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Alteration of Hexosaminidaseisozymesin ICANCER RESEARCH 39, i 829-i 834, May 1979] 0008-5472/79/0039-0000502.00 Alterationof HexosaminidaseIsozymesin HumanRenalCarcinoma' Toshikazu Okochi,2 Hiromasa Seike, Kazuya Higashino, Toshikazu Hada, Shinichiro Watanabe, Yuichi Yamamura, Fumlo Ito, Minoru Matsuda, Masao Osafune, Toshihiko Kotake, and Takao Sonoda TheThirdDepartmentofInternalMedicine(T.0., H. S., K. H., T.H., S. W.,V. V.)andDepartmentofUrology(M.M., M. 0., T.K.. T.S.],OsakaUniversityMedical School, Fukushima-ku, Osaka 553, and Health Administration Center (T. 0. , F. I.], Osaka University, Toyonaka, Osaka 560. Japan ABSTRACT been well studied to our knowledge. Consequently, the present study was undertaken in order to compare the isozyme patterns The activity and isozyme patterns of hexosaminidase in of hexosaminidase and the enzymatic properties of human human renal carcinoma were studied in comparison with those renal carcinoma with those of normal kidney. of normal kidney. Hexosaminidase in extracts from normal kidney and renal carcinoma tissue could be separated into two MATERIALSAND METHODS major forms [hexosaminidase A (Hex A) and hexosaminidase B (Hex B)] by Ceilogel electrophoresis or by diethylaminoethyl Materials. The renal carcinoma tissues obtained at operation cellulose column chromatography. All of i 0 renal carcinoma from the Department of Urology, Osaka University Hospital, tissues showed a low activity ratio of Hex A to Hex B, as were frozen immediately after removal. Normal human kidneys compared with the ratio in normal kidney; the ratio in renal were postmortem specimens obtained within 8 hr after death carcinoma tissue was between O.6i and 2.2i (mean, 1.30), at the Department of Forensic Pathology, Osaka University while that in normal kidney was between 2.50 and 4.52 (mean, Medical School. Placentas at the time of delivery were used. 3.46). Hexosaminidase activity and the ratio of Hex A to Hex B Cultured renal carcinoma cells were established from a fresh in renal carcinoma tissue were independent of the cell type and renal carcinoma tissue as described by Matsuda et a!. (i 4). the differentiation grade of carcinoma tissue. Hex A and Hex B Cultured cells were morphoiogically similar to cells of the of renal carcinoma tissue differed from each other in physico original carcinoma tissue. The cells cultured serially for about chemical properties such as pH dependence of enzyme activ 240 days were used for the studies presented here. Tissues ity, thermostability, and Km'Sfor two synthetic substrates, but and cells were stored frozen at —70°untilused. The cancerous each isozyme maintained its same physicochemicai properties tissue was carefully separated from normal renal tissue and whether from normal or from carcinoma tissue. The isozyme connective tissue. The tissues were minced and homogenized patterns of cultured renal carcinoma cells and placenta were in 5 volumes of ice-cold 25 m@sodium phosphate buffer (pH similar to those of the carcinoma tissue. The results presented 6.0) containing 0.i % Triton X-i 00, using a Potter-Eivehjem here indicate that hexosaminidase isozymes in renal carcinoma homogenizer. After centrifugation at i 5,000 x g for 40 mm at tissue express at least oncoplacental patterns. 40, the supernatants were used as the source of enzyme. Chemicals. p-Nitrophenyl-N-acetyl-$-D-glucosaminide,p-ni INTRODUCTION trophenyl-N-acetyl-/3-D-galactosaminide, and 4-methyiumbelii feryi-N-acetyi-$-D-glucosaminide were purchased from Sigma Human hexosaminidase (2-acetamido-2-deoxy-f3-D-gluco Chemical Co., St. Louis, Mo. Ceilogel was purchased from side acetamidodeoxy-giucohydrolase, EC 3.2. i .30) was Chemetron Co., Milan, Italy, and DEAE-celiuiose was from shown by Robinson and Stirling (20) to exist in 2 major forms, Brown Co., Berlin, N. H. Other chemicals were obtained from acidic (Hex A3) and basic (Hex B). Hexosaminidase isozymes Wako Pure Chemical industries, Osaka, Japan. have been studied in normal human brain, placenta, and kidney EnzymeAssay.Hexosaminidaseactivitywasmeasuredus tissues. Their possible clinical significance was first suggested ing the method of Brattain et a!. (2) with a slight modification. by the work of Okada and O'Brien (i 5). They were able to The substrate solution was prepared by dissolving p-nitro show that Hex A activity was deficient in the classical form of phenyl-N-acetyl-f1-D-glucosaminide in 0.i M citric acid-O.2 M Tay-Sachs disease. Abnormalities in hexosaminidase isozymes sodium phosphate buffer, pH 4.5. The incubation mixture, in a have also been demonstrated in tumor tissues. Weber et a!. total volume of i .0 ml, contained 0.9 ml of substrate solution (25) reported that hexosaminidase isozyme patterns of fast and 0.1 ml of enzyme solution of an appropriate