Neurochemical Research, Vol. 28, Nos. 3/4, April 2003 (© 2003), pp. 645–651
Palmitoylcarnitine Modulates Palmitoylation of Proteins: Implication for Differentiation of Neural Cells* Dorota Szczepankowska1 and Katarzyna A. Nal e˛cz1,2 √
(Accepted August 12, 2002)
[3H]Palmitic acid accumulates in neuroblastoma NB-2a cells, being incorporated in lipids (90%) and proteins (10%) fractions. Addition of palmitoylcarnitine, known to modulate activity of protein kinase C and to promote differentiation of neurons, was observed to decrease incorporation of palmitic acid to sphingomyelin, phosphatidylserine, and phosphatidylcholine, with a parallel increase of palmitic acid bound to proteins through a thioester bond (palmitoylation). In the presence of palmitoylcarnitine, one of the palmitoylated proteins expressed at growing neural cones, GAP-43, was observed to co-localize with caveolin-1, what was correlated with the beginning of differentiation. A new function of palmitoylcarnitine in controlling palmitoylation of proteins and their targeting to cholesterol-rich domains has been proposed.
KEY WORDS: Palmitoylcarnityne; protein palmitoylation; caveolin; neuroblastoma.
additional role for carnitine has been proposed, namely control and modulation of acylCoA/CoASH pools in various compartments of the cell (2). Although in adult brain glucose and lactate are the main energetic substrates (3) and the level of fatty acid -oxidation is relatively low in neurons (4), carnitine accumulates in the brain (5). It is transported through the blood-brain barrier, released from the brain microcapillary endothelial cells as free carnitine (6,7), and taken up by neural cells (8,9). Carnitine and its esters were successfully applied as pharmacological agents for treatment of chronic degenerative diseases of senile brain (10) and for slowing down the progression of mental deterioration in Alzheimer’s disease (11). The pioneering works of Professor Stanislav Tuˇcek and his co-workers allowed us to understand the mechanism leading to beneficial effects of acetylcarnitine administration, because it was demonstrated that acetylcarnitine can be a source of acetyl groups for acetylcholine synthesis (12–15), moreover, being a better precursor than citrate (16–18). It was also
INTRODUCTION Carnitine (4-N-trimethylammonium-3-hydroxybutyric acid) is known to be involved in peripheral tissues in a process of long-chain fatty acids transfer from the cytosol to the mitochondrial matrix, where these acids are further metabolized. The pathway for carnitine-dependent transport of fatty acids (so called “carnitine shuttle”) consists of several enzymes synthesizing acylcarnitine derivatives on the cytosolic side of the outer mitochondrial membrane, the carnitine carrier in the inner mitochondrial membrane, and the enzymes transferring the acyl moieties to CoASH inside mitochondria (for review, see 1). Because carnitine acyltransferases use acylCoA as substrates, an * Special issue dedicated to Dr. Stanislav Tuˇcek. 1 Nencki Institute of Experimental Biology, 3 Pasteur Street, 02-093 Warszawa, Poland. 2 Address reprint requests to: Tel: ⫹48-22-6686216; Fax: ⫹48-228225342; E-mail:
[email protected]
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646 shown that, upon depletion of acetylcholine content, synthesis of this neurotransmitter was stimulated on addition of glucose, acetylcarnitine, or carnitine itself, leading to a conclusion that all these compounds could increase the availability of acyCoA (19–21). Our studies on the reconstituted carnitine carrier isolated from brain mitochondria demonstrated that carnitine exchange was strongly inhibited by short-chain acylcarnitines, mainly acetylcarnitine (22), therefore it was postulated that the main function of this carrier is export of acetylcarnitine from matrix to cytoplasm, thus promoting acetylcholine synthesis, a hypothesis confirmed in the experiments on acetylcholine synthesis in the presence of glucose, carnitine, and choline in isolated neurons and neuroblastoma cells (23,24). Carnitine, when accumulated in neurons, becomes a substrate of several carnitine acyltransferases, what, apart from the synthesis of acetylcarnitine, leads also to the synthesis of long-chain acylcarnitines, which constitute