One step isolation of bovine asialoglycoprotein receptor and its characterization by sequence analysis and MALDI mass spectrometry.
(73/1861)
The asialoglycoprotein receptor (ASGP-R), which is responsible for the uptake of partially deglycosylated serum glycoproteins was isolated from bovine liver. The receptor was purified in one step from solubilized plasma membranes by affinity chromatography on 6-(beta-D-lactosyl)-n-hexylamine coupled to N-hydroxysuccinimide activated Sepharose with a coupling degree of 7.6 micromol/ml gel. The preparation yielded two distinct polypeptides with apparent molecular weights of 48 and 43 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A polyclonal antibody raised against the human ASGP-R recognized the bovine 43 kDa protein in Western blot analysis. The 48 and 43 kDa polypeptides were digested by trypsin and the digests were subsequently analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Sequence analysis of four tryptic fragments, two each of the 48 kDa and of the 43 kDa polypeptides revealed that these were highly homologous to ASGP-R subunits from man, mouse and rat. (+info)
Energy-dependent binding of dansylgalactosides to the beta-galactoside carrier protein.
(74/1861)
Fluorescent beta-galactosides (1-(N-dansyl)amino-beta-D-galactopyranoside (DG0), 2'-(N-dansyl)aminoethyl-beta-D-thiogalactopyranoside (DG2), 2'-(N-dansyl)aminoethyl-beta-D-galactopyranoside (oxy-DG2), and 6'-(N-dansyl)aminohexyl-beta-D-thiogalactopyranoside (DG6)) competitively inhibit lactose transport by membrane vesicles from Escherichia coli ML 309-225, but are not actively transported. An increase in the fluorescence of these dansylgalactosides is observed upon addition of D-lactate, imposition of a membrane diffusion potential (positive outside), or dilution-induced, carrier-mediated lactose efflux. The increase is not observed with 2'-(N-dansyl)aminoethyl-beta-D-thioglucopyranoside nor with membrane vesicles lacking the beta-galactoside transport system. Moreover, the D-lactate-induced fluorescence increase is blocked or rapidly reversed by addition of beta-galactosides, sulfhydryl reagents, inhibitors of D-lactate oxidation, or uncoupling agents. The fluorescence increase exhibits an emission maximum at 500 nm and excitation maxima at 345 nm and at 292 nm. The latter excitation maximum is absent unless D-lactate is added, indicating that the bound dansylgalactoside molecules are excited by energy transfer from the membrane proteins. Titration of vesicles with dansylgalactosides in the presence of D-lactate demonstrates that the lac carrier protein constitutes 3 to 4% oof the total membrane protein, and that the affinity of the carrier for substrate is directly related to the length of the alkyl chain between the galactosidic and the dansyl moieties of the dansylgalactosides. In addition, there is excellent agreement between the affinity constants of the various dansylgalactosides as determined by fluorimetric titration and their apparent Kis for lactose transport (KDs and/or apparent Kis are approximately 550, 3o, 40, and 5 muM FOR DG0, DG2, oxy-DG2, and DG6, respectively). Polarization of fluorescence measurements with DG2 and DG6 demonstrate a dramatic increase in polarization on addition of D-lactate which is reversed by addition of lactose or anaerobiosis. These findings provide strong evidence for the contention that the fluorescence changes observed on "energization" of the membrane are due to binding of the dansylgalactosides per se, rather than binding followed by transfer into the hydrophobic interior of the membrane (+info)
Photoinactivation of the beta-galactoside transport system in Escherichia coli membrane vesicles with 2-nitro-4-azidophenyl-1-thio-beta-D-galactopyranoside.
(75/1861)
2-Nitro-4-azidophenyl-1-thio-beta-D-galactopyranoside (azidophenylgalactoside) is a competitive inhibitor of lactose transport in membrane vesicles isolated from Escherichia coli ML 308-225, exhibiting an apparent Ki of 75 muM. The initial rate and steady state level of [3H]azidophenylgalactoside accumulation are markedly stimulated by the addition of D-lactate to vesicles containing the lac transport system, and kinetic studies reveal an apparent Km of 75 muM. Membrane vesicles devoid of the lac transport system do not take up significant amounts of azidophenylgalactoside in the presence or absence of D-lactate. When exposed to visible light in the presence of D-lactate, azidophenylgalactoside irreversibly inactivates the lac transport system. Strikingly, photolytic inactivation is not observed in the absence of D-lactate. Kinetic studies of the inactivation process yield a KD of 77 muM. Since lactose protects against inactivation and azidophenylgalactoside does not inactivate amino acid transport, it is apparent that these effects are specific for the lac transport system. The results are consistent with the proposal that the lac carrier protein is inaccessible to substrate in the absence of energy coupling. (+info)
Chromosome transfer in Proteus mirabilis mediated by hybrid plasmid.
