Fatty acid and glycerolipid synthesis in abscised daffodil flowers after acetate feeding

The coronae of Narcissus pseudonarcissus flowers incorporated [1-¹⁴C]acetate almost exclusively into the fatty acid moieties of glycerolipids. After a 4 h incubation, the newly synthesized acids were: stearate plus palmitate (50%); oleate (17%); linoleate (32%); and linolenate (0.5%). Phosphatidylcholine and diacylglycerol were the principal labelled lipids. In pulse experiments these acids were further desaturated, with time, to an appreciable extent and, concurrently, transferred essentially from phosphatidylcholine to diacylglycerol, diacylgalactosylglycerol, and diacylgalabiosylglycerol. The labelling of diacylgalactosylglycerol and diacylgalabiosylglycerol paralleled the appearance of linolenate. The distribution of labelled acids in phosphatidylcholine, diacylgalactosylglycerol, and diacylgalabiosylglycerol was very different. The results were compared with those obtained in vitro with isolated coronae chromoplasts and discussed in relation to current schemes of fatty acid and glycerolipid synthesis in plant cells.     

Enzymatic aspects of the reduction of microsomal glycerolipid biosynthesis after perfusion of the liver with dibutyryl adenosine-3',5'-monophosphate.

Livers from fed male rats were perfused in a nonrecycling system for 60 min with a medium containing 100 mg/dl glucose, 3 g/dl bovine serum albumin, and ~0.5 mm oleic acid, with or without 20 μm dibutyryl cyclic adenosine-3′,5′-monophosphate (Bt₂cAMP). At the termination of the experiment, microsomes were isolated from these livers. In agreement with data reported previously, Bt₂cAMP decreased output of triacylglycerol, but stimulated ketogenesis and output of glucose; uptake of free fatty acid was unaffected by the nucleotide. Perfusion with Bt₂AMP decreased the biosynthesis of triacylglycerol, diacylglycerol, and phosphatidate from sn-[U-¹⁴C]glycerol-3-phosphate by microsomes isolated from these livers. Perfusion with Bt₂cAMP also decreased incorporation of sn-glycerol-3-phosphate into phosphatidate by microsomes isolated from the livers, when the microsomes were incubated with NaF to inhibit phosphatidate phosphohydrolase, and when fatty acid, coenzyme A and ATP were replaced by the acyl coenzyme A derivative; the formation of phosphatidate under these conditions was used as an estimate of the activity of sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15). However, the activities of microsomal phosphatidate phosphohydrolase (EC 3.1.3.4) and diacylglycerol acyltransferase (EC 2.3.1.20), measured with microsomal bound substrate, were increased by Bt₂cAMP. These data have been interpreted to mean that Bt₂cAMP inhibits hepatic microsomal synthesis of triacylglycerol at a step prior to the formation of phosphatidate, presumably at the glycerophosphate acyltransferase (EC 2.3.1.15) step(s).     

Seasonal changes in critical enzymes of lipogenesis and triacylglycerol synthesis in the marmot (Marmota flaviventris)

Fatty acid metabolism and triacylglycerol synthesis are critical processes for the survival of hibernating mammals that undergo a prolonged fasting period. Fatty acid synthase, fatty-acid-CoA ligase, diacylglycerol acyltransferase, and monoacylglycerol acyltransferase activities were measured in liver and in white and brown adipose tissue, in order to determine whether enzymes of lipogenesis and triacylglycerol synthesis vary seasonally during hibernation in the yellow-bellied marmot (Marmota flaviventris). Compared with mid-winter hibernation, fatty acid synthase activity was higher in all three tissues during early spring when marmots emerged from hibernation and in mid-summer when they were feeding, consistent with the synthesis of fatty acids from the carbohydrate-rich summer diet. Fatty-acid-CoA ligase and diacylglycerol acyltransferase activities were highest in summer in white adipose tissue when triacylglycerol synthesis would be expected to be high; diacylglycerol acyltransferase activity was also high in brown adipose tissue during spring and summer. In liver, however, diacylglycerol acyltransferase specific activity was highest during hibernation, suggesting that triacylglycerol synthesis may be prominent in liver in winter. Monoacylglycerol acyltransferase activity, which may aid in the retention of essential fatty-acids, was 80-fold higher in liver than in white or brown adipose tissue, but did not vary seasonally. Its dependence on palmitoyl-CoA suggests that a divalent cation might play a role in enzyme activation. The high hepatic diacylglycerol acyltransferase activity during hibernation suggests that the metabolism of very low density lipoprotein may be important in the movement of adipose fatty acids to brown adipose tissue and muscle during the rewarming that occurs periodically during hibernation. These studies suggest that enzymes of lipid metabolism vary seasonally in the marmot, consistent with requirements of this hibernator for triacylglycerol synthesis and metabolism.Abbreviations BAT brown adipose tissue - DGAT diacylglycerol acyltransferase - FAS fatty acid synthase - K _m Michaelis constant - MGAT monoacylglycerol acyltransferase - RQ respiratory quotiant - VLDL very low density lipoprotein - WAT white adipose tissue     

