Donor-Cell Origin High-Risk Myelodysplastic Syndrome Synchronous with an Intracranial Meningioma-Like Tumor, 8 Years after Allogeneic Hematopoietic Stem Cell Transplantation for Chronic Lymphocytic Leukemia.

Secondary neoplasias are well known consequences of radiotherapy or chemotherapy for a primary cancer. In this report, we describe two rare secondary neoplasias occurring in the same patient: a meningioma-like intracranial tumor and high-risk myelodysplastic syndrome (MDS) of donor-cells origin, both diagnosed simultaneously, 8 years after an allogeneic hematopoietic stem cell transplantation (allo-HSCT) for chronic lymphocytic leukemia (CLL). Due to an engraftment failure during the first allo-HSCT of a matched related donor for CLL treatment, the salvage treatment was a second allo-HSCT. At the moment of meningioma-like tumor diagnosis, the patient was pancytopenic due to high-risk MDS, so it was decided to postpone a surgical intervention until hematological improvement. For the high-risk MDS of donor-cells origin the chosen treatment was induction with intensive chemotherapy. Due to refractory disease, the patient was treated with 5-azacitidine and donor-lymphocytes infusion with no response and, finally, a third allo-HSCT of a matched unrelated donor was performed. The patient died 6 months after the third allo-HSCT, in cytogenetic remission but without hematological recovery, due to an intracranial hemorrhage with origin in the meningioma-like tumor.

Charge Stabilized Silver Nanoparticles Applied as Antibacterial Agents.

Monodisperse silver nanoparticle sols were synthesized via chemical reduction processes in aqueous environment without using polymeric stabilizing agents or surfactants. The sols obtained using various reducing agents; inorganic cell permeabilizers and organic phenolic compounds; inter alia gallic acid (GA) and tannin (TA) were thoroughly characterized by various physicochemical methods such as TEM, SEM, AFM, DLS and micro-electrophoresis. The antibacterial activity of the sols against two E. coli strains was characterized via the determination of the Minimum Bactericidal Concentration (MBC). All sols exhibited a pronounced bactericidal effect against the standard K12 strain, especially the GA and TA sols showing MBC concentration as low as 1-5 mg L(-1). In the case of the antibiotic resistant strain the highest activity (MBC of 10 mg L(-1)) was observed for the sol synthesized using sodium hypophosphate and sodium tripolyphosphate. Additionally, interactions of silver nanoparticles with bacteria cell were studied using TEM and AFM imaging. It was shown that the silver particles attach to the bacteria surface inducing disintegration, which enables their penetration inside the bacteria. Our measurements confirmed that the surface chemistry of silver nanoparticles can play a decisive role.

Pressure dependence of butyl nitrate formation in the reaction of butylperoxy radicals with nitrogen oxide.

The yield of 1- and 2-butyl nitrates in the gas-phase reactions of NO with n-C4H9O2 and sec-C4H9O2, obtained from the reaction of F atoms with n-butane in the presence of O2, was determined over the pressure range of 100-600 Torr at 298 K using a high-pressure turbulent flow reactor coupled with a chemical ionization quadrupole mass spectrometer. The yield of butyl nitrates was found to increase linearly with pressure from about 3% at 100 Torr to about 8% at 600 Torr. The results obtained are compared with the available data concerning nitrate formation from NO reaction with other small alkylperoxy radicals. These results are also discussed through the topology of the lowest potential energy surface mainly obtained from DFT(B3LYP/aug-cc-pVDZ) calculations of the RO2 + NO reaction paths. The formation of alkyl nitrates, due essentially to collision processes, is analyzed through a model that points out the pertinent physical parameters of this system.

The gas phase tropospheric removal of fluoroaldehydes (CxF2x+1CHO, x = 3, 4, 6).

