Stability of the glycerol facilitator in detergent solutions

Understanding membrane protein folding and stability is required for a molecular explanation of function and for the development of interventions in membrane protein folding diseases. Stable aqueous detergent solutions of the Escherichia coli glycerol facilitator in its native oligomeric state have been difficult to prepare as the protein readily unfolds and forms nonspecific aggregates. Here, we report a study of the structure and stability of the glycerol facilitator in several detergent solutions by Blue Native and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), circular dichroism (CD), and fluorescence. Protein tetramers were prepared in neutral dodecyl maltoside (DDM) and in zwitterionic lysomyristoylphosphatidylcholine (LMPC) detergent solutions that are stable during SDS-PAGE. Thermal unfolding experiments show that the protein is more stable in LMPC than in DDM. Tertiary structure unfolds before quaternary and some secondary structure in LMPC, whereas unfolding is more cooperative in DDM. The high stability of the protein in DDM is evident from the unfolding half-life of 8 days in 8 M urea, suggesting that hydrophobic interactions contribute to the stability. The protein unfolds readily in LMPC below pH 6, whereas the tetramer remains intact at pH 4 in DDM. At pH 4 in DDM, the protein is more sensitive than at neutral pH to unfolding by SDS and the effect is reversible. At pH 3 in DDM, the tetramer unfolds, losing its tertiary structure but retaining native helical structure which melts at significantly lower temperatures than in the native tetramer. The glycerol facilitator prepared in SDS is mainly monomeric and has ~10% less alpha-helix than the native protein. CD suggests that it forms a condensed structure with non-native tertiary contacts highly similar to the state observed in LMPC at low pH. The implications of the results for in vitro and in vivo folding of the protein are discussed.     

Stability of ATP in Antarctic mineral soils

The stability of exogenous ATP in Antarctic Ross desert soils has been assessed using bioluminescence monitoring of ATP-supplemented samples. Under typical east Antarctic dry valley summer conditions (−3 to +15°C), exogenous ATP was degraded with a half-life of between 0.5 and 30 h. The rate of degradation was affected, in order of significance, by soil biomass levels, temperature and water content. Such rapid removal of exogenous ATP strongly suggests that extracellular ATP from lysed cells in cold desiccated soils does not make a significant contribution to the standing ATP titre     

X-Ray structure of glycerol kinase complexed with an ATP analog implies a novel mechanism for the ATP-dependent glycerol phosphorylation by glycerol kinase.

Glycerol kinase (GK) catalyzes the Mg-ATP-dependent phosphorylation of glycerol which yields glycerol 3-phosphate. The 2.8 A new crystal structure of GK complexed with an ATP analog revealed an unexpected position of the gamma-phosphoryl group, which was 7.2 A distant from the 3-hydroxyl group of glycerol, 5.5 A away from the 3-phosphate of the product (glycerol 3-phosphate) and is stabilized by a beta-hairpin structure. Based on the presented crystal structure and the previously determined structures of GK product complexes, we propose a 3-D model of a nucleophilic in-line transfer mechanism for the ATP-dependent phosphorylation of glycerol by GK.     

Stability of chromatophores during ATP regeneration in an enzyme reactor

ATP regeneration from ADP and inorganic phosphate catalysed by chromatophores from Rhodo-spirillum rubrum has been coupled to the production of glucose-6-phosphate from glucose catalysed by hexokinase. Different ADP derivatives were tested in respect of their function as substrates for the chromatophores, but only 3′NH₂-ADP was phosphorylated. Several factors causing inactivation of the chromatophores during operational conditions were studied. The addition of radical scavengers resulted in an increased stability of the chromatophores, and it was concluded that free radicals had been formed in the presence of oxygen and caused damage to the membranes. In the presence of catalase, a stable ATP production was obtained for 6 h at an activity of 4.5 mol ATP × (mol bacteriochlorophyll)⁻¹ min⁻¹. In a nitrogen flushed reaction medium, the optimum concentration of the redox buffering ascorbic acid changed during the course of several hours of illumination, while it did not change in an air atmosphere.     

