Steady-state electrodiffusion. Scaling, exact solution for ions of one charge, and the phase plane.

This is the first of two papers dealing with electrodiffusion theory (the Nernst-Planck equation coupled with Gauss's law) and its application to the current-voltage behavior of squid axon. New developments in the exact analysis of the steady-state electrodiffusion problem presented here include (a) a scale transformation that connects a given solution to an infinity of other solutions, suggesting the po-sibility of direct comparison of electrical data for membranes with different thicknesses and other properties; (b) a first-integral relation between the electric field and ion densities more general than analogous relations previously reported, and (c) an exact solution for the homovalent system, i.e., a membrane system permeated by various ion species of the same charge. The latter is a generalization of the known one-ion solution. The properties of the homovalent solution are investigated analytically and graphically. In particular we study the phase-plane curves, which reduce to the parabolas discussed by K. S. Cole in the special case in which the current-density parameter (a linear combination of the ionic current densities) is zero.     

Inhibition, by an amphiphilic substance, niflumic acid, of the inward rectification of the crustacean muscle fiber

Agents such as TEA+ or CS+ ions, these last ions instead of K+ ions in poor K extracellular solution, known to reduce or abolish the inwardly rectifying channel in many preparations produced no effect in crayfish muscle membrane By contrast, poor Cl extracellular solution (Cl- ions were replaced by CH3OSO3- ions) blocked the inward current activated by hyperpolarizing pulses and produced an increase of the resting potential. Niflumic acid is a agent which inhibited the inward going rectification of the crayfish muscle membrane. Apparent dissociation constant of niflumic acid with membrane sites was equal to about 6 X 10(-8) M; this value corresponds to that given by Cousin & Motais (1979) concerning translocation of Cl- ions in the membrane of red cells. Activation of the inward going rectification in the crayfish membrane is responsible of an inward current carried by Cl- ions.     

Effects of albumin, dextran, and hyaluronidase on pulmonary interstitial conductivity

The fluid conductivity of albumin solutions of various concentrations relative to that of saline was measured in the interstitium surrounding a short segment of a large (1.5- to 3-mm-diam) blood vessel of an isolated rabbit lung of which air spaces and vasculature were filled with silicon rubber. At a constant driving pressure, the flow of the following solutions was measured sequentially: normal saline and albumin solution (3, 5.5, 8, or 15 g/100 ml saline), hyaluronidase solution (0.02 g/100 ml), and albumin solution (same concentration used before hyaluronidase solution). The albumin-to-saline flow ratios averaged 1.00 +/- 0.23 (SD), 1.01 +/- 0.21, 1.32 +/- 0.63, and 1.54 +/- 0.36 for albumin concentrations of 3, 5.5, 8, and 15 g/100 ml, respectively. These ratios were higher than the corresponding values of 0.88, 0.78, 0.72, and 0.5 expected if the flow of albumin solution were to depend only on fluid viscosity. The flow of dextran and hyaluronan solutions was more viscosity dependent than the flow of albumin solutions. The increased flow of albumin solution could be the result of a reduced excluded volume of albumin caused by collagen and glycosaminoglycans with an increased albumin concentration. The flow of hyaluronidase solution was 24 +/- 22 (SD)-fold (n = 36) larger than the flow of albumin solution. Thus hyaluronan was responsible for most of the hydraulic resistance of the interstitium to bulk flow. After its degradation, the flow of albumin solution became more viscosity dependent. The interaction between plasma proteins and glycosaminoglycans in the pulmonary interstitium could serve to enhance clearance of microvascular filtrate, particularly under conditions of large protein leaks.     

Influence of colloid, preservation medium and trimetazidine on renal medulla injury

In organ transplantation, preservation injury is an important factor which could influence short-term and long-term graft outcome. The renal medulla is particularly sensitive to oxidant stress and ischemia-reperfusion injury (IRI). Using an autotransplant pig kidney model, we investigated renal function and medullary damage determined between day 1 and week 2 after 24- or 48-h cold storage in different preservation solutions: University of Wisconsin solution (UW), Hopital Edouard Herriot solution (a high Na+ version of UW), ECPEG (high Na+ preservation solution with PEG) and ICPEG (a high K+ version of ECPEG) with or without trimetazidine (TMZ). TMZ improved renal preservation and increased renal function when added in each preservation solution (particularly HEH and ECPEG). Medullary damage led to the early appearance of trimethylamine-N-oxide (TMAO) followed by 1H-NMR in urine and plasma. TMZ and ECPEG is the most efficient association to reduce medullary damage. This study clarifies the role of colloid and polarity solution and the role of mitochondrial protection by TMZ.     

