2. Materials and Methods
Perfusion buffers
(a) buffered saline, with 10 mM potassium phosphate, pH 6.8, (b) buffered saline containing 0.5 mM MgCl2 and 0.5 mM MgS04, (c) buffered saline (100 ml) containing 66 mg collagenase, 80 mg hyaluronidase, and 2 g of albumin, (d) MSB, pH 6.9 buffer consisting of 0.1 Pipes, pH 6.9, 2.0 M glycerol, 1mM Mg acetate, 0.5 mM EGTA and mixture of protease inhibitors consisting of leupeptin, aprotinin and PMSF, (e) MSB buffer containing 0.2 % Triton X100, (f) 50 mM TRIS-HCl, pH 7.4 containing 0.25 M sucrose, 10 mM MgCl2 1 mM DTT,10 mg/ml leupeptin and 2 mM PMSF.
Perfusion of rat liver and isolation of hepatocytes
The abdomen of anestetized rat was open and liver cannulated and perfused with 50 ml of ice cold Hanks Balanced Salts (Sigma) without Ca2+containing 20 mg collagenase, type IV, 20 mg hyaluronidase, 1 g defatted albumin, and 3g of heparin. After initial perfusion, the liver was removed from animal abdomen, cut into slices and incubated with cold Hanks solution. Thus prepared slices were subjected to incubation in tissue incubator in 95% 02 and 5% C02 for 40 min, at 37°C. The slices were broken up with rubber policemen, incubated for additional 10 min and filtered through nylon mesh that separated single cells from larger debris. The cells were then centrifuged at 50xg for 2min, washed twice with the enzyme-free Hanks medium, twice with the Minimum Essential medium and counted in hemocytometer. Thus prepared cells were used for preparation of nuclei [28] subcellular organelles and cell cytosol [13-17] and lipid rafts [29]. In the experiments dedicated to the determination of lipid synthesis with and without cell cytosol, the preparations of intact nuclei were additionally rinsed with PBS and urea-PBS in order to remove the residual cytosolic proteins from the nuclear membranes. Thus prepared intact Nuclei (IN) were used for preparation of outer nuclear membrane (ONM) and the Inner Nuclear Membrane (INM). The DNAse treated nuclear matrix, the nuclear contents [28] was used in the set of experiments where instead of the CC, and the nuclear contents was checked for the phospholipids synthesizing activity. The synthesis of phosphatidylinositides and phospholipids was determined using radiolabeled [3H] inositol and arachidonate and [3H]choline [13-17]. The synthesis of transport vesicles (ER, Golgi transport vesicles) was performed in medium containing cytosol at concentration of 5 mg protein/ml of incubation mixture enriched with 50 mM ATP, 250 mM, 50 mM GTP, 5 mM creatine phosphate, 8.0 IU/ml creatine kinase, and where indicated 25 mg/ml RNase, 10 mM UDP-Glc and 10 mM palmitoyl CoA [13-18]. The same preparation of cytosol was used in the experiments with intact nuclei and nuclear membranes.
Isolation and separation of outer and inner nuclear membranes
One volume of the isolated hepatocytes or the IN was homogenized in 8 volumes of 10 mM potassium phosphate buffer, pH 6.8, 1.3 M sucrose and 1mM MgCl2 to rupture at least 80 % of nuclei [28]. The unbroken cells were removed by centrifugation at 50xg for 3 min, and the homogenate centrifuged for 15 min at 1000Xg. The soluble cellular material was saved for experiments evaluating its impact on membrane synthesis, and the nuclear pellet processed further [28]. The nuclear fractions were suspended in a minimum volume of buffered sucrose, adjusted to 2.2 M buffered sucrose with 2.4 M sucrose in the same buffer, followed by centrifugation at 100,000xg (27,000rpm in Beckman 45 Ti rotor) for 1h. The resultant pellet was suspended in 20 mM Tris-HCl, pH 7.5, centrifuged at 1000xg for 15 min, and the recovered nuclei suspended in a buffer (f) at concentration of 2 mg protein/ml. The preparation was then adjusted to 1% (w/v) with sodium citrate and incubated on ice with gentle stirring for 30 min and then centrifuged at 500xg for 15 min. The obtained supernatant contained the outer nuclear membranes ONM whereas the pellet contained the INM [28]. The pellet was suspended in buffer (f) at concentration of 5 mg/ml and digested with DNase 1 (250 mg/ml for 14 h at 4°C). The digested material was separated on sucrose gradient of 0.25-1.6- 2.4 M sucrose by centrifugation at 10,000xg for 2h. The inner nuclear membranes were recovered at 1.6 M sucrose boundary. The fraction of ONM was collected from citrate supernatant by centrifugation at 100,000xg for 20 min. The membrane pellet was suspended in buffer (f) and subjected to the same treatment as INM. The resultant 100 000Xg pellet of ONM was suspended in buffer (f). On the average, the preparation initiated with material obtained from two rat livers yielded 4.7 mg of inner and 3.1 mg outer nuclear membranes.
In the experiments employing IN, the incubation with CC followed by separation of the ONM and INM, the IN samples were subjected to additional sucrose gradient purification. The samples of IN were suspended in 40% buffered sucrose by adding 80% sucrose in 150 mM NaCl, 25 mM TRIS, pH 7.5 buffer and overlaid with 10-30% sucrose gradient in the same buffer and centrifuged at 29,000xg for 21 h. Following gradient centrifugation, the nuclei were recovered from 40 % bottom layer by diluting out sucrose with the buffer and centrifugation at 1,000xg for 10 min. The nuclear pellet was then suspended at a protein concentration of 2mg/ml in 1% citrate and proceeded with separation of outer and inner nuclear membranes. The material recovered from the gradient, was subjected to lipid extraction and SDS-PAGE. This treatment separated any cell membranes that were trapped with nuclei, and allowed us to determine whether incubation of IN with CC produced extranuclear radiolabeled membranes replacing ER membranes used in transport.
Cell surface and cell cytosol labeling with NIP
An aliquot of purified cells corresponding to 1 mg of cell membrane proteins or 1 mg of CC protein/ml of PBS, pH 7.4 was incubated with 10 μl 4-hydroxy-5-iodo-3-nitrophenyl acetate (NIP) in DMSO for 1h at 37°C. The excess of the reagent was removed on Sephadex G-25 column. The protein collected in three 1ml aliquots was used for incubation with individual lipids applied to microplates (ELISA) or with IN, ONM, INM or the respective lipid extracts. Where indicated the rat anti NIP IgG was also used. Protein recovery was calculated by multiplying the concentration of protein by the volume of the derivatized protein fraction recovered from the separation column. On the average, the concentration of NIP hapten was 1.0 x 10-5 mmoles/ml protein and 5.0 moles of NIP hapten/mol IgG. The absorbance of the derivatized protein was read at 280nm and 430nm.
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