Metabolic networks to generate pyruvate,PEP and ATP from glycerol in Pseudomonas fluorescens

Glycerol is a major by-product of the biodiesel industry. In this study we report on the metabolic networks involved in its transformation into pyruvate, phosphoenolpyruvate (PEP) and ATP. When the nutritionally-versatile Pseudomonas fluorescens was exposed to hydrogen peroxide (H₂O₂) in a mineral medium with glycerol as the sole carbon source, the microbe reconfigured its metabolism to generate adenosine triphosphate (ATP) primarily via substrate-level phosphorylation (SLP). This alternative ATP-producing stratagem resulted in the synthesis of copious amounts of PEP and pyruvate. The production of these metabolites was mediated via the enhanced activities of such enzymes as pyruvate carboxylase (PC) and phosphoenolpyruvate carboxylase (PEPC). The high energy PEP was subsequently converted into ATP with the aid of pyruvate phosphate dikinase (PPDK), phosphoenolpyruvate synthase (PEPS) and pyruvate kinase (PK) with the concomitant formation of pyruvate. The participation of the phospho-transfer enzymes like adenylate kinase (AK) and acetate kinase (ACK) ensured the efficiency of this O₂-independent energy-generating machinery. The increased activity of glycerol dehydrogenase (GDH) in the stressed bacteria provided the necessary precursors to fuel this process. This H₂O₂-induced anaerobic life-style fortuitously evokes metabolic networks to an effective pathway that can be harnessed into the synthesis of ATP, PEP and pyruvate. The bioconversion of glycerol to pyruvate will offer interesting economic benefit.     

The role of compartmentation and glycerol kinase in the synthesis of ATP within the glycosome of Trypanosoma brucei

Glycosomes, purified from trypomastigote forms of Trypanosoma brucei, contained all the enzymes necessary to convert glucose to alpha-glycerophosphate and 3-phosphoglycerate. The multienzyme reaction which produces 2 alpha-glycerophosphate, 2 ADP, and 2 NAD+ from 1 glucose, 2 ATP, and 2 NADH was studied spectrophotometrically. Intact glycosomes, suspended with 5.6 mM alpha-glycerophosphate and 2 mM ADP, produced ATP inside the glycosomes for glucose phosphorylation at a rate of 0.7 mumol/min/mg protein, so confirming the feasibility of producing ATP from alpha-glycerophosphate and ADP catalyzed by glycosomal glycerol kinase, and coupling this ATP production to the ATP-requiring stages of glycolysis. No evidence was found for direct channeling of the ATP generated by glycerol kinase and either hexokinase or phosphofructose kinase in glycosomal enzyme complexes cross-linked by dimethyl suberimidate treatment of intact glycosomes prior to solubilization of their membrane. Compartmentation of glycolytic intermediates, enzymes, and ATP inside isolated glycosomes was demonstrated by their inaccessibility to exogenous enzymes. We conclude that the compartmentation of the glycosome and the efficient production of ATP in the glycosome from whole cell concentrations of sn-glycerol 3-phosphate and ADP account for the observed whole cell production of equimolar glycerol from glucose with net ATP synthesis by T. brucei under anaerobic conditions.     

The ATP pool in relation to the production of glycerol and heat during growth of the halotolerant yeast Debaryomyces hansenii

In a study of the halotolerant yeast Debarymyces hansenii cultured in 4 mM and 2.7 M NaCl the intracellular ATP pool, the heat production, the oxygen uptake, and, in the high culture salinity also, the intracellular glycerol concentration were found to be correlated. The intracellular ATP in the 2.7 M NaCl culture had a constant concentration of 3.5 mM ATP during the second half of the lag phase, while in 4 mM NaCl it rose to a maximum of 3.1 mM during the late log phase. The intracellular glycerol concentration in 2.7 M NaCl was about 1.3M during the entire exponential growth phase. Sine the glycerol concentration of the medium was not more than 0.23 mM, glycerol must contribute to the osmotic balance of the cells in high salinity. The corresponding maximum values for the 4 mM NaCl culture were 0.16 M and 0.08 mM. The experimental enthalpy changes were approximately the same for the two salinities, viz. about-1200 kJ per mole consumed glucose. The Y _m-values for the 4 mM and 2.7 M NaCl cultures were 91 and 59, respectively, the difference being a consequence of the decreased efficiency of growth in high salinity.Abbreviations CFU colony-forming units - PCA perchloric acid - TCA trichloroacetic acid     

Enzymatic production of glycerol acetate from glycerol

In this study, we report the enzymatic production of glycerol acetate from glycerol and methyl acetate. Lipases are essential for the catalysis of this reaction. To find the optimum conditions for glycerol acetate production, sequential experiments were designed. Type of lipase, lipase concentration, molar ratio of reactants, reaction temperature and solvents were investigated for the optimum conversion of glycerol to glycerol acetate. As the result of lipase screening, Novozym 435 (Immobilized Candida antarctica lipase B) was turned out to be the optimal lipase for the reaction. Under the optimal conditions (2.5 g/L of Novozym 435, 1:40 molar ratio of glycerol to methyl acetate, 40 °C and tert-butanol as the solvent), glycerol acetate production was achieved in 95.00% conversion.     