The rate coefficient of the OH reaction with the perfluoroaldehydes C(3)F(7)CHO and C(4)F(9)CHO have been determined in the temperature range 252-373 K using the pulsed laser photolysis-laser induced fluorescence (PLP-LIF) method: k(C(3)F(7)CHO+OH) = (2.0 +/- 0.6) x 10(-12) exp[-(369 +/- 90)/T] and k(C(4)F(9)CHO+OH) = (2.0 +/- 0.5) x 10(-12) exp[-(356 +/- 70)/T] cm(3) molecule(-1) s(-1), corresponding to (5.8 +/- 0.6) x 10(-13) and (6.1 +/- 0.5) x 10(-13) cm(3) molecule(-1) s(-1), respectively, at 298 K. The UV absorption cross sections of these two aldehydes and CF(3)(CF(2))(5)CH(2)CHO have been measured over the range 230-390 nm at 298 K and also at 328 K for CF(3)(CF(2))(5)CH(2)CHO. The obtained results for C(3)F(7)CHO and C(4)F(9)CHO are in good agreement with two recent determinations but the maximum value of the absorption cross section for CF(3)(CF(2))(5)CH(2)CHO is over a factor of two lower than the single one recently published. The photolysis rates of C(3)F(7)CHO, C(4)F(9)CHO and CF(3)(CF(2))(5)CHO have been measured under sunlight conditions in the EUPHORE simulation chamber in Valencia (Spain) at the beginning of June. The photolysis rates were, respectively, J(obs) = (1.3 +/- 0.6) x 10(-5), (1.9 +/- 0.8) x 10(-5) and (0.6 +/- 0.3) x 10(-5) s(-1). From the J(obs) measurements and calculated photolysis rate J(calc), assuming a quantum yield of unity across the atmospheric range of absorption of the aldehydes, quantum yields J(obs)/J(calc) = (0.023 +/- 0.012), (0.029 +/- 0.015) and (0.046 +/- 0.028) were derived for the photodissociation of C(3)F(7)CHO, C(4)F(9)CHO and CF(3)(CF(2))(5)CHO, respectively. The atmospheric implication of the data obtained in this work is discussed. The main conclusion is that the major atmospheric removal pathway for fluoroaldehydes will be photolysis, which under low NO(x) conditions, may be a source of fluorinated carboxylic acids in the troposphere.

Reaction of Cl atoms with C6F13CH2OH, C6F13CHO, and C3F7CHO.

The Cl atom initiated oxidation of C(6)F(13)CH(2)OH, C(6)F(13)CHO, and C(3)F(7)CHO was investigated at 298 K and 1000 mbar pressure of air in a photoreactor using in situ Fourier transform infrared (FTIR) analysis. The rate coefficient for the reaction Cl + C(6)F(13)CH(2)OH (reaction 2) was measured using a relative method: k(2) = (6.5 +/- 0.8) x 10(-13) cm(3) molecule(-1) s(-1). C(6)F(13)CHO was detected as the major primary product, while CO and CF(2)O were found to be the major secondary products. A fitting procedure applied to the concentration-time profiles of C(6)F(13)CHO provided a production yield of (1.0 +/- 0.2) for this aldehyde in reaction 2, and the rate coefficient for the reaction Cl + C(6)F(13)CHO (reaction 4) was k(4) = (2.8 +/- 0.7) x 10(-12) cm(3) molecule(-1) s(-1). A high CO yield observed in the oxidation of C(6)F(13)CH(2)OH, (52 +/- 1)%, is attributed to the Cl atom initiated oxidation of C(6)F(13)CHO. High CO yields, (61 +/- 2)% and (85 +/- 5)%, were also measured in the Cl atom initiated oxidation of C(3)F(7)CHO in air and nitrogen, respectively. These high CO yields suggest the occurrence of a decomposition reaction of the perfluoroacyl, C(6)F(13)CO, and C(3)F(7)CO radicals to form CO which will compete with the combination reaction of these radicals with oxygen to form perfluoroacyl peroxy radicals in the presence of air. The latter radicals C(n)F(2)(n)(+1)CO(O)(2) (n = 6-12), through their reaction with HO(2) radicals, are currently considered as a possible source of persistent perfluorocarboxylic acids which have been detected in the environment. The consequences of the present results would be a reduction of the strength of this potential source of carboxylic acids in the atmosphere.

