The 1.25 A resolution refinement of the cholera toxin B-pentamer: evidence of peptide backbone strain at the receptor-binding site.

Crystals of the 61 kDa complex of the cholera toxin B-pentamer with the ganglioside GM1 receptor pentasaccharide diffract to near-atomic resolution. We have refined the crystallographic model for this complex using anisotropic displacement parameters for all atoms to a conventional crystallographic residual R=0.129 for all observed Bragg reflections in the resolution range 22 A to 1.25 A. Remarkably few residues show evidence of discrete conformational disorder. A notable exception is a minority conformation found for the Cys9 side-chain, which implies that the Cys9-Cys86 disulfide linkage is incompletely formed. In all five crystallographically independent instances, the peptide backbone in the region of the receptor-binding site shows evidence of strain, including unusual bond lengths and angles, and a highly non-planar (omega=153.7(7) degrees) peptide group between residues Gln49 and Val50. The location of well-ordered water molecules at the protein surface is notable reproduced among the five crystallographically independent copies of the peptide chain, both at the receptor-binding site and elsewhere. The 5-fold non-crystallographic symmetry of this complex allows an evaluation of the accuracy, reproducibility, and derived error estimates from refinement of large structures at near-atomic resolution. We find that blocked-matrix treatment of parameter covariance underestimates the uncertainty of atomic positions in the final model by approximately 10% relative to estimates based either on full-matrix inversion or on the 5-fold non-crystallographic symmetry.

Construction and characterization of versatile cloning vectors for efficient delivery of native foreign proteins to the periplasm of Escherichia coli.

Induction of the wild type cholera toxin operon (ctxAB) from multicopy clones in Escherichia coli inhibited growth and resulted in low yields of cholera toxin (CT). We found that production of wild type CT or its B subunit (CT-B) as a periplasmic protein was toxic for E. coli, but by replacing the native signal sequences of both CT-A and CT-B with the signal sequence from the B subunit of E. coli heat-labile enterotoxin LTIIb we succeeded for the first time in producing CT holotoxin in high yield in E. coli. Based on these findings, we designed and constructed versatile cloning vectors that use the LTIIb-B signal sequence to direct recombinant native proteins with high efficiency to the periplasm of E. coli. We confirmed the usefulness of these vectors by producing two other secreted recombinant proteins. First, using phoA from E. coli, we demonstrated that alkaline phosphatase activity was 17-fold greater when the LTIIb-B signal sequence was used than when the native leader for alkaline phosphatase was used. Second, using the pspA gene that encodes pneumococcal surface protein A from Streptococcus pneumoniae, we produced a 299-residue amino-terminal fragment of PspA in E. coli in large amounts as a soluble periplasmic protein and showed that it was immunoreactive in Western blots with antibodies against native PspA. The vectors described here will be useful for further studies on structure-function relationships and vaccine development with CT and PspA, and they should be valuable as general tools for delivery of other secretion-competent recombinant proteins to the periplasm in E. coli.

Cyanobacteria carrying an smt-lux transcriptional fusion as biosensors for the detection of heavy metal cations.

The metal-responsive smt operator/promoter region of Synechococcus PCC7942 was fused to the luxCDABE genes of Vibrio fischeri. Plasmid DNA (pJLE23) carrying this fusion conferred metal ion-inducible luminescence to transformed cyanobacteria. Synechococcus PCC7942 (pJLE23) was sensitive to ZnCl2 concentrations within a range of 0.5-4 microM as demonstrated by induction of luminescence. Trace levels of CuSO24 and CdCl2 were also detected.

Expression of mouse metallothionein in the cyanobacterium Synechococcus PCC7942.

A cDNA encoding mouse metallothionein was cloned into the shuttle vector pUc303, creating a translational fusion with the bacterial chloramphenicol acetyltransferase gene. The resulting fusion protein has been expressed in the cyanobacterium Synechococcus PCC7942. Cyanobacterial transformants expressed mouse metallothionein-specific mRNA species as detected by RNA slot blots. In addition, the transformants expressed a unique cadmium ion-binding protein corresponding to the predicted size of the mouse metallothionein fusion protein. Expression of this fusion protein conferred a two- to five-fold increase in cadmium ion tolerance and accumulation on Synechococcus PCC7942.