Absence of the glutamine-synthetase-linked methylammonium (ammonium)-transport system in the cyanobiont of Cycas-cyanobacterial symbiosis

Using the ammonium analogue ¹⁴CH₃NH ₃ ⁺ , ammonium transport was studied in the cyanobiont cells freshly isolated from the root nodules of Cycas revoluta. An L-methionine-dl-sulphoximine (MSX)-insensitive ammonium-transport system, which was dependent on membrane potential (), was found in the cyanobiont. However, the cyanobiont was incapable of metabolizing exogenous ¹⁴CH₃NH ₃ ⁺ or NH ₄ ⁺ because of the absence of another ammonium-transport system responsible for the uptake of ammonium for assimilation via glutamine synthetase (EC 6.3.1.2). Such a modification seems to be the result of symbiosis because the free-living cultured isolate, Anabaena cycadeae, has been shown to possess both the ammonium-transport systems.Abbreviations and symbol ATS/ATSs ammonium transport system/systems - Chl chlorophyll - GS glutamine synthetase - MSX L-methionine-dl-sulphoximine - membrane potential     

Comparative studies on S-adenosyl-l -methionine binding sites of protein N-methyltransferases, using 8-azido-S-adenosyl-l -methionine as photoaffinity probe

Employing a photoaffinity labeling procedure with 8-azido-S-adenosyl-l-[methyl-³H]methionine (8-N₃-Ado[methyl-³H]Met), the binding sites for S-adenosyl-l-methionine (AdoMet) of three protein N-methyltransferases [AdoMet:myelin basic protein-arginine N-methyltransferase (EC2.1.1.23); AdoMet:histone-arginin N-methyltransferase (EC2.1.1.23); and AdoMet:cytochromec-lysine N-methyltransferase (EC2.1.1.59)] have been investigated. The incorporation of the photoaffinity label into the enzymes upon UV irradiation was highly specific. In order to define the AdoMet binding sites, the photolabeled enzymes were sequentially digested with trypsin, chymotrypsin, and endoproteinase Glu-C. After each proteolytic digestion, radiolabeled peptide from each enzyme was resolved on HPLC first by gradient elution and further purified by an isocratic elution. Retention times of the purified radiolabeled peptides from the three enzymes from the corresponding proteolysis were significantly different, indicating that their sizes and compositions were different. Amino acid composition analysis of these peptides confirmed further that the AdoMet binding sites of these protein N-methyltransferases are quite different.     

Inositol bisphosphate and inositol trisphosphate inhibit cell-to-cell passage of carboxyfluorescein in staminal hairs ofSetcreasea purpurea

pH-buffered carboxyfluorescein (Buffered-CF) alone (control), or Buffered-CF solutions containing one of the following: (1)d-myo-inositol (I); (2)d-myo-inositol 2-monophosphate (IP₁); (3)d-myo-inositol 1,4-bisphosphate (IP₂); (4)d-myo-inositol 1,4,5-trisphosphate (IP₃); (5)d-fructose 2,6-diphosphate (F-2,6P₂) were microinjected into the terminal cells of staminal hairs ofSetcreasea purpurea Boom. Passage of the CF from this terminal cell along the chain of cells towards the filament was monitored for 5 min using fluorescence microscopy and quantified using computer-assisted fluorescence-intensity video analysis. Cell-to-cell transport of CF in hairs microinjected with Buffered-CF containing either I, IP₁ or F-2,6P₂ was similar to that in hairs microinjected with Buffered-CF only. On the other hand, cell-to-cell transport of CF in hairs microinjected with Buffered-CF containing either IP₂ or IP₃ was inhibited. These results indicate that polyphosphoinositols may be involved in the regulation of intercellular transport of low-molecular-weight, hydrophilic molecules in plants.Abbreviations CF 5(6)Carboxyfluorescein - DG diacylglycerol - F2, 6P₂ d-fructose 2,6-diphosphate - I d-myo-inositol - IP₁ d-myo-inositol 2-monophosphate - IP₂ d-myo-inositol 1,4-bisphosphate - IP₃ d-myo-inositol 1,4,5-trisphosphate     