(76/1861)
A previously-described fused plasmid, P-lacRIdrd19, was found to mediate chromosomal transfer between cells of Proteus mirabilis strain PM5006; PM5006-(P-lacRIdrd19) was usually the donor and various auxotrophs of PM5006 resistant to nalidixic acid and/or streptomycin were recipients. The donor was usually counterselected with nalidixic acid and/or high concentrations of streptomycin. Recombination experiments with single markers indicated a 40-fold variation in recombination frequencies for different markers. Mapping double-auxotrophic markers by their gradient of transmission confirmed this variation and placed each of two independent isolates of eight markers in a linkage group his-ser-ura-pyrB-trp-cys-ade-ilv. Some donor markers did not register. Despite low recombination frequencies, interrupted mating experiments showed a polarity of early marker transfer. The segregation of unselected markers confirmed the order of some markers and showed that genetic material passed from the presumptive donor to the recipient. Recipients with two auxotrophic markers which could not be cotransduced by phage 5006M were converted to prototrophy by conjugation. The plasmid transferred to recipients at high frequency and all recombinants carried it. Recombinants could act as donors in further matings. Recombinants were fully susceptible to phage 5006M, unlike transductants of PM5006 by this phage. Direct involvement of the plasmid was indicated by drastically diminished recombination frequencies in crosses with recipients carrying P-lac as resident. P-lac had previously been shown to reduce the frequency of transfer of the hybrid plasmid to cells harbouring it. The histidine region was the first to register in recipients and recombined at the highest frequency of 5 times 10-6/donor cell. Some temporary association of plasmid and perhaps only the histidine region of the chromosome is favoured as the mechanism of chromosomal transfer. This could explain why not all donor markers could be mapped. Transduction and transformation were excluded as the cause of results. (+info)
Detection and growth of enteropathogenic Escherichia coli in soft ripened cheese.
(77/1861)
The organism most frequently encountered during the 1971 outbreak of enteropathogenic Escherichia coli (EPEC) in soft ripened cheese was a strain that failed to ferment lactose broth within 48 h. Since existing methods for E. coli are dependent upon fermentation of this sugar, such strains can remain undetected, particularly when present in low numbers. Therefore a cultural testing procedure was developed to insure isolation of both lactose-positive and -negative strains. This method used GN broth, modified by substituting lactose and arabinose for glucose and D-mannitol, as an enrichment medium. MacConkey agar, used as a plating medium, was modified by substituting arabinose for half the lactose. The cultural procedure was used in conjunction with a fluorescent antibody method to screen cheese for the presence of presumptive enteropathogenic E. coli. Suspected isolates were subjected to further biochemical and serological testing and identified as members of specific serogroups. These methods were used for the analysis of over 2,000 wheels of cheese; over 10% of the samples tested were found to contain strains belonging to six different serogroups associated with diarrheal diseases. No attempt was made to confirm pathogenicity by in vivo tests. Enumeration of E. coli in cheese showed that numbers increased during storage. Cheese with less than 10 organisms/g initially increased to over 10-5 at room temperature and over 10-3 at 4 C within 10 days. With higher initial counts, levels up to 10-9 were found at 4 C. These studies showed that the high levels of E. coli encountered in these products cannot be used as a direct indicator of post-processing contamination. (+info)
Recombination and the Escherichia coli K-12 sex factor F.
(78/1861)
Recombination between two Flac tra minus elements to give Flac tra plus recombinants was measured in Rec plus and Rec minus strains of Escherichia coli K-12. Polar tra mutations were used to increase the proportion of tra plus recombinants among the parental Flac tra minus elements transferred by complementation. The kinetics, measured in a rec plus strain, showed that recombination began about 1 h after the initiation of mating and was completed about 1 h later. Recombination was abolished in a recA minus strain, reduced by two-thirds in a recF minus strain, and unaffected in recB minus and recC minus strains. It is proposed that the part not due to the RecF pathway results from a RecBC- and RecF-independent system for formation of single-stranded joins. One such join could be followed either by transfer and a site-specific recombination event, or by a second single-stranded join and then transfer: in either case replication and inheritance of the recombinant molecule would be dependent upon the F transfer replication system. Chromosome mobilization by an F' element was normal in a recB plus recF minus strain, and was reduced only fourfold in a recB minus recF plus strain: in the latter strain, both the RecF pathway and the system for single-stranded joins may have contributed to mobilization. Measurement of post-conjugational chromosomal recombination in exponential-phase recipient cells carrying surface exclusion-deficient Flac mutants indicated that F does not itself determine a generalized recombination system able to replace the RecA plus product or the RecBC and RecF pathways. (+info)
Changing the donor cofactor of bovine alpha 1, 3-galactosyltransferase by fusion with UDP-galactose 4-epimerase. More efficient biocatalysis for synthesis of alpha-Gal epitopes.
