928
BIOCHEMICAL SOCIETY TRANSACTIONS
Isolation of Nuclear-Pore Complexes from Triton X-100-Extracted Rat Liver Nuclear Envelope PETER MARSHALL and JAMES R. HARRIS Biomembrane Unit, Division of Biochemistry, North East London Polytechnic, Romford Road, London E l 5 4LZ, U.K. Previous reports have shown that the nuclear-pore complex is structurally more stable than the surrounding inner and outer nuclear membrane (Yo0 & Bayley, 1967; Gall, 1967; Harris, 1974, 1977; Harris & Marshall, 1977). Disruption of the nuclear envelope by ultrasonication has been used for isolating a nuclear-pore-complex fraction (Harris, 1977), but this was contaminated by adhering membrane and fragments of approximately the same mass as the pore complexes. Subsequently, the procedure has been modified by introducing Triton X-100, which removes the outer nuclear membrane and the lipid of the inner nuclear membrane, before ultrasonication. Electron microscopy, using negative staining with ammonium molybdate, and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis have been used to monitor the fractionation obtained. Rat liver nuclear envelope, in the form of intact nuclear ‘ghosts’ and large torn sheets, was prepared by the method of Harris & Milne (1974). This nuclear envelope (5-10mg of envelope protein) was suspended in 40ml of 2 % Triton X-100 in IOmM-Tris/HCI buffer (pH7.4) for 15min at 22°C. The Triton X-100-insoluble material was pelleted at 5000g,,. for 5min at 4°C in the Beckman JA-20 fixed-angle rotor, and subsequently washed three times with 40ml of IOmM-Tris/HC1 buffer (pH7.4). Negative staining reveals that the pelleted material is still in the form of intact nuclear ‘ghosts’ and large fragments, both of which show the presence of nuclear-pore complexes, but the outer nuclear membrane is absent. This loss of nuclear-envelope protein, as well as lipid, is also indicated by the deletion of specific polypeptide bands on sodium dodecyl sulphatel polyacrylamide-gel electrophoretograms of the Triton X-100-extracted material. The Triton X-100-extracted nuclear envelope (3-5 mg of protein/ml) was ultrasonicated for 5min in a bath-type ultrasonicator (Brenray Industries Ltd., Twickenham, Middx., U.K.) maintained at 2-4°C by the addition of ice, and monitored continuously with a digital thermometer. The partly clarified suspension was then centrifuged for 2min in the Beckman Microfuge (approx. 5000g,,.) and the pellet ultrasonicated again for 5min and centrifuged as before. Electron microscopy of the material after ultrasonication indicated that the Triton X-100-extracted nuclear envelope had broken down into a few large pieces of undisrupted material (comprising the bulk of the Microfuge pellets), and a large amount of finely fragmented material together with apparently free nuclear-pore complexes (comprising the Microfuge supernatants). Pooled Microfuge supernatants (1.0-2.0ml) were layered on a linear 0.25-1 .OOM-sucrose density gradient buffered with IOmM-Tris/HCI (pH7.4), and centrifuged at 30000rev./min (lOOOOOg,,.) for 1 h at 4°C in the MSE 3 x25ml swing-out rotor. The tube was then pierced and the contents were monitored by the U.V. absorption at 280nm. Fractions were dialysed against two 5-litre changes of IOmM-Tris/HC1 buffer (pH7.4) to remove the sucrose, and concentrated to approx. 400p1 in a pre-boiled length of Visking tubing surrounded by Aquacide 11 (Calbiochem, Bishops Stortford, Herts., U.K.). Negative staining of the gradient fractions revealed that the denser region of the sucrose gradient contained mainly undisrupted pieces of the Triton X-100-extracted nuclear envelope, and that with decreasing density there was a diminution of fragment size, with the bulk of the smaller fragments being present in the least-dense region. Between approx. 0 . 6 ~ and - 0.8 M-sucrose there was an enrichment with nuclear-pore complexes, which comprised most of the material in this fraction (see Fig. 1 ) . Although many of the nuclear-pore complexes appeared to be intact, others showed varying degrees of ultrastructural damage. Electrophoretically, some degree of protein fractionation was observed, but generally the gradient fractions appeared to contain most of the polypeptide bands of the Triton X-100-extracted nuclear envelope, and there did not appear to be a major enrichment of any polypeptide band in the fraction known to be 1979
583rd MEETING, CAMBRIDGE
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Fig. 1, Nuclear-pore-complex-rich fraction from a 0.25-1 .Owsucrose density gradient of ultrasonicated Triton X- 1 00-extracted rat liver nuclear envelope Negatively stained with 2 % ammonium molybdate (pH7.0). Magnification x 30000.
