Conformation of the glucotriose unit in the lipid-linked oligosaccharide precursor for protein glycosylation.

The conformation of the glucotriose unit of the protein glycosylation precursor Glc3Man9GlcNAc2 was assessed by deuterium exchange studies on the model tetrasaccharide alpha Glc----2 alpha Glc----3 alpha Glc----3 alpha Man----OCH2CH2CH3 dissolved in deuterated dimethyl sulfoxide. The hydroxyl proton on C-2 of the nonreducing end glucose and on C-4 of the glucose attached to mannose both show dramatic isotope shifts indicative of a strong hydrogen bond between these two hydroxyl groups. Such a hydrogen bond requires a fixed conformation of the glucotriose unit that brings these hydroxyl groups within 3 A of each other, a conformation that is supported by molecular modeling based on hard-sphere exo-anomeric (HSEA) calculations. The temperature dependence of the hydroxyl proton chemical shifts supports the postulated hydrogen bond, and the torsional angles between the three glucose units derived from the HSEA calculations are consistent with results from related studies on other saccharides. The results support a model for biochemical function in which the glucotriose unit could modulate the activity of the oligosaccharyltransferase by binding in a fixed conformation to a specific effector site in the enzyme.     

Saccharomyces cerevisiae structural cell wall mannoprotein

A novel mannoprotein fraction with an average molecular weight of 180 000 has been isolated from Saccharomyces cerevisiae mnn9 mutant cell wall that was solubilized by beta-glucanase digestion. The same material could be extracted from purified wall fragments with 1% sodium dodecyl sulfate. The protein component, 12% by weight, is rich in proline, whereas the carbohydrate, mainly mannose, is about evenly distributed between asparagine and hydroxyamino acids. Endoglucosaminidase H digestion of the isolated mannoprotein reduced its average molecular weight to 150 000, but the mannoprotein, while still embedded in the cell wall, was inaccessible to the enzyme. Biosynthesis and translocation of the mannoprotein were investigated by following incorporation of [3H]proline into this fraction. In the presence of tunicamycin, both mnn9 and wild-type X2180 cells made a mannoprotein fraction with an average molecular weight of 140 000, whereas in the absence of the glycosylation inhibitor, the mnn9 mutant made material with a molecular weight of 180 000 and the mannoprotein made by wild-type cells was too large to penetrate the polyacrylamide gel. Although the cell wall mannoprotein was resistant to heat and proteolytic enzymes, attempts to isolate the carbohydrate-free component failed to yield any characteristic peptide material.     

Plasmodium falciparum: elicitation by peptides and recombinant circumsporozoite proteins of circulating mouse antibodies inhibiting sporozoite invasion of hepatoma cells

The immunodominant epitope region of the circumsporozoite protein of Plasmodium falciparum sporozoites contains 37 tandem repeats of the tetrapeptide Asn-Ala-Asn-Pro and 4 repeats of Asn-Val-Asp-Pro. Synthetic peptides and recombinant proteins of the repeat region were used to immunize mice using different doses and adjuvants. Antisera were tested for inhibition of sporozoite invasion of cultured human hepatoma cells. Synthetic peptides and recombinant proteins elicited high levels of antibodies that inhibited sporozoite invasion when emulsified with complete Freund's adjuvant. Since recombinant proteins with alum elicited a better antibody response to sporozoite invasion than they did without adjuvant, it may be that a recombinant protein containing 32 tandem copies of the tetrapeptide repeat combined with alum could be a candidate malarial vaccine suitable for human trials.     

Purification and characterization of phthalate oxygenase and phthalate oxygenase reductase from Pseudomonas cepacia