(79/1861)
Two fusion enzymes consisting of uridine diphosphogalactose 4-epimerase (UDP-galactose 4-epimerase, EC ) and alpha1, 3-galactosyltransferase (EC ) with an N-terminal His(6) tag and an intervening three-glycine linker were constructed by in-frame fusion of the Escherichia coli galE gene either to the 3' terminus (f1) or to the 5' terminus (f2) of a truncated bovine alpha1, 3-galactosyltransferase gene, respectively. Both fusion proteins were expressed in cell lysate as active, soluble forms as well as in inclusion bodies as improperly folded proteins. Both f1 and f2 were determined to be homodimers, based on a single band observed at about 67 kDa in SDS-polyacrylamide gel electrophoresis and on a single peak with a molecular mass around 140 kDa determined by gel filtration chromatography for each of the enzymes. Without altering the acceptor specificity of the transferase, the fusion with the epimerase changed the donor requirement of alpha1, 3-galactosyltransferase from UDP-galactose to UDP-glucose and decreased the cost for the synthesis of biomedically important Galalpha1,3Gal-terminated oligosaccharides by more than 40-fold. For enzymatic synthesis of Galalpha1,3Galbeta1,4Glc from UDP-glucose and lactose, the genetically fused enzymes f1 and f2 exhibited kinetic advantages with overall reaction rates that were 300 and 50%, respectively, higher than that of the system containing equal amounts of epimerase and galactosyltransferase. These results indicated that the active sites of the epimerase and the transferase in fusion enzymes were in proximity. The kinetic parameters suggested a random mechanism for the substrate binding of the alpha1, 3-galactosyltransferase. This work demonstrated a general approach that fusion of a glycosyltransferase with an epimerase can change the required but expensive sugar nucleotide to a less expensive one. (+info)
Chemical characterization of milk oligosaccharides of the polar bear, Ursus maritimus.
(80/1861)
Two trisaccharides, three tetrasaccharides, two pentasaccharides, one hexasaccharide, one heptasaccharide, one octasaccharide and one decasaccharide were isolated from polar bear milk samples by chloroform/methanol extraction, gel filtration, ion exchange chromatography and preparative thin-layer chromatography. The oligosaccharides were characterized by 1H-NMR as follows: the saccharides from one animal: Gal(alpha1-3)Gal(beta1-4)Glc (alpha3'-galactosyllactose), Fuc(alpha1-2)Gal(beta1-4)Glc (2'-fucosyllactose), Gal(alpha1-3)[Fuc(alpha1-2)]Gal(beta1-4)Glc (B-tetrasaccharide), GalNAc(alpha1-3)[Fuc(alpha1-2)]Gal(beta1-4)Glc (A-tetrasaccharide), Gal(alpha1-3)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc, Gal(alpha1-3)[Fuc(alpha1-2)]Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Gl c, Gal(alpha1-3)Gal(beta1-4)GlcNAc(beta1-3)[Gal(alpha1-3)Gal(beta1-4)Glc NAc(beta1-6)]Gal(beta1-4)Glc; the saccharides from another animal: alpha3'-galactosyllactose, Gal(alpha1-3)Gal(beta1-4)[Fuc(alpha1-3)]Glc, A-tetrasaccharide, GalNAc(alpha1-3)[Fuc(alpha1-2)]Gal(beta1-4)[Fuc(alpha1-3)]Glc (A-pentasaccharide), Gal(alpha1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)Gl c, Gal(alpha1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)[F uc(alpha1-3)]Glc (difucosylheptasaccharide) and Gal(alpha1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3) inverted question markGal(alpha1-3) Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-6) inverted question markGal(beta1-4)Glc (difucosyldecasaccharide). Lactose was present only in small amounts. Some of the milk oligosaccharides of the polar bear had alpha-Gal epitopes similar to some oligosaccharides in milk from the Ezo brown bear and the Japanese black bear. Some milk oligosaccharides had human blood group A antigens as well as B antigens; these were different from the oligosaccharides in Ezo brown and Japanese black bears. (+info)