Stability of the topoisomerase II closed clamp conformation may influence DNA-stimulated ATP hydrolysis

Type II DNA topoisomerases catalyze changes in DNA topology and use nucleotide binding and hydrolysis to control conformational changes required for the enzyme reaction. We examined the ATP hydrolysis activity of a bisdioxopiperazine-resistant mutant of human topoisomerase II alpha with phenylalanine substituted for tyrosine at residue 50 in the ATP hydrolysis domain of the enzyme. This substitution reduced the DNA-dependent ATP hydrolysis activity of the mutant protein without affecting the relaxation activity of the enzyme. A similar but stronger effect was seen when the homologous mutation (Tyr28 --> Phe) was introduced in yeast Top2. The ATPase activities of human TOP2alpha(Tyr50 --> Phe) and yeast Top2(Tyr28 --> Phe) were resistant to both bisdioxopiperazines and the ATPase inhibitor sodium orthovanadate. Like bisdioxopiperazines, vanadate traps the enzyme in a salt-stable closed conformation termed the closed clamp, which can be detected in the presence of circular DNA substrates. Consistent with the vanadate-resistant ATPase activity, salt-stable closed clamps were not detected in reactions containing the yeast or human mutant protein, vanadate, and ATP. Similarly, ADP trapped wild-type topoisomerase II as a closed clamp, but could not trap either the human or yeast mutant enzymes. Our results demonstrate that bisdioxopiperazine-resistant mutants exhibit a difference in the stability of the closed clamp formed by the enzyme and that this difference in stability may lead to a loss of DNA-stimulated ATPase. We suggest that the DNA-stimulated ATPase of topoisomerase II is intimately connected with steps that occur while the N-terminal domain of the enzyme is dimerized.     

Distribution of glycerol dehydrogenase and glycerol kinase activity in halophilic archaea

Abstract A constitutive NAD⁺-dependent glycerol dehydrogenase activity was detected in Halobacterium salinarium and Halobacterium cutirubrum . Optimal activity was found at 3 M KCl and pH 8–10. No glycerol dehydrogenase activity could be demonstrated in representatives of the genera Haloferax and Haloarcula , even when grown in the presence of glycerol, or in Halobacterium saccharovorum and Halobacterium sodomense . Glycerol kinase activity was shown to be present constitutively in all halophilic archaea examined. The finding that glycerol dehydrogenase is found only in part of the halophilic arachaea makes dihydroxyacetone an improbable candidate as the precursor for the glycerol moiety of halobacterial lipids.     

Potentiometric quantitation of glycerol using immobilized glycerol dehydrogenase

Glycerol dehydrogenase was immobilized in polyacrylamide gel layered over a small platinum screen and used to catalyze the oxidation of glycerol. In the presence of NAD(+) and potassium ferricyanide, the coupling reaction generated a measurable electrical potential which was found to be Nernstian with respect to the glycerol concentration range of 10(-4)M to 10(-1)M. The reproducibility of the measurement and the optimal conditions for glycerol determination were described.     

Interaction of aluminum ion with ATP. Mechanism of the aluminum inhibition of glycerol kinase and its reversal by spermine

Aluminum ion inhibited yeast glycerol kinase competitively with respect to the substrate MgATP. The K value of the enzyme for aluminum ion was about 3 M. Spermine at physiological concentrations prevented glycerol kinase from the inhibition by aluminum ion. Nuclear magnetic resonance spectroscopy showed the specific elimination by spermine of aluminum from the metal-ATP complex, but no dissociation of MgATP complex by spermine. Inhibition by aluminum ion of glycerol kinase as well as hexokinase can reduce the utilization of energy fuel in yeast. Change in polyamine concentration may control energy production in vivo, and is responsible for the development of age-related aluminum toxicity. © Rapid Science 1998.     