rich in nuclear-pore complexes. This suggests that a significant number of pore complexes may be disrupted by ultrasonication, the smaller breakdown products then being located in the less-dense gradient fractions. The nuclear-pore-complex-rich fraction nevertheless possesses a significantly different polypeptide composition from the initial Triton X-100-extracted nuclear envelope. Gall, J. G. (1 967) J . Cell B i d . 32, 391-400 Harris, J. R. (1974) Philos. Trans. R . Soc. London, Ser. B 268, 109-117 Harris, J. R. (1977) in Methodological Surveys, Vol. 6 1 Membranous Elements and Mocernent of Molecules: Techniques (Reid, E., ed.), pp. 245-250, Horwood, Chichester Harris, J. R. & Marshall, P. (1977) Micron 8, 217-219 Harris, J. R. & Milne, J. F. (1974) Biochent. Soc. Trans. 2, 1251-1253 Yoo, B. Y.& Bayley, S. T. (1967)J. Ultrastruct. Res. 18,651-660
The Fate of Liposomes in the Rat Small Intestine DAVID A. WHITMORE and KENNETH P. WHEELER Biochemistry Laboratory, School of Biological Sciences, University of Sussex, Brighton BNl 9QG, U.K. The oral administration of insulin entrapped in liposomes, or of insulin in the presence of ‘empty’ liposomes, produces a hypoglycaemic effect in rats (Patel & Ryman, 1976, 1977a). Entrapped insulin is protected from proteolytic digestion (Patel & Ryman, 1977b), but the reported absorption of intact liposomes from the gastrointestinal tract (Dapergolas & Gregoriadis, 1976) has not been confirmed (Patel & Ryman, 1977b). We have therefore studied the absorption of liposomes by the intestine in vitro, using a simple everted-sac preparation, and also examined the stability of liposomes in the presence of bile, protein and lipids.
Vol. 7
BIOCHEMICAL SOCIETY TRANSACTIONS
Isolation of Nuclear-Pore Complexes from Triton X-100-Extracted Rat Liver Nuclear Envelope PETER MARSHALL and JAMES R. HARRIS Biomembrane Unit, Division of Biochemistry, North East London Polytechnic, Romford Road, London E l 5 4LZ, U.K. Previous reports have shown that the nuclear-pore complex is structurally more stable than the surrounding inner and outer nuclear membrane (Yo0 & Bayley, 1967; Gall, 1967; Harris, 1974, 1977; Harris & Marshall, 1977). Disruption of the nuclear envelope by ultrasonication has been used for isolating a nuclear-pore-complex fraction (Harris, 1977), but this was contaminated by adhering membrane and fragments of approximately the same mass as the pore complexes. Subsequently, the procedure has been modified by introducing Triton X-100, which removes the outer nuclear membrane and the lipid of the inner nuclear membrane, before ultrasonication. Electron microscopy, using negative staining with ammonium molybdate, and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis have been used to monitor the fractionation obtained. Rat liver nuclear envelope, in the form of intact nuclear ‘ghosts’ and large torn sheets, was prepared by the method of Harris & Milne (1974). This nuclear envelope (5-10mg of envelope protein) was suspended in 40ml of 2 % Triton X-100 in IOmM-Tris/HCI buffer (pH7.4) for 15min at 22°C. The Triton X-100-insoluble material was pelleted at 5000g,,. for 5min at 4°C in the Beckman JA-20 fixed-angle rotor, and subsequently washed three times with 40ml of IOmM-Tris/HC1 buffer (pH7.4). Negative staining reveals that the pelleted material is still in the form of intact nuclear ‘ghosts’ and large fragments, both of which show the presence of nuclear-pore complexes, but the outer nuclear membrane is absent. This loss of nuclear-envelope protein, as well as lipid, is also indicated by the deletion of specific polypeptide bands on sodium dodecyl sulphatel polyacrylamide-gel electrophoretograms of the Triton X-100-extracted material. The Triton X-100-extracted nuclear envelope (3-5 mg of protein/ml) was ultrasonicated for 5min in a bath-type ultrasonicator (Brenray Industries Ltd., Twickenham, Middx., U.K.) maintained at 2-4°C by the addition of ice, and monitored continuously with a digital thermometer. The partly clarified suspension was then centrifuged for 2min in the Beckman Microfuge (approx. 