Further purification and characterization are reported on rat cytosol cornin (RLCC), an antimitotic substance. Fraction I (purified RLCC) was purified more than 10-fold from crude RLCC with Sephadex G-50 column chromatography and showed a remarkable inhibitory effect on division of inseminated sea urchin eggs and mouse fibroblast cells. Fraction I was observed as one spot, and the molecular weight was estimated to be about 25,000 by thin layer gel filtration. Fraction I contained protein (92%) and RNA (8%), but the antimitotic activity was scarcely affected by treatment by pancreatic RNase. The protein of Fraction I was separated into two bands by SDS-polyacrylamide gel electrophoresis, and the molecular weight was estimated as 10,000 and 15,000, respectively. The 50% inhibition dose of Fraction I on the first division of inseminated sea urchin eggs and on proliferation of mouse L cells was about 2.5 X 10(-5) g/ml and 5 X 10(-4) g/ml, respectively. The yield of fraction I was about 35 mg from 100 g rat liver.</p

Doi, Akitaka

Inaba, Kozo

Nisida, Isamu

Okayama University Scientific Achievement Repository

Acta Medica OkayamaVolume 31, Issue 3 1977 Article 5JUNE 1977Purification and some characteristics of livercytosol cornin, an antimitotic substance fromrat liver cytosolKozo Inaba∗ Akitaka Doi†Isamu Nisida‡∗Okayama University,†Okayama University,‡Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.Purification and some characteristics of livercytosol cornin, an antimitotic substance fromrat liver cytosol∗Kozo Inaba, Akitaka Doi, and Isamu NisidaAbstractFurther purification and characterization are reported on rat cytosol cornin (RLCC), an antim-itotic substance. Fraction I (purified RLCC) was purified more than 10-fold from crude RLCCwith Sephadex G-50 column chromatography and showed a remarkable inhibitory effect on divi-sion of inseminated sea urchin eggs and mouse fibroblast cells. Fraction I was observed as onespot, and the molecular weight was estimated to be about 25,000 by thin layer gel filtration. Frac-tion I contained protein (92%) and RNA (8%), but the antimitotic activity was scarcely affectedby treatment by pancreatic RNase. The protein of Fraction I was separated into two bands bySDS-polyacrylamide gel electrophoresis, and the molecular weight was estimated as 10,000 and15,000, respectively. The 50% inhibition dose of Fraction I on the first division of inseminatedsea urchin eggs and on proliferation of mouse L cells was about 2.5 X 10(-5) g/ml and 5 X 10(-4)g/ml, respectively. The yield of fraction I was about 35 mg from 100 g rat liver.∗PMID: 144419 [PubMed - indexed for MEDLINE] Copyright c©OKAYAMA UNIVERSITYMEDICAL SCHOOLActa Med. Okayama 31, 203~209 (1977)PURIFICATION AND SOME CHARACTERISTICS OFLIVER CYTOSOL CORNIN, AN ANTIMITOTICSUBSTANCE FROM RAT LIVER CYTOSOLKozo INABA, Akitaka DOl and Isamu NISIDADepartment of Physiology, Okayama University Medical School,Okayama 700, JapanReceived March 28, 1977Abstract. Further purification and characterization are reported onrat cytosol cornin (RLCC) , an antimitotic substance. Fraction I (purifiedRLCC) was purified more than lO-fold from crude RLCC with Sepha-dex G-50 column chromatography and showed a remarkable inhibitoryeffect on division of inseminated sea urchin eggs and mouse fibroblastcells. Fraction I was observed as one spot, and the molecular weightwas estimated to be about 25,000 by thin layer gel filtration. FractionI contained protein (92%) and RNA (8 %), but the antimitotic activitywas scarcely affected by treatment by pancreatic RNase. The proteinof Fraction I was separated into two bands by SDS-polyacrylamide gelelectrophoresis, and the molecular weight was estimated as 10,000 and15,000, respectively. The 50% inhibition dose of Fraction Ion the firstdivision of inseminated sea urchin eggs and on proliferation of mouseL cells was about 2.5x 10-5 glml and 5x 10-4 glml, respectively. Theyield of Fraction I was about 35 rug from 100 g rat liver.Nisida and Murakami (1) reported that a pupillo-contracting substance,cornin (2), extracted from bovine cornea or rabbit muscle showed retardingeffects on mitosis of inseminated sea urchin eggs. It has been reported that corninfraction extracted from bovine liver suppressed the growth of cultured mamma-lian cells (3) and that injection of cornin fraction extracted from canine intestineprolonged the survival time of mice inoculated with Ehrlich ascites tumor cells(4).This paper reports on further purification and characterization of the corninfraction extracted from liver cytosol.MATERIALS AND METHODSExtraction of corninfraction. Livers from adult male rats (Wistar strain) werehomogenized with 4 volumes of cold 0.25M sucrose. The homogenate was filtered through 4 layers of gauze, and the filtrate was then centrifuged at 10,000 >( gfor 10 min. The resulting supernatant was centrifuged at 100,oooxg for 60 minto eliminate the microsomal fraction. The cornin fraction was extracted fromthis liver cytosol fraction by a modification of the method of Nisida and Mura-2031Inaba et al.: Purification and some characteristics of liver cytosol corninProduced by The Berkeley Electronic Press, 1977204 K. INABA, A. DOl and I. NISIDAkami (1). The liver cytosol fraction was diluted with 4 volumes of distilledwater and boiled for 3 min and cooled immediately in a water bath and thencentrifuged at 1O,OOOxg for 10 min. Cold ethanol was added to the resultingsupernatant to a final concentration of 70%. The mixture was placed for 2 hrin an ice bath and then centrifuged at 5,000 X g for 10 min. Cold ethanol wasadded to the resulting supernatant to a final concentration of 90%, and themixture was placed in a cold room (0° to 4°C) overnight and then centrifugedat 5,OOOxg for 10 min. The resulting precipitate was mixed with 10 volumes ofcold acetone, centrifuged and washed with cold ether and then dried in air.This acetone powder was designated as the crude liver cytosol cornin (RLCC)fraction.Purification of RLCe. Crude RLCC (100 mg) was dissolved in 2 ml of dis-tilled water and charged on top of a Sephadex G-50 column (3x50 cm) and theneluted with 300 ml of distilled water. The eluate at 3 ml fractions was collected,and the ultraviolet (280 nml-absorbing fractions were lyophilized.Cell assay using inseminated sea urchin eggs. Matured eggs of sea urchin(Temnopleurus toreumaticus) were collected by an intracoelomic injection of 0.5M KCl, The shed eggs were then immediately washed with sea water and thefertilization rate was examined. Intact eggs showing high rates (above 95% lwere used in this experiment. Test materials were dissolved in sea water andmixed with the egg cell suspension. After treatment for 10 min at 25°C, the eggcells were inseminated. The cleaved cells were counted at 5 min intervals..Cell assay using mammalian cultured cells. L cells (3xlO4, mouse fibroblaststrain) in stationary state were subcultured at 37°C in tube containing 1 ml ofEagle's minimum essential medium with 10% bovine serum. For assay of cellgrowth inhibition, the test material dissolved in 1 m1 of culture medium wasadded to the culture 48 hr after subculture. Cells were detached from the tubewall by treatment with 0.05 % trypsin-0.025% EDTA in phosphate-bufferedsaline, pH 7.2, 24 hr and 48 hr after the addition of test materials and countedby a Coulter counter.Amino acid analysis. Fraction 1(2 mg) purified from crude RLCC was hydro-lyzed by 4 ml of 6 N HCI at 105°C for 12 hr. The hydrolysate was evaporatedto dryness under reduced pressure, and dissolved in 2 m1 of 0.01 N HCI andanalyzed by an amino acid analyzer (Model jLC-6AH, JEOL Ltd., japan).Protein determination. Protein was estimated by the method of Lowry et al.