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Phospholipid n-methyltransferases in rat brain myelin

Tsvetnitsky, Vadim; (1994) Phospholipid n-methyltransferases in rat brain myelin. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Despite considerable effort, the pathogenesis of many demyelination diseases including multiple sclerosis is unknown. There are reasons to suggest that the activities of some lipid-metabolizing enzymes in myelin, if affected during demyelinating disease, could contribute to progressing myelin degradation. The carried out research has established the presence of phospholipid N-methyltransferase (PLMTase) in highly purified rat brain myelin and characterized it, while phosphatidylserine decarboxylation was shown to be undetectable in the same preparations. Ion-exchange chromatography revealed a charge heterogeneity of myelin PLMTase. The major form of myelin PLMTase was shown to have molecular weight of ca 65 kDa by column SDS-PAGE and gel-filtration chromatography. While at least four peaks of phospholipid methylating activity, heterogeneous in protein composition, were detected following IEF of myelin proteins, all these preparations contained only ca 65 kDa proteinthe after being subjected to SAH-affinity chromatography, suggesting presence of differently charged isoforms of PLMTase in myelin. At least one detected isoform catalysed formation of appreciable amount of PC, while others formed predominantly monomethylated PE. The ca 65 kDa protein with associated PLMTase activity has been successfully solubilised with either Triton X-100 or CHAPS detergents and purified from myelin by 2300-fold (Triton X-100) or 4600-fold (CHAPS) using combination of ion-exchange chromatography, isoelectric focusing (IEF) and affinity chromatography. The exact nature of the observed charge heterogeneity of myelin PLMTase is still unclear and the undergoing sequencing of the purified protein should provide better understanding of the roles that the putative isoforms of PLMTase play in myelin and their possible relation to demyelination diseases and signal transduction in myelin. A study was undertaken on the direct effect of S-adenosyl-methionine (SAM) and some of its metabolites on the agonist binding to GABA-benzodiazepine receptor complex. The results showed that S-adenosyl-homocysteine (SAH) and methylthioadenosine (MTA) were able to inhibit the specific binding of [3H]flunitrazepam to rat brain membrane preparations with Ki values of 7.9 μM and 15.8 μM, respectively. Computer sequence homology search revealed a 100% homology between certain sequences of the bacterial PSDase and glutamine synthetase and also NADP-specific glutamate dehydrogenase. This observation suggested that the amino acid string (NN 257-264) of the E.Coli PSDase may participate in glutamate or serine head group binding. Equally, the identical amino acid string of the glutamate-binding enzymes may bind PS or serine. Very high degree of homology was revealed between amino acids sequence from mammalian PSDase (NN 206-214) and a sequence from H+-transporting ATP synthase from very diverse organisms. This is hardly a coincidence and may suggest that this homologous sequence is responsible for either PS recognition on a ATP-synthase or is a site for ADP or, perhaps, other adenosine derivative binding on PSDase.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Phospholipid n-methyltransferases in rat brain myelin
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Health and environmental sciences
URI: https://discovery.ucl.ac.uk/id/eprint/10102493
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