Glycosylation of the phosphate binding protein, PstS, in Streptomyces coelicolor by a pathway that resembles protein O-mannosylation in eukaryotes

Previously mutations in a putative protein O -mannosyltransferase (SCO3154, Pmt) and a polyprenol phosphate mannose synthase (SCO1423, Ppm1) were found to cause resistance to phage, φC31, in the antibiotic producing bacteria Streptomyces coelicolor A3(2). It was proposed that these two enzymes were part of a protein O-glycosylation pathway that was necessary for synthesis of the phage receptor. Here we provide the evidence that Pmt and Ppm1 are indeed both required for protein O-glycosylation. The phosphate binding protein PstS was found to be glycosylated with a trihexose in the S. coelicolor parent strain, J1929, but not in the pmt ⁻ derivative, DT1025. Ppm1 was necessary for the transfer of mannose to endogenous polyprenol phosphate in membrane preparations of S. coelicolor . A mutation in ppm1 that conferred an E218V substitution in Ppm1 abolished mannose transfer and glycosylation of PstS. Mass spectrometry analysis of extracted lipids showed the presence of a glycosylated polyprenol phosphate (PP) containing nine repeated isoprenyl units (C₄₅-PP). S. coelicolor membranes were also able to catalyse the transfer of mannose to peptides derived from PstS, indicating that these could be targets for Pmt in vivo .     

Phosphorus nutrition of jarrah (Eucalyptus marginata) seedlings

Summary Effects of calcium phosphate supply on plant dry matter and phosphorus concentrations of parts of jarrah (Eucalyptus marginata) seedlings grown in a lateritic topsoil from the jarrah forest were examined in two glasshouse trials. Phosphorus deficiency depressed root and shoot dry weights and severely deficient leaves were smal and purple with prominent red major veins. Phosphorus deficiency severely reduced stem phosphorus levels (0.5% to 0.02%, experiment 1). Phosphorus concentrations were higher in bark than wood and the amount of phosphorus in the bark was sensitive to stem age and phosphate supply. Phosphorus adequate plants had bark phosphorus concentrations in the range 0.2–0.9% compared to <0.1% in deficient plants (experiment 2). Jarrah leaves accumulated dry matter up to 80 days after expansion and some leaves exported phosphorus during this period. Bark analysis may therefore be preferable to leaf analysis for detecting phosphorus deficiency in this species.     

Zinc nutrition and leaf carbonic anhydrase activity ofEucalyptus maculata seedlings andTrifolium subterraneum

The competitive ability of eight strains ofBradyrhizobium on Vigna was examined. It was found that strains S24, M10, and M11 occupied a greater percent of nodules when introduced as mixed inoculum of two strains. Growth rate of strains did not affect competitive ability of the strains. Two hydrogen-uptake positive (Hup⁺) strains, S24 and M10, were found to be good competitors while another Hup⁺ strain GR4 was not so. Influence of the host in competition was observed in the case of strain GR4.     

Bacterial siderophores : structure and NMR assignment of pyoverdins Pa,siderophores ofPseudomonas aeruginosa ATCC 15692

Summary In iron-deficient conditions,Pseudomonas aeruginosa ATCC 15692 synthesizes two major siderophores, pyoverdins Pa and pyoverdin Pa B. Two other compounds, pyoverdin Pa A (occurring from hydrolysis of pyoverdin Pa during the culture) and pyoverdin Pa C (occurring artifactually during the purification procedure) were also isolated. All these compounds possess the same partly cyclic peptide chain wherel-Orn(OH · HCO) isN -formyl,N -hydroxy-l-ornithine. The chain is bound to a chromophore derived from 2,3-diamino-6,7-dihydroxyquinoline and having the (S) configuration. The four pyoverdins differ only in the acyl substituent bound to the nitrogen atom bound to carbon C3 of the chromophore. This is succinamide (pyoverdin Pa), succinic acid (pyoverdin Pa A), methyl succinate (pyoverdin Pa C) and 2-oxoglutaric acid (pyoverdin Pa B). The complete¹H- and¹³CNMR assignments, using two-dimensional total correlation NMR spectroscopy (TOCSY) and rotating-frame Overhauser enhancement spectroscopy (ROESY) procedures, as well as¹H-¹³C correlations, are reported. The complete sequence of the peptide using CH-NH correlations was achieved by NMR and confirmed the partly cyclic structure earlier reported using fast-atom-bombardment mass spectrometry (FAB-MS) on the siderophores and their dansylated fragments [Briskot G, Taraz K, Budzikiewicz H (1989)Liebigs Ann Chem: 375–384]. The use of these NMR procedures appears to be a tool of choice and a complementary approach to FAB-MS in the structure determination of some complex pyoverdins.Abbreviations Ser serine - Arg arginine - Thr ethreonine - Lys lysine - OHOrn N -hydroxyornithine - Chr chromophore     

Canine alpha-L-fucosidase in relation to the enzymic defect and storage products in canine fucosidosis.

Canine liver alpha-L-fucosidase was purified to apparent homogeneity by affinity chromatography on agarose-epsilon-aminohexanoyl-fucopyranosylamine. It is composed of multiple forms of a common active subunit of 45-50 kDa, which can aggregate in different combinations to form polymers, predominantly dimers. Antiserum was raised against the purified enzyme. There is negligible residual alpha-L-fucosidase in the tissues of English springer spaniels with the lysosomal storage disease fucosidosis. Although no alpha-L-fucosidase protein was detected by Western blotting or by the purification procedure in the affected tissues, some enzymically inactive cross-reacting material was detected in both normal and affected tissues. This suggests that another protein without alpha-L-fucosidase activity was co-purified with the enzyme. Dog liver alpha-L-fucosidase was precipitated by goat anti-(human liver alpha-L-fucosidase) IgG, indicating homology between the enzymes in the two species. Two purified storage products isolated from the brain of a dog with fucosidosis were used as natural substrates for various preparations of canine liver alpha-L-fucosidase. Analysis of the digestion mixtures by t.l.c. and fast-atom-bombardment mass spectrometry suggests that canine alpha-L-fucosidase acts preferentially on the alpha-(1-3)-linked fucose at the non-reducing end and that removal of alpha-(1-6)-linked asparagine-linked N-acetylglucosamine is rate-limiting in the lysosomal catabolism of fucosylated N-linked glycans.     