Photolysis and OH-Initiated oxidation of glycolaldehyde under atmospheric conditions.

The photolysis and OH-initiated oxidation of glycolaldehyde (HOCH(2)CHO), which are relevant atmospheric processes, have been investigated under different conditions using complementary methods in three different laboratories. The UV absorption cross sections of glycolaldehyde determined in two of the laboratories are in excellent agreement. The photolysis of glycolaldehyde in air has been investigated in a quartz cell with sunlamps and in the EUPHORE chamber irradiated by sunlight. The mean photolysis rate measured under solar radiation was (1.1 +/- 0.3) x 10(-5) s(-1) corresponding to a mean effective photolysis quantum yield of (1.3 +/- 0.3). The major products detected were HCHO and CO, whereas CH(3)OH was also observed with an initial yield around 10%. Evidence for OH production was found in both experiments using either OH scavenger or OH tracer species. Photolysis of glycolaldehyde was used as the OH source to measure the reaction rate constants of OH with a series of dienes by the relative method and to identify and quantify the oxidation products of the OH-initiated oxidation of 2-propanol. The different experiments suggest that OH is produced by the primary channel: HOCH(2)CHO + hnu --> OH + CH(2)CHO (1). The rate constant of the OH reaction with glycolaldehyde has been measured at 298 K using the relative method: k(glyc) = (1.2 +/- 0.3) x 10(-11) cm(3) molecule(-1) s(-1). The product study of the OH-initiated oxidation of glycolaldehyde in air has been performed using both a FEP bag and the EUPHORE chamber. HCHO was observed to be the major product with a primary yield of around 65%. Glyoxal (CHOCHO) was also observed in EUPHORE with a primary yield of (22 +/- 6)%. This yield corresponds to the branching ratio ( approximately 20%) of the H-atom abstraction channel from the CH(2) group in the OH + HOCH(2)CHO reaction, the major channel ( approximately 80%) being the H-atom abstraction from the carbonyl group. The data obtained in this work, especially the first determination of the photolysis rate of glycolaldehyde under atmospheric conditions, indicate that the OH reaction and photolysis can compete as tropospheric sinks for glycolaldehyde. Since glycolaldehyde is a significant oxidation product of isoprene whereas the photolysis of glycolaldehyde is a significant source of methanol, isoprene might contribute a few percent of the global budget of methanol.

Formation of nitric acid in the gas-phase HO2 + NO reaction: effects of temperature and water vapor.

A high-pressure turbulent flow reactor coupled with a chemical ionization mass spectrometer was used to investigate the minor channel (1b) producing nitric acid, HNO3, in the HO2 + NO reaction for which only one channel (1a) is known so far: HO2 + NO --> OH + NO2 (1a), HO2 + NO --> HNO3 (1b). The reaction has been investigated in the temperature range 223-298 K at a pressure of 200 Torr of N2 carrier gas. The influence of water vapor has been studied at 298 K. The branching ratio, k1b/k1a, was found to increase from (0.18(+0.04/-0.06))% at 298 K to (0.87(+0.05/-0.08))% at 223 K, corresponding to k1b = (1.6 +/- 0.5) x 10(-14) and (10.4 +/- 1.7) x 10(-14) cm3 molecule(-1) s(-1), respectively at 298 and 223 K. The data could be fitted by the Arrhenius expression k1b = 6.4 x 10(-17) exp((1644 +/- 76)/T) cm3 molecule(-1) s(-1) at T = 223-298 K. The yield of HNO3 was found to increase in the presence of water vapor (by 90% at about 3 Torr of H2O). Implications of the obtained results for atmospheric radicals chemistry and chemical amplifiers used to measure peroxy radicals are discussed. The results show in particular that reaction 1b can be a significant loss process for the HO(x) (OH, HO2) radicals in the upper troposphere.

