Comparative importance in vivo of conserved glutamate residues in the EX7E motif retaining glycosyltransferase Gpi3p, the UDP-GlcNAc-binding subunit of the first enzyme in glycosylphosphatidylinositol assembly.

Saccharomyces cerevisiae Gpi3p is the UDP-GlcNAc-binding and presumed catalytic subunit of the enzyme that forms GlcNAc-phosphatidylinositol in glycosylphosphatidylinositol biosynthesis. It is an essential protein with an EX7E motif that is conserved in four families of retaining glycosyltransferases. All Gpi3ps contain a cysteine residue four residues C-terminal to EX7E. To test their importance for Gpi3p function in vivo, Glu289 and 297 in the EX7E motif of S. cerevisiae Gpi3p, as well as Cys301, were altered by site-specific mutagenesis, and the mutant proteins tested for their ability to complement nonviable GPI3-deleted haploids. Gpi3p-C301A supported growth but membranes from C301A-expressing cells had low in vitro N-acetylglucosaminylphosphatidylinositol (GlcNAc-PI) synthetic activity. Haploids harboring Gpi3p-E289A proved viable, although slow growing but Gpi3-E297A did not support growth. The E289D and E297D mutants both supported growth at 25 degrees C, but, whereas the E289D strain grew at 37 degrees C, the E297D mutant did not. Membranes from E289D mutants had severely reduced in vitro GlcNAc-PI synthetic activity and E297D membranes had none. The mutation of the first Glu in the EX7E motif of Schizosaccharomyces pombe Gpi3p (Glu277) to Asp complemented the lethal null mutation in gpi3+ and supported growth at 37 degrees C, but the E285D mutant was nonviable. Our results suggest that the second Glu residue of the EX7E motif in Gpi3p is of greater importance than the first for function in vivo. Further, our findings do not support previous suggestions that the first Glu of an EX7E protein is the nucleophile and that Cys301 has an important role in UDP-GlcNAc binding by Gpi3ps.     

Evidence for the presence of β-lactamase inStreptomyces glaucescens and its inhibition by sodium clavulanate

Streptomyces glaucescens is shown to possess -lactamase activity which is inhibitable by clavulanate. This is important in regard to its use as a cloning host for enzymes of \-lactam biosynthesis.     

Nutrients in the shadow-nutrients of substance

While the dietary importance of proteins, essential fatty acids, vitamins and trace elements has been well recognised, the role of shadow nutrients, a class of metabolites, which are biosynthesized in the body and serve vital functions, such as lipoic acid, choline, inositol, taurine and carnitine, has not been adequately appreciated. There are reasons to believe that during infancy and in ageing, biosynthesis of these metabolites may be limited. The objective of this review is to highlight the essentiality of these nutrients and the need for their supplementation in the diets of infants and in elderly people. Provision of shadow nutrients where the necessary biosynthetic machinery might not have developed to full stature or might have slowed down, is a new concept in nutrition which needs attention.     

Inhibition of methanogenesis and its reversal during biogas formation from cattle manure

The composition of volatile fatty acids in the biogas digester based on cattle manure as substrate and stabilised at 25°C showed that it contained 87–88% branched chain fatty acids, comprising of isobutyric and isovaleric acids, in comparison to 38 % observed in the digester operating at 35°C. Mixed cellulolytic cultures equilibrated at 25°C (C-25) and 35‡C (C-35) showed similar properties, but rates of hydrolysis were three times higher than that observed in a standard biogas digester. The proportion of isobutyric and isovaleric were drastically reduced when C-25 was grown with glucose or filter paper as substrates. The volatile fatty acids recovered from C-25 (at 25°C) inhibited growth of methanogens on acetate, whereas that from C-35 was not inhibitory. The inhibitory effects were due to the branched chain fatty acids and were observed with isobutyric acid at concentrations as low as 50 ppm. Addition of another micro-organismRhodotorula selected for growth on isobutyric completely reversed this inhibition. Results indicate that the aceticlastic methanogens are very sensitive to inhibition by branched chain fatty acids and reduction in methane formation in biogas digester at lower temperature may be due to this effect.     

Cyclic nucleotide phosphodiesterases from rat anterior pituitary. Characterization of multiple forms and regulation by protein activator and Ca+.