Stachyose synthesis in leaves of Cucurbita pepo

An enzyme synthesizing stachyose, galactinol-raffinose galactosyltransferase (EC2.4.1.67), has been purified ca 40-fold from mature leaves of Cucurbita pepo using ammonium sulphate precipitation, Sephadex gel filtration and DEAE-Sephadex gel chromatography. The purified enzyme fraction was separated from all but 2 % of the total,α-galactosidase activity extracted from the tissue. The enzyme was optimally active at pH 6.9 and was stable for at least a month at 4° in the presence of 20 mM 2-mercaptoethanol. The enzyme displayed high specificity for the donor galactinol (K_m 7.7 mM) and the acceptor raffinose (K_m 4.6 mM) and was unable to effect synthesis of any other member of the raffinose series of galactosyl-sucrose oligosaccharides. Co²⁺, Hg²⁺, Mn²⁺ and Ni²⁺ ions were particularly inhibitory; no metal ion promotion was observed and 5 mM EDTA was ineffective. Myo-inositol was strongly inhibitory (K_i 2 mM), melibiose weakly so. Tris buffer (0. 1 M) was also inhibitory. Galactinol hydrolysis occurred in the absence of the acceptor raffinose but there was no hydrolysis of either raffinose or stachyose in the absence of the donor galactinol. The reaction was readily reversible and exchange reactions were detected between substrates and products. It is proposed that the synthesis of stachyose in mature leaves ofC. pepo proceeds via this galactosyltransferase and not via α-galactosidase.     

Fixation of [13N]N2 and transfer of fixed nitrogen in the Anthoceros-Nostoc symbiotic association

The initial product of fixation of [¹³N]N₂ by pure cultures of the reconstituted symbiotic association between Anthoceros punctatus L. and Nostoc sp. strain ac 7801 was ammonium; it accounted for 75% of the total radioactivity recovered in methanolic extracts after 0.5 min and 14% after 10 min of incubation. Glutamine and glutamate were the primary organic products synthesized from [¹³N]N₂ after incubation times of 0.5–10 min. The kinetics of labeling of these two amino acids were characteristic of a precursor (glutamine) and product (glutamate) relationship. Results of inhibition experiments with methionine sulfoximine (MSX) and diazo-oxonorleucine were also consistent with the assimilation of N₂-derived NH ₄ ⁺ by Anthoceros-Nostoc through the sequential activities of glutamine synthetase (EC 6.3.1.2) and glutamate synthase (EC 1.4.7.1), with little or no assimilation by glutamate dehydrogenase (EC 1.3.1.3). Isolated symbiotic Nostoc assimilated exogenous ¹³NH ₄ ⁺ into glutamine and glutamate and their formation was inhibited by MSX, indicating operation of the glutamine synthetase-glutamate synthase (GS-GOGAT) pathway: However, relative to free-living cultures, isolated symbiotic Nostoc assimilated 80% less exogenous ammonium into glutamine and glutamate, implying that symbiotic Nostoc could assimilate only a fraction of N₂-derived NH ₄ ⁺ . This implication was tested by using Anthoceros associations reconstituted with wild-type or MSX-resistant strains of Nostoc incubated with [¹³N]N₂ in the presence of MSX. The results of these experiments indicated that, in situ, symbiotic Nostoc assimilated about 10% of the N₂-derived NH ₄ ⁺ and that NH ₄ ⁺ was made available to Anthoceros tissue where it was apparently assimilated by the GS-GOGAT pathway. Since less than 1% of the fixed N₂ was lost to the suspension medium, it appears that transfer of NH ₄ ⁺ from symbiont to host tissue was very efficient in this extracellular symbiotic association.Abbreviations DON 6-diazo-5-oxo-l-norleucine - GDH glutamate dehydrogenase - GOGAT glutamate synthase - GS glutamine synthetase - MSX l-methionine-dl-sulfoximine     

Triacylglycerol biosynthesis in developing seeds of Tropaeolum majus L. and Limnanthes douglasii R. Br.

