Microbial Production of Abscisic Acid by Botrytis cinerea

The effect of oryzalexin D, which has been isolated as a group of novel phytoalexins of rice plant, on DNA, RNA, protein, lipid and chitin biosyntheses, respiration and cell membrane permeability was investigated in Pyricularia oryzae. The concentration for 50% inhibition (ED₅₀) by oryzalexin D of the mycelial growth of P. oryzae was 230 ppm. At this concentration, oryzalexin D inhibited equally the incorporation of [2–¹⁴C]thymidine, [2–¹⁴C]uridine, l-[U-¹⁴C]amino acid mixture, l-[methyl-¹⁴C]methionine and d-[l-¹⁴C]glucosamine into DNA, RNA, protein, lipid and chitin in intact cells, but did not inhibit these systems in a homogenate of the mycelia of P. oryzae. Oryzalexin D scarcely inhibited the respiration of the homogenate and mitochondria at ED₅₀. On the other hand, oryzalexin D at ED₅₀ caused leakage of potassium and inhibited the uptake of glutamate by mycelial cells of P. oryzae. These results suggest that interference with the cell membrane function is responsible for the primary mode of action.of oryzalexin D against P. oryzae.     

A possible role of Golgi membrane-associated galactosyltransferase in the formation of zymogen granule glycoproteins

A galactosyltransferase activity in smooth microsomes and Golgi membrane-rich fractions from rat pancreas glycosylated endogenous acceptors during incubation with UDP-[¹⁴C]galactose in the absence of exogenous glycoproteins. To evaluate the role of this activity in secretion, the endogenous products were partially characterized. Galactose-labeled fractions were sequentially extracted in 0.2 m NaHCO₃ and 0.25 m NaBr to prepare membranes and soluble acceptors. Bound radioactivity was equally distributed between these two fractions. Analysis by polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated that the particulate galactose-labeled polypeptides were distinct from the soluble galactose acceptors. Rabbit antisera against highly purified zymogen granule membranes precipitated approximately 40% of the radioactivity of the particulate fraction when solubilized in nonionic detergents. In polyacrylamide gels, the galactose-labeled species of the immunoprecipitate migrated with zymogen granule membrane glycoproteins. Rabbit antisera against secretory proteins cross-reacted with less than 5% of the galactose-labeled soluble acceptors. Mature zymogen granule membranes neither contained detectable galactosyltransferase activity nor served as galactosyltransferase acceptors. These results suggest that galactosyltransferase activity associated with membranes derived from the Golgi complex glycosylated zymogen granule membrane precursors. Analysis of [¹⁴C]galactolipids did not implicate lipid intermediates in this process.     

Identification of a novel sugar-H+ symport protein, FucP, for transport of L-fucose into Escherichia coli

l -Fucose (6-deoxy-l -galactose) is used as sole carbon source by many microorganisms, and its transport into Escherichia coli is mediated by An l -fucose-H⁺ symport activity, in order to determine the nature of a putative transporter encoded by the E. coli fucP gene and Identify its protein product it was cloned downstream of the inducible T7 RNA polymerase and lambda O_l P_l promoters, induction of the T7 promoter resulted in the expression of [¹⁴C]-l -fucose uptake activity and the concomitant expression of a [³⁵S]-Met-labelled 32 kDa protein at levels too tow for detection by staining with Coomassie briiiiant blue or for protein sequencing, induction of the lambda O_l P_l promoter caused the appearance of l -fucose-H₊ symport activity and of a Coomassie brilliant blue-stained 32 kDa membrane protein expressed at high levels sufficient for identification as FucP by N-terminal protein sequencing. The FucP protein is, therefore, a sugar-H₊ symporter different in amino acid sequence from any other known transporter. These and other results illustrate the general unpredictability of cloning strategies for attempting the amplified expression of membrane transport proteins.     