Structural properties of AMP-activated protein kinase: dimerization, molecular shape, and changes upon ligand binding

Heterotrimeric AMP-activated protein kinase (AMPK) is crucial for energy homeostasis of eukaryotic cells and organisms. Here we report on (i) bacterial expression of untagged mammalian AMPK isoform combinations, all containing gamma(1), (ii) an automated four-dimensional purification protocol, and (iii) biophysical characterization of AMPK heterotrimers by small angle x-ray scattering in solution (SAXS), transmission and scanning transmission electron microscopy (TEM, STEM), and mass spectrometry (MS). AMPK in solution at low concentrations (~1 mg/ml) largely consisted of individual heterotrimers in TEM analysis, revealed a precise 1:1:1 stoichiometry of the three subunits in MS, and behaved as an ideal solution in SAXS. At higher AMPK concentrations, SAXS revealed concentration-dependent, reversible dimerization of AMPK heterotrimers and formation of higher oligomers, also confirmed by STEM mass measurements. Single particle reconstruction and averaging by SAXS and TEM, respectively, revealed similar elongated, flat AMPK particles with protrusions and an indentation. In the lower AMPK concentration range, addition of AMP resulted in a significant decrease of the radius of gyration by approximately 5% in SAXS, which indicates a conformational switch in AMPK induced by ligand binding. We propose a structural model involving a ligand-induced relative movement of the kinase domain resulting in a more compact heterotrimer and a conformational change in the kinase domain that protects AMPK from dephosphorylation of Thr(172), thus positively affecting AMPK activity.     

Main-chain length control of conformation, membrane activity, and antibiotic properties of lipopeptaibol sequential analogues

To investigate the effect of backbone length and amphiphilicity on the 3D structure, membrane permeability, and antibacterial properties of trichogins, a subclass of lipopeptaibols, we prepared, by the segment condensation approach in solution and chemically characterized, a set of N(alpha)-1-octanoylated -X-(GLUG)(n)-I-L- ( X=G or U where U=Aib; n=1-4) sequential peptide esters. In parallel, the 12-mer (UGGL)(3) aneurism peptide, an analogue of the 11-mer sequential peptide (n=2) with an amino acid insertion was also synthesized and studied. By FT-IR absorption technique, we clearly showed that, in CDCl(3) solution, all peptides essentially populate intramolecularly H-bonded, helical conformations. Moreover, CD spectroscopy indicates that all peptides, with the single exception of the shortest oligomer (the heptamer), adopt mixed 3(10)-/alpha-helical structures, to an extent approximately correlating with main-chain length, in MeOH solution and sodium dodecylsulfate (SDS) micelles. Significant membrane permeability properties were found only for the three longest GLUG-based peptides, with the 15-mer oligomer (n=4) resulting the most active. The lack of activity exhibited by the aneurism peptide in this experiment strongly suggests a relevant role for the sequence amphiphilicity. In addition, antibacterial activity and selectivity were highlighted and demonstrated to be dependent on peptide main-chain length and amphiphilicity, in the sense that the two shortest GLUG-based homologues are active against Gram-positive strains, whereas the two longest homologues are able to penetrate the membranes of the Gram-negative strains, and the UGGL-based aneurism peptide is inactive.     

Evaluation of the intramolecular stacking of the fluorosulfonylbenzoyl derivatives of 1,N6-ethenoadenosine, adenosine, and guanosine

