Alteration of Hexosaminidaseisozymesin

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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 â€”70Â°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 37Â° 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 37Â°. 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 4Â°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 â€˜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 â€˜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 37Â°,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) â€˜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 Â±S.D., i i .0 Â±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 Â±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. In i 0 cases of renal separated by DEAE-ceiluiose column [email protected] carcinoma, the specific enzyme activity of the supernatants peaks of enzyme activity were eluted from the column, the first ranged from 7.5 to 28.0 units/mg protein (18.4 Â±7.9). The eluting with the equilibrating buffer and the second eluting with enzyme activity in renal carcinoma tissue was scattered in a the equilibrating buffer containing 0.2 M NaCI. The fractions wide range. The ratio of Hex A to Hex B ranged from 0.6i to containing the hexosaminidase activity of each peak were 2.2i (i .30 Â±0.50), and the ratio of Hex A to Hex B in renal pooled. About 80% of the enzyme activity loaded onto the carcinoma tissue was consistently low compared with that in column was recovered in the 2 pooled fractions. Representa normal kidney. The enzyme activity and the ratio of Hex A to tive elution profiles of both normal kidney and renal carcinoma Hex B in renal carcinoma tissue were related to neither cell are shown in Chart i . The pooled fractions thus obtained were type nor cell differentiation grade of carcinoma tissue. concentrated and subjected to electrophoretic analysis, which Hexosaminidase Activity and Ratio of Hex A to Hex B in clearly showed that hexosaminidase isozymes of the first and Placenta and Cultured Renal Carcinoma Cells. The specific second peaks corresponded to Hex B and Hex A, respectively enzyme activity in placenta was found to be lower than that in (Fig. i B). Therefore, the ratio of Hex A to Hex B in the normal kidney (Table i ). However, the specific enzyme activity supernatants of tissue homogenates could be calculated from in cultured renal carcinoma cells was highest (40.4 units/mg the total enzyme activities of 2 enzyme peaks separated protein) among all tissues examined, and the original carci through DEAE-cellulose column. noma tissue (Table 1, Renal Carcinoma 4) of the cultured cells Hexosaminidase Activity and Ratio of Hex A to Hex B In also showed high enzyme activity (28.0 units/mg protein). Normal Kidney and Renal Carcinoma Tissue. Normal and The ratio of Hex A to Hex B in placenta ranged from 0.89 to cancerous renal tissues were extracted with 5 volumes of 25 i .24, while the ratios in cultured renal carcinoma cells were mM sodium phosphate buffer, pH 6.0, containing 0.1 % Triton i .02 and 1.06 for 2 different culture lots. The ratio of Hex A to i830 CANCERRESEARCHVOL. 39

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1979 American Association for Cancer Research. Hexosaminidase !soenzymes in Human Rena! Carcinoma Table Hexosaminidase activity and the ratio of HexcellsSpecific A to Hex B in norma! adult kidney, renal carcinoma, placenta, and cultured rena! carcinoma activityHexosaminidase DifferentiationSpecimen (units/mg pro- gradecRenal activity' (units/mi) tein) Hex A/Hex Bâ€• Age Sex Cell type carcinoma1 22 317.8 27.5 0.61 67 F Granular 33 84.9 17.2 0.71 47 M Mixed 24d 146.3 18.8 0.98 55 F Clear 25 I 78.2 28.0 1 .06 32 F Clear 26 107.7 17.2 1.24 74 M Clear 37 205.3 22.9 1.31 48 M Mixed 38 72.9 8.5 1.34 68 M Clear 29 63.5 7.5 1.72 61 M Clear 210 72.4 9.6 1.79 65 M Clear 3Placenta1 240.7 27.0 2.21 64 M Mixed

0.892 71.7 8.3 0.913 69.4 11.4 1.24Cultured 55.0 8.4

renal car cinomacells1 1.022 ND 40.4 1.06Normal ND 40.4 kidney1 F2 105.6 9.5 2.50 44 F3 165.1 12.3 2.88 63 M4 168.5 10.6 3.03 34 F5 148.3 12.0 3.71 52 F6 138.0 11.2 3.72 21 M7 150.9 11.0 3.87 44 Ma 163.2 10.4 4.52 46

The enzyme activity of supernatant from the tissue extracted with 5 volumes of 25 m@ phosphate buffer, pH 6.0, containing 0. 1 % Triton X 100. b Ratio of Hex A to Hex B in the tissues was calculated from pooled fractions following separation by DEAE-cellulose column chromatography.

C Classified from Grade 1 to 4 in the order of cell differentiation according to the criteria of Skinner et al. (21). d The original tissue used for cell culture. 0 ND, not determined.

carcinoma tissue were examined. The pH dependence of hex osaminidase isozymes was determined using 0. 1 M citric acid 0 0.2 M sodium phosphate buffer, pH 3.0 to 7.0. The pH optimum of Hex A in normal kidney and renal carcinoma tissue ranged from 4.0 to 4.7. The pH optimum of Hex B in both tissues I-' > ranged from 4.0 to 4.5. While there was little difference found I- C.) in the pH optima of hexosaminidase isozymes, a marked dif w ,1) ference in their activity at different pH was evident. Hex A 0 z activity at pH 3.0 decreased to i 2.7% of maximum activity, while the activity of Hex B at this pH was 66.0% of maximum U) 0 activity. Furthermore, Hex B activity decreased gradually at pH wâ€˜C I 4.4 to 4.7, but Hex A maintained maximum activity at the same pH range (Chart 2). The finding that Hex B is more stable at pH low pH (3.0 to 4.0) than is Hex A is in agreement with the reports on human colon carcinoma (2) and placenta (24). Chart 2. Effect of pH on Hex A and Hex B in both normal kidney and renal carcinoma tissue, with the use of 0. 1 N citric acid-0.2 M phosphate buffer. Thermostabiiity of hexosaminidase isozymes was examined Oâ€”o, HexAinnormalkidney;0â€”â€”â€”0,HexBinnormalkidney;S 5, Hex at 58Â°for varying length of time using the enzyme solution A in renalcarcinomatissue;5â€”â€”â€”S.HexB in renalcarcinomatissue. containing 0.1 % human albumin in either 25 m@sodium phos phate buffer, pH 6.0, or 0.i M citric acid-O.2 M sodium phos Hex B in cultured cells was closely similarto the value (i .06) phate buffer, pH 4.5 (Chart 3). Since both Hex A and Hex B in the original carcinoma tissue. were more heat labile at pH 4.5 than at pH 6.0, a comparison Some PropertIes of Hex A and Hex B In Normal Kidney of the thermostabiiity of these isozymes was made at the latter and Renal Carcinoma Tissue. Because it was shown that pH. As can be seen in Chart 3, the thermostability of Hex B hexosaminidase was comprised in renal carcinoma tissue at a was substantially greater than that of Hex A at pH 6.0. low ratioof Hex A to Hex B differingfrom normalkidney, some Km'S of hexosaminidase isozymes were determined for 2 properties of Hex A and Hex B in normal kidney and renal synthetic substrates. Km'Sfor p-nitrophenyi-N-acetyl-,8-D-glu