Triacylglycerols of both Tropaeolum majus L. and Limnanthes douglasii R. Br. are predominantly esterified with very long-chain acyl groups at each position of the glycerol backbone. In order to elucidate whether these acyl groups are directly chanelled into the triacylglycerols via the stepwise acylation of glycerol-3-phosphate, seed oil formation has been investigated in developing embryos of both plant species. [1-¹⁴C]Acetate labelling experiments using embryos at different stages of development, as well as the determination of the properties of the microsomal acyl-CoA:sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15) and acyl-CoA:sn-1-acylglycerol-3-phosphate acyltransferase (EC 2.3.1.51), revealed differences between the two plant species, especially with respect to the incorporation of very longchain acyl groups into the C2 position of the triacylglycerols. In microsomal fractions of developing embryos of L. douglasii both a glycerol-3-phosphate and a 1-acylglycerol-3-phosphate acyltransferase were detected which utilize very long-chain acyl-CoA thioesters as substrates. Thus, in seeds of L. douglasii very long-chain acyl groups can enter not only the C1, but also the C2 position of the triacylglycerols in the course of de-novo biosynthesis. A comparison of the properties of the acyltransferases of developing embryos with those of the corresponding activities of leaves indicates an embryo specific expression of an erucoyl-CoA-dependent microsomal 1-acylglycerol-3-phosphate acyltransferase in L. douglasii. The microsomal glycerol-3-phosphate acyltransferase of developing embryos of T. majus displayed properties very similar to those of the corresponding activity of L. douglasii. On the other hand, the microsomal 1-acylglycerol-3-phosphate acyltransferases of the two plant species showed strikingly different substrate specificities. Irrespective of the acyl groups of 1-acylglycerol-3-phosphate and regardless of whether acyl-CoA thioesters were offered separately or in mixtures, the enzyme of T. majus, in contrast to that of L. douglasii, was inactive with erucoyl-CoA. These results of the enzyme studies correspond well with those of the [1-¹⁴C]acetate labelling experiments and thus indicate that T. majus has developed mechanisms different from those of L. douglasii for the incorporation of erucic acid into the C2 position of its triacylglycerols.Abbreviations GPAT acyl-CoA:sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15) - LPAT acyl-CoA:sn-1-acylglycerol-3-phosphate acyltransferase (EC 2.3.1.51) This work was supported by the Bundesministerium für Forschung und Technologie (Förderkennzeichen 0316600A).     

Purification and properties of glutamine synthetase from Bacillus polymyxa

Glutamine synthetase (EC 6.3.1.2) was purified to homogeneity from a free-living nitrogen fixing bacteria, Bacillus polymyxa. The holoenzyme, relative molecular mass (M_r) of 600 000 is composed of monomeric sub-units of 60 000 (M_r). The isoelectric point of the sub-units was 5.2. The pH optimum for the biosynthetic and transferase enzyme activity was 8.2 and 7.8, respectively. The apparent K _m values (K _m ^app ) in the biosynthetic reaction for glutamate, NH₄Cl and ATP were 3.2, 0.22 and 1 mM, respectively. In the transferase reaction the K _m values for glutamine, hydroxylamine and ADP were 6.5, 3.5 and 8×10^-4 mM respectively. L-Methionine-D-L-sulfoximine was a very potent inhibitor in both biosynthetic and transferase reactions. Similar to most Gram positive bacteria there was no evidence of in vivo adenylylation and the enzyme seemed to be mainly regulated by feed-back mechanism.Abbreviations PMSF phenylmethylsulfonylfluoride - TCA trichloroacetic acid - GS glutamine synthetase - MSO L-Methionine-D-L-sulfoximine - SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis - SVPDE snake venum phosphodiesterase     

Glycerol-3-phosphatase and the control of glycerol metabolism in Dunaliella tertiolecta cells

Glycerol-3-phosphatase (EC 3.1.3.2.1) was studied by following the release of radioactive glycerol from L-(U-¹⁴C)glycerol-3-phosphate in Dunaliella tertiolecta enzyme extracts. The reaction showed a neutral pH optimum and had an absolute requirement for Mg²⁺. The substrate saturation curve was hyperbolic with an apparent K _m value for glycerol-3-phosphate of 0.7 mM in the absence of phosphate. Inorganic orthophosphate was a competitive inhibitor of the enzyme with an estimated K _j of 0.1 mM. The glycerol-3-phosphatase reaction was blocked nearly completely by millimolar Ca²⁺ concentrations. Ca²⁺ inhibition did not depend on the presence of calmodulin in the reaction medium. The characteristics of glycerol-3-phosphatase are discussed in relation to the regulation of the cyclic glycerol metabolism in Dunaliella cells during periods of osmotic stress.     

Sites of phospholipid biosynthesis during induction of intracytoplasmic membrane formation in Rhodopseudomonas sphaeroides

