The characterization of a membrane-bound protein carboxylmethylation system in brain
The membrane-bound component of the cerebral protein carboxylmethylation system, consisting of the membrane-bound enzyme protein carboxylmethyltransferase II (PCMT) and of selected membrane-bound methyl accepting proteins (MAP), is described. The cellular localization of this membrane-bound protein...
Gespeichert in:
Veröffentlicht in: | Neurochemistry international 1987, Vol.10 (2), p.155-166 |
---|---|
Hauptverfasser: | , , , |
Format: | Artikel |
Sprache: | eng |
Schlagworte: | |
Online-Zugang: | Volltext |
Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
Zusammenfassung: | The membrane-bound component of the cerebral protein carboxylmethylation system, consisting of the membrane-bound enzyme protein carboxylmethyltransferase II (PCMT) and of selected membrane-bound methyl accepting proteins (MAP), is described. The cellular localization of this membrane-bound protein carboxylmethylation system is shown to include, in addition to nerve cell bodies and purified synaptosomes, astrocytes and oligodendroglia. The membrane-bound nature of the protein carboxylmethylation system was investigated and these studies revealed a tight association which exposure to several detergents could only partially solubilize. The membrane-bound PCMT could be shown to undergo activation after treatment with Na-deoxycholate and CHAPs, while after its detergent-induced solubilization PCMT activation was observed after Na-deoxycholate, Nonidet P-40 and Lubrol-PX. Solubilization of the carboxylmethylation system in CHAPS appeared to be more effective at 0°C than at 25°C or 37°C. Detergent treatment was shown to be deleterious to the MAPs as PCMT substrates, particularly when the exposure was extended to more than 1 h. These observations prompted exposure of the brain membranes and of their Lubrol-PX and Nonidet P-40 extracts to NH4OH, treatment which promotes the conversion of protein asparagine residues to atypical l-isoaspartate residues, recently shown (in synthetic peptides) to be the single most effective residue recognized for carboxylmethylation by PCMT. We found up to a 400% enhancement of the carboxylmethylation of solubilized membrane MAPs by the equally solubilized PCMT (which resisted the alkaline treatment virtually unscathed) after 90 min at 37°C in 0.05 M NH4OH. However, when brain membrane Lubrol-Px extracts were first subjected to bis(I,I-trifluoroacetoxy)-iodobenzene, a reagent which converts the carboxyamide group of protein-bound asparagine to the corresponding primary amine, the amount of MAPs susceptible to be acted upon by 0.05 M NH4OH became greatly reduced. Finally, acidic slab gel electrophoresis of membrane-bound MAPs, carboxyl-[3H]-methylated by the membrane-bound PCMT, revealed the presence of about 12 radioactive protein bands, ranging in MW from under 20 KDa to about 90 KDa. |
---|---|
ISSN: | 0197-0186 1872-9754 |
DOI: | 10.1016/0197-0186(87)90122-7 |