In response to salinity and drought, some higher plants accumulate the secondary
metabolite glycinebetaine which functions as a compatible osmolyte (Rhodes and
Hanson, 1993). Choline, a precursor of glycinebetaine (Rhodes and Hanson, 1993), is
also a component of a primary metabolite phosphatidylcholine, an ubiquitous membrane
phospholipid (Moore, 1982). In leaves of the glycinebetaine accumulator spinach,
choline is synthesized as phosphocholine (PCho) and PCho is synthesized by three
sequential N-methylations of phosphoethanolamine (PEA) ~ phosphomethyl-EA
(PMEA) ~ phosphodimethyl-EA (PDEA) ~ PCho. The methyl group donor is Sadenosyl-
L-methionine, SAM (Summers and Weretilnyk, 1993). The enzyme SAM:
PMEA N-methyltransferase (PMEAMeT) is suggested to N-methylate PMEA ~ PDEA
~ PCho (Weretilnyk and Summers, 1992).
A four-step strategy was developed for the partial purification of PMEAMeT on
the basis of PMEA-dependent methylations (PMEAMeT activity) that involved the
extraction of soluble leaf protein, ammonium sulfate precipitation, and column
chromatography on DEAE Sepharose, Phenyl Sepharose and High Q Anion matrices.
PDEA-dependent methylating activity co-purified with PMEAMeT activity which
suggested PMEAMeT may N-methylate PMEA ~ PDEA ~ PCho. PMEAMeT was
purified 43-fold and has specific activities of 14.7 and 18.0 nmol•min-1•mg-1 protein with
PMEA and PDEA as substrates, respectively. Thin layer chromatography was used to
identify the reaction products formed during the 30 minute assay incubation: with PMEA
as the substrate, PDEA and PCho were detected in a ratio of 9: 1 as products; and with
PDEA as the substrate, PCho was detected as the only product.
PMEAMeT was estimated to have a native molecular mass of 76 kDa by HPLC
gel filtration chromatography. Both PMEA and PDEA N-methylating activities have an
alkaline pH optimum between 8.5 and 9.0 in 0.1 M Tris-HCl buffer. Neither activity was
iii
affected by the omission of Na2-EDT A from the assay. The addition of 10 mM Mg2+ to
the assay inhibited PMEA and PDEA-dependent methylation by approximately 49% and
32%, respectively; whereas, the addition of 1 and 10 mM Mn2+ to the assay completely
inhibited both activities.
Both activities were inhibited by the reaction products S-adenosyl-Lhomocysteine
by over 90% at 0.2 mM and PCho by approximately 80% at 10 mM. Of
the products ofPCho hydrolysis, choline inhibited PMEA-dependent methylation by 10%
at 10 mM; whereas, Pi inhibited PMEA and PDEA-dependent methylation by 38 and
19%, respectively at 10 mM. The compatible osmolyte glycinebetaine inhibited PMEA
and PDEA-dependent methylation by between 20 and 30% at 140 mM; however, the
inibition of PMEA-dependent methylation can be partly accounted for by the presence of
cr ions in the assay. If present at these concentrations in the same subcellular
compartment, these metabolites could serve as regulators ofPMEAMeT activities in vivo.
Study of PMEAMeT contributed to identifying the number of enzymes that Nmethylate
PEA ~ PMEA ~ PDEA ~ PCho and possible regulatory metabolites for
choline biosynthesis in vivo. These data are pertinent to basic research and also to
genetic-engineering studies aimed at introducing the glycinebetaine-accumulating trait
into crop plants as an approach to enhancing osmotic-stress resistance. / Thesis / Master of Science (MSc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/22469 |
| Date | 11 1900 |
| Creators | Dhadialla, Sharonpal Kaur |
| Contributors | Weretilnyk, Elizabeth A, Biology |
| Source Sets | McMaster University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis |
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