The OH-initiated oxidation of hexylene glycol and diacetone alcohol.

The OH-initiated oxidation of two VOCs directly emitted to the atmosphere through their use as industrial solvents, hexylene glycol (HG, (CH3)2C(OH)CH2CH(OH)CH3) and diacetone alcohol (DA, (CH3)2C(OH)CH2C(O)CH3), has been studied in two photoreactors: a 140 L Teflon bag irradiated by lamps at CNRS-Orleans and the 200 m3 European photoreactor, EUPHORE, irradiated by sunlight. The rate constants for the reactions of HG and DA with OH radicals have been determined at (298 +/- 3) K using a relative rate method: k(HG) = (1.5 +/- 0.4) x 10(-11) and k(DA) = (3.6 +/- 0.6) x 10(-12) cm(3) molecule(-1) s(-1) and have been found in good agreement with estimations from structure-reactivity relationships. The study at Orleans and EUPHORE of the OH-initiated oxidation of hexylene glycol showed the formation of diacetone alcohol, acetone, and PAN as the principal products. The branching ratio of the H-atom abstraction from the > CH- group of HG has been estimated to be (47 +/- 4)% corresponding to the measured formation yield of DA. The formation yields of acetone and PAN lead to the determination of a lower limit of (33 +/- 7)% for the branching ratio of the H-atom abstraction of the -CH2- group of HG. For diacetone alcohol, studies at EUPHORE have shown negligible photolysis under atmospheric conditions (J < 5 x 10(-6) s(-1)) and the formation of acetone, PAN, HCHO, and CO in the OH-initiated oxidation experiments. The molar yield of acetone, close to 100%, corresponds to the branching ratio of the H-atom abstraction from the -CH2- group of DA. The present study has allowed the identification of the nature and the fate of the oxy radicals as intermediates in the oxidation mechanism of both HG and DA. The atmospheric implication of these results, especially the ozone formation potential of HG and DA, is discussed.

The NADH oxidase of Streptococcus pneumoniae: its involvement in competence and virulence.

A soluble flavoprotein that reoxidizes NADH and reduces molecular oxygen to water was purified from the facultative anaerobic human pathogen Streptococcus pneumoniae. The nucleotide sequence of nox, the gene which encodes it, has been determined and was characterized at the functional and physiological level. Several nox mutants were obtained by insertion, nonsense or missense mutation. In extracts from these strains, no NADH oxidase activity could be measured, suggesting that a single enzyme encoded by nox, having a C44 in its active site, was utilizing O2 to oxidize NADH in S. pneumoniae. The growth rate and yield of the NADH oxidase-deficient strains were not changed under aerobic or anaerobic conditions, but the efficiency of development of competence for genetic transformation during growth was markedly altered. Conditions that triggered competence induction did not affect the amount of Nox, as measured using Western blotting, indicating that nox does not belong to the competence-regulated genetic network. The decrease in competence efficiency due to the nox mutations was similar to that due to the absence of oxygen in the nox+ strain, suggesting that input of oxygen into the metabolism via NADH oxidase was important for controlling competence development throughout growth. This was not related to regulation of nox expression by O2. Interestingly, the virulence and persistence in mice of a blood isolate was attenuated by a nox insertion mutation. Global cellular responses of S. pneumoniae, such as competence for genetic exchange or virulence in a mammalian host, could thus be modulated by oxygen via the NADH oxidase activity of the bacteria, although the bacterial energetic metabolism is essentially anaerobic. The enzymatic activity of the NADH oxidase coded by nox was probably involved in transducing the external signal, corresponding to O2 availability, to the cell metabolism and physiology; thus, this enzyme may function as an oxygen sensor. This work establishes, for the first time, the role of O2 in the regulation of pneumococcal transformability and virulence.