An ATP/ADP-Dependent Molecular Switch Regulates the Stability of p53-DNA Complexes

Interaction with DNA is essential for the tumor suppressor functions of p53. We now show, for the first time, that the interaction of p53 with DNA can be stabilized by small molecules, such as ADP and dADP. Our results also indicate an ATP/ADP molecular switch mechanism which determines the off-on states for p53-DNA binding. This ATP/ADP molecular switch requires dimer-dimer interaction of the p53 tetramer. Dissociation of p53-DNA complexes by ATP is independent of ATP hydrolysis. Low-level ATPase activity is nonetheless associated with ATP-p53 interaction and may serve to regenerate ADP-p53, thus recycling the high-affinity DNA binding form of p53. The ATP/ADP regulatory mechanism applies to two distinct types of p53 interaction with DNA, namely, sequence-specific DNA binding (via the core domain of the p53 protein) and binding to sites of DNA damage (via the C-terminal domain). Further studies indicate that ADP not only stabilizes p53-DNA complexes but also renders the complexes susceptible to dissociation by specific p53 binding proteins. We propose a model in which the DNA binding functions of p53 are regulated by an ATP/ADP molecular switch, and we suggest that this mechanism may function during the cellular response to DNA damage.     

Renal glycerol metabolism and the distribution of glycerol kinase in rabbit nephron.

Glycerol and dihydroxyacetone are metabolized by rabbit kidney-cortex tubules, isolated by collagenase treatment. Half-maximal concentrations of both substrates were determined with regard to uptake rates and product formations. Maximal uptake rates were 643 and 329 mumol/h per g of protein for dihydroxyacetone and glycerol respectively. Glucose and lactate were found as major metabolic products. Glycerol kinase, the enzyme catalysing the first step in renal glycerol and dihydroxyacetone metabolism, was measured radiochemically as described by Newsholme, Robinson & Taylor [(1967) Biochim, Biophys. Acta 132, 338-346] and adapted for studies of the localization of this enzyme along the different structures of rabbit nephron. The results show that glycerol kinase is located exclusively in the proximal segments, i.e. the proximal convoluted tubules and the pars recta, but is negligible in the other structures studied. The activities were close to the maximal dihydroxyacetone uptake rates measured in tubule suspensions.     

Assembly of pure tubulin in the absence of free GTP: effect of magnesium, glycerol, ATP, and the nonhydrolyzable GTP analogues

We describe in vitro microtubule assembly that exhibits, in bulk solution, behavior consistent with the GTP cap model of dynamic instability. Microtubules assembled from pure tubulin in the absence of free nucleotides could undergo one cycle of assembly, but could not sustain an assembly plateau. After the initial peak of assembly was reached and bound E-site GTP hydrolyzed to GDP, the microtubules gradually disassembled. We studied buffer conditions that maximized this disassembly while still allowing robust assembly to take place. While both glycerol and glutamate increased the rate of initial assembly and then slowed disassembly, magnesium promoted initial assembly and, surprisingly, enhanced disassembly. After cooling, a second cycle of assembly was unsuccessful unless GTP or the hydrolyzable GTP analogue GMPCPOP was readded. The nonhydrolyzable GTP analogues GMPPNP and GMPPCP could not support the second assembly cycle in the absence of E-site GTP. Analysis using HPLC found no evidence that GMPPNP, GMPPCP, or ATP could bind to free tubulin, and these nucleotides did not compete with GTP for the E-site. We have, however, demonstrated that the nonhydrolyzable GTP analogues and ATP do have an important effect on microtubule assembly. GMPPNP, GMPPCP, and ATP could each enhance the rate of assembly and stabilize the plateau of assembled microtubules against disassembly, while not binding appreciably to free tubulin. We conclude that these nucleotides, as well as GTP itself, enhance assembly by binding to a site on microtubules that is not present on free, unpolymerized tubulin. We estimate the affinity (KD) of the polymeric site for nucleotide triphosphates to be approximately 10(-4)M.     

Biosynthesis of glycerol carbonate from glycerol by lipase in dimethyl carbonate as the solvent

Glycerol carbonate was synthesized from renewable glycerol and dimethyl carbonate using lipase in solvent-free reaction system in which excess dimethyl carbonate played as the reaction medium. A variety of lipases have been tested for their abilities to catalyze transesterification reaction, and Candida antartica lipase B and Novozyme 435 exhibited higher catalytic activities. The silica-coated glycerol with a 1:1 ratio was supplied to prevent two-phase formation between hydrophobic dimethyl carbonate and hydrophilic glycerol. Glycerol carbonate was successfully synthesized with more than 90% conversion from dimethyl carbonate and glycerol with a molar ratio of 10 using Novozyme 435-catalyzed transesterification at 70 °C. The Novozyme 435 [5% (w/w) and 20% (w/w)] and silica gel were more than four times recycled with good stability in a repeated batch operation for the solvent-free synthesis of glycerol carbonate.