about 16%–20% of all the carnitine derivatives in cerebral cortex neurons (24,25). Their accumulation, mainly in the form of palmitoylcarnitine, can reach, however, even 60%–70% in neuroblastoma NB-2a cells (9), and this high content was correlated with stimulation of cell differentiation and inhibition of protein kinase C activity (26,27). An increased palmitoylcarnitine content in NB-2a cells was further correlated with diminished phosphorylation of GAP-43 (named also B-50, F1, pp45, neuromodulin) (28), a protein involved in neuronal development, neuroplasticity, and neurotransmission and located at the cross-road of several signaling pathways (29). Moreover, under conditions leading to increased intracellular content of palmitoylcarnitine, a higher amount of GAP43 was detected in the membrane fraction (28). Because GAP-43 is known to be attached to the membrane due to its palmitoylation (30) and many palmitoylated proteins involved in signal transduction have been reported to be localized in cholesterol-rich domains (31), so-called rafts, the present study has focused on a possibility of regulation by palmitoylcarnitine of palmitoylation of proteins and localization of GAP-43. EXPERIMENTAL PROCEDURE Protein Palmitoylation. Neuroblastoma NB-2a cells were grown in 10% fetal bovine serum and 90% Minimum Essential Medium with Earle’s salts, as described elsewhere (9). The cell monolayer (106 cells, ⬃1 mg protein) was washed with 0.005% bovine serum albumin (BSA) in phosphate buffered saline (PBS). Palmitic acid [9,10-3H] of a specific activity 60 Ci/mmol was 10fold concentrated by evaporation of ethanol (32). Palmitoylcarnitine
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was dissolved as described in (26). Cells were labeled at 37°C with 1 mCi of palmitic acid in PBS (final concentration 3.4 M) either in the absence or presence of 100 M palmitoylcarnitine, for the time indicated in the figure legends. After removal of the incubation medium they were harvested by scrapping in PBS and washed with ice-cold PBS by centrifugation. The pellet was suspended in 150 mM NaCl, 1% Triton X-100, 50 mM Tris-Cl, pH 7.4, supplemented with proteases’ inhibitors (chymostatin, leupeptin, antipain, pepstatin, 20 g/ml each) and homogenized by passing through a 25G needle. After removal of nuclei by centrifugation at 1000 ⫻ g for 10 min, the supernatant was subjected to protein precipitation with methanol and chloroform according to Wessel and Flügge (33). The supernatant was taken for lipid analysis, whilst the pellet was dissolved in 20% sodium dodecyl sulfate (SDS) and subjected to protein estimation (34) and to separation by polyacrylamide gel electrophoresis on 12% gel in the presence of SDS, according to Laemmli (35), with the omission of -mercaptoethanol. To determine the amount of palmitic acid covalently bound through thioester bond, the protein bands stained with Coomassie brilliant blue were excised from parallel lanes and treated either with methanol (total incorporation) or with 0.2 M KOH in methanol (sample after hydrolysis of thioester bond) (36,37). The level of palmitoylation was quantified from the calculated difference in radioactivity. Western Blot Analysis. After separation of proteins by electrophoresis, they were electrotransferred to the nitrocellulose membrane in 192 mM glycine, 20% methanol, 0.05% SDS, and 25 mM Tris at pH 8.3 for 70 min at 400 mA. The membranes were blocked with 4% BSA and 0.2% Tween 20, and exposed to the monoclonal anti-GAP-43 antibody. Further washing, exposure to secondary antibody, and the chemiluminescence detection, after exposure to the Hyper EXL film, were done according to Amersham protocol for the ECL Kit (27). Separation of Lipids. The supernatant after protein precipitation was subjected to thin-layer chromatography in chloroformmethanol-water (65:25:4, v/v) (38). The position of various lipids was defined by comparison with the respective markers run in parallel and visualized by iodine. The amount of radioactive palmitate incorporated into lipids was quantified by scratching of the silica gel and counting the radioactivity. Immunocytochemistry. The cells were fixed with 4% paraformaldehyde in PBS, rinsed four times with PBS, and nonspecific sites were blocked with