Effect of acceptor membrane phosphatidylcholine on the catalytic activity of bovine liver phosphatidylcholine transfer protein

Protein-mediated transfer of phosphatidylcholine (PC) by bovine liver phosphatidylcholine transfer protein (PC-TP) was examined using a vesicle-vesicle assay system. Donor and acceptor membranes were prepared from Escherichia coli phospholipids and limiting amounts of egg yolk PC. PC transfer between vesicles of E. coli lipid/egg PC was markedly higher than transfer of PC from vesicles of E. coli lipid/egg PC to vesicles of E. coli lipid. Kinetic parameters of the interaction between PC-TP and E. coli lipid vesicles with or without PC was investigated. The apparent dissociation constants of the complex formed between PC-TP and these vesicles were determined kinetically and from double-reciprocal plots of intrinsic PC-TP fluorescence intensity increase versus vesicle concentration. The magnitude of the dissociation constant decreased as the PC content of the vesicles increased from 0 to 5 mol%. In addition, kinetic analysis revealed that the presence of PC in acceptor vesicles increased both the association and dissociation of PC-TP from vesicles. The effect of membrane PC molecules on transfer rates was examined using bis-phosphatidylcholine, a dimeric PC molecule which is not transferred by PC-TP. Rates of PC transfer to acceptor vesicles comprised of E. coli lipid/bis-PC were virtually identical to rates observed with acceptors vesicles prepared from E. coli lipid. The results suggest that transfer of PC by PC-TP is enhanced only when insertion of protein-bound PC occurs concurrently with the extraction of a molecule of membrane PC, i.e., a concerted, one-step catalytic mechanism for phospholipid exchange.     

Biosynthesis of trans,trans,trans-geranylgeranyl diphosphate by the cytosolic fraction from rat tissues.

The cytosolic fractions from rat liver, brain, kidney, spleen and testis demonstrate the capacity to synthesize two products from [3H]isopentenyl diphosphate, i.e., farnesyl diphosphate and geranylgeranyl diphosphate. The highest rate of geranylgeranyl diphosphate synthesis was found in brain, testis and spleen, accounting for up to 30% of the total incorporation of radioactivity under optimal conditions. In all tissues examined the geranylgeranyl diphosphate formed was identified as the trans,trans,trans-isomer. The ratio of geranylgeranyl diphosphate to farnesyl diphosphate produced was specific for the tissue investigated and could be altered by the addition of divalent cations. The results in this study demonstrate the presence of a specific trans,trans,trans-geranylgeranyl diphosphate synthetase showing high affinity for farnesyl diphosphate.     

Interactions ofCandida albicans with bacteria and salivary molecules in oral biofilms

The yeastCandida albicans coaggregates with a variety of streptococcal species, an interaction that may promote oral colonization by yeast cells.C. albicans andCandida tropicalis are the yeasts most frequently isolated from the human oral cavity and our data demonstrate that both these species bind toStreptococcus gordonii NCTC 7869 while two otherCandida species (Candida krusei andCandida kefyr) do not. Adherence ofC. albicans was greatest when the yeast had been grown at 30° C to mid-exponential growth phase. For 21 strains ofC. albicans there was a positive correlation between the ability to adhere toS. gordonii and adherence to experimental salivary pellicle. Whole saliva either stimulated or slightly inhibited adherence ofC. albicans toS. gordonii depending on the streptococcal growth conditions. The results suggest that the major salivary adhesins and coaggregation adhesins ofC. albicans are co-expressed.     

Control of cell volume in the J774 macrophage by microtubule disassembly and cyclic AMP