Selective Acylation of Plasma Membrane Proteins of Mycoplasma mycoides subsp. mycoides SC,the Contagious Bovine Pleuropneumonia Agent

The plasma membrane of Mycoplasma mycoides subsp. mycoides SC (strain KH₃J) contains over 160 polypeptides with apparent molecular masses ranging from 14 to 125 kDa and isoelectric point values (pIs) from 5 to 9. In vivo labeling with [¹⁴C]-fatty acids revealed about 35 acylated polypeptides including the two major components p42 and p65 and displaying pIs between 5.5 and 9.0, with a majority between 6.5 and 8. The amphiphilic nature of most of these acyl proteins was confirmed by Triton X-114 phase partitioning. Gas-liquid chromatography analyses showed that the order of preference for protein acylation was 16:0 > 18:2c > 18:1c > 18:0 > 14:0, with 16:0 being the major O-ester-bound fatty acyl chain and 18:2c the major N-linked chain. The presence of S-glycerylcysteine and a ratio of [O-ester-bound acyl chains + N-linked chains]/O-ester bound chains of ≈ 1.2 in M. mycoides subsp. mycoides SC membrane proteins are consistent with a lipid modification similar to that occurring in lipoproteins of Gram-negative eubacteria that contain an N-terminal acyl S-glycerylcysteine.     

N-[1-C]acetylimidazole as a reagent for tyrosyl modification in protein NMR studies: Acetylation of cytochrome c

The reaction of N-[1-¹³C] acetylimidazole with cytochrome c and guanidinated cytochrome c was evaluated as a means of introducing NMR-detectable groups as conformation-dependent probes. Resonances from both N-[1-¹³C]acetyl lysyl and O-[1-¹³C]acetyl tyrosyl groups were observed when ferricytochrome c was acetylated. However, only O-[1-¹³C]acetyl tyrosyl resonances were seen with acetylated guanidinated ferricytochrome c. Chemical shifts of the four O-[1-¹³C]acetyl tyrosyl groups were conformation dependent and ranged from 172 to 176 ppm. A convenient method for the preparation of N-[1-¹³C]acetylimidazole is described.     

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.     

Transamidination of [1-14C]-guanidinoacetic acid to 14C-glycine and decarboxylation to 14CO2 in white spruce shoot primordia entering winter dormancy

Feeding [1-¹⁴C]-guanidinoacetic acid to shoot primordia, O₂ uptake was inhibited and major products were ¹⁴C-glycine, ¹⁴CO₂ and ¹⁴C-serine. The direct decarboxylation of [1-¹⁴C]-guanidinoacetic acid to ¹⁴CO₂ and N-methylguanidine, the methylation of [1-¹⁴C]-guanidinoacetic acid to ¹⁴C-creatine, and the lytic cleavage to urea and ¹⁴C-glycine were all ruled out. Enzymatic transamidinations of [1-¹⁴C]-guanidinoacetic acid with amino acid acceptors occurred as arginine-rich storage proteins were being turned over and new proteins synthesized containing ¹⁴C-glycine and ¹⁴C-serine. The products of transamidination were recycled as substrates until ¹⁴C-glycine was metabolized in different directions and transported to mitochondria and peroxisomes. ¹⁴C-Glycine was decarboxylated by a glycine decarboxylase multienzyme complex resulting in a net carbon loss and a sharp decline in total protein rich in arginine N. Under these conditions, unlabelled arginine and ornithine contributed as substrates for reversible transamidination reactions. Peroxisomes and mitochondria are hypothesized as providing arginine-derived nitric oxide to maintain redox homeostasis in response to the stresses imposed by [1-¹⁴C]-guanidinoacetic acid and to protect against the inhibitory activity of sulfhydryls on transamidinase activity. The destruction of a respiratory inhibitor by transamidination may comprise a mechanism associated with the awakening from of dormancy and the mobilization of storage protein reserves in conifers.     