Metalloregulation of the cyanobacterial smt locus: identification of SmtB binding sites and direct interaction with metals.

The smtB gene of Synechococcus PCC 7942 encodes a trans-acting repressor of the metal-regulated smtA gene that encodes a class II metallothionein. Recombinant SmtB has been expressed in Escherichia coli and purified. Electrophoretic mobility shift assays using recombinant SmtB or a protein extract from Synechococcus PCC 6301 reveal the concentration-dependent formation of three specific complexes with the smt operator/promoter. SmtB is also capable of direct interaction with metals as evidenced by 65Zn binding to the SmtB protein as well as the inhibition of repressor-DNA complex formation in the presence of various metal ions. Methylation interference analysis of such complexes identifies four protein contact points within the smt operator/promoter DNA. The points of contact appear to represent two pairs of binding sites, one pair in each of two inverted repeats (nt 548-563, 589-602). The contact points within each pair lie on opposing DNA strands and are separated by 10 bp, placing the repressor binding sites on opposite sides of the DNA helix. Based on electrophoretic mobility shift assays, methylation interference and molecular size calculations we propose that recombinant SmtB binds to the smt operator/promoter in multimeric fashion.

Para-aminobenzoate synthase gene of Saccharomyces cerevisiae encodes a bifunctional enzyme.

The Saccharomyces cerevisiae gene for para-aminobenzoate (PABA) synthase has been identified based upon its ability to confer sulfonamide resistance when present on a multicopy episomal vector. The 3840 bp DNA sequence fragment reported here contains a 2199 bp open reading frame encoding a 733 amino acid protein with similarity to the two components of PABA synthase described for prokaryotes (Escherichia coli PabA and PabB), suggesting that PABA synthase is bifunctional in yeast. The cloned sequence was confirmed to be PABA synthase by gene disruption. Chromosome gel analysis places the gene for PABA synthase on chromosome XIV.

Cytolytic T lymphocytes specific for tumors and infected cells from mice with a retrovirus-induced immunodeficiency syndrome.

LP-BM5 retrovirus complex-infected C57BL/6 mice develop immunodeficiency, somewhat analogous to AIDS, termed murine AIDS (MAIDS). After secondary stimulation with syngeneic B-cell lymphomas from LP-BM5-infected mice, C57BL/6 mice produced vigorous CD8+ cytotoxic T lymphocytes specific for MAIDS-associated tumors. An anti-LP-BM5 specificity was suggested because spleen and lymph node cells from LP-BM5-infected mice served as target cells in competition assays, and cells from LP-BM5, but not ecotropic, virus-infected mice functioned as secondary in vitro stimulators to generate cytotoxic T lymphocytes to MAIDS tumors.

Nucleoside transport in human erythrocytes. A simple carrier with directional symmetry and differential mobility of loaded and empty carrier.

Rapid kinetic techniques were employed to measure the transport of uridine and thymidine in human erythrocytes in zero-trans entry and exist and equilibrium exchange procedures. The kinetic parameters of transport were computed by fitting appropriate integrated rate equations to time courses of transmembrane equilibration of radiolabeled substrate. Transport of uridine and thymidine conformed to the simple carrier model with directional symmetry, but differential mobility of loaded and empty carrier. As was apparent from comparison of zero-trans influx and equilibrium exchange flux, the loaded carrier moved 3 to 18 times faster than the empty carrier in batches of erythrocytes obtained from different individuals. The maximum equilibrium exchange velocities also differed for different batches of erythrocytes. Storage of the cells at 4 degrees C for 4 days or treatment of cells with oxidizing or reducing agents or suspension in hypotonic solutions had no effect on the kinetic properties of the nucleoside transporter. All natural ribo- and deoxyribonucleosides tested when present on the trans side at concentrations well above the Michaelis-Menten constant for transport accelerated the influx and efflux of uridine and inhibited uridine influx when present at these concentrations on the cis side, but nucleobases had no significant effect.

