ABSTRACT
The somatic isoform of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC1.2.1.12) is involved in such crucial for cancer cells development pathways as induction of apoptosis and glycolytic regulation. At the same time, sperm-specific isoform (GAPDHS) does not exhibit all the same functions as somatic enzyme. The expression of sperm-specific GAPDH without N-terminal domain in some melanoma cells along with somatic isoenzyme, shown in our previous work, has led to the proposal of this unusual enzyme's possible role in regulation of cancer cells glycolysis. In the presented work we have tested production of GAPDHS in 13 additional melanoma cell lines by immunoblotting. We have also gathered data on energy metabolism in 5 selected cell lines by evaluation of glucose uptake and lactate production in differing conditions. We have demonstrated that in standard cultivation media glucose uptake by MelP cells, producing substantial amounts of GAPDHS protein was higher than in MelKor cells, producing lesser amounts of GAPDHS. All other analyzed cell lines that do not produce GAPDHS (MelMS, MelSi and Malme3M) had even a lower glucose uptake rate.
Subject(s)
Melanoma , Energy Metabolism , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Humans , Isoenzymes/genetics , Isoenzymes/metabolism , Male , Melanoma/genetics , Melanoma/metabolism , Spermatozoa/metabolismABSTRACT
The effect of hexacyanoferrate(III) on the catalytic activity of transketolase has been studied. This oxidant inactivates only one of two active sites of the enzyme, the one with a higher affinity to the coenzyme (thiamine diphosphate). The second active site does not lose its catalytic activity. These observations indicate that the active sites of holotransketolase, being indiscernible by data of X-ray analysis, exhibit functional nonequivalence.
Subject(s)
Ferricyanides/pharmacology , Transketolase/antagonists & inhibitors , Catalysis , Catalytic Domain , Ferricyanides/chemistry , Kinetics , Thiamine Pyrophosphate/chemistry , Thiamine Pyrophosphate/metabolism , Transketolase/chemistry , Transketolase/metabolismABSTRACT
Catalytic activity of two active sites of transketolase and their affinity towards the substrates (xylulose-5-phosphate and ribose-5-phosphate) has been studied in the presence of Ca2+ and Mg2+. In the presence of Ca2+, the active sites exhibit negative cooperativity in binding both xylulose-5-phosphate (donor substrate) and ribose-5-phosphate (acceptor substrate) and positive cooperativity in the catalytic transformation of the substrates. In the presence of Mg2+, nonequivalence of the active sites is not observed.
Subject(s)
Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/enzymology , Transketolase/chemistry , Catalytic Domain , Kinetics , Pentosephosphates/metabolism , Ribosemonophosphates/metabolism , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Substrate Specificity , Transketolase/metabolismABSTRACT
Two new optical methods for transketolase activity assay using only one substrate, xylulose 5-phosphate or glycol aldehyde, have been developed. For transketolase activity assay in the first method, it is necessary to add auxiliary enzyme, glyceraldehyde phosphate dehydrogenase. It is not needed in the second method. The range of transketolase concentration in the activity assay is 0.036-0.144 U/ml for the first method and 1.8-6.8 U/ml for the second one.
Subject(s)
Methods , Transketolase/metabolism , Glyceraldehyde 3-Phosphate/biosynthesis , Glyceraldehyde 3-Phosphate/chemistry , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Kinetics , Molecular Structure , Pentosephosphates/chemistry , Pentosephosphates/metabolism , Ribosemonophosphates/metabolism , Saccharomyces cerevisiae/enzymology , Substrate SpecificityABSTRACT
Transketolase (TK) is a homodimer, the simplest representative of thiamine diphosphate (ThDP)-dependent enzymes. It was first ThDP-dependent enzymes the crystal structure of which has been solved and revealed the general fold for this class of enzymes and the interactions of the non-covalently bound coenzyme ThDP with the protein component. Transketolase is a convenient model to study the structure(s) of the active center and the mechanism of action of ThDP-dependent enzymes. This review summarizes the results of studies on the kinetics of the interaction of ThDP with TK from Saccharomyces cerevisiae as well as the generation of the catalytically active form of the coenzyme within the holoenzyme and formation of the enzyme's active center.
Subject(s)
Catalytic Domain/physiology , Coenzymes/chemistry , Coenzymes/metabolism , Saccharomyces cerevisiae/enzymology , Thiamine Pyrophosphate/metabolism , Transketolase/chemistry , Transketolase/metabolism , Animals , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Humans , Thiamine Pyrophosphate/chemistry , Transketolase/physiologyABSTRACT
The optical properties of thiamine diphosphate-dependent enzymes change significantly on their interaction with cofactors (thiamine, bivalent metal ions) and substrates. These changes are connected with structural alterations of the active site and the mechanism of its functioning, and in some cases they reflect changes in the optical properties of the coenzyme itself within the protein. The use of optical characteristics, especially together with model systems, appeared to be a rather promising approach for investigation of the active site of thiamine diphosphate-dependent enzymes and the mechanism of its functioning. So, it seemed to be useful to summarize the literature data concerning the optical characteristics of thiamine (thiamine diphosphate) in model systems and the efficiency of their application for study of thiamine diphosphate-dependent enzymes.