(5).RNA determination. RNA was estimated by the method of Brown (6) afterRLCC was treated with cold 5% perch10ric acid.SDS-polyacrylamide gel electrophoresis. To a sample (500 l~g/0.3 ml of 10 mMTris-HC1 buffer, pH 8.0), 0.03 m1 of 10% SDS (sodium dodecy1 sulfate) and 3drops of glycerol were added, and the mixture was heated in a boiling waterbath for one min. The mixture (0.05 ml) was then charged on the top of a 7.5%SDS-po1yacry1amide gel column (0.5x 8.5 cm). Electrophoresis was carried outat 5 rnA/tube for 5 hr and then stained with Coomassie Brilliant Blue. Cyto-chromec and hiStone fractions (Sigma Chemical) were used as marker proteins.2Acta Medica Okayama, Vol. 31 [1977], Iss. 3, Art. 5http://escholarship.lib.okayama-u.ac.jp/amo/vol31/iss3/5Antimitotic Substance, Liver Cytosol Cornin 205RESULTSThe mitosis rate of inseminated sea urchin eggs was retarded by pretreatmentof 0.025 % crude RLCC for 10 min (Fig. I).100~~c0 50III>040 50 60 70Ti me after insemination (mi n)Fig. 1. Effect of crude RLCC on the division of inseminated sea urchin eggs.Ordinate, Percentage of divided eggs to initial eggs. Abscissa, Time afterinsemination. Open circles, control. Solid circles, crude RLCC (2.5 X 1O-4g/ m l).This crude liver cytosol cornin was further separated into three fractio s bySephadex G-50 column chromatography (Fig. 2).Fr.mFr. I0.50Ec 0400roN--0 0.30Q)()c0.Q 0,20~0III.Q«0,100.050 200 40 60 80 100 I 20Tube numberFig. 2. Sephadex C-50 column chromatography of crude RLCC. Ordinate,Absorption ofUV (280 nm). Abscissa, Tube number.3Inaba et al.: Purification and some characteristics of liver cytosol corninProduced by The Berkeley Electronic Press, 1977206 K. INABA, A. Dor and I. NrsIDA100~~c0 50(/)>0o L--/f--,l4o-=------l.L~....,,;5~O:::----"===--J.---::6~O--L----=7-=-O----l.­Time after insemination (min)Fig. 3. Effect of fractions separated from crude RLCC on division of inseminatedsea urchin eggs. Ordinate, Percentage of divided eggs to initial eggs. Abscissa, Timeafter insemination. Open circles, control. Solid circles, Fraction I (2.5 X 1Q-5g/ m l).Solid triangles, Fraction II (2.5 X 1Q-4g/m l). Open triangles, Fraction III (2.5 X 1Q-4g/ mll.Fig. 4. Effect of RNase on the antImItotic actIvIty of Fraction I isolated from crudeRLCC. Ordinate, Cell numbers per tube. Abscissa, Incubation time at 37°C. Open circles,control. Solid circles, Fraction I (I x 1Q-3g/ ml). Half solid circles, Fraction I (5 X 1Q-4g/ m l).Open triangles, Fraction I (RNA free, 5x 1Q-4g/ m l). Solid triangles, Fraction I (RNA free,I X 1Q-3g/ m l). Fraction I free of RNA was prepared from crude RLCC which was previouslytreated with pancreatic RNase (25pg/50mg crude RLCC/ml) for 60 min at 37°C. Arrowshows the time when an equal volume of the culture medium containing test material wasadded to the culture.4Acta Medica Okayama, Vol. 31 [1977], Iss. 3, Art. 5http://escholarship.lib.okayama-u.ac.jp/amo/vol31/iss3/5Antimitotic Substance, Liver Cytosol Comin 207The effect of each fraction separated from crude RLCC was examined onmitosis of inseminated sea urchin eggs (Fig. 3). The mitosis was suppressed by60% and 96% with pretreatment of 0.0025% and 0.025%, respectively, ofFraction 1. Fractions II and III had no effect on mitosis of inseminated sea urchineggs. The yield of Fraction I was about 35 mg from 100 g of rat liver.As Fraction I contains protein (92 % ) and RNA (8 % ), the effect of RNaseon antimitotic activity of Fraction I was examined by the cultured cell assaysystem (Fig. 4). The cell growth inhibitory activity of Fraction I was not affectedsignificantly by RNase treatment. RNA isolated from Fraction I by the SDS-phenol method had no effect on mitosis. The antimitotic activity of Fraction Iwas eliminated with digestion using b3cterial protease (Nagarse, Katayama Che-mical Co., Osaka) at 200 flg or 160 PUN/10 mg of Fraction I, at 3r C for 1 hr.Fraction I was shown to be a single spot and its molecular weight was calcu-lated as about 25,000 by thin layer gel filtration on Sephadex G-IOO. Fraction Iwas separated into two bands by SDS-polyacrylamide gel electrophoresis, andFig. 