SummaryIt is accepted that Saccharomyces cerevisiae genome arose from complete duplication of eight ancestral chromosomes; functionally normal ploidy was recovered because of the massive loss of 90% of duplicated genes. There is evidence that indicates that part of this selective conservation of gene pairs is compelling to yeast facultative metabolism. As an example, the duplicated NADP-glutamate dehydrogenase pathway has been maintained because of the differential expression of the paralogous GDH1 and GDH3 genes, and the biochemical specialization of the enzymes they encode. The present work has been aimed to the understanding of the regulatory mechanisms that modulate GDH3 transcriptional activation. Our results show that GDH3 expression is repressed in glucose-grown cultures, as opposed to what has been observed for GDH1 , and induced under respiratory conditions, or under stationary phase. Although GDH3 pertains to the nitrogen metabolic network, and its expression is Gln3p-regulated, complete derepression is ultimately determined by the carbon source through the action of the SAGA and SWI/SNF chromatin remodelling complexes. GDH3 carbonmediated regulation is over-imposed to that exerted by the nitrogen source, highlighting the fact that operation of facultative metabolism requires strict control of enzymes, like Gdh3p, involved in biosynthetic pathways that use tricarboxylic acid cycle intermediates.
Glutamate plays an important role in osmoprotection in various bacteria. In these cases, increased intracellular glutamate pools are not attributable to the NADP-dependent glutamate dehydrogenase (NADP-GDH) or the glutamate synthase, which do not increase their activities under hyperosmotic conditions, but rather to changes in other enzymes involved in glutamate metabolism. We performed a study which indicates that, as opposed to what happens in bacteria, the activity of NADP-GDH is fivefold higher when the halotolerant yeast Debaryomyces hansenii is grown in the presence of 1 M NaCl, compared with growth in media with no added salt. Since purified NADP-GDH activity in vitro was not enhanced by the presence of salt and was more sensitive to ionic strength than the two isoenzymes from S. cerevisiae, increased enzyme synthesis is the most plausible mechanism to explain our results. We discuss the possibility that increased NADP-GDH activity in D. hansenii plays a role in counteracting the inhibitory effect of high ionic strength on the activity of this enzyme.
Rabbit ceruloplasmin (Cp) was purified after solid ammonium sulfate precipitation to 60% final saturation by a two-step column chromatography procedure, utilizing DEAE-Sephadex A-50 and changing the NaCl concentration in the buffer to 0.16 M to achieve the isolation of the protein. The purified Cp was used to prepare antibodies in guinea pigs that were used afterwards to determine the Cp concentration in normal rabbits and in rabbits with an experimentally induced chronic anemia. The molecular weight of rabbit Cp determined by SDS-PAGE was 125,000 and a high molecular weight Cp of 200,000 comprising 8% of the total purified protein was also found. An optical density ratio (610 nm/280 nm) of 0.0475 and a molar extinction coefficient of 7625 were obtained. Copper determinations yielded a value of 0.24% that corresponds to 5 copper atoms per molecule. The staining of Cp following discelectrophoresis in polyacrylamide gels also showed a two band pattern.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.