Summary
Antisera were raised to cholinergic presynaptic plasma membranes and synaptic vesicles isolated from the electric organ of Torpedo marmorata and tested by immunochemical and immunohistochemical methods. The antisera responded to many antigens not specific to nerve endings, but it was possible to eliminate these antibodies by means of simple absorption procedures with fractions containing the unwanted antigens. After absorption, staining of thin sections of electric organ by immunofluorescence was limited to the region of nerve endings in the tissue.
The remaining antibodies responded in the case of the plasma membrane antisera predominantly to a 33,000 molecular-weight polypeptide and a chloroform/methanol-soluble antigen. In cross reactivity studies it was found that this antiserum not only stains cholinergic nerve endings in Torpedo but also those in mammalian tissue. The antigen responsible for the cross reactivity is restricted to the chloroform/methanol-soluble material.
The vesicle antiserum labels cholinergic nerve endings in mammalian tissue as well; the relevant antigen in this case is different from the one described above and is likely to be a glycosaminoglycan. The antisera provide valuable markers for cholinergic nerve terminals. In addition, the vesicle antiserum may now be used to study axonal transport and the life cycle of this organelle in the cholinergic neurone.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- SDS:
-
sodium dodecyl sulphate
- PAGE:
-
polyacrylamide gel electrophoresis
- EGTA:
-
ethylenebis (oxoethylenenitrilo) tetra-acetic acid
- MW:
-
apparent molecular weight
References
Bjerrum OJ, Bøg-Hansen TC (1976) Immunochemical gel precipitation techniques in membrane studies. In: Maddy AH (ed) Biochemical analysis of membranes. Chapman and Hall, London, pp 378–426
Blomberg F, Perlman P (1971) Immunochemical studies of detergent-soluble nucleoside phosphatases in rat liver plasma membranes. Biochim Biophys Acta 233:53–60
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Anal Biochem 72:248–254
Brogren C-H, Bøg-Hansen TC (1975) Enzyme characterisation in quantitative immunoelectrophoresis. An enzymological study of human serum esterases. Scand J Immunol 4: Suppl 2:37–51
Dowe GHC, Kilbinger A, Whittaker VP (1980) Isolation of cholinergic synaptic vesicles from the myenteric plexus of guinea-pig small intestine. J Neurochem 35:993–1003
Dowdall MJ, Zimmermann H (1976) The preparation and properties of “nerve terminal sacs” from the electric organ of Torpedo. Exp Brain Res 24:8–9
Dowdall MJ, Zimmermann H (1977) The isolation of pure cholinergic nerve terminal sacs (T-sacs) from the electric organ of juvenile Torpedo. Neuroscience 2:405–421
Jones RT, Walker JH, Richardson PJ, Fox GQ, Whittaker VP (1981) Immunohistochemical localisation of cholinergic nerve terminals. Cell Tissue Res 218:355–373
Klingenberg M (1976) The ADP-ATP carrier in mitochondrial membranes. In: Martonose AN (ed) The enzymes of biological membranes: Membrane transport. Plenum Press, New York 3:383–438
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond) 227:680–685
Laskey RA, Mills AD (1975) Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem 56:335–341
Luqmani YA (1981) Nucleotide uptake by isolated cholinergic synaptic vesicles: evidence for a carrier of adenosine 5′-triphosphate. Neuroscience, in press
Mayer RJ, Walker JH (1980) Immunochemical methods in the biological sciences: Enzymes and proteins. Academic Press, London, p 118
Ohsawa K, Dowe GHC, Morris SJ, Whittaker VP (1979) The lipid and protein content of cholinergic synaptic vesicles from the electric organ of Torpedo marmorata purified to constant composition: Implications for vesicle structure. Brain Res 161:447–457
O'Farrel PH (1975) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 246:6328–6334
Rice RH, Means GE (1971) Radioactice labelling of proteins in vitro. J Biol Chem 246:881–832
Richardson PJ (1981) Quantitation of cholinergic synaptosomes from guinea pig brain. J Neurochem 37:258–260
Richardson PJ, Walker JH, Jones RT, Whittaker VP (1981) Identification of a cholinergic antigen Chol-1 as a ganglioside. J Neurochem: accepted for publication
Schaffner W, Weissmann C (1973) A rapid, sensitive and specific method for the determination of protein in dilute solution. Anal Biochem 56:502–514
Stadler H, Tashiro T (1979) Isolation of synaptosomal plasma membranes from cholinergic nerve terminals and a comparison of their proteins with those of synaptic vesicles. Eur J Biochem 101:171–178
Stadler H, Whittaker VP (1978) Identification of vesiculin as a glycosaminoglycan. Brain Res 153:408–413
Tashiro T, Stadler H (1978) Chemical composition of cholinergic synaptic vesicles from Torpedo marmorata based on improved purification. Eur J Biochem 90:479–487
Walker JH, Betts SA, Manning R, Mayer RJ (1976) Absorption of antisera for studies on specific enzyme turnover. Biochem J 159:355–362
Weeke B (1973a) Rocket immunoelectrophoresis. Scand J Immunol 4:Suppl 1:37–46
Weeke B (1973b) Crossed immunoelectrophoresis. Scand J Immunol 4:Suppl 1:47–56
Whittaker VP (1973) The biochemistry of synaptic transmission. Naturwissenschaften 60:281–289
Whittaker VP, Zimmermann H (1976) The innervation of the electric organ of Torpedinidae: A model cholinergic system. In: Malins CD, Sargent JR (eds) Biochemical and biophysical perspectives in marine biology. Academic Press, New York, Vol 3, pp 67–116
Zimmermann H (1979) Vesicle recycling and transmitter release. Neuroscience 4:1773–1804
Author information
Authors and Affiliations
Additional information
Enzymes. Na+, K+-activated ATPase (EC 3.6.1.3); acetylcholine esterase (EC 3.1.1.7); choline acetyl-transferase (EC 2.3.1.6)
Rights and permissions
About this article
Cite this article
Walker, J.H., Theresa Jones, R., Obrocki, J. et al. Presynaptic plasma membranes and synaptic vesicles of cholinergic nerve endings demonstrated by means of specific antisera. Cell Tissue Res. 223, 101–116 (1982). https://doi.org/10.1007/BF00221502
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00221502