Endotoxic lipid A interaction with human platelets. Structure-function analysis of lipid A homologs obtained from Salmonella minnesota Re595 lipopolysaccharide

We previously reported that human blood platelets are directly stimulated by endotoxic Lipid A via the protein kinase C pathway (Grabarek, J., Timmons, S., and Hawiger, J. (1988) J. Clin. Invest. 82, 964-971). To study the relationship between the molecular structure of Lipid A and its ability to activate human platelets, we used Lipid A homologs derived from Salmonella minnesota Re595 lipopolysaccharide. Preparations of Lipid A are heterogeneous in regard to the degree of substitution of fatty acids which result in multiple homologs. These were separated by thin-layer chromatography and characterized by fast atom bombardment spectroscopy and related techniques (Johnson R. S., Her, G.-R., Grabarek, J., Hawiger, J., and Reinhold, V. N. (1990) J. Biol. Chem. 265, 8108-8116). The homologs of monophosphoryl Lipid A (MLA) present in fractions TLC-8 (heptaacyl MLA ion, m/z 1953), TLC-7 (three hexaacyl species with predominant MLA ion m/z 1715), and TLC-6 (four pentaacyl homologs with predominant MLA ion, m/z 1505) induced secretion of [14C]serotonin and aggregation of platelets. Lipid A homologs in fractions TLC-5 (three tetraacyl MLA ions, m/z 1323, 1307, and 1279), TLC-4 (one major triacyl MLA ion, m/z 1097), TLC-3 (tetraacyl MLA ion, m/z 1278), TLC-2 (a diphosphoryl hexaacyl Lipid A ion, m/z 1795, and several ions of low abundance), and TLC-1 (two ions, m/z 1097 and 666) were not active in regard to human platelet aggregation and [14C]serotonin secretion. The most active homolog was heptaacyl MLA ion, m/z 1953, present in TLC-8, while homologs present in TLC-7 and TLC-6 were 5 and 10 times less active, respectively. Rapid phosphorylation of a human platelet protein of Mr 40,000-47,000 (P47), a substrate for protein kinase C activation, preceded secretion of serotonin when platelets were triggered by the most active heptaacyl MLA ion, m/z 1953. These events were time-dependent, with half-maximal response of phosphorylation of P47 at 30 s and [14C]serotonin secretion at 45 s. A marked difference in the degree of phosphorylation of P47 was observed with heptaacyl MLA homolog present in TLC-8 inducing complete phosphorylation (97%), whereas less acylated Lipid A homologs present in TLC-1 caused marginal phosphorylation (20%). These results indicate that the degree of acylation of monophosphoryl Lipid A determines its functional properties toward human platelets in regard to secretion of [14C]serotonin, aggregation, and activation of protein kinase C.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification and structural determination of nontoxic lipid A obtained from the lipopolysaccharide of Salmonella typhimurium

Endotoxin extracted from the heptose-less mutant of Salmonella typhimurium was hydrolyzed in 0.1 N HCl in methanol/water (1:1, v/v) at 100 degrees C to yield lipid A, which was then fractionated on a Sephadex LH-20 column to yield a major monophosphoryl lipid A fraction. The monophosphoryl lipid A was further fractionated by preparative thin layer chromatography. This process yielded three major bands (TLC-1, -3, and -5) and two minor bands (TLC-7 and -9). The purity of these fractions was established by ion exchange and reverse phase high performance liquid chromatography. The thin layer fractions were analyzed by fast atom bombardment mass spectrometry. TLC-1 and -3 gave molecular ions (M-H)- at m/e 1730 and 1716, respectively. Both of these fractions contained beta-hydroxymyristic, lauric, and 3-myristoxymyristic acids in O-acyl linkages. The molecular formula and Mr of TLC-1 are C95H179O22N2P and 1731.16; those of TLC-3 are C94H177O22N2P and 1717.15. TLC-1 was a methyl homolog of TLC-3. The major component of TLC-5 (C80H151O22N2P and Mr = 1506.99) gave a molecular ion at m/e 1506 and contained two beta-hydroxymyristic acids and a lauric acid in the O-acyl linkages. The major component of TLC-7 (C66H125O19N2P and Mr = 1280.83) and the single component of TLC-9 gave molecular ions at m/e 1280 and 1098, respectively. TLC-7 contained lauric and beta-hydroxymyristic acids in the O-acyl linkages. TLC-9 (C54H103O18N2P and Mr = 1098.69) contained a single O-acylated beta-hydroxymyristate group. TLC-1 and -3 were nontoxic in the chick embryo lethality test and regressed established tumors in the syngeneic guinea pigs.     

Characterization of a structural series of lipid A obtained from the lipopolysaccharides of Neisseria gonorrhoeae. Combined laser desorption and fast atom bombardment mass spectral analysis of high performance liquid chromatography-purified dimethyl derivatives

Monophosphoryl lipid A (MLA) obtained from the lipopolysaccharides of serum-sensitive strains of Neisseria gonorrhoeae was fractionated on a silicic acid column to yield the hexaacyl and pentaacyl MLAs. The dimethyl derivative of the hexaacyl MLA was analyzed by proton nuclear magnetic resonance spectroscopy. The dimethyl esters of hexaacyl and pentaacyl MLAs were further purified by reverse-phase high performance liquid chromatography, and all of the peaks were analyzed by laser desorption mass spectrometry. Considerable structural information was obtained by laser desorption mass spectrometry due to three kinds of specific fragmentations of the sugar at the reducing end. Two major fractions were also analyzed by positive ion fast atom bombardment mass spectrometry. High performance liquid chromatography was able to separate the dimethyl MLA according to number, nature, and position of the fatty acyl groups. Since almost no structural information is available, the mass spectra of the samples were interpreted on the basis of the established structure of a model lipid A (hexaacyl MLA derived from Salmonella minnesota). Thirteen different structures of dimethyl MLA were identified. The four prominent dimethyl MLAs found in the fractionated samples were M1 (Mr = 1463), M2 (Mr = 1479), M3 (Mr = 1661), and M4 (Mr = 1677). These MLAs appear to have a 1'----6 linked glucosamine disaccharide backbone. The most prominent hexaacyl MLA was M3. We propose that it contains hydroxylaurate at the 3- and 3'-positions in ester linkage and lauroxymyristate at the 2- and 2'-positions in amide linkage of the glucosamine disaccharide. The most abundant pentacyl MLA was M2. We propose that it contains hydroxylaurate at the 3- and 3'-positions in ester linkage, lauroxymyristate at the 2'-position in amide linkage, and hydroxymyristate at the 2-position in amide linkage of the disaccharide. The lipid A of N. gonorrhoeae appeared to differ from that of the Salmonella strains by the presence of shorter-chain fatty acids and by the normal fatty acid distribution in the reducing and distal subunits.     

Bilin attachment sites in the alpha, beta, and gamma subunits of R-phycoerythrin. Structural studies on singly and doubly linked phycourobilins

Three unique bilin peptides, a beta subunit peptide bearing a doubly linked phycourobilin (PUB), and two gamma subunit peptides with singly linked PUB groups, were obtained by enzymatic degradation of Gastroclonium coulteri R-phycoerythrin. These peptides were shown to have the sequences (Klotz, A. V., and Glazer, A. N. (1985) J. Biol. Chem. 260, 4856-4863): (Formula: see text) The sequence of peptide beta-3T was identical to that previously established for a doubly linked phycoerythrobilin (PEB) peptide derived from a B-phycoerythrin (Lundell, D. J., Glazer, A. N., DeLange, R. J., and Brown, D. M. (1984) J. Biol. Chem. 259, 5472-5480). Secondary ion mass spectrometry of beta-3T yielded a protonated molecular ion of 1629 mass units, the same as that given by the doubly linked PEB peptide (Schoenleber, R. W., Lundell, D. J., Glazer, A. N., and Rapoport, H. (1984) J. Biol. Chem. 259, 5481-5484), indicating that the doubly linked PUB and PEB tetrapyrroles were isomeric structures. High resolution 1H NMR analyses of peptides beta-3T, gamma-BV8, and gamma-DP provided unambiguous structural assignments for the singly and doubly linked PUB chromophores and indicated that the peptides in gamma-BV8 and gamma-DP were linked to ring A. The determination of which peptide fragment is linked to ring A and which to ring D in peptide beta-3T was not achieved in this study. 1H NMR analyses of three PEB-peptides from G. coulteri R-phycoerythrin--alpha-1 Cys(PEB)-Tyr-Arg, alpha-2 Leu-Cys(PEB)-Val-Pro-Arg, and beta-1 Met-Ala-Ala-Cys(PEB)-Leu-Arg--showed that they were identical to previously described corresponding chromopeptides from Porphyridium cruentum B-phycoerythrin, with the peptide linked to ring A of PEB in each instance (Schoenleber, R. W., Lundell, D. J., Glazer, A. N., and Rapoport, H. (1984) J. Biol. Chem. 259, 5485-5489). This is the first documented report on the structure of singly or doubly linked phycourobilins.     

Purification and characterization of the isoleucyl-tRNA synthetase component from the high molecular weight complex of sheep liver: a hydrophobic metalloprotein

Native isoleucyl-tRNA synthetase and a structurally modified form of methionyl-tRNA synthetase were purified to homogeneity following trypsinolysis of the high molecular weight complex from sheep liver containing eight aminoacyl-tRNA synthetases. The correspondence between purified isoleucyl-tRNA synthetase and the previously unassigned polypeptide component of Mr 139 000 was established. It is shown that dissociation of this enzyme from the complex has no discernible effect on its kinetic parameters. Both isoleucyl- and methionyl-tRNA synthetases contain one zinc ion per polypeptide chain. In both cases, removal of the metal ion by chelating agents leads to an inactive apoenzyme. As the trypsin-modified methionyl-tRNA synthetase has lost the ability to associate with other components of the complex [Mirande, M., Kellermann, O., & Waller, J. P. (1982) J. Biol. Chem. 257, 11049-11055], the zinc ion is unlikely to be involved in complex formation. While native purified isoleucyl-tRNA synthetase displays hydrophobic properties, trypsin-modified methionyl-tRNA synthetase does not. It is suggested that the assembly of the amino-acyl-tRNA synthetase complex is mediated by hydrophobic domains present in these enzymes.     

Purification and characterization of multiple isoforms of tropomyosin from rat cultured cells

We have previously shown that rat cultured cells contain five isoforms of tropomyosin (Matsumura, F., Yamashiro-Matsumura, S., and Lin, J. J.-C. (1983) J. Biol. Chem. 258, 6636-6644) and that these tropomyosins are differentially expressed upon cell transformation (Matsumura, F., Lin, J. J.-C., Yamashiro-Matsumura, S., Thomas, G. P., and Topp, W. C. (1983) J. Biol. Chem. 258, 13954-13964). To examine functions of tropomyosin in microfilament organization, we have purified and partially separated the multiple isoforms of tropomyosin by chromatography on hydroxylapatite. Analyses of cross-linked dimers produced by air oxidation have revealed that all isoforms except the tropomyosin isoform with apparent Mr of 35,000 form homodimers. Although these tropomyosins share many properties characteristic of tropomyosin, structural analyses at a peptide level and immunological analyses have shown that the five isoforms can be classified into two groups, i.e. tropomyosins with higher apparent Mr (Mr = 40,000, 36,500, and 35,000) and tropomyosins with lower apparent Mr (Mr = 32,400 and 32,000). The low Mr tropomyosins show less ability for head-to-tail polymerization and lower affinity to actin than the high Mr tropomyosins. We suggest that these differences in properties may be related to the changes in microfilament organization observed in transformed cells.     

Molecular architecture of a light-harvesting antenna. Core substructure in Synechococcus 6301 phycobilisomes: two new allophycocyanin and allophycocyanin B complexes

Two new allophycocyanin-containing complexes were found among the products of partial dissociation of the phycobilisomes of Synechococcus 6301 strain AN112. These complexes were purified to homogeneity and characterized with respect to composition, stability, and spectroscopic properties. The structures of the complexes were established to be (alpha AP beta AP)3 . 10.5K and (alpha 1APB alpha 2AP beta 3AP) . 10.5 K, where alpha AP and beta AP are subunits of allophycocyanin, and alpha APB is the subunit of allophycocyanin B (see Lundell, D. J., and Glazer, A. N. (1981) J. Biol. Chem. 256, 12600-12606), and 10.5K is an uncolored polypeptide of 10.5-kilodaltons. These complexes are derived from the core substructure of the phycobilisome. Electron microscopic studies of the morphology of the core of strain AN112 phycobilisomes (Yamanaka, G., Glazer, A. N., and Williams, R. C. (1980) J. Biol. Chem. 255, 11004-11010) as well as structural studies of an 18 S subassembly derived from the phycobilisomes by partial dissociation (Yamanaka, G., Lundell, D. J., and Glazer, A. N. (1982) J. Biol. Chem. 257, 4077-4086) indicated that the core assembly consisted of two cylindrical elements each made up of the same four distinct "trimeric" biliprotein-containing complexes. Two such core components, (alpha AP beta AP)3 and alpha 2AP beta 2AP. 18.3K . 75K (where 18.3K and 75K are polypeptides of 18.3- and 75-kilodaltons), were shown to be contained within the 18 S subassembly (Lundell, D. J., and Glazer, A. N. (1983) J. Biol. Chem. 258, 894-901). The isolation of the two allophycocyanin-containing complexes described here completes the characterization of the four types of components in the Synechococcus 6301 phycobilisome core. Two lines of evidence indicate that each of the four complexes is present twice in the core: comparison of the compositions (and yields) of the complexes with that of the intact AN112 phycobilisome, and near-coincidence of the molar absorption spectrum of the phycobilisome with that generated by summing the spectra of the constituent complexes taken in appropriate molar proportions.     

Physical, immunochemical, and functional properties of Acanthamoeba profilin

Acanthamoebe profilin has a native molecular weight of 11,700 as measured by sedimentation equilibrium ultracentrifugation and an extinction coefficient at 280 nm of 1.4 X 10(4) M-1cm-1. Rabbit antibodies against Acanthamoeba profilin react only with the 11,700 Mr polypeptide among all other ameba polypeptides separated by electrophoresis. These antibodies react with a 11,700 Mr polypeptide in Physarum but not with any proteins of Dictyostelium or Naeglaria. Antibody-binding assays indicate that approximately 2% of the ameba protein is profilin and that the concentration of profilin is approximately 100 mumol/liter cells. During ion exchange chromatography of soluble extracts of Acanthamoeba on DEAE-cellulose, the immunoreactive profilin splits into two fractions: an unbound fraction previously identified by Reichstein and Korn (1979, J. Biol. Chem., 254:6174-6179) and a tightly bound fraction. Purified profilin from the two fractions is identical by all criteria tested. The tightly bound fraction is likely to be attached indirectly to the DEAE, perhaps by association with actin. By fluorescent antibody staining, profilin is distributed uniformly throughout the cytoplasmic matrix of Acanthamoeba. In 50 mM KCl, high concentrations of Acanthamoeba profilin inhibit the elongation rate of muscle actin filaments measured directly by electron microscopy, but the effect is minimal in KCl with 2 MgCl2. By using the fluorescence change of pyrene-labeled Acanthamoeba actin to assay for polymerization, we confirmed our earlier observation (Tseng, P. C.-H., and T. D. Pollard, 1982, J. Cell Biol. 94:213-218) that Acanthamoeba profilin inhibits nucleation much more strongly than elongation under physiological conditions.     

Primary structure of rat brain prostaglandin D synthetase deduced from cDNA sequence

The amino acid sequence of rat brain prostaglandin D synthetase (Urade, Y., Fujimoto, N., and Hayaishi, O. (1985) J. Biol. Chem. 260, 12410-12415) was determined by a combination of cDNA and protein sequencing. cDNA clones specific for this enzyme were isolated from a lambda gt11 rat brain cDNA expression library. Nucleotide sequence analyses of cloned cDNA inserts revealed that this enzyme consisted of a 564- or 549-base pair open reading frame coding for a 188- or 183-amino acid polypeptide with a Mr of 21,232 or 20,749 starting at the first or second ATG. About 60% of the deduced amino acid sequence was confirmed by partial amino acid sequencing of tryptic peptides of the purified enzyme. The recognition sequence for N-glycosylation was seen at two positions of amino acid residues 51-53 (-Asn-Ser-Ser-) and 78-80 (-Asn-Leu-Thr-) counted from the first Met. Both sites were considered to be glycosylated with carbohydrate chains of Mr 3,000, since two smaller proteins with Mr 23,000 and 20,000 were found during deglycosylation of the purified enzyme (Mr 26,000) with N-glycanase. The prostaglandin D synthetase activity was detected in fusion proteins obtained from lysogens with recombinants coding from 34 and 19 nucleotides upstream and 47 and 77 downstream from the first ATG, indicating that the glycosyl chain and about 20 amino acid residues of N terminus were not essential for the enzyme activity. The amino acid composition of the purified enzyme indicated that about 20 residues of hydrophobic amino acids of the N terminus are post-translationally deleted, probably as a signal peptide. These results, together with the immunocytochemical localization of this enzyme to rough-surfaced endoplasmic reticulum and other nuclear membrane of oligodendrocytes (Urade, Y., Fujimoto, N., Kaneko, T., Konishi, A., Mizuno, N., and Hayaishi, O. (1987) J. Biol. Chem. 262, 15132-15136) suggest that this enzyme is a membrane-associated protein.     

Purification and primary sequences of the major arginine-containing antifreeze glycopeptides from the fish Eleginus gracilis

An Arg-containing antifreeze glycoprotein from the polar fish Eleginus gracilis was isolated, and the major components were purified to homogeneity. The general protocol for purification was chromatography of serum on DEAE-cellulose, followed by chromatography on a cation exchanger. DEAE-cellulose chromatography resulted in two fractions, A and B. Fraction A contained most of the antifreeze glycoprotein found in E. gracilis (approximately 80% by weight) and consisted of 13 distinct components. Unlike antifreeze glycoproteins from other previously studied polar fish, Fraction A contained both low and high molecular weight antifreeze glycoprotein components. The two major components of Fraction A were sequenced and compared with the sequence of antifreeze glycoproteins 7 and 8 from both Boreogadus saida and Pagothenia borchgrevinki. The antifreeze glycoproteins from E. gracilis were shown to have a similar composition to those previously studied, except for an additional Ala-Arg dipeptide at the carbon terminal in the major components of Fraction A and the position of Pro in the low molecular weight components. The activity of E. gracilis antifreeze glycoproteins is the subject of a companion article (Burcham, T. S., Osuga, D. T., Yeh, Y., and Feeney, R. E. (1986) J. Biol. Chem. 261, 6390-6397).     

Revision of the blocked N terminus of rat heart fatty acid-binding protein by liquid secondary ion mass spectrometry

The primary structure of rat heart muscle fatty acid-binding protein was investigated by liquid secondary ion mass spectrometry. The protein was digested with trypsin, chymotrypsin, and Staphylococcus aureus V8 protease and the resulting peptides were separated by reverse phase high performance liquid chromatography. The masses of the protonated molecular ions (MH+) of the tryptic, chymotryptic, and S. aureus protease peptides were determined by liquid secondary ion mass spectrometry analysis using 20-500 pmol of material. From the tryptic digest, two peptides with MH+ 1036 and 861 were initially found that did not match the published primary sequence (Sacchettini, J. C., Meininger, T. A., Lowe, J. B., Gordon, J. I., and Banaszak, L. J. (1987) J. Biol. Chem. 262, 5428-5430). The amino acid sequences of these two peptides were determined by a combination of mass spectrometry, B/E-linked scanning, and high performance tandem mass spectrometric techniques to be: (Formula: see text). These new data require that corrections be made to the previously published sequence, involving residues 1-4 and 51-52. The corrected amino sequence for rat m-FABP reveals greater homology with myelin P2, mouse adipocyte p422 protein, and intestinal fatty acid-binding protein than was previously demonstrated.     

Evidence for a guanine nucleotide-binding regulatory protein in invertebrate and mammalian sperm. Identification by islet-activating protein-catalyzed ADP-ribosylation and immunochemical methods

The abalone sperm adenylate cyclase does not appear to be regulated by guanine nucleotides, but has a Mg2+-supported catalytic activity similar to other hormone- and guanine nucleotide-regulated enzymes (Kopf, G. S., and Vacquier, V. D. (1984) J. Biol. Chem. 259, 7590-7596; Kopf, G. S., and Vacquier, V. D. (1985) Biol. Reprod. 33, 1094-1104). The present studies were undertaken to ascertain whether the abalone enzyme has associated guanine nucleotide-binding regulatory proteins. Membrane fractions were incubated with either islet-activating protein (IAP) or cholera toxin and analyzed by sodium dodecyl sulfate SDS-polyacrylamide gel electrophoresis for the presence of toxin-catalyzed ADP-ribosylated proteins. The supernatant from a Lubrol PX-extracted 48,000 X g pellet fraction contained a Mr = 41,000 IAP substrate. This substrate could not be ADP-ribosylated prior to detergent extraction. Lubrol PX-solubilized fractions of membrane preparations from mouse, bovine, and human sperm also contained a Mr = 41,000 IAP substrate. These proteins co-migrated on sodium dodecyl sulfate-polyacrylamide gels with the Mr = 41,000 alpha i-subunit of the inhibitory guanine nucleotide-binding regulatory protein (Gi) from transformed chicken embryo fibroblast and mouse S-49 lymphoma membrane extracts. The sperm IAP substrates displayed similar protease digest patterns to alpha i of mouse S-49 lymphoma cells. Sea urchin sperm analyzed in a similar manner contained a Mr = 39,000 IAP substrate. Cholera toxin-catalyzed ADP-ribosylation of specific sperm membrane proteins was not observed in any of the sperm preparations tested. The presence of the beta-subunit common to both the stimulatory and inhibitory guanine nucleotide-binding regulatory heterotrimers was confirmed in sperm using an antiserum directed against the purified beta-subunit of the guanine nucleotide-binding regulatory proteins from bovine brain. It is concluded that all of the sperm tested, with the possible exception of sea urchin sperm, contain a Gi-like protein. Additional properties of these proteins and their role(s) in sperm function are currently being examined.     

Stoichiometric analysis of the specific interaction of the glucocorticoid receptor with DNA

Purified preparations of activated glucocorticoid X receptor complex (GR) contain a Mr 94,000 hormone-binding polypeptide co-purifying together with a Mr 72,000 non-hormone-binding polypeptide (Wrange, O., Okret, S., Radojcic, M., Carlstedt-Duke, J., and Gustafsson, J.-A. (1984) J. Biol. Chem. 259, 4534-4541). GR binds selectively to discrete regions of DNA in mouse mammary tumor virus (Payvar, F., DeFranco, D., Firestone, G.L., Edgar, B., Wrange, O., Okret, S., Gustafsson, J.-A., and Yamamoto, K. R. (1983) Cell 35, 381-392). Such GR-binding DNA fragments were used to measure the stoichiometry of GR to DNA. Quantitative DNaseI protection "footprinting" analysis was used to ensure that saturation conditions for specific DNA-binding were achieved. Glycerol density gradient centrifugation was used to quantitate Mr 94,000 binding to specific and nonspecific DNA sites. One Mr 94,000 entity was bound per specific DNA site. A modified GR purification procedure resulted in increased amounts of Mr 72,000 polypeptide (1.6:1, 94,000:72,000 molar ratio), compared to previous GR preparations. Glycerol gradient centrifugation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the specific GR X DNA complex contained similar amounts of Mr 94,000 and Mr 72,000 polypeptide. It is as yet uncertain if the Mr 72,000 polypeptide is a functional subunit of GR or a co-purifying contaminant only.     

The chitin disaccharide, N,N'-diacetylchitobiose, is catabolized by Escherichia coli and is transported/phosphorylated by the phosphoenolpyruvate:glycose phosphotransferase system

We have previously reported that wild type strains of Escherichia coli grow on the chitin disaccharide N,N'-diacetylchitobiose, (GlcNAc)(2), as the sole source of carbon (Keyhani, N. O., and Roseman, S. (1997) Proc. Natl. Acad. Sci., U. S. A. 94, 14367-14371). A nonhydrolyzable analogue of (GlcNAc)(2,) methyl beta-N, N'-[(3)H]diacetylthiochitobioside ([(3)H]Me-TCB), was used to characterize the disaccharide transport process, which was found to be mediated by the phosphoenolpyruvate:glycose phosphotransferase system (PTS). Here and in the accompanying papers (Keyhani, N. O., Boudker, O., and Roseman, S. (2000) J. Biol. Chem. 275, 33091-33101; Keyhani, N. O., Bacia, K., and Roseman, S. (2000) J. Biol. Chem. 275, 33102-33109; Keyhani, N. O., Rodgers, M., Demeler, B., Hansen, J., and Roseman, S. (2000) J. Biol. Chem. 275, 33110-33115), we report that transport of [(3)H]Me-TCB and (GlcNAc)(2) involves a specific PTS Enzyme II complex, requires Enzyme I and HPr of the PTS, and results in the accumulation of the sugar derivative as a phosphate ester. The phosphoryl group is linked to the C-6 position of the GlcNAc residue at the nonreducing end of the disaccharide. The [(3)H]Me-TCB uptake system was induced only by (GlcNAc)(n), n = 2 or 3. The apparent K(m) of transport was 50-100 micrometer, and effective inhibitors of uptake included (GlcNAc)(n), n = 2 or 3, cellobiose, and other PTS sugars, i.e. glucose and GlcNAc. Presumably the PTS sugars inhibit by competing for PTS components. Kinetic properties of the transport system are described.     

Isolation and characterization of thioredoxin f from the filamentous cyanobacterium, Anabaena sp. 7119

Two thioredoxin fractions had previously been reported to occur in Anabaena 7119 by Buchanan and co-workers (Yee, B. C., dela Torre, A., Crawford, N. A., Lara, C., Carlson, D. E., and Buchanan, B. B. (1981) Arch. Microbiol. 130, 14-18). These proteins were detected by their ability to activate spinach fructose-1,6-bisphosphatase (Fru-P2-ase). The partially purified proteins resembled similar thioredoxins found in spinach chloroplasts and were designated thioredoxin f (Tf) for the fraction most effective in activating spinach Fru-P2-ase and thioredoxin m (Tm) for the fraction most effective in activating spinach NADPH-malate dehydrogenase. Using the assay system of Yee and co-workers, we were able to separate and purify to homogeneity two thioredoxin fractions from Anabaena extracts. Tm corresponded to the thioredoxin fraction we had isolated and studied previously (Gleason, F. K., and Holmgren, A. (1981) J. Biol. Chem. 256, 8301-8309). The other fraction, Tf, was characterized further. Unlike the thioredoxins found in higher plants, the cyanobacterial thioredoxins do not appear to be related. Anabaena thioredoxin f has a Mr = 25,500 as compared to the more usual Mr = 12,000 for Tm. From a comparison of the amino acid composition, Tf is not obviously a dimer or otherwise related to Tm. Tf has one active center cystine disulfide. Anabaena Tf activates spinach Fru-P2-ase very efficiently but has very little activity with spinach malate dehydrogenase. Anabaena Tf, unlike Tm, does not reduce the homologous ribonucleotide reductase. Anabaena Tf also does not activate a partially purified preparation of Anabaena Fru-P2-ase. We conclude that the cyanobacterial Tf is a unique protein with no structural or functional properties in common with other thioredoxins.     

Isolation and characterization of eight lipid A precursors from a 3-deoxy-D-manno-octylosonic acid-deficient mutant of Salmonella typhimurium

Temperature-sensitive mutants of Salmonella typhimurium that are defective in the biosynthesis of 3-deoxy-D-manno-octulosonate are known to accumulate disaccharide precursor(s) of lipid A at 42 degrees C (Rick, P. D., Fung, L. W.-M., Ho, C., and Osborn, M. J. (1977) J. Biol. Chem. 252, 4904-4912). We have devised new methods for purifying this material by chromatography on DEAE-cellulose and silicic acid columns and have fractionated it into eight related anionic components that fall into four sets, as judged by their charge. Substances IA and IB have an apparent net charge of -1, IIA and IIB of -2, IIIA and IIIB of -3, and IVA and IVB of -4. Negative ion fast atom bombardment mass spectrometry reveals that the simplest component is IVA [( M - H]- at m/z 1404). Compound IVA is also the most abundant, representing 30-50% of the accumulated lipids after 3 h at 42 degrees C. Structural studies of IVA, including NMR spectroscopy described in the accompanying paper, reveal that it consists of O-(2-amino-2-deoxy-beta-D-glucopyranosyl)-(1----6)-2-amino-2-deoxy-alpha - D-glucose, acylated at positions 2, 3, 2', and 3' with beta-hydroxymyristoyl moieties and bearing phosphate groups at positions 1 and 4'. Compound IIIA ([M - H]- at m/z 1527) contains an additional phosphoethanolamine residue, while IIA ([M - H]- m/z 1535) bears an aminodeoxypentose substituent, presumably 4-amino-4-deoxy-L-arabinose. Compound IA ([M - H]- at m/z 1658) bears both a phosphoethanolamine and an aminodeoxypentose. The compounds of the less abundant B series are further derivatized with an ester-linked palmitoyl moiety. Our results demonstrate that these precursors are far more heterogeneous than previously suspected.     

Protein kinase C-stimulated phosphorylation in vitro of a Mr 80,000 protein phosphorylated in response to phorbol esters and growth factors in intact fibroblasts. Distinction from protein kinase C and prominence in brain

In previous studies in intact 3T3-L1 fibroblasts and adipocytes, we demonstrated that the phosphorylation state of an acidic, multicomponent Mr 80,000 protein appeared to be a specific and useful marker for the activation state of protein kinase C (Blackshear, P.J., Witters, L.A., Girard, P.R., Kuo, J.F., and Quamo, S.N. (1985) J. Biol. Chem. 260, 13304-13315). In the present studies, we demonstrate that the Mr 80,000 protein from rat adipose tissue was a substrate for protein kinase C in vitro, and co-migrated on two-dimensional gels with the analogous protein from murine 3T3-L1 adipocytes labeled by exposure of intact cells to 32Pi and phorbol 12-myristate 13-acetate. Partial proteolytic maps of the two 32P-proteins were nearly identical, supporting the postulate that the sites phosphorylated by protein kinase C in vitro, and in response to phorbol 12-myristate 13-acetate in vivo, were similar or identical. Despite their similar apparent molecular weights, we were able to distinguish between the Mr 80,000 protein and protein kinase C by several physical criteria. The Mr 80,000 protein kinase C substrate was found in fractions of all rat tissues examined, but was most prominent in rat brain. Phorbol 12-myristate 13-acetate also stimulated phosphorylation of the Mr 80,000 protein in several types of cultured neuronal cells, suggesting a possible role for this protein in cholinergic neurotransmission. The Mr 80,000 protein appears to be a useful marker for protein kinase C activation in a variety of cell types.     

Membrane topology of ATP synthase from bovine heart mitochondria and Escherichia coli

The polypeptides exposed to lipids in the membranous F0 sector of the mitochondrial and Escherichia coli ATP synthases were labelled with radioactive photoreactive lipids. Highly resolving gel electrophoretic conditions were used in order to separate all the eighteen components forming the bovine heart mitochondrial enzyme. The hydrophobic labelling was performed on fully active and inhibitor-sensitive ATP synthases. In the mitochondrial enzyme prepared according to Serrano et al. (1976) [J. Biol. Chem. 251, 2453-2461] seven polypeptides of Mr 30500; 11500; 10500; 10000; 9500; 8500 and 4500 were labelled. The major amount of radioactivity was associated with the 30500-Mr component, which is thought to be the adenine nucleotide carrier. In the preparation of Galante et al., (1979) which almost completely lacks this component [J. Biol. Chem. 254, 12372-12378] nine polypeptides of Mr 25000; 21000; 11500; 10500; 10000; 9500; 9200; 8500 and 4500 were labelled. In the ATPase synthase from E. coli the major amount of labelling was associated with subunit b and only a minor portion with subunit c.     

Interaction of the mitochondrial ATPase complex with phospholipids

The interaction of bovine heart mitochondrial oligomycin-sensitive ATPase (Serrano, R., Kranner, B. L., and Racker, E. (1976) J. Biol. Chem. 251, 2453-2461) with phospholipids has been examined by labeling the subunits exposed to lipids with photoreactive radioactive phospholipids. A subunit of Mr = 29,000 and some polypeptides in the range of 6,000 to 13,000 daltons were labeled. F1-ATPase subunits did not interact with the photoactive probes. This result is compared with the different pattern of labeling obtained with another mitochondrial ATPase preparation (Galante, Y.M., Wong, S. Y., and Hatefi, Y. (1979) J. Biol. Chem. 254, 12372-12378), which is devoid of the 29,000 component.     

Structural variability of rabbit secretory components. Allotype-associated differences in the third, fourth, and fifth domains

We have previously shown (Frutiger, S., Hughes, G. J., Hanly, W. C., Kingzette, M., and Jaton, J.-C. (1986) J. Biol. Chem. 261, 16673-16681) that limited tryptic digestion of the high Mr form of rabbit secretory component of allotypes t61, t62, and t63 generates two major fragments, the NH2-terminal domain and a 40-kDa fragment encompassing domains 3, 4, and 5. Similarly, from the low Mr form of secretory component, (SC) the NH2-terminal domain, together with a 30-kDa fragment containing domains 4 and 5, were released. These fragments were used as inhibitors in a sensitive competitive binding radioimmunoassay with noncross-reactive rabbit alloantisera to study the distribution and localization of the major allotype-specific allotopes within the SC polypeptide. The 40-kDa fragments were shown to inhibit the 125I-labeled intact SC/anti-SC allotype reaction to the extent of 90%, i.e. nearly as well as the intact homologous high Mr SC form. In contrast, the NH2-terminal fragments (domain 1) were not inhibitory. The low Mr SC of each allotype was less inhibitory on a molar basis than the homologous high Mr SC polypeptide, an observation compatible with the deletion of domains 2 and 3 in the smaller polypeptide (Deitcher, D. L., and Mostov, K. E. (1986) Mol. Cell. Biol. 6, 2712-2715; Frutiger, S., Hughes, G. J., Fonck, Ch., and Jaton, J.-C. (1987) J. Biol. Chem. 262, 1712-1715). The structural correlates of the allotypic specificities were evaluated by comparative peptide mapping of the 40-kDa fragments (allotypes t61, t62, and t63). The data suggest that the t61 allotype structure differs significantly from the t62 and t63 structures, the latter two being much more related to each other than to t61. These findings are in full agreement with the serological data. The inhibition results suggest that the major allotype-specific, noncross-reactive allotopes of SC are distributed throughout domains 3, 4, and 5, even though domain 4 appears to be more conserved than domains 3 and 5 between the allotypes t61 and t63. Seven amino acid substitutions between t61 and t63 have been detected within domains 3, 4, and 5.