MAY 1979 i 83i

aminidase activity in renal carcinoma tissue was considerably variable, the ratio of Hex A to Hex B in all cases of renal carcinoma examined was consistently low (0.61 to 2.2i ). Hex osaminidase isozymes of renal carcinoma were identical with those in normal kidney in some physicochemical properties. These results indicate that a relative increase of Hex B in >. I-. hexosaminidase isozymes has occurred in renal carcinoma > I- tissue, and these results are similar to those obtained from 0 human colon carcinoma by Brattain et a!. (2). -J z Hexosaminidase isozyme patterns of human renal carcinoma (5 were found to be similar to those of placenta. These results 0 indicate that hexosaminidase isozymes in human renal carci 0 B 58' pH 4.5 zC- noma have at least expressed the oncoplacental alterations, w 80 0 although a possibility that they may express oncofetal altera (hi tions is not yet ruled out. Srivastava et a!. (23) and Beutler et 60 a!. (i ) have shown that Hex A is a heteropolymer composed of 40 both a- and fl-subunits and that Hex B is a homopolymer composed of only a-subunit. Therefore, the results presented 20 here could be due to a decrease in the synthesis of the a- subunit and/or an increased synthesis of the /1-subunit in renal 0 carcinoma. Co 20 30 TIME N MIN An increase in total hexosaminidase activity coupled with a Chart 3. Thermostabillty of Hex A and Hex B in both normal kidney and renal relative increase of Hex B activity was reported to be associ carcinoma tissue. Heat treatment was carried out at 58Â°in 2 different pH's (6.0 ated with a higher lipid content in the human brain (7). In the and 4.5). Symbols are given in Chart 2. light of the results presented here, it is interesting to note that the lipid content in renal carcinoma has been reported to be cosaminide were obtained from 4 sets of assays. The Km'Sof about 3.5 times that of normal kidney (i 8). The association of Hex A in both tissues were 0.7 mM, while those of Hex B in a high lipid content with a relative increase in Hex B remains to both tissues were 0.5 [email protected] in Km'Sbetween Hex be determined. A and Hex B is statistically significant for normal kidney (p < Having now delineated the pattern of hexosaminidase iso 0.005) and renal carcinoma (p < 0.001 ). Km'S of Hex A and zymes in renal carcinoma, it will be important for the diagnostic Hex B for p-nitrophenyl-N-acetyl-/1-D-galactosaminide were ob aid to determine if an altered isozyme pattern can be detected tamed from 2 sets of assays and were the same (0. i mM) in in the urine of the patients with renal carcinoma. both tissues. The results presented here indicate that Hex A could be REFERENCES discriminated from Hex B in some physicochemical properties. 1. Beutler, E., Yoshida, A., KuhI, W.. and Lee, J. E. S. The subunits of human However, each isozyme maintained its own physicochemical hexosaminidase A. Biochem. J., 159: 54i â€”543,1976. 2. Brattain, M. G., Kimball, P. M., and Pretlow, T. G., II. fl-Hexosamlnldase properties whether from normal or from cancerous renal tissue. Isozymes in human colonic carcinoma. Cancer Res., 37: 731â€”735,1977. 3. Dance, N., Price, R. G.. Robinson,D., and Stirling,J. L. $-Galactosidase, $-glucosidase and N-acetyl-$-glucosamlnidase in human kidney. Clin. Chim. DISCUSSION Acta,24:189â€”197,1969. 4. Ellis, R. B., Rapson, N. T., Patrick, A. D., Greaves, M. F., and Path, N. R. C. Several isozymes, as exemplified by aldolase (i 7), arylami Expression of hexosaminidase isoenzymes In childhood leukemia. N. EngI. dase (8), â€˜y-glutamyitranspeptidase(6),and dehydrogenases J. Med., 298: 476â€”480,1978. 5. Fluharty, A. L, Lassila, E. L, Porter, M. T., and Kihara, H. The electropho (lactic dehydrogenase, glucose-6-phosphate dehydrogenase, retic separation of human $-galactosidases on cellulose acetate. Biochem. malate dehydrogenase, and malic enzyme) (10), have been Med., 5: 158-164, i97i. 6. Hada, T., Higashino,K.. Yamamoto,H., Yamamura,V., Matsuda,M.. Osa studied in human renal carcinoma. To the best of our knowl fune, K., Kotake, T., and Sonoda, T. A novel â€˜y-glutamyltranspeptidasein edge, a study of hexosaminidase isozymes of human renal renal carcinoma in comparison with normal kidney enzyme. Clin. Chim. Acta, carcinoma, however, has not been reported. The results of our 85: 267â€”277,1978. 7. Harzer, K., and Sandhoff, K. Age-dependentvariationsof the human N- study for hexosaminidase isozyme patterns and their physico acetyl-/3-o-hexosaminidases.J. Neurochem., 18: 2041 -2050, i 971. chemical properties in renal carcinoma and cultured renal 8. Hlwada,K., Torso, M., and Kokubu,T. Placentalformof membrane-bound carcinoma cells are presented here. Hexosaminidase isozymes neutral arylamidase found in renal cell carcinoma. Clin. Chim. Acta, 79: 569-573,1977. have been separated into 2 major forms in normal human 9, Lee, J. E. S., and Yoshida, A. Purification and chemical characterization of tissues such as brain (7, i 9), kidney (3, i 3, 26), liver (i i), human hexosaminidase A and B. Biochem. J., 159: 535-539. 1976. 10. U. J. J., U, S. A., Klein, L A., and villee, C. A. Dehydrogenase isozymes in colon (2), placenta (9, 22), spleen (20), and ieukocytes (4, 15) the hamster and human renal adenocarcinoma. In: C. Markert (ed), Isozyme by electrophoresis, ion-exchange column chromatography, III, InternationalConferenceon Isozymes,pp. 837â€”853.NewYork: Ace and isoelectric focusing. Hex A is the predominant isozyme in demic Press, Inc., 1975. 11. Li, Y.-T., Mazzotta, M. V., Wan, C-C., Orth, R., and LI, 5.-C. Hydrolysis of almost all tissues of normal adult humans studied thus far (3, Tay-Sachs ganglloside by $-hexosaminldase A of human liver and urine. J. 19, 20). in the experiments reported here, the ratio of Hex A to Blol. Chem., 248: 75i2â€”7515, 1973. Hex B in normal adult kidney ranged from 2.50 to 4.52 (mean, 12. Lowry. 0. H., Rosebrough, N. J., Fart, A. L, and Randall, R. J. Protein measurementwiththeFolinphenolreagent.J.Biol.Chem.,193:265â€”275, 3.46). The value was quite similar to the value of 3.7 calculated i95i. from the results of Marinkovic et a!. (I 3). Although the hexos 13. Marinkovic, D. V., and Marinkovic, J. N. Purification of two hexosaminidases

