Isolation and Characterization of Lytechinastatin - Journal of Natural

Nov 1, 1981 - George R. Pettit, John A. Rideout, Julia A. Hasler, Dennis L. Doubek, Peter ... George R. Pettit , Youichi Fujii , Julia A. Hasler , Jea...
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IS0LXT I 0N A 3 D CH ARXCTE R I ZXT I O r\; 0F LTT E CH I S -isTXT I N 1 GEORGER . PETTIT,'JOHS Ai,RIDEOL-T, JKLI.I-1.HASLER,DESSISL. DOKBEK, and PETER R . REI-CROFT Cuncer Reseiirch Insiiiiite nnd D e p o r t m e n t qf Chernistry, riconu S i o f e lTni?ersity, Teiiipe, d ri-ona 85287 .IBsTRAcT.-The venomous marine animal Lytechimts rariegaizis (Lamarck) h a s been found t o contain an antineoplastic glycoprotein. Separation of an aqueous extract of this Weste-rn ;itlantic sea urchin b y a series of macroreticular resin (or centrifugation) and gel permeation chromatographic sequences led t o t h e new glycoprotein designated lytechinastatin,

The sea urchin family Tosopneuctidae (Echinodermata phylum. Echinacea superorder) ii n ell Snon-n for its venomous specie.:. e-pecially the lethal Western Pacific (Japan) Tosopileiisles elegaiis and T . pileolus ( 2 ) . The ubiquitous Caribbean (range- from Brazil to S o r t h Carolina) L y t e c k i u u s Tariegatus (Lamarck), another toxic member of this famill. also containc a pedicellarial (1) venom. I n this ca>e the venom n as found to be dialyzable and presented an acetylcholinetype pliarmacological respon-e (3). Other biochemical btudies of this species have been limited to the sperm ribo-omal DS-1 ( 4 - i ) , egg jelly coat (8)' a tubulinlike protein [regulation of microtubule function (9) ], and a secretion with fertilization-inhibiting properties (10). Marine invertebratee such a i L . 'i ariegatzrs have been of conbiderable interest to us during the past fifteen years (11) for evaluation as new sources of potentially useful cancer chemotherapeutic agents. For example, n-e recently described (1.12) the isolation of ctrongylostatin. 1 and 2 from the venomous green sea urchin StroizgyloceIzfrotzis droebacliieiisis (Muller). Strongylostatin 1 n a. found t o be a glycoprotein of exceedingly high molecular weight ( 2 4 x 10') which exhibited significant antineoplastic activity (3543% life extension a t 3-10 nig kg do-e levels) in the Sational Cancer Institute's ( S C I ) murine P38S lymphocytic leukemia (PS) i i i ezio sjstem (13). Strongyloqtatin 2 proved to be a related, albeit loner molecular TT eight. gl) coprotein TI ith qimilar anticancer activity (1). In 1965 n e began to invectigate L. iariegatus collected in the Gulf of l\lexico (-1palachee Bay, Florida) and an ethanol extract reached the confirmed active level (PS T C 150 at 400 mg kg) in the S C I exploratory biological program. The antineoplactic activity (Pi3 T C 1 2 i a t GO mg Sg) mas next observed in an aqueouq fraction obtained from a 1971 recollection. In subsequent recollection3 made in 1 9 i 5 and 197 the activitj na. found to again vary betneen the ethanol (1975) and aqueous ( 1 9 7 ) extract-repeating t h e procedure) of the excliided band recovered by evaporation of the Tvater i i ~ i~ : c i t o )was dissolved in water (50 m l ; and applied t o a column (5 x 7 5 cmi of Sephacryl S-200 superfine. T h e first band (0.70 g, lightbrown void volume fraction) eluted by water was found to contain the P S iti Liz'o active (T C 129 at 5 nig kg component. .1 portion (0.25 g) of this fraction vias chromatographed in water 130 ml) on a column (5 x 75 e m ) of Sepharose 2B t o yield three fractions (monitored with a iiv detector 1 . .ifter desalting (a Bio-filter 80 beaker was used 1 and subsequent lyophilization, t h e first fraction yielded t h e antineoplastic ( P S T C 121-122 at 12-25 mg kg) glycoprotein lytechinastatin (83 mg) as a t a n or beige fluffy powder slightly soluble (0.6 m g , m l j in water upon remaining 1 h r at room temperature. I n a large-scale isolation of lytechinastatin, t h e aqueous extract (GO g) was triturated with methanol, the insoluble portion was dissolved in water 1400 ml) and centrifuged a t 37,000 g, and t h e supernatant solution was chromatographed with a Pharmacia Kl00 100 column (7 liter volume) packed with Sephadex G-50. The excluded band (3.8 gi was finally chroniatographed (as described above) on Sepharose 2B. However, the yield (0.15 g ) of lytechinastatin was reduced by this simplified procedure. Based on the w&ght of aqueous extract employed, this corresponds t o a yieldof 0.25cc (by weight) of lytechinastatin. Because of lytechinastatin's molecular size, polyacrylamide gel electrophoresis could not be used t o evaluate t h e extent of purification achieved by these methods. CHARACTERIZATIOS OF LTTECHISASTATIS .--An analytical column (1 s 40 cm was prepared containing Sepharose 2B in 311 guanidine hydrochloride. T h e fractionation range of this column was calibrated for upper and lower limits by chromatography of molecular weight markers (monitored with a uv detector). Particles removed from the methanol insoluble portion of the aqueous extract b y centrifugation proved to be a convenient marker for the exclusion limit 12 s 10' for carbohydrate) and column void volume. Low molecular weight material from the G-50 columns (see above) provided a useful marker for the lower end of the fractionation range (105) and t o t a l bed volume. Chromatograph)- of lytechinastatin (1 mg) on this column produced an elution curve which fell between the two calibrated limits. Assun;ing t h a t lytechinastatin obeyed the usual linear relationship between elution volume i's. molecular weight (and t h a t it behaved as a carbohydrate rather than a protein) it corresponded t o a maximum molecular weight of approximately 2 x lo6. Lytechinastatin was found t o be a glycoprotein ( 1 8 7 carbohydrate, based on a glucose standard, 14) and amino acid analvses (1%17) s h o x e d t h e following composition: Ala 9.36, h r g 3.41) Asx 12.15, Glx 11.24, Gly 10.72, His 1.44, Ile 5.17, Leu 7:49, Lys 1.33, Met 0.59, P h e 3.96, P r o 5.69, Ser 6.13, T h r i.79, T y r 2.49, J-a1 7.73 in mole rc amino acid. Anal. found: C, 45.05: H , 6 . 5 2 : S , 5 . 2 5 : S, 1.68: P, 0.54. S o significant amounts of any metal were detected. T h e composition of 5.25$ nitrogen corresponds t o a protein content of about 3 3 5 1a nitrogen content of 1005 pfotein was approximated t o be 16$). The results of a Lowry protein assay indicated 2 7 7 protein. AICKSOWLEDGIIESTS