PBS containing 2.5% BSA and 50% goat serum. Cells were incubated overnight with primary antibodies raised against GAP-43 and caveolin-1 at final dilution of 1:100 in 2.5% BSA, 50% goat serum in PBS (4°C). After four washes with PBS, the cells were incubated with fluorochrome-conjugated secondary antibodies at a final dilution of 1:100 for 1 h and rinsed four times with PBS. The samples were mounted in Kaiser’s glycerol gelatin. Scanning in the confocal Zeiss microscope was performed with argon and HeNe lasers, with illumination and emission for fluorescein isothiocyanate (FITC) 470 nm and 525 nm and tetramethylrhodamine isothiocyanate (TRITC) 525–540 nm and 625 nm, respectively. The Laser Scanning Microscope LSM 510 program was used to analyze the images. Materials. Palmitic acid [9,10-3H] was from American Radiolabeled Chemicals (St. Louis, MO). Palmitoylcarnitine, protease inhibitors, molecular weight markers for gel electrophoresis, markers for separation of lipids, anti-rabbit IgG TRITC-conjugate, antimouse IgG FITC-conjugate, and mouse monoclonal anti-GAP-43 (clone GAP-7B10) were from Sigma (St. Louis, MO, USA). Rabbit polyclonal anti-caveolin-1 (N-20) antibody was delivered by Santa Cruz Biotechnology. Hyperfilm TM-MP, Hybond ECL nitrocellu-
Palmitoylcarnitine and Protein Palmitoylation lose membrane, and ECL Kit were purchased from Amersham. Components of cell culture medium were from Life Technologies. Thin-layer chromatography silica gel 60 aluminium sheets were from Merck. All other reagents were of analytical grade.
RESULTS Neuroblastoma NB-2a cells accumulate palmitic acid. As presented in Fig. 1, this process was observed to increase with time, reaching maximal values at 30 and 60 min and to decrease slightly at longer times.
Fig. 1. Time dependence of palmitic acid incorporation into protein and lipid fraction in neuroblastoma NB-2a cells. The incorporation of palmitic acid [9,10-3H] was measured as described in Experimental Procedure. A, Total incorporation (䊲), incorporation into lipids (䊱), and in the protein fraction (䊏). The results present the means from three independent experiments. B, Incorporation of palmitic acid in total protein fraction. The results represent the mean ⫾ SD from three independent experiments.
647 Accumulation of palmitic acid in the lipid fraction corresponds to 90% of the total radioactivity measured in the cells. To have enough of radioactive compound accumulated in the cells for further, more detailed analysis, a time of 60 min has been chosen, which corresponded to the highest binding of palmitic acid to the protein fraction (Fig. 1B). Administration of palmitoylcarnitine doubled the amount of radioactivity detected in both lipid and protein fractions (not shown). It has to be noted, however, that in the presence of this long-chain acylcarnitine, palmitic acid remained mainly in its free form (Fig. 2, upper panel) and a significant decrease of its incorporation in such lipids as sphingomyelin, phosphatidylserine, and phosphatidylcholine was observed. This effect was the most dramatic in case of phosphatidylcholine, the synthesis of which was inhibited by 90% in the presence of palmitoylcarnitine. Palmitic acid was found to bind covalently through a KOH-sensitive thioester bond to several proteins. In the presence of palmitoylcarnitine, palmitoylation of several proteins
Fig. 2. Effect of palmitoylcarnitine on lipid synthesis and protein palmitoylation. NB-2a cells were incubated with palmitic acid [9,10-3H] either in the absence (filled bars) or presence (open bars) of 100 M palmitoylcarnitine. Upper panel: Chromatogram of lipid separation, where the positions of following markers have been indicated: SPM, sphingomyelin; PS, phosphatidylserine; PC, phosphatidylcholine; PALM, palmitic acid. Lower panel: Protein separation after polyacrylamide gel electrophoresis. The numbers (in kD) indicate positions of the following marker proteins: 116, -galactosidase; 97, phosphorylase-b; 66, bovine serum albumin; 55, glutamate dehydrogenase; 45, ovalbumin; 36, glyceraldehyde-3phosphate dehydrogenase; 29, carbonic anhydrase; 14.2, bovine ␣lactalbumin. The panels represent the means ⫾ SD from three independent experiments and the results were normalized to 1 mg of cellular protein.