A rapid, gratuitous and cell-division uncoupled induction of intracytoplasmic photosynthetic membrane formation was demonstrated in low-aeration suspensions of chemotrophically grown Rhodopseudomonas sphaeroides. Despite a nearly 2-fold increase in phospholipid levels, no significant increases were detected in the specific activities of CDP-1,2-diacyl-sn-glycerol:sn-glycerol-3-phosphate phosphatidyltransferase (phosphatidylglycerophosphate synthase, EC 2.7.8.5) and CDP-1,2-diacyl-sn-glycerol:L-serine O-phosphatidyltransferase (phosphatidylserine synthase, EC 2.7.8.8), the first committed enzymes of anionic and zwitterionic phospholipid biosyntheses, respectively. The distribution of phosphatidylglycerophosphate and phosphatidylserine synthase activities after rate-zone sedimentation of cell-free extracts indicated that intracytoplasmic membrane phospholipids were synthesized mainly within distinct domains of the conserved cytoplasmic membrane. Labeling studies with ³²P_i and L-[³H]phenylalanine suggested that preexisting phospholipid was utilized initially as the matrix for insertion of intracytoplasmic membrane protein that was synthesized and assembled de novo during induction.Abbreviations BChl bacteriochlorophyll a - B800-850, B875 peripheral and core light-harvesting BChl-protein complexes, respectively, identified by near-IR absorption maxima This paper is dedicated to Professor Gerhart Drews on the occasion of his sixtieth birthday     

Enzymatic Characteristics of UDP-sulfoquinovose: Diacylglycerol Sulfoquinovosyltransferase from Chloroplast Envelopes*

Envelope membranes from chloroplasts contain UDP-sulfoquinovose: diacylglycerol sulfoquinovosyltransferase which catalyses the final step in sulfolipid assembly. In situ produced diacylglycerol served as radioactive acceptor to measure enzymatic activity. With this assay, several enzymatic parameters were investigated. The enzyme, which has maximal activity at pH 7.5, was stimulated by magnesium ions due to a decrease of the K_m for uridine 5′-diphospho-sulfoquinovose from 80 pM (no magnesium) to 10 μM (5 mM magnesium). This stimulation had a K_m of 0.7 mM magnesium and may be relevant in light/dark modulation of the enzymatic activity. The lower efficiency of guanosine 5′-diphospho-sulfoquinovose observed before can be ascribed to a higher K_m of this sugar nucleotide (400 μM). Under optimized and linearized conditions the sulfoquinovosyltransferase displayed about 10% of the activity of the UDP-galactose: diacylglycerol galactosyltransferase which competes in the same membrane system for diacylglycerols. Addition of acidic lipids, such as sulfolipid and phosphatidylglycerol, to envelope membranes resulted in an inhibition of the sulfoquinovosyltransferase, whereas the galactosyltransferase was not affected. In vivo this may contribute to an adjustment of the sulfolipid proportion in plastid membranes. In contrast to the galactosyltransferase the sulfoquinovosyltransferase did not discriminate against the dipalmitoyl molecular species of diacylglycerol when offered together with the oleoyl-palmitoyl species. Under conditions when oleoyl-palmitoyl-and dipalmitoyl-diacylglycerols were synthesized with concurrent conversion to monogalactosyl and sulfoquinovosyl diacylglycerol, the sulfolipid was highly enriched in the fully saturated species. This may explain the occurrence of dipalmitoyl species in sulfolipids, as found in many plants.     

Synthesis of mono- and digalactosyldiacylglycerol in isolated spinach chloroplasts

Purified, intact chloroplasts of Spinacia oleracea L. synthesize galactose-labeled mono- and digalactosyldiacylglycerol (MGDG and DGDG) from UDP-[U-¹⁴C]galactose. In the presence of high concentrations of unchelated divalent cations they also synthesize tri- and tetra-galactosyldiacylglycerol. The acyl chains of galactose-labeled MGDG are strongly desaturated and such MGDG is a good precursor for DGDG and higher oligogalactolipids. The synthesis of MGDG is catalyzed by UDP-Gal:sn-1,2-diacylglycerol galactosyltransferase, and synthesis of DGDG and the oligogalactolipids is exclusively catalyzed by galactolipid:galactolipid galactosyltransferase. The content of diacylglycerol in chloroplasts remains low during UDP-Gal incorporation. This indicates that formation of diacylglycerol by galactolipid:galactolipid galactosyltransferase is balanced with diacylglycerol consumption by UDP-Gal:diacylglycerol galactosyltransferase for MGDG synthesis. Incubation of intact spinach chloroplasts with [2-¹⁴C]acetate or sn-[U-¹⁴C]glycerol-3-P in the presence of Mg²⁺ and unlabeled UDP-Gal resulted in high ¹⁴C incorporation into MGDG, while DGDG labeling was low. This de novo made MGDG is mainly oligoene. Its conversion into DGDG is also catalyzed, at least in part, by galactolipid:galactolipid galactosyltransferase.     

