During conditions of osmotic stress, some plants accumulate compatible
osmolytes such as glycine betaine or choline-0-sulphate. Choline is required
as a precursor for synthesis of both osmolytes and choline is also required by
all plants as a component of phospholipids. In the betaine accumulator
spinach, choline synthesis requires three sequential N-methylations of
phosphoethanolamine (PEA) to generate phosphocholine (PCho), with the first
N-methylation being catalyzed by S-adenosyi-L-methionine: PEA Nmethyltransferase
(PEAMeT). Choline synthesis and, more particularly the
activity of PEAMeT, are up-regulated by salinity (Summers and Weretilnyk,
1993). This thesis reports on the partial purification and preliminary
characterization of PEAMeT from spinach.
A variety of column chromatography matrices including DEAE Sepharose,
phenyl Sepharose, w-aminohexyl agarose, hydroxylapatite, phenyl Superose,
Mono Q and adenosine agarose, have been used to purify PEAMeT. A 5403-
fold purified preparation yielded a specific activity of 189 nmol· min-1
• mg-1
protein. SDS-PAGE analysis of this preparation revealed a number of
polypeptide bands but only one which photoaffinity cross-linked to [3H]SAM.
The estimated native molecular weight (MW) of PEAMeT was found to be 77
kDa by gel filtration chromatography and an estimated MW of 54 kDa was determined by SDS-PAGE. SDS-PAGE analysis of samples photoaffinity crosslinked
to [3H]SAM gave a slightly higher estimated MW of 57 kDa.
Effects of various factors on PEAMeT assay conditions were evaluated
using partially purified PEAMeT preparations. PEAMeT activity as a function
of pH gave a unimodal curve with an apparent pH optimum at 7.8 with 1 00
mM HEPES-KOH buffer. In vitro PEAMeT activity was inhibited by phosphate,
PCho, S-adenosyi-L-homocysteine, ca+ 2, Mn+2 and co+2 but not by choline,
betaine, ethanolamine, mono- and dimethylethanolamine or Mg+2
•
Phosphobase N-methyltransferase activities present in preparations enriched
for PEAMeT activity can catalyse the reaction sequence PEA- PMEA - PDEA
- PCho. Under optimized assay conditions using PEA as the sole substrate,
PMEA, PDEA and PCho were quantified and were detected in the order: PMEA
(77%) > PDEA (17%) > PCho (6%). Thus a single enzyme, PEAMeT, is
capable of converting PEA to PCho in leaves of spinach. The existence of a
second enzyme which converts PMEA to PCho has also been reported for
leaves and roots of spinach (Weretilnyk and Summers, 1992). The presence
of two enzymes with overlapping activities raises questions regarding the roles
of these two enzymes in choline metabolism. For example, do these enzymes
also have overlapping functions in choline synthesis, particularly under
conditions of osmotic stress? / Thesis / Master of Science (MSc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/22572 |
| Date | 09 1900 |
| Creators | Smith, David Delmar |
| Contributors | Weretilnyk, Elizabeth A., Biology |
| Source Sets | McMaster University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis |
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