Effect of temperature on kinetics and symmetries of the hexose transporter of Novikoff rat hepatoma cells.

We have used rapid kinetic techniques to measure the accumulation of radioactively labeled 3-O-methyl-D-glucose to transmembrane equilibrium in Novikoff cell suspensions as a function of temperature. Arrhenius plots of the maximum velocities of isotopic exchange and zero-trans entry were continuous between 6 and 39 degrees C (Ea = 15-19 kcal/mol). Equilibrium exchange and zero-trans entry of 3-O-methyl-glucose at six concentrations (1-30 mM) at 16, 27, and 35 degrees C conformed to appropriate integrated rate equations derived for a single transporter. The hexose transporter exhibited directional symmetry, but the loaded carrier moved about 2 times faster than the empty carrier at all three temperatures investigated. Thus, the differential mobilities of loaded and empty carrier are not affected by temperature in this range. The Michaelis--Menten constant for equilibrium exchange increased about 2-fold with increase in temperature between 16 and 35 degrees C.

Broad specificity hexose transport system with differential mobility of loaded and empty carrier, but directional symmetry, is common property of mammalian cell lines.

Rapid kinetic techniques were employed to measure the transport of the nonmetabolizable hexose, 3-O-methyl-D-glucose, in suspensions of human HeLa cells, mouse L- and P388 leukemia cells, and Chinese hamster ovary cells in zero-trans entry and exit and equilibrium exchange procedures. The kinetic parameters of transport were computed by fitting appropriate integrated rate equations to time courses of transmembrane equilibration of radiolabeled substrate. Transport of all four lines, as in Novikoff rat hepatoma cells, conformed to a simple carrier model with directional symmetry but differential mobility of loaded and empty carrier. As was apparent from a comparison of influx and exchange flux, the loaded carrier of all cell types moved between 4 and 14 times faster than the empty carrier. ATP depletion of the cells by incubation in glucose-free medium containing KCN and iodoacetate had no significant effect on the kinetic properties of the transporter. ATP-depleted cells were used to measure the transport of D-glucose, 2-deoxyglucose, D-galactose, and D-glucosamine in the absence of intracellular metabolism. The differential mobilities of empty carrier and carrier loaded with these hexoses and the efficiency of their transport were equivalent to those observed with 3-O-methylglucose, but the Michaelis-Menten constants for the transport of D-galactose and D-glucosamine were 5-8-fold higher than those for D-glucose, 2-deoxy-D-glucose, and 3-O-methylglucose, which were about equivalent.

Facilitated transport of inosine and uridine in cultured mammalian cells is independent of nucleoside phosphorylases.

The zero-trans uptake of uniformly and base-labeled inosine and uridine was measured a 25 degrees C in suspensions of Novikoff rat hepatoma cells, Chinese hamster ovary cells, mouse L cells, mouse S49 lymphoma cells and a purine-nucleoside phosphorylase-deficient subline thereof (NSU-1), and in monolayer culture of mouse 3T3 and L cells. The initial velocities of uptake of both nucleosides were about the same in all cell lines investigated, regardless of the position of the label or of the substrate concentration between 3 and 300 microM or whether or not the cells possessed uridine or purine-nucleoside phosphorylase activity. The kinetic parameters for the facilitated transport of uridine and inosine were also similar in phosphorylase positive and negative cell lines (K = 120--260 microM and V = 6--40 pmol/microliters cell water per s) and the transport activities of the cells exceeded their total phosphorylase activities by at least 10-fold for uridine and 1--2-fold for inosine. Chromatographic fractionation of the intracellular contents and of the culture fluid showed that the free nucleosides appeared intracellularly prior to and more rapidly than their phosphorolysis products. During the initial 20--60 s of uptake of U-14C-labeled nucleosides the rates of intracellular appearance of ribose-1-P and base were about the same. After several minutes of incubation, on the other hand, the main intracellular component was ribose-1-P whereas the base attained a low intracellular steady-state concentration and accumulated in the medium due to exit transport. Other nucleosides, dipyridamole and nitrobenzylthioinosine, specifically inhibited the transport of uridine and inosine, and depressed the intracellular accumulation of ribose-1-P and the formation of base commensurate with that inhibition. The data indicate that the metabolism of inosine and uridine by the various cell lines can be entirely accounted for by the facilitated transport of unmodified nucleoside into the cell followed by intracellular phosphorolysis.