We have explored the possibilities that cell volume is regulated by the status of microtubule assembly and cyclic AMP metabolism and may be coordinated with shape change. Treatment of J774.2 mouse macrophages with colchicine caused rapid microtubule disassembly and was associated with a striking increase (from 15-20 to more than 90 percent) in the proportion of cells with a large protuberance at one pole. This provided a simple experimental system in which shape changes occurred in virtually an entire cell population in suspension. Parallel changes in cell volume could then be quantified by isotope dilution techniques. We found that the shape change caused by colchicine was accompanied by a decrease in cell volume of approximately 20 percent. Nocodozole, but not lumicolchicine, caused identical changes in both cell shape and cell volume. The volume loss was not due to cell lysis nor to inhibition of pinocytosis. The mechanism of volume loss was also examined. Colchicine induced a small but reproducible increase in activity of the ouabain-sensitive Na(+), K(+)-dependent ATPase. However, inhibition of this enzyme/transport system by ouabain did not change cell volume nor did it block the colchicines-induced decrease in volume. One the other hand, SITS (4’acetamido, 4-isothiocyano 2,2’ disulfonic acid stilbene), an inhibitor of anion transport, inhibited the effects of colchicines, thus suggesting a role for an anion transport system in cell volume regulation. Because colchicine is known to activate adenylate cyclase in several systems and because cell shape changes are often induced by hormones that elevate cyclic AMP, we also examined the effects of cyclic AMP on cell volume. Agents that act to increase syclic AMP (cholera toxin, which activates adenylate cyclase; IBMX, and inhibitor of phosphodiesterase; and dibutyryl cyclic AMP) all caused a volume decrease comparable to that of colchicine. To define the effective metabolic pathway, we studied two mutants of J774.2, one deficient in adenylate cyclase and the other exhibiting markedly reduced activity of cyclic AMP-dependent protein kinase. Cholera toxin did not produce a volume change in either mutant. Cyclic AMP produced a decrease in the cyclase-deficient line comparable to that in wild type, but did not cause a volume change in the kinase- deficient line. This analysis established separate roles for cyclic AMP and colchicine. The volume decrease induced by cyclic AMP requires the action of a cyclic AMP-dependent protein kinase. Colchicine, on the other hand, induced a comparable volume change in both mutants and wild type, and thus does not require the kinase.     

Membrane behavior of exocytic vesicles: II in fate of the trichocyst membranes in paramecium after induced trichocyst discharge

A specific exocytic process, the discharge of spindle trichocysts of paramecium caudatum was examined by means of the electron microscope. This exocytosis is induced by an electric shock simultaneously in nearly all of the trichocysts (ca. 6,000-8,0000 of a single cell. Single paramecia were subjected to the shock and then fixed at defined times after the shock so that the temporal sequence of the pattern of changes of the trichocyst membranes after exocytosis could be studied. The trichocyst vacuoles fuse with the plasma membrane only for that length of time required for expulsion to take place. After exocytosis, the membrane of the vacuole does not become incorporated into the plasma membrane; rather, the collapsed vacuole is pinched off and breaks up within the cytoplasm. The membrane vesiculates into small units which can no longer be distinguished from vesicles of the same dimensions that exist normally within the cell's cytoplasm. the entire process is completed within 5-10 min. These results differ from the incorporation of mucocyst membranes into the plasma membrane as proposed for tetrahymena.     

Dispersal,fragmentation, and isolation shape the phylogeography of the European lineages of Polyommatus (Agrodiaetus) ripartii (Lepidoptera: Lycaenidae)

Polyommatus ripartii is a biogeographically and taxonomically poorly understood species of butterfly with a scattered distribution in Europe. Recently, it has been shown that this species includes several European endemic and localized taxa (galloi, exuberans, agenjoi) that were previously considered species and even protected, a result that poses further questions about the processes that led to its current distribution. We analysed mitochondrial DNA and the morphology of P. ripartii specimens to study the phylogeography of European populations. Three genetically differentiated but apparently synmorphic lineages occur in Europe that could be considered evolutionarily significant units for conservation. Their strongly fragmented and counterintuitive distribution seems to be the result of multiple range expansions and contractions along Pleistocene climatic oscillations. Remarkably, based on the 79 specimens studied, these genetic lineages do not seem to extensively coexist in the distributional mosaic, a phenomenon most evident in the Iberian Peninsula. One of the important gaps in the European distribution of P. ripartii is reduced by the discovery of new Croatian populations, which also facilitate a better understanding of the biogeography of the species. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109 , 817‐829.     

Eight new microsatellite markers in Atlantic cod (Gadus morhua L.) derived from an enriched genomic library

One hundred and fifty random clones from an enriched genomic library of Atlantic cod were sequenced. Primer pairs were designed for 15 microsatellites containing perfect di‐, tri‐, tetra‐ and hexanucleotide repeats and characterized in 96 unrelated fish. Eight markers were successfully amplified, with the number of alleles ranging from two to nine per locus and observed heterozygosity ranging from 0.341 to 0.977. Loci Gmo‐G13 and Gmo‐G14 had a significant excess of homozygotes. All loci conformed to the Hardy–Weinberg equilibrium. Genetic linkage disequilibrium analysis between all pairs of the loci showed no significant departure from the null hypothesis between any of the loci.     

