Under conditions of osmotic stress such as drought and salinity, many plants accumulate compatible organic solutes such as glycine betaine (Rhodes and Hanson, 1993). The primary metabolite choline is a precursor for glycine betaine synthesis in addition to being a component of phospholipids. In spinach leaves, choline synthesis involves three sequential N-methylations of phosphoethanolamine (PEA) in order to generate phosphocholine (PCho) via the pathway PEA -+ phosphomethylethanolamine (PMEA) -+ phosphodimethylethanolamine (PDEA) -+ PCho. The S-adenosyl-L-methionine (SAM) dependent N-methyltransferase phosphomethylethanolamine N-methyltransferase (PMEAMeT) can catalyze two of the three sequential steps: PMEA -+ PDEA -+ PCho (Dhadialla, 1999). This thesis describes a seven-step strategy for PMEAMeT purification from spinach leaves and provides evidence for the existence oftwo distinct enzymes with apparently overlapping capacities to use both PMEA and PCho as substrates. A seven step purification strategy was used in this project which included the initial four-step strategy used by Dhadialla ( 1999) to partially purify PMEAMeT approximately 70-fold. The seven steps included precipitation of soluble leaf protein from spinach leaves by 1.8-2.6M (NH^4)^2SO^4 fractionation followed by open column chromatography on DEAE Sepharose CL-6B, Phenyl Sepharose CL-4B, Macro-Prep® High Q, and Sephacryl S-100, then high performance chromatography on Mono QHR 5/5 and Protein Pak SW-300. The highest fold purifieation achieved for PMEAMeT was 7,507-fold and yielded a specific activity of 3,243 nmol min^-1 mg^-1 protein. SDS-PAGE analysis of this sample and silver staining of the polyacrylamide gel revealed that approximately 15 polypeptide bands are present in this sample and included two polypeptides with estimated molecular masses of 30 and 50 kDa. Anion exchange chromatography on a Mono Q matrix followed by photoaffinity cross-linking with aliquots of individual fractions shown to have high PMEAMeT activity, shows that the 30 and 50 kDa photoaffinity cross-linked species can be partially resolved from each other by this matrix. This observation is best explained if the two polypeptides are not subunits of the same methyltransferase enzyme. Evidence that both the 30 and 50 kDa [^3H]SAM-binding polypeptides contribute to PMEAMeT activity was provided by showing that the inclusion of either PMEA or PDEA in the photoaffinity cross-linking assay prevented the binding of [3H]:;AM to either polypeptide; a result consistent with PMEA and PDEA serving as substrates for the enzyme(s) associated with both polypeptides. In addition, the presence of PEA in the photoaffinity cross-linking assay did not prevent the binding of [^3H]SAM to either polypeptide showing that in these species cross-linking is not prevented by phosphobases that are not suitable substrates for PMEAMeT. This report describes for the first time the existence of two enzymes in spinach leaves that possess PMEAMeT activity. The role of these enzyme(s) in spinach might involve the maintenance of low PMEA and PDEA pool sizes. Low pools for these metabolites may be required to prevent the incorporation of PMEA and PDEA into the polar head groups of phospholipids in place of PCho. Whether the substitution of PMEA or PDEA for PCho is deleterious to phospholipid structure or function in plant membranes is unknown. / Thesis / Master of Science (MSc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/22471 |
| Date | 09 1900 |
| Creators | Burian, Thomas |
| Contributors | Weretilnyk, Elizabeth, Biology |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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