Exit transport of a cyclic nucleotide from mouse L-cells.

At a concentration of 30 mum, 1,4,5,6,8-pentazaacenaphthylene, 3-amino-1,5-dihydro-5-methyl-1-beta-D-[5-14C]ribofuranosyl (NSC-154020), a tricyclic, 7-deazapurine nucleoside (TCN) is rapidly taken up by cultured mouse L-cells and converted to intracellular TCN-monophosphate, but not further metabolized. The TCN-monophosphate is also excreted by the cells into the medium. It is released by a saturable process against a concentration gradient and the release is inhibited by various inhibitors of energy production. This inhibition correlates with a depletion of the cells of ATP. Thus TCN-monophosphate excretion probably involves an active transport system. This transport system is highly temperature-dependent (the Q10 falls between 3 and 4) and is inhibited by papaverine, theophylline, Persantin, Probenecid, phenethyl alcohol and p-chloromercuribenzoate, but not by 500 muM cyclic AMP, AMP, or adenosine. Significant amounts of various natural phosphorylated intermediates (AMP, ATP, UTP, UMP, UDP-hexoses, and phosphorylcholine) are not released from the cells under similar experimental conditions either in the absence or presence of 30 muM TCN.

G2+M arrest of cultured mammalian cells after incorporation of tritium-labeled nucleosides.

Novikoff rat hepatoma cells were propagated in suspension cultures containing 0.5 to 10 muC of 3H-methyl-thymidine, 3H-5-uridine, 3H-G-adenosine or 3H-8-adenine. The presence of the 3H-labeled precursors caused an inhibition of cell replication which was due to a delay or arrest of the cells in G2 and M. The degree of inhibition was proportional to the amount of radioactivity incorporated into nucleic acids. Almost immediate and complete inhibition resulted from incubation with 10 muC 3H-thymidine/ml. The presence of 0.5 muC 3H-thmidine/ml caused a significant increase in the relative proportion of cells in G2+M, even though the population doubling time of the culture appeared to be unaltered.

Transport and countertransport of thymidine in ATP depleted and thymidine kinase-deficient Novikoff rat hepatoma and mouse L cells: evidence of a high Km facilitated diffusion system with wide nucleoside specificity.

Incubation of cultured Novikoff rat hepatoma and mouse L cells in a glucose-free basal medium containing 5 mM KCN and 5 mM iodoacetate for about 10 minutes resulted in a complete depletion of the cells of ATP. ATP-depleted wild type cells or thymidine kinase-deficient sublines of Novikoff or L cells took up thymidine rapidly from the medium without concentrating it intracellularly, and exhibited countertransport of thymidine. Thus uptake was by facilitated diffusion. This transport system differs from the substrate-specific, low-Km (0.5 muM] thymidine transport system previously described for various types of cultured cells in that it exhibits an at least 100-fold higher Km and transports equally well various ribo- and deoxyribonucleosides. The results suggest that the rate-limiting step in thymidine incorporation into the nucleotide pool by wild type cells is phosphorylation rather than transport, or that the cells possess two transport systems, a facilitated diffusion system with low substrate specificity and a second system which involves substrate phosphorylation by thymidine kinase.

Membrane effects of cytochalasin B. Competitive inhibition of facilitated diffusion processes in rat hepatoma cells and other cell lines and effect on formation of functional transport sites.