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648 was increased (Fig. 2B), the strongest effect being observed in case of proteins in the range of 25–30 and about 50 kD. Many proteins are known to undergo palmitoylation, a process known to anchor them in the plasma membrane, moreover, frequently in cholesterol-rich domains (31). One of the palmitoylated proteins involved in the process of neuronal differentiation is GAP-43. As analyzed by Western blot analysis (Fig. 3), anti GAP-43 antibodies recognize mainly a band of 43 kD, which could correspond to the group of proteins migrating with Rf of 0.35 during electrophoretic separation. It has to be added that GAP-43, although its molecular weight is 23 kD, is also known to migrate as a band of Mr about 43,000 – 46,000 in the polyacrylamide gels (39). Because palmitoylcarnitine was observed to promote differentiation of NB-2a cells (26) and transfer of GAP-43 to the membrane fraction (28), the effect of this acylcarnitine on localization of GAP43 was further studied. As presented in Fig. 4, both caveolin and GAP-43 are expressed in NB-2a cells and some points with concentrated localization of either protein could have been observed (panels A1, A2), although there is no co-localization. Addition of BSA with ethanol, a medium usually applied to suspend palmitoylcarnitine, stimulated slightly expression of both proteins (panels B1, B2). Palmitoylcarnitine itself promotes concentration of GAP-43 in certain areas of the plasma membrane that look like the beginning of growing cone, the co-localization of both proteins could be detected (panel C3). Palmitic acid itself promotes slightly differentiation and appearance of GAP43 at the plasma membrane rather in broad areas
Fig. 3. Detection of GAP-43 in neuroblastoma NB-2a cells. Proteins from neuroblastoma NB-2a cells were isolated by electrophoresis and either stained with Commassie blue (lane a) or electrotransferred and subjected to Western blot analysis with anti-GAP-43 antibodies (lane b), as described in Experimental Procedure. The numbers (in kD) indicate positions of ovalbumin (45) and glyceraldehyde-3-phosphate dehydrogenase (36).
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(panel D2). When both palmitic acid and palmitoylcarnitine were added together, GAP-43 was detected at the concentrated domains, moreover, very often colocalized with caveolin-1 (panels E2, E3).
DISCUSSION Accumulation of palmitic acid in neuroblastoma NB-2a cells was optimal at 30–60 min, an observation reported already for other cells and tissues (32). It should be notified that the transformed NB-2a cells use glycolysis as the main energy supplying pathway and the loss of radioactivity due to -oxidation is most probably low, a conclusion drawn from almost a 10-fold higher accumulation of palmitate in these cells when compared with fibroblasts 3T3 (not shown). This observation is also with agreement with capability of NB-2a cells to accumulate high amounts of palmitoylcarnitine (9,26). A prevailing incorporation of palmitic acid in the lipid fraction already has been reported for cultured cells and the brain tissue (32); however, what was quite surprising was the observed decrease of radioactivity in certain lipids in the presence of palmitoylcarnitine. It remains obscure if this phenomenon is due to a replacement of palmitic acid by a palmitoyl moiety from palmitoylcarnitine at the same level of lipid synthesis or if it reflects a real decrease of lipids synthesis. Taking into account co-localization of GAP43 and caveolin, one could have been expecting that administration of palmitoylcarnitine increased synthesis of sphingomyelin, one of the main lipids in rafts, especially that such a dramatic decrease of palmitic acid incorporation in phosphatidylcholine was detected. Because the amount of sphingomyelin decreased, we would rather postulate that the presence of palmitoylcarnitine does not increase the amount of cholesterolrich domains, it rather leads to their reorganization, due to their dynamic character (40). Palmitic acid was also shown to bind through a thioester bond to several proteins. It seems too preliminary to speculate about their identity; it has to be emphasized, however, that the biggest differences between proteins palmitoylated in the presence and absence of palmitoylcarnitine were detected for proteins of molecular weight in the ranges 25–30 kD, as well as slightly below 55 kD and at 66 kD. Among the known palmitoylated proteins involved in signal transduction, one can find in the higher molecular weight range such important proteins as tyrosine kinases (Fyn, Lyn) and serpentine receptors. On the other hand, palmitoylated proteins at lower molecular weight range can contain
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Fig. 4. Localization of GAP-43 and caveolin-1 in neuroblastoma NB-2a cells. Cells were fixed and immunocytochemistry was performed as described in Experimental Procedure. Detection of caveolin-1 (left panels, index 1), GAP-43 (middle panels, index 2) or both protein (right panels, index 3) was performed after exposure of the cells for 1 h to medium without any additions (A), BSA (B), 100 M palmitoylcarnitine (C), 3.4 M palmitic acid (D) and palmitoylcarnitine with palmitic acid (E).