The differences in conformation in solution of fluorosulfonylbenzoyl nucleosides were analyzed by fluorescence and proton nuclear magnetic resonance spectroscopy. The quantum yield of 5'-p-fluorosulfonylbenzoyl-1,N6-ethenoadenosine (5'-FSB epsilon A) in aqueous solution is low (? = 0.01) as compared to that of its parent nucleoside, ethenoadenosine (? = 0.54), and increases approximately 5-fold when measured in a series of solvents of decreasing dielectric constant. The quantum yield of 5'-p-sulfonylbenzoyl-1,N6-ethenoadenosine covalently bound to glutamate dehydrogenase and pyruvate kinase is also 0.01, suggesting that the analogue may exist in the same conformation when enzyme-bound as when free in solution. In D2O, the resonances of the purine ring protons on 5'-FSB epsilon A, 5'-p-fluorosulfonylbenzoyl adenosine (5'-FSBA), and 5'-p-fluorosulfonylbenzoyl guanosine (5'-FSBG) are shifted upfield by about 0.1-0.3 ppm relative to the corresponding protons of their parent nucleosides. The calculated difference in chemical shift (delta delta) decreases as the dielectric constant of the solvent decreases. The delta delta decreases with increasing temperature. These data indicate that 5'-FSB epsilon A, 5'-FSBA, and 5'-FSBG exist in aqueous solution in a conformation in which the purine ring is intramolecularly stacked with the benzoyl moiety. From the magnitude of change in delta delta for 5'-FSB epsilon A, 5'-FSBA, and 5'-FSBG as a function of solvent, it appears that the three analogues differ in their sensitivity to disruption of stacking. The solution conformation of these three fluorosulfonylbenzoyl nucleoside analogues may be an important determinant of their reaction with various enzymes and may explain differences among the analogues in their reaction with a single enzyme.     

Cryopreservation of Preimplantation Embryos of Cattle, Sheep, and Goats

Preimplantation embryos from cattle, sheep, and goats may be cryopreserved for short- or long-term storage. Preimplantation embryos consist predominantly of water, and the avoidance of intracellular ice crystal formation during the cryopreservation process is of paramount importance to maintain embryo viability. Embryos are placed into a hypertonic solution (1.4 – 1.5 M) of a cryoprotective agent (CPA) such as ethylene glycol (EG) or glycerol (GLYC) to create an osmotic gradient that facilitates cellular dehydration. After embryos reach osmotic equilibrium in the CPA solution, they are individually loaded in the hypertonic CPA solution into 0.25 ml plastic straws for freezing. Embryos are placed into a controlled rate freezer at a temperature of -6°C. Ice crystal formation is induced in the CPA solution surrounding the embryo, and crystallization causes an increase in the concentration of CPA outside of the embryo, causing further cellular dehydration. Embryos are cooled at a rate of 0.5°C/min, enabling further dehydration, to a temperature of -34°C before being plunged into liquid nitrogen (-196°C). Cryopreserved embryos must be thawed prior to transfer to a recipient (surrogate) female. Straws containing the embryos are removed from the liquid nitrogen dewar, held in room temperature air for 3 to 5 sec, and placed into a 37°C water bath for 25 to 30 sec. Embryos cryopreserved in GLYC are placed into a 1 M solution of sucrose for 10 min for removal of the CPA before transfer to a recipient (surrogate) female. Embryos cryopreserved in EG, however, may be directly transferred to the uterus of a recipient.     

Structural study of novel antimicrobial peptides, nigrocins, isolated from Rana nigromaculata.

Novel cationic antimicrobial peptides, named nigrocin 1 and 2, were isolated from the skin of Rana nigromaculata and their amino acid sequences were determined. These peptides manifested a broad spectrum of antimicrobial activity against various microorganisms with different specificity. By primary structural analysis, it was revealed that nigrocin 1 has high sequence homology with brevinin 2 but nigrocin 2 has low sequence homology with any other known antimicrobial peptides. To investigate the structure-activity relationship of nigrocin 2, which has a unique primary structure, circular dichroism (CD) and homonuclear nuclear magnetic resonance spectroscopy (NMR) studies were performed. CD investigation revealed that nigrocin 2 adopts mainly an alpha-helical structure in trifluoroethanol (TFE)/H(2)O solution, sodium dodecyl sulfate (SDS) micelles, and dodecylphosphocholine micelles. The solution structures of nigrocin 2 in TFE/H(2)O (1:1, v/v) solution and in SDS micelles were determined by homonuclear NMR. Nigrocin 2 consists of a typical amphipathic alpha-helix spanning residues 3-18 in both 50% TFE solution and SDS micelles. From the structural comparison of nigrocin 2 with other known antimicrobial peptides, nigrocin 2 could be classified into the family of antimicrobial peptides containing a single linear amphipathic alpha-helix that potentially disrupts membrane integrity, which would result in cell death.     