5000g,,.) and the pellet ultrasonicated again for 5min and centrifuged as before. Electron microscopy of the material after ultrasonication indicated that the Triton X-100-extracted nuclear envelope had broken down into a few large pieces of undisrupted material (comprising the bulk of the Microfuge pellets), and a large amount of finely fragmented material together with apparently free nuclear-pore complexes (comprising the Microfuge supernatants). Pooled Microfuge supernatants (1.0-2.0ml) were layered on a linear 0.25-1 .OOM-sucrose density gradient buffered with IOmM-Tris/HCI (pH7.4), and centrifuged at 30000rev./min (lOOOOOg,,.) for 1 h at 4°C in the MSE 3 x25ml swing-out rotor. The tube was then pierced and the contents were monitored by the U.V. absorption at 280nm. Fractions were dialysed against two 5-litre changes of IOmM-Tris/HC1 buffer (pH7.4) to remove the sucrose, and concentrated to approx. 400p1 in a pre-boiled length of Visking tubing surrounded by Aquacide 11 (Calbiochem, Bishops Stortford, Herts., U.K.). Negative staining of the gradient fractions revealed that the denser region of the sucrose gradient contained mainly undisrupted pieces of the Triton X-100-extracted nuclear envelope, and that with decreasing density there was a diminution of fragment size, with the bulk of the smaller fragments being present in the least-dense region. Between approx. 0 . 6 ~ and - 0.8 M-sucrose there was an enrichment with nuclear-pore complexes, which comprised most of the material in this fraction (see Fig. 1 ) . Although many of the nuclear-pore complexes appeared to be intact, others showed varying degrees of ultrastructural damage. Electrophoretically, some degree of protein fractionation was observed, but generally the gradient fractions appeared to contain most of the polypeptide bands of the Triton X-100-extracted nuclear envelope, and there did not appear to be a major enrichment of any polypeptide band in the fraction known to be 1979
583rd MEETING, CAMBRIDGE
929
Fig. 1, Nuclear-pore-complex-rich fraction from a 0.25-1 .Owsucrose density gradient of ultrasonicated Triton X- 1 00-extracted rat liver nuclear envelope Negatively stained with 2 % ammonium molybdate (pH7.0). Magnification x 30000.
rich in nuclear-pore complexes. This suggests that a significant number of pore complexes may be disrupted by ultrasonication, the smaller breakdown products then being located in the less-dense gradient fractions. The nuclear-pore-complex-rich fraction nevertheless possesses a significantly different polypeptide composition from the initial Triton X-100-extracted nuclear envelope. Gall, J. G. (1 967) J . Cell B i d . 32, 391-400 Harris, J. R. (1974) Philos. Trans. R . Soc. London, Ser. B 268, 109-117 Harris, J. R. (1977) in Methodological Surveys, Vol. 6 1 Membranous Elements and Mocernent of Molecules: Techniques (Reid, E., ed.), pp. 245-250, Horwood, Chichester Harris, J. R. & Marshall, P. (1977) Micron 8, 217-219 Harris, J. R. & Milne, J. F. (1974) Biochent. Soc. Trans. 2, 1251-1253 Yoo, B. Y.& Bayley, S. T. (1967)J. Ultrastruct. Res. 18,651-660
The Fate of Liposomes in the Rat Small Intestine DAVID A. WHITMORE and KENNETH P. WHEELER Biochemistry Laboratory, School of Biological Sciences, University of Sussex, Brighton BNl 9QG, U.K. The oral administration of insulin entrapped in liposomes, or of insulin in the presence of ‘empty’ liposomes, produces a hypoglycaemic effect in rats (Patel & Ryman, 1976, 1977a). Entrapped insulin is protected from proteolytic digestion (Patel & Ryman, 1977b), but the reported absorption of intact liposomes from the gastrointestinal tract (Dapergolas & Gregoriadis, 1976) has not been confirmed (Patel & Ryman, 1977b). We have therefore studied the absorption of liposomes by the intestine in vitro, using a simple everted-sac preparation, and also examined the stability of liposomes in the presence of bile, protein and lipids.
Vol. 7