Susceptibility of infant mice to F5 (K99)E. coli infection: Differences in glycosyltransferase activities in intestinal mucosa of inbred CBA and DBA/2 strains

EnterotoxigenicEscherichia coli (ETEC) strains expressing F5 (K99) fimbriae cause diarrhoea in the young animal through adhesion to specific sialoglycolipids of the small intestine surface. We studied here an infant mouse diarrhoea model, as CBA infant mice are susceptible to F5-positive ETEC infection, whereas DBA/2 ones are resistant. In an attempt to determine an enzymatic basis for susceptibility and resistance, we investigated the intestine ganglioside pattern in relation to the activity of glycosyltransferases responsible for the globo- and ganglio-series. We observed that the intestine of susceptible CBA infant mice displayed a characteristic sialoglycolipid pattern containing mainly the F5 receptors. The two murine strains differed in the relative activities of galactosyltransferases (GbOse₃Cer and GM1 synthases),N-acetylgalactosylaminyltransferases (GA2 and GM2 synthases) and sialyltransferases (GM3 and GD3 synthases). An elevated GM3-synthase activity was observed in the intestine of susceptible CBA infant mice, at the age of high susceptibility. Hence, we conclude that the marked specificity of mouse type correlated with susceptibility and resistance to F5-positive ETEC infection which could be controlled through the regulation of glycosyltransferase activities.Abbreviations NeuAc N-acetylneuraminic acid - NeuGc N-glycolylneuraminic acid - Glc glucose - GalNAc N-acetylgalactosamine - Gal galactose - Car ceramide - LacCer lactosylceramide (Galß-4Glcß1-1Cer) - GA2 asialo-GM2 (GgOse₃Cer) - GA1 asialo-GM1 (GgOse₄Cer) - NeuAc/NeuGc-GMla II³ NeuAc/NeuGc-GgOse₄Cer - NeuAc/NeuGc-GM1a IV³ NeuAc/NeuGc-GgOse₄Cer - NeuAc/NeuGc-GM2 II³ NeuAc/neuGc-GgOse₃Cer - NeuAc/NeuGc-GM3, II³ NeuAc/NeuGc-LacCer; NeuAc/NeuGc-GD1a, IV³ NeuAc/NeuGc, II³ NeuAc/NeuGc-GgOse₄Cer; NeuAc/NeuGc-GD1b II³ (NeuAc/NeuGc)₂-GgOse₄Cer - NeuAc/NeuGc-GD1c IV³ (NeuAc/NeuGc)₂-GgOse₄Cer - NeuAc/NeuGc-GD2, II³ (NeuAc/NeuGc)₂-GgOse₃Cer; NeuAc/NeuGc-GD3, II³ (NeuAc/NeuGc)₂-Lac Cer; NeuAc/NeuGcGT1a IV³ (NeuAc/NeuGc)₂, II³ NeuAc/NeuGc-GgOse₄Cer - NeuAc/neuGc-GT1b IV³ NeuAc/NeuGc, II³ (NeuAc/NeuGc)₂-GgOse₄Cer - NeuAc/NeuGc-GT1c II³ (NeuAc/NeuGc)₃-GgOse₄Cer; NeuAc/NeuGc-GT2, II³ (NeuAc/NeuGc)₃-GgOse₃Cer - NeuAc/NeuGc-GT3 II³ (NeuAc/NeuGc)₃-Lac Cer - NeuAc/NeuGc-GQ1b IV³ (NeuAc/NeuGc)₂, II³ (NeuAc/NeuGc)₂-GgOse₄Cer - NeuAc/NeuGc-GQ1c IV³ NeuAc/NeuGc, II³ (NeuAc/NeuGc)₃-GgOse₄Cer - NeuAc/NeuGc-GP1c IV³ (NeuAc/NeuGc)₂, II³ (NeuAc/NeuGc)₃-GgOse₄Cer - GD, GT and GQ di-, tri- and tetra-sialoglangliosides. NeuGc-SPG, IV³ NeuGc-nLcOse₄Cer. Glycosyltransferases assayed in this work areN-acetylgalactosaminyltransferases - UDP-GalNAc lactosylceramide 1-4N-acetylgalactosaminyltransferase or GA2 synthase (EC 2.4.1-) and UDP-GalNAc:(N-acetylneuraminyl)-lactosylceramide 1-4N-acetylgalactosaminyltransferase or GM2 synthase (EC 2.4.1.92) - sialyltransferases CMP-N-acetylneuraminate: lactosylceramide 2–3 sialyltransferase (sialyltransferases I and IV) or GM3 synthase (EC 2.4.99.-) and CMP-N-acetylneuraminate:(N-acetylneuraminyl) lactosylceramide 2-8 sialyltransferase (sialyltransferase II) or GD3 synthase (EC 24.99.8) - galactosyltransferases UDP-galactose:N-acetylgalactosaminyl-(N-acetylneuraminyl) lactosylceramide 1-3 galactosyltransferase (galactosyltransferase II) or GM1a synthase (EC 2.4.1.62) and UDP-galactose:lactosylceramide 1-4 galactosyltransferase or GbOse₃Cer synthase (EC 2.4.1-)     

