576th MEETING, LONDON

1019

Goldberg, A. L. (1969)J. Biol. Chenr. 244,3223-3229 Goldberg, A. L. & Goodman, H.M. (1969)J. Physiol. (London) 200,667-675 Goldspink, D.F. (1977)J . Physiol. (London) 264, 267-282 Goldspink, D.F. (1978)Biochem. J . 174,595-602 Kostyo, J. L. & Redmond, A. F. (1966)Endocrinology 79, 531-540 Lowry, 0.H.,Rosebrough, N. J.,Farr, A. L. &Randall, R. J. (1951)J.Biol. Cheni. 193,265-275 Sola, 0 . M.,Christensen, D. L. & Martin, A. W.(1973)Exp. Neurol. 41, 76-100 Thesleff, S.(1963)in The Effect of Use and Disuse on Neuromuscrrlar Functions (Gutmann, E. & Hnik, P., eds.), pp. 41-51,Elsevier, Amsterdam Williams, P. E. & Goldspink, G.(1978)J. Anat. in the press

Colchicine Persistence and Binding in Goldfish Brain G R A H A M CLINGBINE and CHRISTINE E. HEADING North-East London Polytechnic, Department of Paramedical Sciences, Romford Road, London E15, U.K. Intracranial injections of the alkaloid colchicine can impair the conditioned avoidance performance of goldfish trained over 5 days, but its isomer, lumicolchicine, has no effect (Clingbine & Heading, 1977). The same authors report that a single intracranial injection of [3H]colchicine results in radioactivity being detected in all brain regions over the 5-day period. The first study reported here investigates whether colchicine persists over 5 days in goldfish brain. Groups of four fish were given a single 1Opl intracranial injection of [3H]colchicine ( I mg/ml; activity 0.06Ci/mmol). The groups were killed at various time intervals u p to 5 days later. The brains were removed and separately homogenized by hand in a mini-homogenizer (Jencons Ltd.) containing 1 ml of chloroform/methanol (2: 1, v/v) into which radioactivity was extracted. The supernatant obtained from an 8OOg centrifugation in an MSE bench instrument was applied to two separate t.1.c. plates (Cam Lab Ltd) typically as a 5p1 spot containing an additional 3pg of unlabelled colchicine. A similar spot of standard [3H]colchicine was applied to each plate. One plate was run for 45min in chloroform/acetone/diethylamine(5:4: 1, by vol.) (solvent system 1) and the other for 75 min in cyclohexane/chloroform/diethylamine (5:4: I , by vol.) (solvent system 2). The plates were cut into 0.5cm x 1 cm pieces and each piece was placed in lOml of scintillation fluid {toluene/2-ethoxyethanol(7: 3, v/v)/0.5% 2,5-diphenyloxazole/0.01% POPOP [1,4-bis-(5-phenyloxazol-2-yl)benzene]}. Radioactivity was measured in a Packard Tricarb liquid-scintillation spectrometer. The counting efficiency (10%) was determined by using an automated sequence with reference to a n external standard. Radioactivity was detected in positions corresponding to that of standard colchicine in samples from all fish killed at all times (45min, 6 and 24h and 5 days). RF values varied slightly from day to day but were typically 0.7 in solvent system 1 and 0.15 in solvent system 2. The proportion of radioactivity in the samples that chromatographed as colchicine ranged from 70 to 95%, unrelated to time of killing. The results indicate that colchicine can persist in goldfish brain over the 5-day period after a single intracranial injection. The second study reported here concerns the possibility that the colchicine might be persisting in a protein-bound form. Colchicine is known t o be capable of binding t o the microtubular protein tubulin, and can be shown t o bind to soluble and particulate fractions of mammalian brains in vitro at 37°C (see Feit & Barondes, 1970). If the compound could bind to tubulin from goldfish brains at 18°C(their body temperature under laboratory conditions), it would be reasonable t o suggest that at least part of the colchicine recovered in the previous studies had been bound t o tubulin. Brains from about seven to eight goldfish (6-8g body wt.) or one mouse (Tyler Original, body wt. 3 5 4 0 g ) were removed, weighed and homogenized with an UltraTurrax instrument in 12ml of ice-cold buffer (0.01 M-sodium phosphate/O.Ol Mmagnesium chloride) at pH6.5.