650 Ras proteins (H- and K-ras), as well as caveolin-1 and monomeric GAP-43. The latter protein is, however, mainly migrating as a band of Rf ⫽ 0.35 (Fig. 3). This band was observed to incorporate 60% more of [3H]palmitate in the presence of palmitoylcarnitine. It has to be stressed that we expect a presence of more than one protein in each band excised from polyacrylamide gel, and the identity of palmitoylated proteins should be further confirmed in experiments applying immunoprecipitation of each of the mentioned proteins and quantification of the change of its palmitoylation status. Although palmitoyl moiety in palmitoylcarnitine is already at the activated state, like in palmitoylCoA, it does not seem to be a donor of substrate for the process of protein pamitoylation. If this were the case, one would observe a decrease in radioactivity incorporation in proteins from labeled palmitic acid. It seems more probable that accumulation of palmitoylcarnitine in the cells directs palmitic acid more to proteins than to lipids. An observed co-localization of GAP-43 and caveolin, upon conditions stimulating palmitoylation of proteins, indicates that GAP-43 is attached, as many other palmitoylated proteins, to cholesterol-rich domains. Because GAP-43 is known to bind calmodulin (41) and to regulate G0 protein in neural growth cones (42), it seems that regulation of its cellular localization by palmitoylcarnitine gives an additional explanation of the observed effects of carnitine derivatives on functioning of neural cells.
ACKNOWLEDGMENTS This study was supported by a Polish State Committee for Scientific Research grant No. 6 PO4A 053 16 (0373/PO4/99/16). The authors are extremely grateful to Professor Jerzy Moraczewski from the Faculty of Biology at the University of Warsaw for the access to a confocal microscope.
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5. Bresolin, N., Freddo, L., Vergani, L., and Angelini, C. 1982. Carnitine, carnitine acyltransferases, and rat brain function. Exp. Neurol. 78:285–292. 6. Mroczkowska, J. E., Galla, H. J., Nal e˛cz, M. J., and Nal e˛cz, K. A. 1997. Evidence for an asymmetrical uptake of L-carnitine in the blood-brain barrier in vitro. Biochem. Biophys. Res. Commun. 241:127–131. 7. Mroczkowska, J. E., Roux, F. S., Nal e˛cz, M. J., and Nal e˛cz, K. A. 2000. Blood-brain barrier controls carnitine level in the brain: A study on a model system with RBE4 cells. Biochem. Biophys. Res. Commun. 267:433–437. 8. Wawrze´nczyk, A., Sacher, A., Mac, M., Nal e˛cz, M. J., and Nal e˛cz, K. A. 2001. Transport of L-carnitine in isolated cerebral cortex neurons. Eur. J. Biochem. 268:2091–2098. 9. Nal e˛cz, K. A., Korzon, D., Wawrze´nczyk, A., and Nal e˛cz, M. J. 1995. Transport of carnitine in neuroblastoma NB-2a cells. Arch. Biochem. Biophys. 322:214–220. 10. Bonavita, E. 1986. Study of the efficacy and tolerability of L-acetylcarnitine therapy in the senile brain. Int. J. Clin. Pharmacol. Ther. Toxicol. 24:511–516. 11. Spagnoli, A., Lucca, U., Menasce, G., Bandera, L., Cizza, G., Forloni, G., Tettamanti, M., Frattura, L., Tiraboschi, P., Comelli, M., et al. 1991. Long-term acetyl-L-carnitine treatment in Alzheimer’s disease. Neurology 41:1726–1732. 