Modification of l-phenylalanine ammonia-lyase in soybean cell suspension cultures by 2-aminooxyacetate and l-2-aminooxy-3-phenylpropionate

Suspension-cultured cells of soybean (Glycine max (L.) Merr. cv. Kanrich) produce large amounts of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), the first enzyme of phenylpropanoid metabolism, during growth. 2-Aminooxyacetic acid (AOA) and l-2-aminooxy-3-phenylpropionic acid (l-AOPP) inhibit the enzyme competitively in vitro and have been used for in vivo studies. The amount of extractable enzyme in the cells and their utilization of NO ₃ ^– and NH ₃ ⁺ are reduced upon the addition of AOA. When AOA was added at various times during growth, the appearance of additional enzyme activity was prevented but enzyme already formed was not inhibited. No evidence was obtained for the presence of an inhibitor in the extracts and AOA inhibition in vitro was readily reversible. It is conculded that AOA acts to inhibit the formation of PAL in suspension-cultured soy bean cells. In vitro inhibition of soybean PAL by l-AOPP could not be reversed; in contrast, the inhibition of maize (Zea mays L.) PAL was readily reversible. Added l-AOPP, which was rapidly taken up by the soybean cells, prevented the large increase in enzyme activity. Although PAL activity was blocked in the cultures, no appreciable increase in phenylalanine content could be detected in cell extracts. The response of soybean cell suspensions to l-AOPP addition thus differs from that of other tissues which in presence of l-AOPP show an increase in PAL activity and an accumulation of phenylalanine.Abbreviations AOA 2-aminooxyacetic acid - l-AOPP l-2-aminoxy-3-phenylpropionic acid - PAL l-phenylalanine ammonialyase (EC4.3.1.5)     

d-Alanine metabolism in the lucinid clam Lucinoma aequizonata

The chemoautotrophic symbiont-bearing clam Lucinoma aequizonata contains very high levels of free d-alanine in all tissues. The possible sources for this amino acid and its involvement in the clams' metabolism were investigated. Very low levels of d-alanine (generally below 1 mol·l^-1) were measured in the sediment porewaters from the habitat of the clams. Experiments with ¹⁴C-labeled tracers demonstrate an active metabolism of d-alanine in the clams rather than a role as inert waste product. d-alanine is metabolized at about 0.12 mol·g fw^-1·h^-1. Label from aspartate, but not glucose and CO₂, is incorporated into d-alanine. Incubation with labeled d-alanine did not result in formation of radioactive l-alanine. Tests for alanine racemase (EC 5.1.1.1) and d-amino acid oxidase (EC 1.4.3.3.) did not show activity in either gill, i.e. symbiont and host, or foot tissue. d-Alanine amino transferase (EC 2.6.1.b.) was demonstrated in gill and foot tissues. Two sources for d-alanine are proposed: a degradation of cell walls of symbiotic bacteria and production by the host using a d-specific alanine transaminase.Abbreviations aa amino acid(s) - fw fresh weight - HPLC high-performance liquid chromatography - MBH methyl benzethonium hydroxyde - NAC N-acetyl-l-cysteine - OPA ortho-phthaldialdehyde - TCA tricarbonic acid     

Purification of UDP-galactose: diacylglycerol galactosyltransferase from chloroplast envelopes of spinach (Spinacia oleracea L.)

Uridine 5-diphosphate(UDP)-galactose: 1,2-diacylglycerol 3-O--d-galactopyranosyltransferase (EC 2.4.1.46) is an integral protein of chloroplast envelope membranes from which it has been partially purified (Covès et al., 1986, FEBS Lett. 208, 401–406). We have worked out a purification procedure which after removal of peripheral membrane proteins, solubilization and two chromotographic steps allowed us to identify a 22-kDa protein as the galactosyltransferase. Enrichment of enzymatic activity was paralleled by an enrichment of this protein and its radioactive derivative obtained by photoaffinity labelling with [-^–32P]UDP which is a potent inhibitor of the enzyme. The purification factor of about 350 is substantially higher than achieved previously and indicates that the enzyme represents less than 0.3% of the envelope proteins. The purified enzyme has a Km of 87 M for UDP-galactose with dioleoylglycerol as acceptor and could not be activated by addition of other lipids.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-propanesulfonate - DTE dithioerythritol - MGD monogalactosyl diacylglycerol - PMSF phenylmethanesulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis This work was supported by the Deutsche Forschungsgemeinschaft.     