G-electrode-loading method for isoelectric focusing, enabling separation of low-abundance and high-molecular-mass proteins

The G-electrode-loading method (GELM) is a technique enabling a large number of proteins from rat liver to enter an immobilized pH gradient (IPG) gel strip for isoelectric focusing (IEF). In this method, three slips containing the sample solution are placed on the cathodic edge of an IPG gel strip and a slip containing Chaps solution, a filtration membrane, and an electrode slip are placed on top. Finally, a G-electrode is placed on these slips. The Chaps solution (an amphoteric compound) is supplied gently to the sample solution during IEF and helps the proteins in the sample solution to enter the IPG gel strips with a high solubilization capacity. This method was compared with traditional slip-loading and in-gel rehydration, and it showed the best results for protein separation, including high-molecular-mass proteins.     

Flexible structures of SIBLING proteins, bone sialoprotein, and osteopontin

Bone sialoprotein (BSP) and osteopontin (OPN) are two members of the SIBLING (Small Integrin-Binding LIgand, N-linked Glycoprotein) family of genetically related proteins that are clustered on human chromosome 4. We present evidence that this entire family is the result of duplication and subsequent divergent evolution of a single ancient gene. The solution structures of these two post-translationally modified recombinant proteins were solved by one dimensional proton NMR and transverse relaxation times. The polypeptide backbones of both free BSP and OPN rapidly sample an ensemble of conformations consistent with them both being completely unstructured in solution. This flexibility appears to enable these relatively small glycoproteins to rapidly associate with a number of different binding partners including other proteins as well as the mineral phase of bones and teeth. These proteins often function by bridging two proteins of fixed structures into a biologically active complex.     

Regulation of fructosyltransferase activity by carbohydrates, in solution and immobilized on hydroxyapatite surfaces

We tested the effect of several carbohydrates on the activity of cell-free fructosyltransferases (FTF) in solution and immobilized onto hydroxyapatite (HA) and found an inhibitory dose-dependent effect of glucose on FTF activity, both on the surface and in solution. Glucose at 160 mM inhibits FTF activity by 75% both on HA and in solution. Fructose at 160 mM inhibited FTF activity by 25% in solution and by 15% on HA. Levan inhibited FTF activity by 30% in solution, while dextrans and inulin had a limited effect on FTF activity. Circular dichroism and infrared analysis demonstrated no major changes in the chemical structure of fructans synthesized by cell-free FTF on HA and in solution, in the presence or absence of glucose. However, as verified by size-exclusion chromatography, glucose inhibited the synthesis of high molecular-weight fructans. The results indicate that glucose, a byproduct of the FTF enzymatic reaction, is the main carbohydrate affecting FTF activity. Selective inhibition of high molecular-weight fructan production by glucose, may indicate that two mechanisms are involved in the synthesis of fructans, both in solution and on the surface.     

pH- and thermal-dependent conformational transition of PGAIPG, a repeated hexapeptide sequence from tropoelastin

The secondary structure of PGAIPG (Pro-Gly-Ala-IIe-Pro-Gly), a repeated hexapeptide of tropoelastin, in buffer solution of different pH was determined by using attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. The thermal-dependent structural change of PGAIPG in aqueous solution or in solid state was also examined by thermal FTIR microspectroscopy. The conformation of PGAIPG in aqueous solution exhibited a pH-dependent structural characterization. A predominant peak at 1614 cm(-1) (aggregated beta-sheet) with a shoulder near 1560 cm(-1) (beta-sheet) appeared in pH 5.5-8.5 buffer solutions. A new broad shoulder at 1651 cm(-1) (random coil and/or alpha-helix) with 1614 cm(-1) was observed in the pH 4.5 buffer solution. However, the broad shoulder at 1651 cm(-1) was converted to a maximum peak at 1679 cm(-1) (beta-turn/antiparallel beta-sheet) when the pH shifted from 4.5 to 3.5, but the original pronounced peak at 1614 cm(-1) became a shoulder. Once the pH was lowered to 2.5, the IR spectrum of PGAIPG was dominated by major absorption at 1679 cm(-1) with a minor peak at 1552 cm(-1) (alpha-helix/random coil). The result indicates that the pH was a predominant factor to transform PGAIPG structure from aggregated beta-sheet (pH 8.5) to beta-turn/intermolecular antiparallel beta-sheet (pH 2.5). Moreover, a partial conformation of PGAIPG with minor alpha-helix/random coil structures was also explored in the lower pH buffer solution. There was no thermal-dependent structural change for solid-state PGAIPG. The thermal-induced formation of aggregated beta-sheet for PGAIPG in aqueous solution was found from 28 to 30 degrees C, however, which might be correlated with the formation of an opaque gel that turned from clear solution. The formation of aggregated beta-sheet structure for PGAIPG beyond 30 degrees C might be due to the intermolecular hydrogen bonded interaction between the hydrophobic PGAIPG fragments induced by coacervation.     