12. Tuˇcek, S. 1985. Regulation of acetylcholine synthesis in the brain. J. Neurochem. 44:11–24. 13. Doleˇzal, V. and Tuˇcek, S. 1981. Utilization of citrate, acetylcarnitine, acetate, pyruvate and glucose for the synthesis of acetylcholine in rat brain slices. J. Neurochem. 36:1323–1330. 14. Tuˇcek, S. 1990. The synthesis of acetylcholine: Twenty years of progress. Prog. Brain Res. 84:467–477. 15. Tuˇcek, S., Riˇceny, J., and Doleˇzal, V. 1990. Advances in the biology of cholinergic neurons. Adv. Neurol. 51:109–115. 16. Tuˇcek, S., Doleˇzal, V., and Sullivan, A. C. 1981. Inhibition of the synthesis of acetylcholine in rat brain slices by (⫺)-hydroxycitrate and citrate. J. Neurochem. 36:1331–1337. 17. Tuˇcek, S. and Cheng, S. C. 1974. Provenance of the acetyl group of acetylcholine and compartmentation of acetyl-CoA and Krebs cycle intermediates in the brain in vivo. J. Neurochem. 22:893–914. 18. Tuˇcek, S. and Cheng, S. C. 1970. Precursors of acetyl groups in acetylcholine in the brain in vivo. Biochim. Biophys. Acta 208:538–540. 19. Riˇcny, J., Tuˇcek, S. and Novakova, J. 1992. Acetylcarnitine, carnitine and glucose diminish the effect of muscarinic antagonist quinuclidinyl benzilate on striatal acetylcholine content. Brain Res. 576:215–219. 20. Tuˇcek, S., Riˇcny, J., and Doleˇzal, V. 1982. Acetylcoenzyme A and the control of the synthesis of acetylcholine in the brain. Acta Neurobiol. Exp. 42:59–68. 21. Tuˇcek, S. 1983. Acetylcoenzyme A and the synthesis of acetylcholine in neurones: Review of recent progress. Gen. Physiol. Biophys. 2:313–324. 22. Kami´nska, J., Nal e˛cz, K. A., and Nal e˛cz, M. J. 1995. Mechanism of carnitine transport catalyzed by carnitine carrier from rat brain mitochondria. Acta Neurobiol. Exp. 55:1–9. 23. Wawrze´nczyk, A., Nal e˛cz, K. A., and Nal e˛cz, M. J. 1994. Synergistic effect of choline and carnitine on acetylcholine synthesis in neuroblastoma NB-2a cells. Biochem. Biophys. Res. Commun. 202:354–359. 24. Wawrze´nczyk, A., Nal e˛cz, K. A., and Nal e˛cz, M. J. 1995. Effect of externally added carnitine on the synthesis of acetylcholine in rat cerebral cortex cells. Neurochem. Int. 26:635–641. 25. Wawrze´nczyk, A., Nal e˛cz, K. A., and Nal e˛cz, M. J. 1995. Effect of gamma-aminobutyric acid on the carnitine metabolism in neural cells. Biochem. Biophys. Res. Commun. 213: 383–388. 26. Nal e˛cz, K. A., Mroczkowska, J. E., Berent, U., and Nal e˛cz, M. J. 1997. Effect of palmitoylcarnitine on the cellular differentiation,
1. Kerner, J. and Hoppel, C. 2000. Fatty acid import into mitochondria. Biochim. Biophys. Acta 1486:1–17. 2. Ramsay, R. R. 2000. The carnitine acyltransferases: Modulators of acyl-CoA-dependent reactions. Biochem. Soc. Trans. 28:182–186. 3. Nehlig, A. and Pereira de Vasconcelos, A. 1993. Glucose and ketone bodies utilization by the brains of neonatal rats. Prog. Neurobiol. 40:163–221. 4. Warshaw, J. B. and Terry, M. L. 1976. Cellular energy metabolism during fetal development: VI. Fatty acid oxidation by developing brain. Dev. Biol. 52:161–166.