Acetylcholine Synthesis and CO2 Production from Variously Labeled Glucose in Rat Brain Slices and Synaptosomes

Abstract: The molecular basis of the close linkage between oxidative metabolism and acetylcholine (ACh) synthesis is still unclear. We studied this problem in slices and synaptosomes by measurement of ACh synthesis from [U-¹⁴C]glucose, and ¹⁴CO₂ production from [3,4-¹⁴C]- and [2-¹⁴C]glucose, an index of glucose decarboxylation by the pyruvate dehydrogenase complex (PDH) and the enzymes of the Krebs cycle, respectively. We examined both under conditions that either inhibited (low O₂ or antimycin) or stimulated (2,4- dinitrophenol [DNP] or 35 mm -K⁺) ¹⁴CO₂ production from [2-¹⁴C]- or [3,4-¹⁴C]glucose. Incorporation of [U-¹⁴C]glucose into ACh was reduced under low O₂ and by antimycin or DNP (by 51-93%) and stimulated by 35 mm -K⁺ (by 30-60%). Under all of these conditions, ACh synthesis and the decarboxylation of [3,4-¹⁴C]- and [2-¹⁴C]glucose were linearly related (r= 0.741 and 0.579, respectively). The difference in the rate of ¹⁴CO₂ production from [3,4-¹⁴C]- and [2-¹⁴C]glucose was used as a measure of the amount of glucose that was not oxidatively decarboxylated (efflux). We found that efflux was reduced (low 0₂ and antimycin), unchanged (DNP in slices), or increased (DNP in synaptosomes and K⁺ stimulation in slices) compared with control values under 100% O₂. ACh synthesis and efflux were more closely related (r= 0.860) than ACh synthesis and ¹⁴CO₂ production from variously labeled glucoses.     

The structure of a fucose-containingO-glycosidic carbohydrate chain of human platelet glycocalicin

The hydrazinolysis procedure currently used for the release ofN-glycosidic carbohydrate chains was applied to glycocalicin. The resulting mixture of oligosaccharide-alditols was fractionated by high-voltage paper electrophoresis into a neutral (5%) and several acidic fractions. The neutral compounds were passed over Bio-Gel P-4. SomeN-glycosidic oligosaccharide-alditols, of theN-acetyllactosamine type as well as of the oligomannoside type, were found to be present. However, oligosaccharide-alditols derived fromO-glycosidic carbohydrate chains were also found, indicating a partial cleavage of GalNAc1-OSer/Thr linkages under the hydrazinolysis conditions applied. One of the neutralO-glycosidic components was characterized, by 500-MHz¹H-NMR spectroscopy in combination with sugar analysis, as the following pentasaccharidealditol: In addition the afuco analogue of this compound was obtained.     

EFFECTS OF SODIUM FLUOROACETATE ON THE METABOLISM OF N-ACETYLASPARTATE AND ASPARTATE IN MOUSE BRAIN

A subconvulsant dose of sodium fluoroacetate inhibited the metabolic utilization of intracerebrally-administered N-acetyl-l -[U-¹⁴C]asparticacid and the labelling of glutamine from this precursor in mouse brain, but not the labelling of glutamate or aspartate. A convulsant dose also inhibited the utilization of l -[U-¹⁴C]aspartic acid. When intraperitoneal injection of a convulsant dose of sodium fluoroacetate was followed by intracerebral injection of N-acetyl-l -[U-¹⁴C]asparticacid, the levels of N-acetylaspartate, aspartate and glutamate in brain were lowered, while the glutamine content was increased. The specific radioactivity of glutamine relative to that of glutamate was much lower when these compounds were labelled from l -[U-¹⁴C]aspartic acid than when N-acetyl-l -[U-¹⁴C]aspartic acid was used as the precursor. Intracerebral injection of tracer amounts of l -[U-¹⁴C]aspartic acid reduced the content of N-acetylaspartate in brain and raised the glutamine content. Sodium fluoroacetate had no additional effect on the relative specific radioactivity of glutamine or the content of N-acetylaspartate, aspartate, glutamate or glutamine when l -[U-¹⁴C]aspartic acid was the precursor. We consider the results to be consistent with a selective inhibition both by sodium fluoroacetate and by exogenous aspartic acid of the tricarboxylic acid cycle in brain associated with the biosynthesis of glutamine. We suggest that the activity of this pathway may regulate the metabolism of N-acetylaspartate and aspartate.     