The DC-module of doublecortin: dynamics, domain boundaries, and functional implications

The doublecortin-like (DC) domains, which usually occur in tandem, constitute novel microtubule-binding modules. They were first identified in doublecortin (DCX), a protein expressed in migrating neurons, and in the doublecortin-like kinase (DCLK). They are also found in other proteins, including the RP1 gene product which-when mutated-causes a form of inherited blindness. We previously reported an X-ray structure of the N-terminal DC domain of DCLK (N-DCLK), and a solution structure of an analogous module of human doublecortin (N-DCX). These studies showed that the DC domain has a tertiary fold closely reminiscent of ubiquitin and similar to several GTPase-binding domains. We now report an X-ray structure of a mutant of N-DCX, in which the C-terminal fragment (residues 139-147) unexpectedly shows an altered, "open" conformation. However, heteronuclear NMR data show that this C-terminal fragment is only transiently open in solution, and assumes a predominantly "closed" conformation. While the "open" conformation may be artificially stabilized by crystal packing interactions, the observed switching between the "open" and "closed" conformations, which shortens the linker between the two DC-domains by approximately 20 A, is likely to be of functional importance in the control of tubulin polymerization and microtubule bundling by doublecortin.     

Hyperpolarizing effect of aminophylline, theophylline, and cAMP on rat diaphragm fibers

We studied the effect of aminophylline and theophylline (0.1-2 mM) on the resting membrane potential (Vm) of rat diaphragm fibers in vitro (25 degrees C). The main findings are the following. 1) Aminophylline and theophylline hyperpolarize the fibers in a dose-dependent manner. This effect is present with 0.1 and 0.25 mM of aminophylline and theophylline, respectively, and the maximum effect is reached with 1 mM of the drug (approximately 5-8 mV in comparison to the normal values). This effect is reversible by washing out the preparation with normal solution. 2) Dibutyryladenosine 3',5'-cyclic monophosphate (DBcAMP, 2 mM) produces a similar increment in the Vm. 3) The hyperpolarizing action observed in the presence of aminophylline, theophylline, and DBcAMP is suppressed by 5 X 10(-4) M ouabain or by lowering the bath temperature to 5 degrees C. These results suggest that the xanthines may directly or indirectly stimulate a Na-K pump. Two possibilities may be considered: 1) an electrogenic effect of the Na-K pump and 2) a reduction in the extracellular K+ concentration in the solution contacting the external side of the cell as a consequence of the activity of the Na-K pump. Alternative mechanisms such as a reduction in Na permeability or an increment in K permeability might collaborate in the hyperpolarizing effect of the drugs tested.     

Application of NMR,molecular simulation,and hydrodynamics to conformational analysis of trisaccharides

The preferred conformations and conformational flexibilities of the trisaccharides alpha-D-Glcp-(1-->2)-beta-D-Glcp-(1-->3)-alpha-D-Glcp-OMe (I) and alpha-D-Glcp-(1-->3)[beta-D-Glcp-(1-->4)]-alpha-D-Glcp-OMe (II) in aqueous solution were determined using nuclear magnetic resonance (NMR) spectroscopy, molecular dynamics (MD) and Langevin dynamics (LD) simulations, and hydrodynamics calculations. Both trisaccharides have a vicinal substitution pattern in which long range (nonsequential) interactions may play an important role. LD simulation at 600 K indicated that the all-syn conformation predominated, though other conformations were apparent. NOE data and MD and LD simulations at 298 K all indicated that trisaccharide I is a single all-syn conformer in solution. Given that previous studies showed evidence of anti-conformers in beta-D-Glcp-(1-->2)-beta-D-Glcp-(1-->3)-alpha-D-Glcp-OMe, this result provides an example of how changing the anomeric configuration of one residue from beta to alpha can make an oligosaccharide more rigid. Discrepancies in inter-ring distances obtained by experiment and by simulation of the all-syn conformer suggest the presence of an anti-psi conformation at the beta-(1-->4)-linkage for II. A combined analysis of measured and calculated translational diffusion constants and (13)C T(1) relaxation times yield order parameters of 0.9 for each trisaccharide. This implies that any interconversion among conformations is significantly slower than tumbling. Anisotropies of approximately 1.6 and 1.3 calculated for I and II, respectively, are consistent with the observed relatively flat T(1) profiles because the tumbling is not in the motional narrowing regime.     