activity. The growing hepatoma in rat were similar to those of brain or fetal final substrate concentration was 4 mM.After incubation at 37° liver. More recently, Brattain et a!. (2) reported that hexosamin for 30 mm, the reaction was terminated by the addition of 2.0 idase isozymes in normal human colon mucosa showed a ml of 0.4 M glycine-NaOH buffer, pH i 0.5. The extinctions of preponderance of Hex A over Hex B, whereas colon carcinoma the developed yellow color were determined at 4i 0 nm. Care tissue contained a higher proportion of Hex B. However, hex was taken that the reaction obeyed zero-order kinetics under osaminidase isozymes of human renal carcinoma have not the assay conditions. One unit of enzyme activity was defined as the amount of enzyme which releases i @zgofp-nitrophenol ,AcompendiumofthisworkwaspresentedattheSixthMeeting,Internationalfrom the substrate per mm at 37°. Research Group for Carcino-Embryonic Proteins, September 17 to 2i , i 978, Protein Assay. Proteinconcentrationwas determinedby the Marburg, West Germany (16). This work was supported in part by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture, Japan. method of Lowry et a!. (12) using bovine serum albumin as a 2 To whom requests for reprints should be addressed. standard. 3 The abbreviations used are: Hex A, hexosaminidase A; Hex B, hexosamini dase B. CellogelElectrophoresis.Electrophoresiswasperformed Received September 7, 1978; accepted February 9, 1979. on Cellogel (5 x i 8 cm) according to the procedure of Fluharty MAY 1979 i 829 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1979 American Association for Cancer Research. T. Okochi et a!. et a!. (5). Electrophoresis was run at a constant current of 1.2 20 ma/cm for 2.5 hr at 4°using 40 mM sodium phosphate (pH A 6.5) as an electrode buffer which usually resulted in a voltage of 220 V. After completion of electrophoresis, the Ceilogel was ‘5 sandwiched between Whatman No. 3MM paper strips which had been soaked with the staining solution, supported on a glass plate, and wrapped in a plastic film. The staining solution E @0 contained 0.5 mM 4-methylumbeliiferyl-N-acetyl-$-D-giucosa ‘I) minide in O.i M citric acid-0.2 M phosphate buffer, pH 4.5. C :@ O2M NaCl After i 0 to 30 mm incubation at 37°,strips of Whatman in buffer paper soaked with 0.4 PAglycine-NaOH buffer (pH 10.5) were >. substituted for strips of the substrate paper. The alkaline so > iution stops the enzyme reaction and produces strongly flu 0 0-0 orescent 4-methylumbeiliferone. Fluorescent bands were vis w -@10 C,) 20 m ualized with a Toshiba Black Lamp (FL-2OBLB), and photo ci z graphs were taken immediately, before the fluorescence dif z B B fused. Co Cl) ‘5 DEAE-cellulose Column Chromatography. One ml of the 0 3 supernatant from tissues or cells was loaded onto a DEAE w I cellulose column (6.5 x 2 cm) previously equilibrated with 25 C0 l0 mM sodium phosphate buffer, pH 6.0. Two major peaks of hexosaminidase activity were obtained by stepwise elution with the equilibrating buffer and 0.2 M NaCi in the same buffer. 5 0.2 M NaCI in buffer RESULTS 0 5 C0 20 2' Eiectrophoretlc Patterns of Hexosaminidase isozymes on Cellogel. Hexosaminidase isozymes from normal kidney, renal FRACTION NUMBER Chart i . Representative elution profiles of hexosaminidase from a DEAE carcinoma, placenta, and cultured renal carcinoma cells were cellulose column. A, normal adult kidney (Table i , Normal Kidney 4); B, renal separated using Ceilogel electrophoresis. Two major isozymes, carcinoma tissue (Table i , Renal Carcinoma 1). Column size, 6.5 x 2 cm; fraction a fast-moving band (Hex A) and a slow-moving band (Hex B), volume, 5 ml. 0, enzyme activity; 5, protein. were obtained by this method. Isozyme patterns of normal kidney showed an apparent preponderance of Hex A activity x-i 00, and the clarified supernatants were tested for hexosa as compared with Hex B. However, those of renal carcinoma minidase activity. Approximately 90% of the enzyme activity tissue showed a lesser preponderance of Hex A, or a prepon present in tissue homogenates was found in the clarified su derance of Hex B over Hex A. Similarly, a relative increase of pernatants. In 7 normal kidneys, the specific enzyme activity of Hex B was observed in placenta and cultured renal carcinoma the supernatants ranged from 9.5 to i 2.3 units/mg protein cells (Fig. i ). In order to obtain Hex A and Hex B activities (mean ±S.D., i i .0 ±i .0), with a ratio of Hex A to Hex B precisely, chromatographic analysis was carried out. between 2.50 and 4.52 (3.46 ±0.69). The enzyme activity Chromatographic Patterns of Hexosaminidase Isozymes and the ratio of Hex A to Hex B in normal kidney were not on DEAE-celiulose. Hexosaminidaseisozymescould also be related to sex or age as shown in Table 1.
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