Phosphodiesterase activities for adenosine and guanosine 3':5'-monophosphates (cyclic AMP and cyclic GMP) were demonstrated in particulate and soluble fractions of rat anterior pituitary gland. Both fractions contained higher activity for cyclic GMP hydrolysis than that for cyclic AMP hydrolysis when these activities were assayed at subsaturating substrate concentrations. Addition of protein activator and CaCl2 to either whole homogenate, particulate or supernatant fraction stimulated both cyclic AMP and cyclic GMP phosphadiesterase activities. Almost 80% of cyclic AMP and 90% of cyclic GMP hydrolyzing activities were localized in soluble fraction. Particulate-bound cyclic nucleotide phosphodiesterase activity was completely solubilized with 1% Triton X-100. Detergent-dispersed particulate and soluble enzymes were compared with respect to Ca2+ and activator requirements and gel filtration profiles. Particulate, soluble and partially purified phosphodiesterase activities were also characterized in relation to divalent cation requirements, kinetic behavior and effects of Ca2+, activator and ethyleneglycol-bis-(2-aminoethyl)-N,N'-tetraacetic acid. Gel filtration of either sonicated whole homogenate or the 10500 X g supernatant fraction showed a single peak of activity, which hydrolyzed both cyclic AMP and cyclic GMP and was dependent upon Ca2+ and activator for maximum activity. Partially purified enzyme was inhibited by 1-methyl-3-isobutylxanthine and papaverine with the concentration of inhibitor giving 50% inhibition at 0.4 muM substrate being 20 muM and 24 muM for cyclic AMP and 7 muM and 10 muM for cyclic GMP, respectively. Theophylline, caffeine and theobromine were less effective. The rat anterior pituitary also contained a protein activator which stimulated both pituitary cyclic nucleotide phosphodiesterase(s) as well as activator-deficient brain cyclic GMP and cyclic AMP phosphodiesterases. Chromatography of the sonicated pituitary extract on DEAE-cellulose column chromatography resolved the phosphodiesterase into two fractions. Both enzyme fractions hydrolyzed cyclic AMP and cyclic GMP and had comparable apparent Km values for the two nucleotides. Hydrolysis of cyclic GMP and cyclic AMP by fraction II enzyme was stimulated 6--7-fold by both pituitary and brain activator in the presence of micromolar concentrations of Ca2+.     

Receptor-mediated gonadotropin action in the ovary. Action of cytoskeletal element-disrupting agents on gonadotropin-induced steroidogenesis in rat luteal cells.

The role of the cellular cytoskeletal system of microtubules and microfilaments on gonadotropin-stimulated progesterone production by isolated rat luteal cells has been investigated. Exposure of luteal cells to human choriogonadotropin resulted in a stimulation of cyclic AMP (4-7-fold) and progesterone (3-4-fold) responses.l Incubation of cells with the microfilament modifier cytochalasin B inhibited the gonadotropin-induced steroidogenesis in a dose- and time-dependent manner. The effect of cytochalasin B on basal production of steroid was less pronounced. Cytochalasin B also inhibited the accumulation of progesterone in response to lutropin, cholera enterotoxin, dibutyryl cyclic AMP and 8-bromo cyclic AMP. The inhibition of steroidogenesis by cytochalasin B was not due to (a) inhibition of 125I-labelled human choriogonadotropin binding to luteal cells, (b) inhibition of gonadotropin-stimulated cyclic AMP formation or (c) a general cytotoxic effect and/or inhibition of protein biosynthesis. Cytochalasin D, like cytochalasin B, inhibited gonadotropin- and 8-bromo cyclic AMP-stimulated steroidogenesis. Although cytochalasin B also blocked the transport of 3-O-methyl-glucose into luteal cells, cytochalasin D was without such an effect. Increasing glucose concentration in the medium, or using pyruvate as an alternative energy source, failed to reverse the inhibitory effect of cytochalasin B. The anti-microtubular agent colchicine failed to modulate synthesis and release of progesterone by luteal cells in response to human choriogonadotropin. These studies suggest that the cellular microfilaments may be involved in the regulation of gonadotropin-induced steroidogenesis. In contrast, microtubules appear to be not directly involved in this process.     

Studies on the chemical modification of potato (Solanum tuberosum) lectin and its effect on haemagglutinating activity

1. Modification of potato (Solanum tuberosum) lectin with acetic anhydride blocked 5.1 amino and 2.7 tyrosyl groups per molecule of lectin and decreased the haemagglutinating activity of the lectin. De-O-acetylation regenerated 2.0 of the tyrosyl groups and resulted in a recovery of activity. 2. Modification with citraconic anhydride or cyclohexane-1,2-dione did not greatly affect activity, although modification of amino and arginyl groups could be demonstrated. 3. Treatment with tetranitromethane nitrated 3.7 tyrosine residues per molecule of lectin with concomitant loss of activity. The presence of 0.1m-NN′N″-triacetylchitotriose (a potent inhibitor of the lectin) in the reaction medium protected all the tyrosyl residues from nitration and the lectin was fully active. 4. Modification of tryptophyl groups with 2-hydroxy-5-nitrobenzyl bromide and 2,3-dioxoindoline-5-sulphonic acid modified 0.9 and 2.6 residues per molecule of lectin respectively with a loss of activity in each case. Reaction of potato lectin with 2,3-dioxoindoline-5-sulphonic acid in the presence of inhibitor protected 2.4 residues of tryptophan from the reagent. Loss of haemagglutination activity was prevented under these conditions. 5. Reaction of carboxy groups, activated with carbodi-imide, with α-aminobutyric acid methyl ester led to the incorporation of 5.3 residues of the ester per molecule of lectin. Presence of inhibitor in this case, although protecting activity, did not prevent modification of carboxy groups; in fact an increase in the number of modified residues was seen. This effect could be imitated by performing the reaction in 8m-urea. In both cases the number of carboxy groups modified was close to the total number of free carboxy groups as determined by the method of Hoare & Koshland [(1967) J. Biol. Chem. 242, 2447–2453]. Guanidination of lysine residues after carboxy-group modification gave less homoarginine than did the unmodified lectin under the same conditions, suggesting the formation of intramolecular cross-links during carbodi-imide activation. 6. It is suggested from the results presented that amino, arginyl, methionyl, histidyl and carboxyl groups are not involved in the activity of the lectin and that tyrosyl and tryptophyl groups are very closely involved. These findings are similar to those reported for other proteins that bind N-acetylglucosamine oligomers and also fit the general trend in other lectins.     

