Methods M 1 Frogs

Xenopus laevis frogs were kept in tanks of water at 19-21°C and fed twice weekly on a diet of blow-fly larvae and minced beef. The frogs were looked after and fed by Mrs C. Kwasnik.

Female frogs were induced to lay by injection of 100-150 i.u. of FSH 1-8 days prior to laying, followed by a * _{second injection of 500-600 i.u . of HCG 8-16 hours prior to}• laying. Both injections were into the dorsal lymph sac of the frog. Iced water was occasionally used after the injecticjp of HCG to slow down the initial production of eggs. Injection of HCG alone without the in itial priming injection of FSH was also found to be effective at inducing laying and this reduced the number of frogs that laid eggs in 'strings' of jelly.

Embryos were obtained by one of two methods: through the natural mating of frogs or by artificial fertilisation of eggs. In the first case the male frog was injected with 100 i.u. of FSH at the same time as the female frog received her injection of HCG. The frogs were then placed in a tank of water and left for between 8 to 24 hours until the mating was complete. An in vitro fertilisation was carried out when a large number of embryos at synchronous stages of development were required. An adult male Xenopus laevis frog was killed by injection of 300^1 of Euthatal into

the dorsal lymph sac. Both testes were then removed and stored at 4°C for up to 2 weeks in Barths saline (see below). In vitro fertilisation of the eggs was carried out as follows: eggs were collected in a petri dish and the medium removed, a piece of testis was stroked over the eggs and a small amount of distilled water added to promote sperm motility. The eggs were then left for a minute before flooding the petri dish with d istilled water. If the fertilisation was successful rotation of the embryos was observed after 15 min. Barths saline (Barth X) was comprised of the following: 88mM NaCl ImM KC1 24mM NaHCO-. 0. 82mM MgSO« O. 33mM Ca( NO:* )» 0.41mM CaCla 15mM Tris.HCl pH 7.6 lOmg/litre Benzyl penicillin lOmg/litre Streptomycin sulphate

Xenopus eggs and embryos were dejellied by swirling in a solution of IX or 2% cystein pH 8.0, for 5-10 min. The eggs and embryos were then washed thoroughly and stored in Barths saline, or in 1/10 diluted Barths saline until use. M. 3 Purification of Crude Membrane from Xenopus Embryos

homogenised in 2ml of homogenisation buffer (10% w/v sucrose, 150mM NaCl, 10mM Mg-acetate, 20mM Tris.HCl pH 7.6, ImM PMSF). The homogenate was spun in an eppendorf centrifuge for a very

brief period to remove yolk. An excessive spin at this stage was found to cause loss of membrane. The supernatant was removed avoiding the superficial lipid layer. This was divided into four aliquots and these were layered onto 1ml of 20% sucrose buffer (20% w/v sucrose, 50mM NaCl, lOmM Mg-acetate, 20mM Tris.HCl pH 7.6) in a centrifuge tube. The gradients were spun at 17,000g for 30 min at 4°C in a Beckmann High Speed 18 centrifuge. The crude membrane was found to pellet at the bottom of the tube and cytoplasmic proteins were present at the interface between the two sucrose layers.

M. 4 TritQn-X114 Purification of Membrane-Proteins

This method is adapted from that described by Bordier (1981). Crude membrane pellets were resuspended in 200-300pl of 500mM KC1, lOOmM Tris.HCl pH 7.2. 50-80pl of recondensed and saturated TX114 (see below) was added to each tube. The samples were vortexed and sonicated on ice using a sonic probe for 15 sec each. They were then le ft at 4°C for between 30 min and an hour. The tubes were spun at full speed in an eppendorf microfuge for 10 min after which the detergent and aqueous phases were mixed and removed into clean tubes. The pigment containing pellets were discarded. The samples were spun again for 10 min, the aqueous and detergent phases were mixed and layered onto 600pl of sucrose containing buffer (6% sucrose, 150mM KC1, lOmM Tris.HCl pH

7.5, 0.0075* TX114) . The tubes were warmed to 30°C for 3 min and spun at high speed for 10 sec. The uppermost 400pl was then removed and mixed with 20)il of TX114 in a clean eppendorf. This was layered over the original sucrose cushion which was warmed -to 30^C and spun for 10 sec at high speed. The uppermost aqueous layer was removed and stored as TX114 insoluble proteins (or TX114 supernatant).

lml of wash 1 <500mM KC1, lOmM Tris.HCl pH 7.2) was added to the TX114 fraction. The tube was whirlimixed, warmed to 30°C for 3 min until cloudy, cooled on ice for 3 min until clear, and warmed again to 30oC for 3 min until cloudy. The mixture was spun for 10 sec and the aqueous phase removed and discarded. This washing procedure was repeated using lOmM Tris.HCl pH 7.2. The final TX114 fraction was stored at -20°C or acetone precipitated.

To recondense TX114, 20g of uncondensed TX114 was added to 16mg of butylated hydroxytoluene and 980ml of buffer

(400mM KC1, lOmM Tris.HCl pH 7.4). The detergent was dissolved at 4°C then left overnight at 37°C. The aqueous phase was removed and replaced with fresh buffer and the method repeated. After the third recondensation the TX114 layer was removed and stored at 4°C.