Cytochalasin B competitively inhibits the transport of 2-deoxy-D-glucose and thymidine in a number of different cell lines (Novikoff rat hepatoma cells, mouse L, S180 and Ki-MSV-transformed BALB/3T3 cells, and human HeLa cells). The apparent Km values for the transport of these substrates as well as the apparent Ki values for the inhibition by cytochalasin B are very similar for the various cell lines, and the effect is readily and completely reversed by removal of the chemical. Thymidine transport by Chinese hamster ovary cells however, is little affected by cytochalasin B, whereas the transport of 2-deoxy-D-glucose, uridine and guanine by these cells is competitively inhibited to about the same extent as in other cell lines. In addition and concomitant with the inhibition of cytokinesis and an alteration in cell shape, cytochalasin B also impairs and delays the formation of functional transport sites for thymidine, guanine and choline in synchronized populations of Novikoff cells without affecting the apparent affinities of the transport systems for their substrates. This effect is unrelated to the direct inhibition of the transport processes, since the drug does not directly inhibit choline transport and has no effect on the formation of 2-deoxy-D-glucose transport sites in spite of the fact that it strongly inhibits the transport of this substrate. The inhibition of functional transport sites may be due to the induction of a structural alteration in the membrane by cytochalasin B which impairs the insertion of new proteins of certain but not all transport systems into the membrane.

Temperature-dependent changes in activation energies of the transport systems for nucleosides, choline and deoxyglucose of cultured Novikoff rat hepatoma cells and effects of cytochalasin B and lipid solvents.

The initial rates of transport of uridine, thymidien, purines, choline and 2-deoxy-D-glucose by cultured Novikoff rat hepatoma cells were determined as a function of temperature between 5 and 41 degrees C. Arrhenius plots of all transport systems exhibited sharp breaks in slope; between 17 and 23 degrees for uridine, thymidine and hypoxanthine-guanine transport and between 29 and 32 degrees for choline and 2-deoxy-D-glucose transport. The activation energies for the transport systems changed from 15-26 kcal/mole below the transition temperatures to 4-9 kcal/mole above the transition temperatures. Propagation of the cells in the presence of cis-6-octadecenoic acid which results in marked changes in the lipid composition of cell membrane, had little effect on the temperature characteristics of the various transport systems. Similarly, propagation of the cells for 24 hr in media containing Tween 40 or nystatin had no effect on the capacity of the cells to transport the various substrates or on the temperature dependence of the transport systems. The presence of ethanol, phenethyl alcohol or Persantin at concentrations that inhibited thymidine and 2-deoxy-D-glucose transport between 40 and 70% also did not alter the transition temperatures or activation energies for the transport of these substrates. Cytochalasin B, on the other hand, shifted the transition temperature for 2-deoxy-D-glucose transport to higher temperatures in a concentration-dependent manner, whereas it had no effect on the temperature dependence of thymidien transport.

Cell cycle and growth stage-dependent changes in the transport of nucleosides, hypoxanthine, choline, and deoxyglucose in cultured Novikoff rat hepatoma cells.

Populations of Novikoff rat hepatoma cells (subline N1S1-67) were monitored for the rates of transport of various substrates and for their incorporation into acid-insoluble material as a function of the age of cultures of randomly growing cells in suspension as well as during traverse of the cells through the cell cycle. Populations of cells were synchronized by a double hydroxyurea block or by successive treatment with hydroxyurea and Colcemid. Kinetic analyses showed that changes in transport rates related to the age of cultures or the cell cycle stage reflecte alterations in the V max of the transport processes, whereas the Km remained constant, indicating that changes in transport rates reflect alterations in the number of functional transport sites. The transport sites for uridine and 2-deoxy-D-glucose increased continuously during traverse of the cells through the cell cycle, whereas those for choline and hypoxanthine were formed early in the cell cycle. Increases in thymidine transport sites were confined to the S phase. Synchronized cells deprived of serum failed to exhibit normal increases in transport sites, although the cells divided normally at the end of the cell cycle. Arrest of the cells in mitosis by treatment with Colcemid prevented any further increases in transport rates. The formation of functional transport sites was also dependent on de novo synthesis of RNA and protein. Inhibition of DNA synthesis in early S phase inhibited the increase in thymidine transport rates which normally occurs during the S phase, but had no effect on the formation of the other transport systems. Transport rates also fluctuated markedly with the age of the cultures of randomly growing cells, reaching maximum levels in the mid-exponential phase of growth. The transport systems for thymidine and uridine were rapidly lost upon inhibition of protein and RNA synthesis, and thus seem to be metabolically unstable, whereas the transport systems for choline and 2-deoxy-D-glucose were stable under the same conditions.