Kitazin P,Inhibitor of Phosphatidylcholine Biosynthesis in Pyricularia oryzae

The effect of organophosphorus fungicide, Kitazin P (IBP, S-benzyl diisopropyl phosphorothiolate), on lipid biosynthesis of Pyricularia oryzae was investigated. Addition of IBP to the mycelial cells suspension of P. oryzae induced a striking decrease in incorporation of methionine-methyl-¹⁴C, S-adenosylmethionine-methyl-¹⁴C, and glycerol-1-¹⁴C into phosphatidylcholine, which is the most abundant phospholipid in P. oryzae, but incorporation of choline-methyl-¹⁴C into phosphatidylcholine and that of methionine-methyl-¹⁴C into simple lipids were not affected. Incorporation of methionine-methyl-¹⁴C into phosphatidylcholine is found to be directly proportional to mycelial cells growth of P. oryzae. Enzymes responsible for the biosynthesis from glycerol to phosphatidylcholine through Greenberg’s pathway, except phospholipid N-mefhyltransferase, were not inhibited by IBP. IBP concentration required for 50% inhibition of phospholipid N-methyltransferase was 40 ppm. IBP had no effect on activities of glycerokinase, glycerophosphate acyltransferase, phosphatidic acid cytidyltransferase, phosphatidylserine synthetase, and phosphatidylserine decarboxylase, respectively. Therefore, the specific inhibition of conversion from phosphatidylethanolamine to phosphatidylcholine by the transmethylation of S-adenosylmethionine might be regarded as one of the modes of action of IBP.     

Endogenous protein mono-ADP-ribosylation in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Protein mono-ADP-ribosylation post-translationally transfers the ADP-ribose moiety from the β-NAD⁺ donor to various protein acceptors. This type of modification has been widely characterized and shown to regulate protein activities in animals, yeast and prokaryotes, but has never been reported in plants. In this study, using [³²P]NAD⁺ as the substrate, ADP-ribosylated proteins in Arabidopsis were investigated. One protein substrate of 32 kDa in adult rosette leaves was found to be radiolabeled. Heat treatment, protease sensitivity and nucleotide derivative competition assays suggested a covalent reaction of NAD⁺ with the 32 kDa protein. [carbonyl-¹⁴C]NAD⁺ could not label the 32 kDa protein, confirming that the modification was ADP-ribosylation. Poly (ADP-ribose) polymerase inhibitor failed to suppress the reaction, but chemicals that destroy mono-ADP-ribosylation on specific amino acid residues could break up the linkage, suggesting that the reaction was not a poly-ADP-ribosylation but rather a mono-ADP-ribosylation. This modification mainly existed in leaves and was enhanced by oxidative stresses. In young seedlings, two more protein substrates with the size of 45 kDa and over 130 kDa, respectively, were observed in addition to the 32 kDa protein, indicating that different proteins were modified at different developmental stages. Although the substrate proteins remain to be identified, this is the first report on the characterization of endogenously mono-ADP-ribosylated proteins in plants.     

Subcellular site of the biosynthesis ofO-acetylated sialic acids in bovine submandibular gland

Bovine submandibular glands were homogenized and fractionated under conditions which yielded subcellular fragments from mainly one cell type, the mucous acinar cell, as judged by morphological analysis of the glands before and after homogenization. The majorN-acetylneuraminate-9(7)-O-acetyltransferase activity was detected in the cytosolic fraction, a result supported by the high specific radioactivity of free sialic acids isolated after [¹⁴C]acetate-labelling experiments. Separation of membranes on a Ficoll density gradient gave six fractions which were analyzed biochemically and morphologically. The particulate activities of acetyltransferase and sialyltransferase were found in fractions containing smooth and mitochondrial membranes. MembraneO-acetyl sialic acids were present at the highest levels in these fractions and also had the highest specific radioactivity after [¹⁴C]acetate-labelling experiments. Significant amounts of theO-acetyltransferase activity also occur in the cytosol and are consistent with a model ofO-acetyl sialic acid biosynthesis involving both cytosolic and smooth membrane sites ofO-acetylation.     