Functional evaluation of serine/threonine residues in the P-Loop of Rhodobacter sphaeroides phosphoribulokinase.

The N-terminal region of phosphoribulokinase (PRK) has been proposed to contain a "P-loop" or "Walker A" motif. In Rhodobacter sphaeroides PRK, four alcohol side chains, contributed by S14, T18, S19, and T20, map within the P loop and represent potential Mg-ATP ligands. Each of these has been individually replaced with an alanine and the impact of these substitutions on enzyme-ATP interactions and overall catalytic efficiency evaluated. Each mutant PRK retains the ability to tightly bind the positive effector, NADH (0.7-0.9 per site), and exhibits allosteric activation, suggesting that the proteins retain a high degree of structural integrity. Similarly, each mutant PRK retains the ability to stoichiometrically (0.7-1.2 per site) bind the alternative substrate trinitrophenyl-ATP. Despite the large size of the PRK oligomer (8 x 32 kDa), (31)P NMR can be used to detect stoichiometrically bound Mg-ATP substrate, which produces markedly broadened peaks in comparison with signals from unbound Mg-ATP. Elimination of alcohol substituents in mutants T18A, S19A, or T20A produces enzymes which retain the ability to form stable PRKMg-ATP complexes. Each mutant complex is characterized by (31)P resonances for alpha- and gamma-phosphoryls of bound Mg-ATP which are narrower than measured for wild-type PRKMg-ATP; signals for the beta-phosphoryl are poorly detectable for mutant PRKMg-ATP complexes. Kinetic characterization indicates that these mutants differ markedly with respect to catalytic activity. T20A exhibits V(m) comparable to wild-type PRK, while V(m) is diminished by 8-fold for T18A and by 40-fold for S14A. In contrast to these modest effects, S19A exhibits decreases in V(m) and V(m)/K(Ru5P) of 500-fold and >15000-fold, respectively. S19A and T18A exhibit only modest (6-7-fold) increases in S(1/2) for ATP but larger (30-45-fold) increases in K(m) for Ru5P. K(I) values for the competitive inhibitor, 6-phosphogluconate, do not significantly change upon mutation of T18 or S19, suggesting that these residues are not crucial to Ru5P binding. A role for the alcohol group of S19, the eighth residue in P-loop motif, as a ligand to the Mg-ATP substrate seems compatible with the characterization data; adjacent alcohols do not efficiently function as surrogates. Such a proposed function for S19 is compatible with its proximity to E131, the acidic residue in a putative Walker B motif and probable second Mg-ATP ligand in PRK's active site.     

Identification of the allosteric regulatory site in bacterial phosphoribulokinase

Bacterial phosphoribulokinases (PRKs) are octameric members of the adenylate kinase family of enzymes. The enzyme is allosterically activated by NADH and allosterically inhibited by AMP. We have determined the crystal structure of PRK from Rhodobacter sphaeroides bound to the ATP analogue AMP-PCP to a resolution of 2.6 A. The structure reveals that the ATP analogue does not bind to the canonical ATP site found in adenylate kinase family members. Rather, the AMP-PCP binds in two different orientations at the interface of three of the monomers in the octamer. This interface was previously characterized as having an unusually large number of arginine residues. Of the five arginine residues that are near the bound nucleotide, one (Arg 221) is highly conserved in both prokaryotic and eukaryotic (nonallosterically regulated) PRKs, two (Arg 234 and Arg 257) are on a second subunit and conserved in only prokaryotic PRKs, and two (Arg 30 and Arg 31) are on a third subunit with only one of them (Arg 31) conserved in prokaryotic PRKs. Each of these arginine residues was converted by site-directed mutagenesis to alanine. Fluorescence binding data suggest that none of these arginines are involved in active site ATP binding and that Arg 234 and Arg 257 on the second subunit are directly involved in NADH binding, while the other arginines have a minimal effect on NADH binding. While the wild-type enzyme exhibits low maximal activity and hyperbolic kinetics with respect to ATP in the absence of NADH and high maximal activity and sigmoidal kinetics in the presence of NADH, the R31A mutant exhibits identical hyperbolic kinetics with respect to ATP in the presence or absence of NADH. Thus, the transmission of allosteric information from one subunit to another is conducted through a single path that includes NADH and Arg 31.