Kinetics of glutaraldehyde fixation of erythrocytes: size, deformability, form, osmotic and hemolytic properties.

Red blood cells interact with glutaraldehyde (GA) in a complex kinetic pattern of events. At a given GA concentration in phosphate buffered saline (PBS), the sequence of cell 'volume' response, as measured by resistive pulse spectroscopy (RPS), includes: an immediate response to the overall solution osmolality; a constant volume, latent phase; a rapid swelling phase; an intermediate constant volume phase; and a shrinkage phase to a final steady state volume. The final volume depends on fixative solution osmolality; for GA concentrations between 0.05% and 0.25% w/v, fixative osmolalities of less than 355 mosM, including 'isotonic', or greater than 355 mosM, lead to final cell volumes greater or less than native, respectively. Cell-membrane deformability decreases continuously and monotonically with time, as assessed by RPS. The rate of fixation is a direct function of GA concentration, in accordance with a derived empirical expression. The measured kinetic responses are related to considerations of cell size, deformability, and form, and to mechanisms involved in abrupt osmotic hemolysis.     

Effects of SNP, ouabain, and amiloride on electrical potential profile of isolated sheep pleura.

The fluid and solute transport properties of pleural tissue were studied by using specimens of intact visceral and parietal pleura from adult sheep lungs. The samples were transferred to the laboratory in a Krebs-Ringer solution at 4 degrees C within 1 h from the death of the animal. The pleura was then mounted as a planar sheet in a Ussing-type chamber. The results that are presented in this study are the means of six different experiments. The spontaneous potential difference and the inhibitory effects of sodium nitroprusside (SNP), ouabain, and amiloride on transepithelial electrical resistance (R(TE)) were measured. The spontaneous potential difference across parietal pleura was 0.5 +/- 0.1 mV, whereas that across visceral pleura was 0.4 +/- 0.1 mV. R(TE) of both pleura was very low: 22.02 +/- 4.1 Omega. cm2 for visceral pleura and 22.02 +/- 3.5 Omega. cm2 for parietal pleura. There was an increase in the R(TE) when SNP was added to the serosal bathing solution of parietal pleura and to the serosal or mucosal bathing solution in visceral pleura. The same was observed when ouabain was added to the mucosal surface of visceral pleura and to either the mucosal or serosal surface of parietal pleura. Furthermore, there was an increase in R(TE) when amiloride was added to the serosal bathing solution of parietal pleura. Consequently, the sheep pleura appears to play a role in the fluid and solute transport between the pleural capillaries and the pleural space. There results suggest that there is a Na+ and K+ transport across both the visceral and parietal pleura.     

Cyclic peptides. XIX. Synthesis, conformation, and biological activity of cyclo(-Pro-Val-Pro-Val-) with L-L-L-L and L-D-L-D sequences

cyclo(-L-Pro-L-Val-L-Pro-L-Val-) (1L) and cyclo(-L-Pro-D-Val-L-Pro-D-Val-) (1D) were synthesized by the conventional method for peptide synthesis. Conformations of 1L and 1D in solution were studied. Compound 1L has a cis-trans-cis-trans backbone conformation with C2 symmetry in CDCl3. This conformation is slightly different from that in crystalline state and in DMSO-d6 solution. Compound 1D has a cis-trans-cis-trans conformation in DMSO-d6 and an all-trans conformation in trifluoroethanol-d3. Compound 1L retarded stem-growth of rice seedlings and, in contrast, compound 1D promoted root-growth of rice seedlings.     

Expression, characterization, and crystallization of the pyrophosphate-dependent phosphofructo-1-kinase of Borrelia burgdorferi.

The two genes for the putative pyrophosphate-dependent phosphofructokinases (PPi-PFKs) of Borrelia burgdorferi were cloned by PCR and expressed in Escherichia coli, and their protein products were purified to near homogeneity. The larger of the two gene products, a 62-kDa protein, is an active PPi-PFK and exists in solution as a dimer. It has apparent K(m) values for fructose 6-P and PPi of 109 and 15 microM, respectively, and a pH optimum of 6.4 to 7.2. The 62-kDa protein was crystallized and subjected to preliminary diffraction analysis. The smaller gene product, a 48-kDa protein, exists in solution as a higher polymer and shows no ATP- or PPi-dependent activity, despite having a secondary structure as estimated by circular dichroism that is not significantly different from that of other PFKs.