An enzymatic system has been isolated that catalyzes dihydroxylation of phthalate to form 1,2-dihydroxy-4,5-dicarboxy-3,5-cyclohexadiene with consumption of NADH and O2. This system is comprised of two proteins: a flavo-iron-sulfur protein with NADH-dependent oxidoreductase activity and a nonheme iron protein with oxygenase activity. Phthalate oxygenase is a large (approximately 217 kDa) protein composed of apparently identical 48-kDa monomers. The active enzyme has one Rieske-type [2Fe-2S] center and one mononuclear iron/monomer. Removal of the mononuclear iron by incubation with EDTA or with o-phenanthroline inhibits oxygenation; ferrous ion completely restores activity. No other metals are effective. Phthalate oxygenase is specific for phthalate or other closely related compounds. However, only phthalate is tightly coupled to NADH oxidation and O2 consumption with a stoichiometry of 1:1:1. Phthalate oxygenase is chemically competent to oxygenate phthalate when artificially supplied with reducing equivalents and O2. Phthalate oxygenase reductase is required, however, for efficient catalytic activity. The reductase is a monomeric 34-kDa flavo-iron-sulfur protein containing FMN and a plant-ferredoxin-type [2Fe-2S] center in a 1:1 ratio. Phthalate oxygenase reductase is specific for NADH but can pass electrons to a variety of acceptors, including: phthalate oxygenase, cytochrome c, ferricyanide, and dichlorophenolindophenol. This system is similar to other bacterial oxygenase systems involved in aromatic degradation including: benzoate dioxygenase, toluene dioxygenase, benzene dioxygenase, and 4-methoxybenzoate demethoxylase. However, phthalate oxygenase can be isolated in large quantities and is more stable than most other such systems.     

Electron-nuclear double resonance spectroscopy of 15N-enriched phthalate dioxygenase from Pseudomonas cepacia proves that two histidines are coordinated to the [2Fe-2S] Rieske-type clusters

We have performed ENDOR spectroscopy at microwave frequencies of 9 and 35 GHz at 2 K on the reduced Rieske-type [2Fe-2S] cluster of phthalate dioxygenase (PDO) from Pseudomonas cepacia. Four samples have been examined: (1) 14N (natural abundance); (2) uniformly 15N labeled; (3) [15N]histidine in a 14N background; (4) [14N]histidine in a 15N background. These studies establish unambiguously that two of the ligands to the Rieske [2Fe-2S] center are nitrogens from histidine residues. This contrasts with classical ferredoxin-type [2Fe-2S] centers in which all ligation is by sulfur of cysteine residues. Analysis of the polycrystalline ENDOR patterns has permitted us to determine for each nitrogen ligand the principal values of the hyperfine tensor and its orientation with respect to the g tensor, as well as the 14N quadrupole coupling tensor. The combination of these results with earlier M?ssbauer and resonance Raman studies supports a model for the reduced cluster with both histidyl ligands bound to the ferrous ion of the spin-coupled [Fe2+ (S = 2), Fe3+ (S = 5/2)] pair. The analyses of 15N hyperfine and 14N quadrupole coupling tensors indicate that the geometry of ligation at Fe2+ is approximately tetrahedral, with the (Fe)2(N)2 plane corresponding to the g1-g3 plane, and that the planes of the histidyl imidazoles lie near that plane, although they could not both lie in the plane. The bonding parameters of the coordinated nitrogens are fully consistent with those of an spn hybrid on a histidyl nitrogen coordinated to Fe. Differences in 14N ENDOR line width provide evidence for different mobilities of the two imidazoles when the protein is in fluid solution. We conclude that the structure deduced here for the PDO cluster is generally applicable to the full class of Rieske-type centers.     

Methanol production by Mycobacterium smegmatis.

Mycobacterium smegmatis cells produce [3H]methanol when incubated with [methyl-3H]methionine. The methanol is derived from S-adenosylmethionine rather than methyltetrahydrofolate. M. smegmatis cells carboxymethylate several proteins, and some of the methanol probably results from their demethylation, but most of the methanol may come from an unidentified component with a high gel mobility. Although methanol in the medium reached 19 microM, it was not incorporated into the methylated mannose polysaccharide, a lipid carrier in this organism.     

Response of 7,12-dimethylbenz(a)anthracene-induced rat mammary tumor cells to tamoxifen, in vitro

Primary cell cultures from a density-defined cell subpopulation of the DMBA-induced rat mammary tumors were exposed to tamoxifen during their log phase of growth. Growth inhibition and the ultrastructure of surviving cells were examined along with the influence of this antiestrogen on the secreted proteins as determined by pulse labeling with [35S]methionine and fluorography. Cell growth was remarkably inhibited at clinically achievable concentrations. However, ultrastructural changes in the surviving cells were minimal, the most noteworthy being the accumulation of myelin bodies. Protein secretion was affected in the defined subpopulation of several tumors by the reduced production of a high-molecular-weight protein. These tumors may represent a population of estrogen-sensitive tumors within the DMBA-induced mammary tumor model.