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fromhumankidney.BiOChern.J.,163:133-140, 1977. spleen. Biochem. J., 107: 321-327, 1968. 14. Matsuda, M., Osafune, K., Kotake, T., Sonoda, T., Watanabe, S., and Hada, 21. Skinner,D. G., Colvin,R. B., VermilIon,C. D., Pfister,R. C., and Leadbetter, T. A fundamentalstudy of renal cell carcinoma I. In vitro long-termcall w. F.Diagnosisandmanagementofrenalcellcarcinoma.Cancer(Phila.), cultures of renal cell carcinomas. Acta Urol. Jpn., 24: 27-33, 1978. 28:i165â€”ii77,1971. 15. Okada. S.. and O'Brien, J. S. Tay-Sachs disease: generalized absence of a 22. Srivastava,S.K., Awasthi,V. C., Yoshida,A.,andBeutler,E. Studieson beta-o-N-acetylhexosaminldase component. Science, 165: 698â€”700,1969. human fl-o-N-acetylhexosamlnidases. I. Purification and properties. J. Biol. 16. Okochi, T., Selke, H., Hada, T., Ito. F., Higashino, K., and Yamamura, V. Chem., 249: 2043-2048, 1974. Car@lno-fetaIafteratlons in hexosaminidase isozymes In human renal card 23. Srlvastava,S.K..Wiktorowlcz,J.E.,andAwasthi,V.C.Interrelationshipof nortia. In: Carcino-Embryonic Proteins, Biology, Chemistry. Clinical Appli hexosaminidases A and B: confirmation of the common and the unique cation. Amsterdam: Elsevier Publishing company, in press, 1979. subunft theory. Proc. Nati. Aced. Sd. U. S. A., 73: 2833-2837, 1976. 17. Pfieiderer,G., Thoner, M., and Wachsmuth,E. D. Histologicalexamination 24. Tallman, J. F., Brady, R. 0., Quirk, J. M., Villalba, M., and Gal, A. E. Isolation of the aldolasemonomercompositionof cells from humankidney and and relationship of human hexosaminidases. J. Blol. Chem., 249: 3489- hypemephroid carcinoma. Beitr. Pathol., 156: 266â€”279,1975. 3499, 1974. 18. Popelier, G., Geers, R., and Verdonk, G. Upid analysis of renal cell card 25. Weber, A., Poenaru, L, Lafarge, C., and Schapira, F. Modification of hex nomas and normal renal cortex. Urol. Res., 4: 183-1 84, 1976. osaminldaeelsozymes in rat hepatoma. Cancer Res., 33: 1925-1 930. 1973. 19. RobInson, D., Jordan, T. W., and Horsburgh, T. The N-acetyl-$-o-hexosa 26. Wiktorowlcz, J. E.. Awasthi, V. C., Kurosky, A., and Srlvastava, S. K. minidases of calf and human brain. J. Neurochern., 19: 1975-1985, 1972. Purification and properties of human kidney-cortex hexosaminidases A and 20. RobInson, D., and Stirling, J. L N-Acetyl-@8-glucosaminidasesinhuman B. Blochem.J.,165:49-53, 1977.

MAY 1979 1833

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1834 CANCERRESEARCHVOL. 39

Toshikazu Okochi, Hiromasa Seike, Kazuya Higashino, et al.

Cancer Res 1979;39:1829-1834.

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