6 research outputs found
Resolution of mitochondrial and chloroplast membrane protein complexes from green leaves of potato on blue-native polyacrylamide gels
59-66Purification
of mitochondria and mitochondrial protein
complexes from green tissues is often severely impaired by the presence of
chloroplasts and their proteins. Here we present a method which
allows analysis of respiratory protein complexes from potato leaves. The
procedure includes the preparation of an organellar fraction specifically
enriched in mitochondria and the
separation of organellar protein complexes by blue-native polyacrylamide gel
electrophoresis (BN-PAGE). For the first time mitochondrial and chloroplast
protein complexes have been resolved simultaneously in a native gel. BNPAGE allowed
the separation of eleven bands, including the mitochondrial NADH-dehydrogenase,
the bc1 complex and the mitochondrial F1-ATP synthase as
well as the chloroplast F1-ATP synthase, the cytochrome b6f
complex, the two photosystems and the light harvesting complex. The resolution
of the protein complexes in the first dimension was good enough to allow
identification of all subunits of individual complexes in the second dimension
under denaturing conditions. Thus, BNPAGE offers an opportunity to analyze
mitochondrial and chloroplast protein complexes from a single preparation from
very small amounts of tissue. The implications of our
findings, for studies on protein expression and turnover in different tissues
and developmental stages, are discussed.
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Photorespiratory nitrogen cycle - a critical evaluation
The photorespiratory nitrogen cycle proposed by Keys et al. (Nature 275: 741-743, 1978) involved formation of glycine by transamination of glyoxylate in the peroxisomes utilizing glutamate. Subsequently, glycine is oxidized to ammonia, serine and CO2 in the mitochondria. The ammonia is reassimilated via the GS/GOGAT pathway generating glutamate. In this article, experimental evidence which suggests the occurrence of alternative mechanisms of glycolate and serine synthesis as well as of CO2 and ammonia evolution is discussed. The problem of utilization of NADH coupled to ATP synthesis during photosynthesis is still unresolved, which complicates the glycine oxidation reaction in light. Further, factors are presented that determine the availability of amino donors in the peroxisomes and of amino acids viz., glycine, serine and glutamate for the operation of the photorespiratory N cycle. Recent evidence regarding the role of formate arising out of the reaction of glyoxylate with H2O2 in the regulation of photosynthetic electron flow in the Hill reaction, as well as of photorespiratory substrates functioning as carbon sources for the citric acid cycle in the light or for export to the growing tissues, suggests that the role of photo-respiration in plant metabolism needs to be reexamined