A novel callose synthase from pollen tubes of Nicotiana

Pollen-tube cell walls are unusual in that they are composed almost entirely of callose, a (1,3)--linked glucan with a few 6-linked branches. Regulation of callose synthesis in pollen tubes is under developmental control, and this contrasts with the deposition of callose in the walls of somatic plant cells which generally occurs only in response to wounding or stress. The callose synthase (uridine-diphosphate glucose: 1,3--d-glucan 3--d-glucosyl transferase, EC 2.4.1.34) activities of membrane preparations from cultured pollen tubes and suspension-cultured cells of Nicotiana alata Link et Otto (ornamental tobacco) exhibited different kinetic and regulatory properties. Callose synthesis by membrane preparations from pollen tubes was not stimulated by Ca²⁺ or other divalent cations, and exhibited Michaelis-Menten kinetics only between 0.25 mM and 6 mM uridine-diphosphate glucose (K _m 1.5–2.5 mM); it was activated by -glucosides and compatible detergents. In contrast, callose synthesis by membrane preparations from suspension-cultured cells was dependent on Ca²⁺, and in the presence of 2 mM Ca²⁺ exhibited Michaelis-Menten kinetics above 0.1 mM uridine-diphosphate glucose (K _m 0.45 mM); it also required a -glucoside and low levels of compatible detergent for full activity, but was rapidly inactivated at higher levels of detergent. Callose synthase activity in pollen-tube membranes increased ten fold after treatment of the membranes with trypsin in the presence of detergent, with no changes in cofactor requirements. No increase in callose synthase activity, however, was observed when membranes from suspension-cultured cells were treated with trypsin. The insoluble polymeric product of the pollen-tube enzyme was characterised as a linear (1,3)--d-glucan with no 6-linked glucosyl branches, and the same product was synthesised irrespective of the assay conditions employed.Abbreviations Ara l-arabinose - CHAPS 3-[(3-cholamidopropyl)dimethylammonia]-1-propane sulphonic acid - DAP diphenylamine-aniline-phosphoric acid stain - Gal d-galactose - Glc d-glucose - Man d-mannose - Mes 2-(N-morpholino)ethane sulphonic acid - Rha d-rhamnose - Rib d-ribose - TFA trifluoroacetic acid - UDPGlc uridine-diphosphate glucose - Xyl d-xylose This research was supported by funds from a Special Research Centre of the Australian Research Council. H.S. was funded by a Melbourne University Postgraduate Scholarship and an Overseas Postgraduate Research Studentship; S.M.R. was supported by a Queen Elizabeth II Research Fellowship. We thank Bruce McGinness and Susan Mau for greenhouse assistance, and Deborah Delmer and Adrienne Clarke for advice and encouragement throughout this project.     

Evidence for an ammonium transport system in free-living and symbiotic cyanobacteria

The free-living cyanobacterium Anabaena variabilis showed a biphasic pattern of ¹⁴CH₃NH ₃ ⁺ uptake. Initial accumulation (up to 60 s) was independent of CH₃NH ₃ ⁺ metabolism, but long-term uptake was dependent on its metabolism via glutamine synthetase (GS). The CH₃NH ₃ ⁺ was converted into methylglutamine which was not further metabolised. The addition of l-methionine-dl-sulphoximine (MSX), to inhibit GS, inhibited CH₃NH ₃ ⁺ metabolism, but did not affect the CH₃NH ₃ ⁺ transport system.NH ₄ ⁺ , when added after the addition of ¹⁴CH₃NH ₃ ⁺ , caused the efflux of free CH₃NH ₃ ⁺ ; when added before ¹⁴CH₃NH ₃ ⁺ , NH ₄ ⁺ inhibited its uptake indicating that both NH ₄ ⁺ and CH₃NH ₃ ⁺ share a common transport system. Carbonylcyanide m-chlorophenylhydrazone and triphenyl-methylphosphonium both inhibited CH₃NH ₃ ⁺ accumulation indicating that the transport system was -dependent. At pH 7 and at an external CH₃NH ₃ ⁺ concentration of 30 mol dm^-3, A. variabilis showed a 40-fold intracellular accumulation of CH₃NH ₃ ⁺ (internal concentration 1.4 mmol dm^-3). Packets of the symbiotic cyanobacterium Anabaena azollae, directly isolated from the water fern Azolla caroliniana, also showed a -dependent NH ₄ ⁺ transport system suggesting that the reduced inhibitory effect of NH ₄ ⁺ on nitrogenase cannot be attributed to the absence of an NH ₄ ⁺ transport system but is probably related to the reduced GS activity of the cyanobiont.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - GS glutamine synthetase - HEPES 4-(2-hydroxyethyl)-1-piperazine ethanesulphonic acid - MSX l-methionine-dl-sulphoximine - membrane potential - pH transmembrane pH difference - TPMP⁺ triphenylmethylphosphonium