Structure of the Sec7 domain of the Arf exchange factor ARNO.

Small G proteins switch from a resting, GDP-bound state to an active, GTP-bound state. As spontaneous GDP release is slow, guanine-nucleotide-exchange factors (GEFs) are required to promote fast activation of small G proteins through replacement of GDP with GTP in vivo. Families of GEFs with no sequence similarity to other GEF families have now been assigned to most families of small G proteins. In the case of the small G protein Arf1, the exchange of bound GDP for GTP promotes the coating of secretory vesicles in Golgi traffic. An exchange factor for human Arf1, ARNO, and two closely related proteins, named cytohesin 1 and GPS1, have been identified. These three proteins are modular proteins with an amino-terminal coiled-coil, a central Sec7-like domain and a carboxy-terminal pleckstrin homology domain. The Sec7 domain contains the exchange-factor activity. It was first found in Sec7, a yeast protein involved in secretion, and is present in several other proteins, including the yeast exchange factors for Arf, Geal and Gea2. Here we report the crystal structure of the Sec7 domain of human ARNO at 2 A resolution and the identification of the site of interaction of ARNO with Arf.

Crystal structures of the small G protein Rap2A in complex with its substrate GTP, with GDP and with GTPgammaS.

The small G protein Rap2A has been crystallized in complex with GDP, GTP and GTPgammaS. The Rap2A-GTP complex is the first structure of a small G protein with its natural ligand GTP. It shows that the hydroxyl group of Tyr32 forms a hydrogen bond with the gamma-phosphate of GTP and with Gly13. This interaction does not exist in the Rap2A-GTPgammaS complex. Tyr32 is conserved in many small G proteins, which probably also form this hydrogen bond with GTP. In addition, Tyr32 is structurally equivalent to a conserved arginine that binds GTP in trimeric G proteins. The actual participation of Tyr32 in GTP hydrolysis is not yet clear, but several possible roles are discussed. The conformational changes between the GDP and GTP complexes are located essentially in the switch I and II regions as described for the related oncoprotein H-Ras. However, the mobile segments vary in length and in the amplitude of movement. This suggests that even though similar regions might be involved in the GDP-GTP cycle of small G proteins, the details of the changes will be different for each G protein and will ensure the specificity of its interaction with a given set of cellular proteins.

Allosteric activation increases the maximum velocity of E. coli phosphofructokinase.

Several mutations that cause a decrease of 25 to 65% of the catalytic activity, were introduced at different positions in the phosphofructokinase from Escherichia coli, and the influence of the allosteric activator GDP on these mutants was measured. In the case of the wild-type enzyme, GDP converts the highly cooperative saturation towards fructose-6-phosphate into a hyperbolic saturation with almost no change in the maximum velocity. The mutants Glu148 --> Leu, Leu178 --> Val and Leu178 --> Trp are still cooperative for fructose-6-phosphate, and their cooperativity is also abolished or markedly decreased by GDP. In addition, GDP acts on these mutants as an activator of maximum velocity, and increases their catalytic rate constants by 35 to 65% depending on the mutation. The Leu178 --> Val mutant is even as active as the wild-type enzyme in the presence of GDP. The Thr125 --> Ser mutation decreases the maximum velocity by 60% and also suppresses the cooperativity towards fructose-6-phosphate. Accordingly, the only effect of GDP on the Thr125 --> Ser mutant is on its maximum velocity and not on its affinity for fructose-6-phosphate. However, the maximum velocity of this mutant is not increased by GDP but reduced by 33%. These results show that GDP affects the maximum velocity of these mutants and suggest that the activation by GDP of the wild-type enzyme measured by steady-state kinetics could be partially due to an increase of the maximum velocity, and not exclusively to an increase of the affinity for fructose-6-phosphate.

Kinetic Studies of OH Reactions with a Series of Methyl Esters.

