Untersuchungen zur Rolle der Phosphoenolpyruvat-Carboxykinase und des NADP+-Malat-Enzyms in der filamentösen Phaeophycee Ectocarpus siliculosus
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Philipps-Universität Marburg
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Abstract
The filamentous brown-alga Ectocarpus siliculosus
shows high activities of phosphoenolpyruvate carboxykinase
(PEPCK) and NADP+-malic enzyme. The putative participation of
both enzymes in a C4-like carbon cycle became questionable
because not all enzymes necessary for this cycle could be
detected and because the relevant metabolite pools in vivo were
too small. This raised the question of their cellular function.
In order to clarify their function, PEPCK and the NADP+-malic
enzyme were purified to homogeneity. The molecular mass of
native PEPCK was determined to be 90 kDa. SDS-PAGE revealed two
polypeptides of 62 kDa and 18 kDa, respectively. The molecular
mass of native malic enzyme was 440 kDa. SDS-PAGE revealed only
one polypeptide of 56 kDa which suggested that the native
molecule might be a homo-octamer. The carboxylating-reaction of
PEPCK displayed typical Michaelis-Menten-Kinetics for each
substrate. The Km was 1,46 mM for CO2, 0,5 mM for PEP and 0,23
mM for ADP. The double-reciprocal plot of the activity of
NADP+-malic enzyme as a function of the concentration of
L-malate yielded a straight line with a Km for L-malate of 0,35
mM. With respect to the binding of NADP+ malic enzyme revealed
positive cooperativity with a Hill coefficient of 2,74. This
indicates at least three binding sites for NADP+. Rabbits were
immunised using the denatured polypeptides after SDS-PAGE.
Specific antibodies were obtained only against the 18 kDa
polypeptide of PEPCK. In immunoblots, the antibodies reacted
with both, the 18 kDa and the 62 kDa polypeptide of the PEPCK,
indicating that they were both degradation products from a
single probably monomeric polypeptide (the native 90 kDa
protein). In immuno-localisation studies this antibody against
PEPCK stained the pyrenoids of E. siliculosus. With antibodies
against native Rubisco from spinach, Rubisco was also found to
be exclusively localised in the pyrenoids. The localisation of
PEPCK in direct neighbourhood to Rubisco is so far the
strongest argument against the existence of a C4 pathway in E.
siliculosus. An anaplerotic role of PEPCK in the alga is
likely.
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Dates
Created: 2003Issued: 2004-03-04Updated: 2011-08-10
Faculty
Fachbereich Biologie
Publisher
Philipps-Universität Marburg
Language
ger
Data types
DoctoralThesis
Keywords
pyrenoidEctocarpusPEPCKcarbon-acquisition , malic-enzyme
DFG-subjects
EctocarpusKohlenstoff-AkquisitionPyrenoidPEPCKMalat-Enzym
DDC-Numbers
570
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Busch, Sylvia (128831863): Untersuchungen zur Rolle der Phosphoenolpyruvat-Carboxykinase und des NADP+-Malat-Enzyms in der filamentösen Phaeophycee Ectocarpus siliculosus. : Philipps-Universität Marburg 2004-03-04. DOI: https://doi.org/10.17192/z2004.0087.
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This item has been published with the following license: In Copyright