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BIOCHEMICAL SOCIETY TRANSACTIONS

l OOOOg (MSE Superspeed 50 centrifuge) at Homogenate (9ml) was centrifuged at O 4°C for 1 h in the l o x lOml rotor. The supernatant was decanted and its volume noted. The pellet was washed twice with 5ml of buffer and was then rehomogenized in 30ml of buffer in an ice bath. Both the supernatant and rehomogenized pellet were diluted with buffer to give a range of protein concentrations. Samples (0.5ml) were digested overnight in 0.5ml of IM-NaOH and protein determined (Lowry et al., 1951) the next day. Samples (lml) of supernatant or rehomogenized pellet were incubated for l t h at 0, 18 or 37°C with 2p1 of [3H]colchicine (sp. activity 4Ci/mmol) or [3H]lumicolchicine (prepared by irradiation of [3H]colchicine with U.V. light and observing changes in the absorption spectrum; see Wilson et al., 1974). Tubes were then plunged into ice t o stop the reaction. Radioactivity in the soluble fraction was assayed by using the method of Weisenberg et al. (1968). A 2.3cm diameter circle of DE 81 filter paper (Whatman) was equilibrated with ice-cold buffer. The 1ml incubated sample was added to the filter disc, which was supported on a sintered-glass disc below a Millipore funnel. Chilled 10mM-cokhicine (1 ml) was added, followed by lOml of ice-cold buffer. Filtration was then allowed to proceed under light vacuum to completion (approx. 6min). The paper was washed five times under vacuum with 5ml of buffer and then assayed for radioactivity directly in scintillation fluid (Wilson, 1970). Radioactivity in the particulate fraction was assayed by layering each 1 ml sample of incubation mixture over 7ml of 10% (w/v) sucrose in buffer. Each tube was centrifuged as described above. The supernatant was aspirated and the pellet washed twice with 5mI of buffer. The pellet was suspended in lOml of scintillation fluid and the radioactivity assayed. Binding of [3H]colchicine occurred in a concentration-dependent manner in the soluble and particulate fractions of homogenates of both mouse and goldfish brain at 37°C. At 18"C, binding occurred at about half the extent of that at 37°C. No binding occurred a t O'C, or with samples incubated with [3H]lumicolchicine. G . C. is in receipt of a Science Research Council Studentship Research Grant. We thank Dr. J. R. Lagnado of Bedford College, University of London, for advice.

Clingbine, G. & Heading, C. E. (1977) Br. J . Pharmacol. 59,449P-45OP Feit, H. & Barondes, S. H. (1970) J . Neurochem. 17, 1355-1364 Lowry,O. H.,Rosebrough,N. J.,Farr, A . L. &Randall, R. J. (1951)J. Biol. Chem. 193,265-275 Weisenberg, R . C., Borisy, G . & Taylor, E. W. (1968) Biochemistry 7, 4466-4479 Wilson, L. (1970) Biochemistry 9, 4999-5007 Wilson, L., Ramburg, J. R., Mizel, S. B., Grisham, L. M. & Creswell, K. M. (1974) Fed. Proc. Fed. Am. SOC.Exp. Biol. 33, 158-166

Is there a Membrane-Bound, Calcium-Dependent Phosphatidylinositol Phosphodiesterase in Rat Brain? ROBIN F. IKVINE and REX M. C. DAWSON A. R . C . Institute of Animal Physiology, Babraham, Cambridge C B 2 4AT, U.K.

Independent evidence from two laboratories (Lapetina & Michell, 1973; Friedel e t al., 1969) suggests that a membrane-bound Ca2+-activatedphospholipase C exists in nervous tissue that specifically hydrolyses phosphatidylinositol. This enzyme has been distinguished from the cytosolic Caz+-dependent phosphatidylinositol phosphodiesterase (EC 3.1.4.10) of brain (Thonison, 1967) by three main differences. ( I ) The niembranebound activity is stimulated by deoxycholate (Lapetina & Michell, 1973), whereas this detergent reportedly inhibits the soluble enzyme (Atherton & Hawthorne, 1968). (2) The p H optima of the membrane-bound and soluble enzyme are 7.0 (Lapetina & Michell, 1973) and 5.5 (Thomson, 1967) respectively. (3) Some brain membrane fractions 1978