The mevalonate pathway in the bloodstream form of Trypanosoma brucei. Identification of dolichols containing 11 and 12 isoprene residues

The major surface antigen of the bloodstream form of Trypanosoma brucei, the variant surface glycoprotein, is attached to the plasma membrane via a glycosylphosphatidylinositol anchor. The biosynthesis of the glycosylphosphatidylinositol anchor, as well as the assembly of the asparagine-linked oligosaccharide chains found on the variant surface glycoproteins, involves polyisoprenoid lipids that act as sugar carriers. Preliminary observations (Menon, A.K., Schwarz, R.T., Mayor, and Cross, G.A.M. (1990) J. Biol. Chem. 265, 9033-9042) suggested that the sugar carriers in T. brucei were short-chain polyisoprenoids containing substantially fewer isoprene residues than polyisoprenols in mammalian cells. In this paper we describe metabolic labeling experiments with [3H]mevalonate, as well as chromatographic and mass spectrometric analyses of products of the mevalonate pathway in T. brucei. We report that cells of the bloodstream form of T. brucei contain a limited spectrum of short chain dolichols and dolichol phosphates (11 and 12 isoprene residues). The total dolichol content was estimated to be 0.28 nmol/10(9) cells; the dolichyl phosphate content was 0.07 nmol/10(9) cells. The same spectrum of dolichol chain lengths was also found in a polar lipid that could be labeled with [3H]mevalonate, [3H]glucosamine, and [3H]mannose, and which was characterized as Man5GlcNAc2-PP-dolichol. The most abundant product of the mevalonate pathway identified in T. brucei was cholesterol (140 nmol/10(9) cells). Ubiquinone (0.09 nmol/10(9) cells) with a solanesol side chain was also identified.     

Developmental variation of glycosylphosphatidylinositol membrane anchors in Trypanosoma brucei. Identification of a candidate biosynthetic precursor of the glycosylphosphatidylinositol anchor of the major procyclic stage surface glycoprotein.

The major surface antigen of the mammalian bloodstream form of Trypanosoma brucei, the variant surface glycoprotein (VSG), is attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. The VSG anchor is susceptible to phosphatidylinositol-specific phospholipase C (PI-PLC). Candidate precursor glycolipids, P2 and P3, which are PI-PLC-sensitive and -resistant respectively, have been characterized in the bloodstream stage. In the insect midgut stage, the major surface glycoprotein, procyclic acidic repetitive glycoprotein, is also GPI-anchored but is resistant to PI-PLC. To determine how the structure of the GPI anchor is altered at different life stages, we characterized candidate GPI molecules in procyclic T. brucei. The structure of a major procyclic GPI, PP1, is ethanolamine-PO4-Man alpha 1-2Man alpha 1-6 Man alpha 1-GlcN-acylinositol, linked to lysophosphatidic acid. The inositol can be labeled with [3H]palmitic acid, and the glyceride with [3H]stearic acid. We have also found that all detectable ethanolamine-containing GPIs from procyclic cells contain acylinositol and are resistant to cleavage by PI-PLC. This suggests that the procyclic acidic repetitive glycoprotein GPI anchor structure differs from that of the VSG by virtue of the structures of the GPIs available for transfer.     

Galactose-containing glycosylphosphatidylinositols in Trypanosoma brucei.

Many eukaryotic surface glycoproteins, including the variant surface glycoproteins (VSGs) of Trypanosoma brucei, are synthesized with a carboxyl-terminal hydrophobic peptide extension that is cleaved and replaced by a complex glycosylphosphatidylinositol (GPI) membrane anchor within 1-5 min of the completion of polypeptide synthesis. We have reported the purification and partial characterization of candidate precursor glycolipids (P2 and P3) from T. brucei. P2 and P3 contain ethanolamine-phosphate-Man alpha 1-2Man alpha 1-6Man alpha 1-GlcN linked glycosidically to an inositol residue, as do all the GPI anchors that have been structurally characterized. The anchors on mature VSGs contain a heterogenously branched galactose structure attached alpha 1-3 to the mannose residue adjacent to the glucosamine. We report the identification of free GPIs that appear to be similarly galactosylated. These glycolipids contain diacylglycerol and alpha-galactosidase-sensitive glycan structures which are indistinguishable from the glycans derived from galactosylated VSG GPI anchors. We discuss the relevance of these galactosylated GPIs to the biosynthesis of VSG GPI anchors.