5. SDS-polyacrylamide gel electrophoresis of Fraction I isolated from crude RLCC.5Inaba et al.: Purification and some characteristics of liver cytosol corninProduced by The Berkeley Electronic Press, 1977208 K. INABA, A. DOl and 1. NISIDAtheir molecular weights were calculated as about 10,000 and 15,000, respectively(Fig. 5).The amino acid composition of the hydrolysate of Fraction I is shown inTable 1. The content of the acidic amino acids was relatively high comparedwith that of basic amino acids. The content of sulfur-containing amino acids inFraction I was very low.T ABLE I. AMINO ACID COMPOSITION OF FRACTION I OF RLCCAmino acidL-ArginineL-HistidineL-LysineL-PhenylalanineL-TyrosineL-LeucineL-IsoleucineL-MethionineL-CysteineL-ValineL-AlanineGlycineL-ProlineL-Glutamic acidL-SerineL-ThreonineL-Aspartic acid---------Percent in moles2.21.65.82.51.16.53.10.70.66.010.79.87.414.08.88.1II. 3DISCUSSIONSince the antimitotic activity of Fraction I purified from crude RLCC waseliminated by protease treatment and was little affected by RNase treatment, theactivity of Fraction I is probably associated with the protein in Fraction I, andthe RNA in Fraction I may be considered a contaminant.Numerous investigators (7-11) have reported that cell growth inhibitingsubstances were extracted from mammalian liver. It was found that some ofthem are closely associated with arginase (10,11). These substances were gen-erally heat labile in contrast with the cornin substance. The antimitotic activityof RLCC does not seem to be tissue-specific in contrast with liver chalone I: 12),though neither is species-specific.Further purification is necessary for critical characterization of RLCC, and6Acta Medica Okayama, Vol. 31 [1977], Iss. 3, Art. 5http://escholarship.lib.okayama-u.ac.jp/amo/vol31/iss3/5Antimitotic Substance, Liver Cytosol Cornin 209the purification of Fraction I with protamine sulfate treatment is under investi-gation.Acknowledgment. The authors wish to thank profoundly Prof. M. Yoshida and his col-laborators of Okayama Marine Laboratory for their kindness in procuring sea urchins forthis investigation.REFERENCESI. Nisida, 1. and Murakami, T. H.: Antimitotic action of cornin as a biologically activepolypeptide. 1. Biochemical properties of cornin. Acta Med. Okayama 19, 1-9, 1965.2. Fukui, M.: Cornin, a pupillo-contracting substance extracted from cornea. j. Yon agoMed. Assoc. 9, 673-681, 1958 (in Japanese).3. Ohtsuki, H.: Studies on the cornin extracted from bovine liver I. Purification of thecornin and its physico-chemical properties. Acta Med. Okaya,na 28, 1-6, 1974.4. Ohya, T: Studies on the effect of tissue substance "cornin" on transplantable malignanttumor in mice. Acta Med. Okayama 21, 227-250, 1967.5. Lowry, O. H., Rosebrough, N. j., Farr, A. L., Randall, R. J.: Protein measurementwith Folin phenol reagent. j. BioI. Chem. 19, 265-275, 1951.6. Brown, A.H.: Determination of pentose in the presence of large quantities of glucose.Arch. Biochem. 11, 269-278, 1946.7. Hori, M. and Ukita, T.: Inhibition in vitro of growth of rat ascites tumor cells bybovine livcr extract. j. Biochem. (Tokyo) 51, 322-334, 1962.8. Lieberman, I. and Ove, P. : Inhibition of cultured mammalian cells by liver extracts.Biochim. Biophy. ,,! ,·ta 38, 153, 1960.9. Suzuki, S. : The growth-inhibiting substances in liver extract on rat ascites hepatomacells in tissue culture. lpn. J. Exp. /'vied. 29, 341353, 1959.10. Holley, R. V.: Evidence that a rat liver "inhibitor" of the synthesis of DNA incultured mammalian cells is arginase. Biochil1l. Bi Iphys. A;ta 145, 525-527, 1967.11. Inaba, K., Doi, A. and Nisida, 1. : Cell growth inhibiting factor in rat liver micro-somes. Cell Struct. Fund. 2, 155-158, 1977.12. Verly, W. G., Deschamps, Y., Pushpathadam, J. and Desrosiers, M.: The hepaticchalone. I. Assay Method for the hormone and purification of the rabbit liver chalone.Can. l. Biochem. 49, 1376-1383, 1971.7Inaba et al.: Purification and some characteristics of liver cytosol corninProduced by The Berkeley Electronic Press, 1977

Purification and some characteristics of liver cytosol cornin, an antimitotic substance from rat liver cytosol

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