrophoresis the gels were stained for protein with Coomassie brilliant blue or for sugar by the periodic acid-Schiff method (19,20). Some gels were also cut into transverse sections and eluted overnight in phosphate-buffered saline to measure sucrase activity and blood group activity. Blood Group Activity -ABO blood group activity was measured by the hemagglutination inhibition test using a microtitration system (21 Further chromatography of the preparations on DEAE-cellulose resulted in increase in the specific activity to gel electrophoresis the purified enzymes from the area of metaplasia and the intestine both gave a single band of glycoprotein which coincided in position with sucrase activity in the lower part of gel but on the upper part of gel showed very minor contaminated glycoprotein (Figs. 1 and 2). Gel filtration on Bio-Gel P-300 was effective for purification of the enzyme, especially that from the area of intestinal metaplasia, and was used instead of the affinity chromatography on Sephadex G-200 used previously (ll), because human sucrase showed low affinity for the latter at 4".
Sucrase from the area of intestinal metaplasia was eluted in the same position as that from the small intestine on both Bio-Gel P-300 and DEAE-cellulose chromatography.

Kinetics and pH Activity
Profiles-With sucrose as substrate the Michaelis constants for the purified enzymes from small intestine (21 mM, M.K.) and an area of intestinal metaplasia (18 mM, T.S.) were similar. Their V,,, values were also similar, being 25.6 units/mg of protein (M.K.) and 23.2 units/ mg of protein (T.S.), respectively. The pH activity profiles of the two enzymes were also essentially the same (Fig. 3).

Blood
Group Activity of Sucrase -Blood group activity was detected in two preparations of sucrase from the small intestine as reported previously by Kelly and Alpers (5). Namely, as shown in Table III, the enzyme from a patient of blood group type A (C.S.) showed hemagglutination inhibition of blood group type A only, that from a patient of type B (F.A.) showed only type B activity. The enzyme from a patient of type 0 (M.K.), as expected, showed neither type A nor type B activity.
Table III also shows the blood group activities of five enzyme preparations from areas of intestinal metaplasia. The preparations from two patients of blood group type A (T.S. and S.K.) showed no type A activity (and no type B activity). One of the three preparations from patients of type B showed neither type B nor type A activity (I.Y.), whereas the other two preparations showed type B activity but not type A activity (F.A. and   Fig. 1. Gels were cut into 2-mm sections and the sucrase activity and blood group activity in each slice were measured as described under "Experimental Procedures." The patients from whom the sucrases were obtained and their blood group types were as follows; a, F.A., type B; b, S. K., and one preparation from the small intestine (F.A.) were subjected to electrophoresis on polyacrylamide gel. Then the sucrase activity and blood group activity in each gel were examined. Fig. 4 shows the distributions of the two activities in the gels. The preparation from the intestine of the patient of blood group type B showed blood group type B activity coinciding with sucrase activity (a). Two preparations of types A and B, respectively, from areas of intestinal metaplasia showed no blood group activity coinciding with sucrase activity (b and d), while the other two preparations from cases of type B showed blood group type B activity (c and e). Fig. 4 also shows that blood group activity was found at the top of the polyacrylamide gel. This might be due to a blood group substance contaminated in the sucrase fraction during its purification. The blood group activities of the sucrase preparations after electrophoresis on polyacrylamide gel are summarized in Table III. All patients of blood group type A or B were found to secrete material with blood group activity in their saliva. Two of the five enzyme preparations from areas of intestinal metaplasia had similar level of blood group activity to those of preparations from the small intestine, namely the end point of hemagglutination inhibition were both 2.1 milliunits of suerase/ml for the former, comparable to 1.4 and 2.8 milliunitsl ml of the latter.

DISCUSSION
In this work, three of five preparations of sucrase from areas of intestinal metaplasia were found to have no blood group activity, but they had similar enzymatic properties to those of sucrase from the small intestine.
The patients from whom the preparations were obtained were all found to be secretors, as shown in Table III, so this phenomenon was not due to the fact that the cases were nonsecretors.
Blood group activity was always found at the top of the polyacrylamide gel when the sucrase was electrophoresed (Fig. 4)