Absolute rate constants have been measured for the gas-phase reactions of hydroxyl radicals with a series of methyl esters: methyl propionate (k 1), methyl butyrate (k 2), methyl valerate (k 3), and methyl caproate (k 4). Experiments were carried out using the pulsed laser photolysis-laser induced fluorescence technique over the temperature range 253-372 K. The obtained kinetic data were used to derive the following Arrhenius expressions: k 1 = (1.45 ± 0.42) × 10-12 exp[-(148 ± 86)/T]; k 2 = (0.96 ± 0.29) × 10-12 exp[(380 ± 91)/T]; k 3 = (1.37 ± 0.64) × 10-12 exp[(401 ± 142)/T]; k 4 = (2.46 ± 1.04) × 10-12 exp[(326 ± 130)/T] (in units of cm3 molecule-1 s-1). At room temperature, the rate constants obtained (in units of 10-12 cm3 molecule-1 s-1) were as follows: methyl propionate (0.83 ± 0.09); methyl butyrate (3.30 ± 0.25); methyl valerate (4.83 ± 0.55); methyl caproate (7.15 ± 0.70). Our results are compared with the previous determinations and discussed in terms of structure-activity relationships.

Tetramer-dimer equilibrium of phosphofructokinase and formation of hybrid tetramers.

Moderate concentrations of KSCN inactivate the allosteric phosphofructokinase from Escherichia coli by dissociating the subunit interface that contains the binding site for the substrate fructose-6-phosphate. At a given KSCN concentration, the activity varies with the concentration of protein as expected from a simple equilibrium between active tetramers and inactive dimers. The equilibrium constants for the dissociation of a tetramer into dimers have been determined in 0.4 M KSCN for the wild-type enzyme and the noncooperative mutant T125S, the hypercooperative mutant E148A-R152A, and the inactive mutant D127S. The stability of the tetrameric structure is decreased by the mutations E148A-R152A that are in the interface and increased by the mutation T125S that does not belong to it. There could be an inverse correlation between the cooperativity of the saturation by fructose-6-phosphate (in absence of any effector) and the stability of the interface that contains its binding site. Hybrid tetramers can be formed upon reassociation of a dimer from an active phosphofructokinase (wild-type, T125S, or E148-R152A) with a dimer from the inactive D127S mutant, and their stability and cooperativity toward fructose-6-phosphate have been measured without purifying them. The results indicate that the formation of a hybrid interface involves some flexibility of the two dimers and that the allosteric coupling between distant sites could be related to the plasticity and instability of the interactions across this interface.

Hypercooperativity induced by interface mutations in the phosphofructokinase from Escherichia coli.

The saturation of the allosteric phosphofructokinase from Escherichia coli by its substrate fructose-6-phosphate is highly cooperative and seems to occur in an "all-or-none" process at all active sites. This cooperativity measured by the Hill coefficient can still be markedly increased by mutation of a single residue located at a subunit interface, Arg152. X-ray crystallography shows that Arg152 forms an ion-pair with Glu148 within an alpha-helix of one subunit. This ion-pair is close to a symmetry axis and interacts with the ion-pair Glu148-Arg152 of the neighbouring chain across the subunit interface. Mutations of Glu148 affect cooperativity much less than those of Arg152. The substitution of Arg152 by lysine increases the Hill coefficient by two-fold to a value larger than the number of substrate binding sites, which exceeds the maximum cooperativity predicted by the two "classical" models, concerted or sequential, of allosteric regulation. This indicates that the steady-state overall hypercooperativity is (at least partly) of kinetic origin. The hypercooperative mutants of Arg152 also show an enhanced cooperativity in their allosteric inhibition by phospho-enol-pyruvate. These results suggest that the allosteric coupling between distant sites involves (1) electrostatic interactions across the subunit interface between residues Glu148 and Arg152 from two adjacent chains, and (2) a relative movement of the alpha-helices containing Glu148 and Arg152 that could propagate and amplify a conformational change between the interface and the active site within each subunit.

