GDP binding to brown-adipose-tissue mitochondria was decreased in obese Zucker rats. Adrenalectomy restored both GDP binding and serum tri-iodothyronine of obese rats to values observed in lean rats. The effects of adrenalectomy on GDP binding and serum tri-iodothyronine were reversed by corticosterone. Decreasing food intake had no effect on brown-adipose-tissue GDP binding in obese rats. Young (5-week-old) obese rats showed a normal increase in brown-adipose-tissue mitochondrial GDP binding after housing at 4 degrees C for 7 days, but this response was attenuated in 10-week-old obese rats. Overfeeding with sucrose increased brown-adipose-tissue thermogenesis in lean, but not in obese, rats. After adrenalectomy, overfeeding with sucrose enhanced brown-adipose-tissue mitochondrial GDP binding in obese rats.

The behaviour of heavy subcellular particles from rat liver during centrifugation in an A-XI1 zonal rotor has been studied with the help of enzymic markers and light and electron microscopy. A simple and reproducible method, not entailing hypotonic conditions, was developed for the isolation of plasma membrane fragments from liver homogenates, after study of factors affecting the yield and purity of such fragments. The livers were perfused with warm 0.25 M sucrose-5 mM NaHCO,, pH 7.5. Homogenisation and resuspension of the nuclear fraction were carried out in the same medium. Zonal centrifugation gave a band of plasma membrane fragments which showed a 14-fold increase in 5'-nucleotidase specific activity over the homogenate. This purification was increased to 30-fold by vigorous rehomogenisation in sucrose of density 1.19 followed by flotation of the plasma membrane fragments. These were now almost free of succinatedehydrogenase activity although still possessing some glucose-6-phosphatase and acid B-glycerophosphatase activity.We have already described the pattern obtained after centrifuging in a zonal A-XI1 rotor a crude nuclear fraction prepared from rat liver homogenised in 0.08M sucrose [l]. Further work has been prompted by the discovery that the requisite release of plasma membrane fragments from nuclei resulted from inadvertent gentle agitation of the homogenised material while it was being transported between this and a collaborating laboratory. We now describe studies on this release which have led to a simple method for preparing plasma membrane fragments with avoidance of a lytic step.The activity of 5'-nucleotidase can be visualised cytochemically and appears to be present only in the plasma membranes of the parenchymal cell [2,3]. Whilst we agree that a small amount of non-sedimen- After gentle bomogenisation, the 5'-nucleotidase activity of rat liver is divided almost equally between the nuclear and microsomal fractions prepared by differential centrifugation [lo]. The large sheets of plasma membrane that are responsible for the nucleotidase activity in the nuclear fraction can be purified by isopycnic centrifugation on sucrose gradients providing that the membranes can be released from the clumps of nuclei. Several methods have been used to obtain this release and to separate the sheets of plasma membrane from the other elements such as mitochondria and endoplasmic reticulum fragments which contaminate the nuclear fraction. I n the method of Neville [ll] and its widely used adaptation by Emmelot and his colleagues [12], a preliminary homogenisation in a hypotonic medium (1 mM sodium bicarbonate) is followed by flotation of the plasma membrane from dense sucrose into a sucrose gradient. Amongst recent methods, that used by Song and his colleagues [I31 to prepare bile

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