Regulation of ketone body production from (14C)palmitate in rat liver mitochondria: effects of cyclic nucleotides and unlabeled fatty acids.

N⁶′, O²′-dibutyryl adenosine 3′, 5′-cyclic monophosphoric acid, but not other cyclic nucleotides stimulates [¹⁴C]ketone body production from [¹⁴C]palmitate in isolated rat liver mitochondria. Butyrate alone, as well as unlabeled acetate, octanoate and palmitate had similar effects. This redistribution of the oxidative products of [¹⁴C]palmitate can best be explained by exceeding the capacity of the Krebs cycle and/or changes in the acetyl coenzyme A/coenzyme A ratio. In contrast to [¹⁴C]palmitate, [¹⁴C]octanoate oxidation to [¹⁴C]O₂ and [¹⁴C]ketone bodies was inhibited by the addition of unlabeled fatty acids. This suggests that an additional mechanism by which unlabeled fatty acids may stimulate [¹⁴C]ketone body production is by enhancing the carnitine-dependent transport of [¹⁴C]palmitate into mitochondria.     

Biosynthesis of β-glucans by cell-free extracts from saccharomyces cerevisiae

Cell-free extracts from Saccharomyces cerevisiae catalyzed the incorporation of glucosyl residues from UDP-[U-¹⁴C]glucose into β-1, 3-glucans which contained a significant proportion of β-1, 6-glycosidic linkages. When GDP-[U-¹⁴C]-glucose was used as substrate only trace amounts of glucose were incorporated. Activity of β-glucan synthetase was distributed among membrane and cell wall fractions, specific activity being higher in this latter. β-Glucan synthesized by membrane and cell wall fractions contained 0.6% and 2.5% of β-1, 6-glycosidic linkages respectively. A marked decrease in the activity of β-glucan synthetase occurred as the cells aged. Significant activity of glycogen synthetase was detected only in cells which had reached the stationary phase of growth.     

The developmental pattern of homologous and heterologous tRNA methylation in rat brain differential effect of spermidine

UsingS-adenosyl-L-[Me-¹⁴C] methionine, rat cerebral cortex methyltransferase activity was determined during the early postnatal period in the absence of addedEscherichia coli tRNA and in its presence. [Me-¹⁴C] tRNA was purified from both systems and its [Me-¹⁴C] base composition determined. The endogenous formation of [Me-¹⁴C] tRNA (homologous tRNA methylation) was totally abolished in the presence of 2.5 mM spermidine, whereasE. coli B tRNA methylation (heterologous methylation) was markedly stimulated. Only [Me-¹⁴C] 1-methyl guanine and [Me-¹⁴C]N ²-methyl guanine were formed by homologous methylation, there being an inverse shift in their relative proportions with age. Heterologous tRNA methylation led, additionally, to the formation of [Me-¹⁴C]N ₂ ² -dimethyl guanine, 5-methyl cytosine, 1-methyl adenine, 5-methyl uracil, 2-methyl adenine, and 1-methyl hypoxanthine. A comparison of heterologous tRNA methylation between the whole brain cortex (containing nerve and glial cells) and bulk-isolated nerve cell bodies revealed markedly lower proportions of [Me-¹⁴C]N ₂-methyl andN ₂ ² -dimethyl guanine and significantly higher proportions of [Me-¹⁴C] 1-methyl adenine in the neurons. The present findings suggest (1) that homologous tRNA methylation may provide developing brain cells with continuously changing populations of tRNA and (2) that neurons are enriched in adenine residue-specific tRNA methyltransferases that are highly sensitive to spermidine.This research was supported by grant NS-06294 of the United States Public Health Service.     