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√
√
√
√
√
√
√
REFERENCES
√
√
√
√
√
√
√
√
√
√
Palmitoylcarnitine and Protein Palmitoylation
27. 28.
proliferation and protein kinase C activity in neuroblastoma nb-2a cells. Acta Neurobiol. Exp. 57:263–274. Sobiesiak-Mirska, J., Nal e˛cz, M. J., and Nal e˛cz, K. A. 2003. Interaction of palmitoylcarnitine with protein kinase C in neuroblastoma NB-2a cells. Neurochem. Int. 42:45–55. Mizgalska, J. A., Berent, U., Mac, M., Oestreicher, A. B., De Graan, P. N. E., Gispen, W. H., Nal e˛cz, M. J., and Nal e˛cz, K. A. 1998. Accumulation of palmitoylcarnitine in neuroblastoma NB-2a cells affects the expression, phosphorylation and localization of B-50 protein. Neurosci. Res. Commun. 22: 73–82. Oestreicher, A. B., De Graan, P. N., Gispen, W. H., Verhaagen, J., and Schrama, L. H. 1997. B-50, the growth associated protein-43: Modulation of cell morphology and communication in the nervous system. Prog. Neurobiol. 53:627–686. Skene, J. H. and Virag, I. 1989. Posttranslational membrane attachment and dynamic fatty acylation of a neuronal growth cone protein, GAP-43. J. Cell Biol. 108:613–624. Simons, K. and Ikonen, E. 1997. Functional rafts in cell membranes. Nature 387:569–572. Patterson, S. I. and Skene, J. H. 1995. Inhibition of dynamic protein palmitoylation in intact cells with tunicamycin. Methods Enzymol. 250:284–300. Wessel, D. and Flügge, U. I. 1984. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal. Biochem. 138:141–143. Nal e˛cz, M. J., Nal e˛cz, K. A., and Azzi, A. 1986. Extraction, partial purification and reconstitution of a mixture of carriers from the inner mitochondrial membrane. Pages 67–75, in Azzi, A., √
√
√
29.
30. 31. 32. 33. 34.
651
√
√
35. 36.
√
37. 38.
39. 40.
41.
42.
Masotti, L., and Vecli, A. (eds.), Membrane Proteins: Isolation and Characterization. Springer-Verlag, Berlin. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. Slomiany, B. L., Takagi, A., Liau, Y. H., Jozwiak, Z., and Slomiany, A. 1984. In vitro acylation of rat gastric mucus glycoprotein with [3H]palmitic acid. J. Biol. Chem. 259:11997– 20000. Agrawal, H. C. and Agrawal, D. 1989. Effect of cycloheximide on palmitylation of PO protein of the peripheral nervous system myelin. Biochem. J. 263:173–177. Kates, M. 1986. Identification of individual lipids and lipid moieties. Pages 326–424, in Burdon, R. H. and Knippenberg, P. H. (eds.), Techniques of Lipidology: Isolation, Analysis and Identification of Lipids. Vol. 3. Elsevier Science B.V., Amsterdam. Benowitz, L. I. and Routtenberg, A. 1997. GAP-43: An intrinsic determinant of neuronal development and plasticity. Trends Neurosci. 20:84–91. Pralle, A., Keller, P., Florin, E. L., Simons, K., and Horber, J. K. 2000. Sphingolipid-cholesterol rafts diffuse as small entities in the plasma membrane of mammalian cells. J. Cell Biol. 148:997–1008. Wakim, B. T., Alexander, K. A., Masure, H. R., Cimler, B. M., Storm, D. R., and Walsh, K. A. 1987. Amino acid sequence of P-57, a neurospecific calmodulin-binding protein. Biochemistry 26:7466–7470. Strittmatter, S. M., Valenzuela, D., Kennedy, T. E., Neer, E. J., and Fishman, M. C. 1990. G0 is a major growth cone protein subject to regulation by GAP-43. Nature 344:836–841.