The cooperativity and allosteric inhibition of Escherichia coli phosphofructokinase depend on the interaction between threonine-125 and ATP.

During the reaction catalyzed by the phosphofructokinase (EC 2.7.1.11) from Escherichia coli, the phosphoryl group transferred from ATP interacts with Thr-125 [Shirakihara, Y. & Evans, P. R. (1988) J. Mol. Biol. 204, 973-994]. The replacement of Thr-125 by serine changes the saturation by fructose 6-phosphate from cooperative to hyperbolic and abolishes the allosteric inhibition by phosphoenolpyruvate. The same changes, a saturation by fructose 6-phosphate that is no longer cooperative and an activity that is no longer inhibited by phosphoenolpyruvate, are observed with wild-type phosphofructokinase when adenosine 5'-[gamma-thio]triphosphate is used instead of ATP as the phosphoryl donor. These two perturbations of the ATP-Thr-125 interaction lead to the suppression of both the allosteric inhibition by phosphoenolpyruvate and the cooperativity of fructose-6-phosphate saturation, as if replacing the neutral oxygen of ATP by sulfur or removing the methyl group of Thr-125 had "locked" phosphofructokinase in its active conformation. The geometry of this ATP-Thr-125 interaction and/or the presence of the methyl group on the beta-carbon of Thr-125 are crucial for the regulatory properties of phosphofructokinase. This interaction could be a hydrogen bond between the neutral oxygen of the gamma-phosphate of ATP and the hydroxyl group of Thr-125.

Lobster enolase crystallized by serendipity.

An unknown protein crystallized from a lobster muscle preparation in which arginine kinase was the majority component. It was identified as enolase by peptide sequencing and activity testing, and a SIRAS electron density map showed its three-dimensional structure to be very similar to that of yeast enolase.

The role of Glu187 in the regulation of phosphofructokinase by phosphoenolpyruvate.

In bacterial phosphofructokinases, either a glutamic or an aspartic residue is present at position 187, and the mechanism of inhibition by phosphoenolpyruvate seems to be correlated to the nature of residue 187. Upon binding phosphoenolpyruvate, only the enzymes with a Glu187 would undergo a major allosteric conformational change from an active into an inactive state, whereas the enzymes with an Asp187 would only show a simple upward shift in their pH-profile of activity. The phosphofructokinase from Spiroplasma citri, which has an Asp187, has been purified and its properties follow this pattern. The behaviour of mutants of the enzyme from Escherichia coli in which Glu187 is replaced by either aspartate or leucine confirms the importance of residue 187. The major allosteric transition of E. coli phosphofructokinase is abolished by the substitution Glu187-->Asp, suggesting that a glutamate at position 187 is necessary (but not sufficient) for the protein to undergo the change from the active into the inactive state induced by phosphenolpyruvate. In addition, the presence of an acidic residue, aspartate or glutamate, at position 187 is required (but not sufficient) for the binding of ADP (or GDP). This requirement of a negative charge for ADP binding could explain the striking conservation of an aspartate residue at position 187 in all the eukaryotic phosphofructokinases.

Crystallization of D-lactate dehydrogenase from Lactobacillus bulgaricus.

The D-lactate dehydrogenase (D-LDH) from Lactobacillus bulgaricus has been purified and co-crystallized with its cofactor NAD+. Crystals suitable for X-ray diffraction experiments have been obtained from an ammonium sulfate solution by the hanging-drop method. The crystals belong to the orthorhombic space group C222 (or C222(1)) with cell dimensions a = 76.5 A, b = 93.3 A, c = 118.4 A and one monomer of 37,000 daltons per asymmetric unit. They diffract beyond 3.0 A resolution. Sequence comparison suggests that D-LDHs have no evolutionary relationship to L-LDHs and belong instead to the family of the D-2-hydroxyacid dehydrogenases. The X-ray crystallographic structure of the D-LDH from Lactobacillus bulgaricus will be a decisive test of this hypothesis.