For support of this investigation, we are pleased to thank t h e S a t i o n a l Cancer Institute (performed pursuant to Contract Sol-ClI-97262 with the Division of Cancer Treatment, S C I , S a t i o n a l Institutes of Health, D H W ) , and t h e S a t i o n a l Cancer Institute, DHW, for G r a n t s Xos. C,1-1604941 through 07; the Fannie E . Rippel Foundation, l l r s . M a r y Dell Pritzlaff, and the Olin Foundation (Spencer T. and Ann I T . ) . F e also wish t o thank l l e s s r s . T . S. Krupa and L . D. T-anell for technical assistance, and Drs. J. Douros and 31. Suffness for other valuable assistance. Receiyed

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LITERATURE CITED 1. G. R . P e t t i t , J. A . Hasler, K . D . Paul1 and C . L . Herald, 1.>Yof.Prod., 4-4,701 (1981). 2 . B. W.Halstead, Poisonotts a n d 17enovious X n r i n e d n i m i i s of the TI.orld, 1 , U.S. Government Printing Office, Washington, D . C . , 1965) p: 549. 3 . E. G . Alendes, L. ;Ibbud and P. Umiji, Sctence, 139, 408 (1963,). 4 . S . K. hlishra, E x p e r i e n t i o , 35, llGl (1979). 5 . F . E . Wilson, X . Blin and D . W .Stafford, Chroniosoniii (Beri.1, 65, 3 i 3 (1978). 6. S . K. l l i s h r a , M o l e c . Biol. R e p . , 4, 211 (1978).

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Pollock, Jr., hl. Swihart and J. H . Taylor, .l’ucZetc A c i d s R e s . , 5 , 4855 (1978). 7. J. ?*I. 8. G. K . SeGall and W. J. Lennarz, Decelopniental Biol., 71, 33 (1979). 9. R. W. Rubin, R. Kahn and J. J. Prior, Tassue and Cell, 8, 209 (1976). 10. R. E. Daniel, T r a n s . K e n t u c k y A c a d . Sca., 28, 53 (1967). 11. G. R. P e t t i t , Y. Kamano, Y. Fujii, C. L. Herald, 11. Inoue, P. Brown, D . Gust, K . Kitahara, J. hl. Schmidt, D. L. Doubek and C. hlichel, J . ,Vut. P r o d . , 44, 482 (1981). 12. G. R. P e t t i t , C. L . Herald and L. D . 1-anell, J . ‘Vat. P r o d . , 42, 407 (1979). 13. R . I. Geran, K. H . Greenberg, 33. hl. Macdonald, A. RI. Schumacher and B. J. Abbott, Cancer Cheniother. R e p . , P a r t 8, 3, S o . 2 (1972). 14. R. G. Spiro, “Methods in Enzymology,” Vol. 8, rlcademic Press, Kew York, S.Y., 196G, pp. 93-95. 15. D. H. Spackman, W . H . Stein and H. More, Anal. Chent., 30, 1190 (1958). 16. R. W. Hubbard and D. hl. Kremen, A n a l . Biochent., 12, 593 (1965). 17. J. T. Benson, Jr. and J. A. Patterson, A n a l . C h e m . , 37, 1108 (1965).