• MeSH
• bakteriální proteiny biosyntéza   MeSH
• dehydratasy metabolismus   MeSH
• enzymová represe MeSH
• Escherichia coli enzymologie   metabolismus   účinky léků   MeSH
• galaktosidasy metabolismus   MeSH
• glukosa farmakologie   MeSH
• hexosafosfáty farmakologie   MeSH
• izotopy uhlíku MeSH
• leucin metabolismus   MeSH
• serin MeSH

Tuberculosis (TB) remains one of the major health concerns worldwide. Mycobacterium tuberculosis (Mtb), the causative agent of TB, can flexibly change its metabolic processes during different life stages. Regulation of key metabolic enzyme activities by intracellular conditions, allosteric inhibition or feedback control can effectively contribute to Mtb survival under different conditions. Phosphofructokinase (Pfk) is one of the key enzymes regulating glycolysis. Mtb encodes two Pfk isoenzymes, Pfk A/Rv3010c and Pfk B/Rv2029c, which are differently expressed upon transition to the hypoxia-induced non-replicating state of the bacteria. While pfkB gene and protein expression are upregulated under hypoxic conditions, Pfk A levels decrease. Here, we present biochemical characterization of both Pfk isoenzymes, revealing that Pfk A and Pfk B display different kinetic properties. Although the glycolytic activity of Pfk A is higher than that of Pfk B, it is markedly inhibited by an excess of both substrates (fructose-6-phosphate and ATP), reaction products (fructose-1,6-bisphosphate and ADP) and common metabolic allosteric regulators. In contrast, synthesis of fructose-1,6-bisphosphatase catalyzed by Pfk B is not regulated by higher levels of substrates, and metabolites. Importantly, we found that only Pfk B can catalyze the reverse gluconeogenic reaction. Pfk B thus can support glycolysis under conditions inhibiting Pfk A function.

• MeSH
• adenosindifosfát metabolismus   farmakologie   MeSH
• adenosintrifosfát metabolismus   farmakologie   MeSH
• alosterická regulace MeSH
• bakteriální proteiny antagonisté a inhibitory   metabolismus   MeSH
• enzymová indukce MeSH
• fosfofruktokinasy antagonisté a inhibitory   metabolismus   MeSH
• fruktosadifosfáty biosyntéza   farmakologie   MeSH
• fruktosafosfáty metabolismus   farmakologie   MeSH
• glukoneogeneze MeSH
• glykolýza MeSH
• hexosafosfáty metabolismus   MeSH
• izoenzymy antagonisté a inhibitory   metabolismus   MeSH
• katalýza MeSH
• kinetika MeSH
• kyslík farmakologie   MeSH
• L-laktátdehydrogenasa metabolismus   MeSH
• Mycobacterium tuberculosis účinky léků   enzymologie   MeSH
• pyruvátkinasa metabolismus   MeSH
• rekombinantní proteiny metabolismus   MeSH
• substrátová specifita MeSH
• zpětná vazba fyziologická MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH

• MeSH
• glukuronáty MeSH
• oxazepam farmakokinetika   MeSH
• uridyltransferasa MeSH
• xenobiotika farmakokinetika   MeSH

• Konspekt
• Farmacie. Farmakologie
• NLK Obory
• farmacie a farmakologie

Heat shock protein 70 kDa (hsp70), a molecular chaperone involved in folding of nascent proteins, has been studied for its ability to activate innate and specific immunity. High purity hsp70 preparation is generally required for immunization experiments, because endotoxins and other immunologically active contaminants may affect immune responses independently of hsp70. We have developed a novel modification of E. coli-expression medium that enabled a simple two-step production and purification method for endotoxin-free recombinant hsp70. During Ni-NTA-based affinity purification of hsp70, a contaminating protein from host E. coli cells, L-glutamine-D-fructose-6-phosphate aminotransferase (GFAT), was identified. By testing various compounds, supplementation of growth medium with a GFAT metabolite, N-acetylglucosamine, was found to reduce GFAT expression and increase the total hsp70 yield five times. The new protocol is based on column purification of His-tagged hsp70 protein produced by E. coli with the modified medium, followed by endotoxin removal by Triton X-114 extraction. This approach yielded hsp70 with high purity and minimal endotoxin contamination, making the final product acceptable for immunization experiments. In summary, a simple modification of growth medium allowed production of recombinant mouse hsp70 in high yield and purity, thus compatible with immunological studies. This protocol may be useful for production of other His.

• MeSH
• acetylglukosamin chemie   metabolismus   MeSH
• chromatografie afinitní MeSH
• endotoxiny biosyntéza   izolace a purifikace   MeSH
• Escherichia coli metabolismus   MeSH
• glutaminfruktosa-6-fosfáttransaminasa (izomerizující) biosyntéza   izolace a purifikace   MeSH
• kultivační média chemie   MeSH
• myši MeSH
• proteiny tepelného šoku HSP70 biosyntéza   izolace a purifikace   MeSH
• průmyslová mikrobiologie metody   MeSH
• rekombinantní proteiny biosyntéza   izolace a purifikace   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH