Glutamate mutase from Clostridium cochlearium. Purification, cobamide content and stereospecific inhibitors.

Both components, E and S, of the adenosylcobalamin-(coenzyme B12)-dependent glutamate mutase from Clostridium cochlearium were purified. Component S (16 kDa) must be added to component E to obtain activity, although the latter contains substoichiometric amounts of component S besides the major 50-kDa subunit. The enzyme proved to be very similar to that of C. tetanomorphum as described by Barker et al. [Barker, H. A., Rooze, V., Suzuki, F. & Iodice, A. A. (1964) J. Biol. Chem. 239, 3260-3266] but component E of C. cochlearium was more stable and led to the first pure preparation. The pink component E showed a cobamide-like absorbance spectrum with a characteristic maximum at 470 nm indicating the presence of a cob(II)amide, probably Co alpha-[alpha-(aden-9-yl)]-cob(II)amide. A typical cob(II)amide signal at g = 2.23 with hyperfine and superhyperfine splitting was observed by EPR spectroscopy. A cobamide content of about 0.43 mol/mol 50-kDa subunit was determined by cyanolysis. Substitution of the migrating hydrogen at C-4 of glutamate by fluorine yielded the potent competitive inhibitor (2S,4S)-4-fluoroglutamate (Ki = 70 microM). (2R,3RS)-3-Fluoroglutamate (Ki = 600 microM) was also inhibitory. The competitive inhibition by 2-methyleneglutarate (Ki = 400 microM) and (S)-3-methylitaconate (Ki = 100 microM) but not by (RS)-2-methylglutarate suggested the transient formation of an sp2 center during catalysis. However, the presence of an N-terminal pyruvoyl residue was excluded and no evidence for the participation of another electrophilic center in the reaction was obtained.     

Oligosaccharides at individual glycosylation sites in glycoprotein 71 of Friend murine leukemia virus

Glycoprotein 71 from Friend murine leukemia virus was digested with proteases and the glycopeptides obtained were isolated and assigned, by amino acid sequencing, to the eight N-glycosylated asparagines in the molecule; only Asn334 and Asn341 could not be separated. The oligosaccharides liberated from each glycopeptide by endo-beta-N-acetylglucosaminidase H, or by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F, were fractionated and subjected to structural analysis by one- and two-dimensional 1H NMR, as well as by methylation/gas-liquid-chromatography/mass-fragmentography. At each glycosylation site, the substituents were found to be heterogeneous including, at Asn334/341 and Asn410, substitution by different classes of N-glycans: oligomannosidic oligosaccharides, mainly Man alpha 1----6(Man alpha 1----3)Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAc beta 1----, were detected at Asn168, Asn334/341 and Asn410. Hybrid species, partially sialylated, intersected and (proximally) funcosylated Man alpha 1----6(Man alpha 1----3)Man alpha 1----6 and Man alpha 1----3Man alpha 1----6 and Man alpha 1----3Man alpha 1----6(Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAc beta 1----, were found at Asn12, as previously published [Schlüter, M., Linder, D., Geyer, R., Hunsmann, H., Schneider, J. & Stirm, S. (1984) FEBS Lett. 169, 194-198] and at Asn334/341. N-Acetyllactosaminic glycans, mainly partially intersected and fucosylated NeuAc alpha 2----3 or Gal alpha 1----3Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6(NeuAc alpha 2----6 or NeuAc alpha 2----3Gal-beta 1----4GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNac beta 1----4GlcNAc beta 1---- with some bifurcation at ----6Man alpha 1----6, were obtained from Asn266, Asn302, Asn334/341, Asn374 and Asn410. In addition, Thr268, Thr277, Thr279, Thr304/309, as well as Ser273 and Ser275, were found to be O-glycosidically substituted by Gal beta 1----3GalNAc alpha 1----, monosialylated or desialylated at position 3 of Gal or/and position 6 of GalNAc.     

Control of Early Gene Expression of Bacteriophage T4: Involvement of the Host rho Factor and the mot Gene of the Bacteriophage

Many early mRNA species of bacteriophage T4 are not synthesized after infection of Escherichia coli in the presence of chloramphenicol. This has been interpreted as a need for T4 protein(s) to be synthesized to allow expression of some early genes, e.g., those for deoxycytidinetriphosphatase, deoxynucleosidemonophosphate kinase and UDP-glucose-DNA beta-glucosyltransferase. In the experiments described here, early mRNA of bacteriophage T4 was allowed to accumulate during chloramphenicol treatment. After the addition of rifampin to inhibit further RNA synthesis, and subsequent removal of chloramphenicol, the accumulated mRNA was permitted to express itself into measured enzyme activities. It was shown that the early mRNA species coding for deoxycytidinetriphosphatase and UDP-glucose-DNA beta-glucosyltransferase could be formed in the presence of chloramphenicol if the E. coli host cell carried a mutation in the structural gene for the RNA chain termination factor rho. This was interpreted to mean that T4 protein(s) with anti-rho activity is normally required for the expression of these two early genes. An altered rho-factor could not, however, relieve the need of phage protein synthesis for the formation of another early mRNA, that coding for deoxynucleosidemonophosphate kinase. In this case the mot gene of T4 seemed to be involved, since the primary infection of E. coli cells with the mot gene mutant tsG1 did not allow subsequent deoxynucleoside monophosphate kinase mRNA synthesis after wild-type phage infection in the presence of chloramphenicol. In control experiments, deoxynucleoside monophosphate kinase mRNA synthesis induced by wild-type phage superinfecting in the presence of chloramphenicol was facilitated by the primary infection with T4 phage containing an unmutated mot gene.     

Copper regulation of ceruloplasmin in copper-deficient rats.

In copper-deficient rats, oral intubation of copper increases the rate of ceruloplasmin synthesis without affecting general synthesis of plasma or liver proteins. It also restores the enzyme from half to full activity. Copper given by injection at doses commonly employed has additional nonspecific effects on protein synthesis and in some strains of rats produces severe hemolysis. In contrast to deficient rats, in normal rats copper does not elevate plasma ceruloplasmin unless hemolysis also occurs. Thus, at least in deficiency, copper availability controls the rate of synthesis, acitvation, and plasma concentration of ceruloplasmin.     

Effect on sphingomyelin-containing liposomes of phospholipase D from Corynebacterium ovis and the cytolysin from Stoichactis helianthus

The toxic, sphingomyelin-specific phospholipase D (phosphatidylcholine phosphatidohydrolase EC 3.1.4.4) from Corynebacterium ovis was purified to near homogeneity. It has a molecular weight of 31 000 and a pI of approx. 9.8. Although not cytolytic itself, it protected red cells from hemolysis by staphylococcal sphingomyelinase (beta-hemolysin) and helianthus toxin. The apparently non-enzymatic cytolysin (helianthus toxin) from the sea anemone Stoichactis helianthus also interacts with membrane sphingomyelin. C. ovis and helianthus toxins were compared with regard to their effects on liposome model membranes, and they were found both to produce changes analogous to those in erythrocytes. Only helianthus toxin caused release of trapped glucose marker, but liposomes could be protected from release by pretreatment with C. ovis toxin. Both toxins demonstrated binding to sphingomyelin-containing liposomes, but only the bacterial sphingomyelinase catalyzed the release of choline from these vesicles.     

Fibroblast surface antigen (SF): Molecular properties,distribution in vitro and in vivo,and altered expression in transformed cells

We have recently described a cell type-specific surface (SF) antigen that is deleted in chick fibroblasts transformed by Rous sarcoma virus. SF antigen is a major surface component and makes up about 0.5% of the total protein on normal cultured fibroblasts. The antigen is shed from normal cells and is present in circulation (serum, plasma), and in vivo, also, in tissue boundary membranes. The molecular equivalents of both cellular and serum SF antigen are distinct, large polypeptides, one of which (SF210, MW 210,000) is glycosylated and, on the cell surface, highly susceptible to proteases and accessible to surface iodination. Immunofluorescence and scanning electron microscopy have indicated that the antigen is located in fibrillar structures of the cell surface, membrane ridges, and processes. Human SF antigen is present in human fibroblasts and in human serum. We have recently shown that human SF antigen is identical to what has been known as the “cold-insoluble globulin” and that it shows affinity toward fibrin and fibrinogen. Our results also indicate that loss of the transformation-sensitive surface proteins is due not to loss of synthesis but to lack of insertion of the protein in the neoplastic cell surface. Both normal and transformed cells produce the SF antigen, but the latter do not retain it in the cell surface. The loss of SF antigen, a major cell surface component, from malignant cells creates an impressive difference between the surface properties of normal and malignant cells. The possible significance of SF antigen to the integrity of the normal membrane and its interaction to surrounding structures is discussed.     

A histological method for the visualization of the intercellular permeability barrier in mammalian stratified squamous epithelia

Summary Mammalian epidermis and oral epithelia possess an intercellular permeability barrier which is located in the superficial region of the tissue. This study reports a staining reaction which appears to demonstrate a histological correlate of this functional property. Specimens of ear skin, palate, buccal and oesophageal mucosa and of cornea and bladder were obtained from adult rabbits and rats, bisected and either incubatedin vitro with 2.5% horseradish peroxidase as a tracer or fixed and processed for light microscopy and stained with a modification of Hart's elastin stain. Examination of specimens prepared by each procedure showed a complementary staining pattern in the intercellular spaces of the stratum corneum or in the superficial region of the non-keratinized tissue. In the epidermis and oral and oesophageal epithelia, the region which excluded the tracer stained with the modified elastin stain. In contrast, the corneal and bladder epithelia neither excluded the tracer nor showed intercellular staining. This relationship between staining of the intercellular space and the exclusion of tracer suggests that the intercellular material in the superficial region of epithelia may be chemically altered to form a barrier substance, possibly as the result of the discharge of the contents of the membrane-coating granules which are present in all the epithelia examined except the cornea and bladder.     

Investigating the evolution of floras: problems and progress — An introduction

The Cape flora of southern Africa is a remarkable hotspot for plant species diversity and endemism. At a meeting in Zurich in 2004 progress in understanding the evolution of this diversity was reviewed. In this symposium, four papers presenting several of the methods used in this investigation were reported. These papers deal with molecular dating methods, the reconstruction of ancestral habitats, with possible speciation scenarios for the Cape flora, and the importance of the correct sampling strategies.