Characterization of bovine aortic protein kinase C with histone and platelet protein P47 as substrates.

A Ca2+- and phospholipid-dependent protein kinase (protein kinase C) was partially purified from the media of bovine aortas by chromatography on DEAE-Sephacel and phenyl-Sepharose. Enzyme activity was characterized with both histone and a 47 kDa platelet protein (P47) as substrates, because the properties of protein kinase C can be modified by the choice of substrate. Both phosphatidylserine and Ca2+ were required for kinase activity. With P47 as substrate, protein kinase C had a Ka for Ca2+ of 5 microM. Addition of diolein to the enzyme assay caused a marked stimulation of activity, especially at low Ca2+ concentrations, but the Ka for Ca2+ was shifted only slightly, to 2.5 microM. With histone as substrate, the enzyme had a very high Ka (greater than 50 microM) for Ca2+, which was substantially decreased to 3 microM-Ca2+ by diolein. A Triton X-100 mixed-micelle preparation of lipids was also utilized to assay protein kinase C with histone as the substrate. Under these conditions kinase activity was almost totally dependent on the presence of diolein; again, diolein caused a large decrease in the Ka for Ca2+, from greater than 100 microM to 2.5 microM. The increased sensitivity of protein kinase C to Ca2+ with P47 rather than histone, and the ability of diacylglycerol to activate protein kinase C without shifting the Ka for Ca2+, when P47 is the substrate, illustrate that the mechanism of protein kinase C activation is influenced by the exogenous substrate used to assay the enzyme.     

Microdistribution and local dosimetry of 226Ra in trabecular bone of the beagle

Sections of lumbar vertebral bodies of young adult beagle dogs have been analyzed autoradiographically to characterize and quantify the local distribution of 226Ra by means of a scanning microscope photometer. The animals received a single injection of 355 kBq/kg body weight and were serially sacrificed at 5 to 1381 days postinjection. Hotspot concentrations decreased from about 51 kBq/g bone at 5 days to 20 kBq/g at 1381 days postinjection. The diffuse concentration changed from 8.3 to 1.9 kBq/g. The mean 226Ra concentration in the trabecular areas scanned was initially higher and at the end of the observation period lower than the average calculated for the whole lumbar vertebral column. Density and area of, and fraction of bone activity in, hotspots virtually remained constant. With time hotspots tended to become translocated into bone volume. Mean dose rates to lining cells from both hotspots and diffuse labels decreased from about 210 mGy/d at early postinjection times to 105 mGy/d. This corresponds to 2.5 to 1.1 times the average skeletal dose rate. A discussion of the level of irradiation in terms of hit frequencies shows that osteoblasts in the initial phase of hotspot formation receive about 60 hits to their nucleus for the duration of bone formation. After about 6 months, however, the 226Ra concentration in new bone and the corresponding hit frequency appears to be low enough that interference with bone formation is unlikely. Morphometric measurements showed that abnormal bone accretion and thickening of trabeculae occurred. This was interpreted as an imbalance between bone formation and resorption. Both formation and resorption seem to be substantially lowered compared to control animals.     

Structures of glycosphingolipids isolated from human embryonal carcinoma cells. The presence of mono- and disialosyl glycolipids with blood group type 1 sequence

Structures of glycolipids present in the human embryonal carcinoma cell PA1, were elucidated by fast atom bombardment-mass spectrometry, methylation analysis, and exo- and endoglycosidase digestion. PA1 cells contain globotriaosylceramide, sialosylgangliotriaosylceramide, sialylated and nonsialylated lacto-N-neotetraosylceramide, and the following glycolipids with a blood group type 1 sequence: (formula; see text) The two former glycolipids, lacto-N-tetraosylceramide and sialosyllacto-N-tetraosylceramide, reacted with monoclonal antibodies, K21 and K4, respectively. K21 and K4 antigens are present in many of the human embryonal carcinoma cells but not in a variety of other cell lines, suggesting that sialylated but not fucosylated blood group type 1 sequences are characteristic markers for human embryonal carcinoma cells and malignant teratocarcinomas.     

A novel mass spectrometric procedure for the rapid determination of the types of carbohydrate chains present in glycoproteins: application to alpha-galactosidase I from Vicia faba seeds

A fast atom bombardment mass spectrometric protocol has been developed to determine the type of oligosaccharide chain present in glycoproteins. The procedure is based on acetolysis of the intact glycoconjugate, extraction of the peracetylated carbohydrate fragments and analysis by fast atom bombardment mass spectrometry. The molecular ions present in the FAB spectra uniquely define the composition of the oligosaccharides with respect to hexose, aminohexose and sialic acid content. High mannose oligosaccharides yield a series of peracetylated hexose oligomers whereas complex-type oligosaccharides afford a series of N-acetyl-lactosamine containing species. Fucosylation is usually not detected but sialylated oligosaccharides are readily identified and the type of sialic acid is also defined. The method has been tested on three glycoproteins of known structure - fetuin, ribonuclease B and erythrocyte Band 3 - and on a glycoprotein of unknown structure - alpha-galactosidase I, an enzyme lectin from Vicia faba. The latter is shown to contain high mannose carbohydrate chains.