IN VITRO STUDIES ON THE INTERACTION OF BRAIN MONOAMINE OXIDASE WITH 5,6- AND 5,7-DIHYDROXYTRYPTAMINE12

[¹⁴C]5,6-Dihydroxytryptamine ([¹⁴C] 5,6-DHT) and [¹⁴C]5,7-dihydroxytryptamine ([¹⁴C]5,7-DHT) were deaminated to toluene-isoamylalcohol extractable products when incubated with homogenates of rat hypothalamus or pons-medulla oblongata. [¹⁴C]5,6-Dihydroxyindole acetic acid ([¹⁴C]5.6-DHIAA) and [¹⁴C]5,7-dihydroxyindole acetic acid ([¹⁴C]5,7-DHIAA) were detected as MAO metabolites by TLC besides non-identified components. The conversion of [¹⁴C]5,6-DHT and [¹⁴C]5,7-DHT obeyed, at least initially, Michaelis-Menten kinetics (K_m 5,7-DHT: 0.5 × 10^?3M; K_m 5,6-DHT: 1.25 × 10^?3M). Inhibition of the reaction by the MAO A inhibitor, clorgyline, resulted in a typical double sigmoidal inhibition curve indicating that both amines are metabolized by both types of MAO (A and B). In deprenyl inhibition studies, however, 5,7- and 5,6-DHT seemed to be preferred substrates of MAO A. Incubation of rat brain homogenates with [¹⁴C]5,6-DHT and [¹⁴C]5,7-DHT or with the MAO metabolites [¹⁴C]5,6-DHIAA and [¹⁴C]5,7-DHIAA caused a time-dependent break-down of the dihydroxylated indole compounds with subsequent binding of radioactivity to perchloric acid insoluble tissue components. 5,6-DHT inactivated MAO in rat brain homogenates parallel to its decomposition and extensive protein binding. The inactivation of MAO by 5,6-DHT and the extensive binding of radioactivity to protein were antagonized by dithiothreitol (DTT), glutathione (GSH) and L-ascorbic acid. Reduction of [O₂] in the incubation medium slightly attenuated the inactivation of MAO by 5,6-DHT. Catalase or superoxide dismutase failed to prevent MAO from being inactivated by 5,6-DHT. The results suggest that oxidation products of 5,6-DHT, e.g. its corresponding o-quinone, are involved in the inactivation of MAO in vitro and mainly responsible for the binding of radioactivity to brain proteins in vitro. Similar mechanisms may also be operative in the in vivo neurotoxicity of 5,6-DHT. The lack of inactivation of MAO by 5,7-DHT in vitro correlated with a low degree of radioactivity binding (from [¹⁴C]5,7-DHT) to homogenate protein pellets; the binding to proteins was barely influenced by GSH, cysteine, DTT and l -ascorbic acid. These latter findings do not provide a plausible explanation for the mechanism(s) involved in the well known in vivo neurotoxicity of 5,7-DHT.     

Differential labeling of the subunits of respiratory Complex III with [3H]succinic anhydride, [14C]succinic anhydride,andp-diazobenzene-[35S]sulfonate

Exposure of antimycin-treated Complex III (ubiquinol-cytochromec reductase) purified from bovine heart mitochondria to [³H]succinic anhydride plus [³⁵S]p-diazobenzenesulfonate (DABS) resulted in somewhat uniform relative labeling of the eight measured subunits of the complex by [³H]succinic anhydride. In contrast, relative labeling by [³⁵S]DABS was similar to [³H]succinic anhydride for the subunits of high molecular mass, i.e., core proteins, cytochromes, and the iron-sulfur protein, but greatly reduced for the polypeptides of molecular mass below 15 kDa. With Complex III depleted in the iron-sulfur protein the relative labeling of core protein I by exposure of the complex to [³H]succinic anhydride was significantly enhanced, whereas labeling of the polypeptides represented by SDS-PAGE bands 7 and 8 was significantly inhibited. Dual labeling of the subunits of Complex III by¹⁴C- and³H-labeled succinic anhydride before and after dissociation of the complex by sodium dodecyl sulfate, respectively, was measured with the complex in its oxidized, reduced, and antimycin-inhibited states. Subunits observed to be most accessible or reactive to succinic anhydride were core protein II, the iron-sulfur protein, and polypeptides of SDS-PAGE bands 7, 8, and 9. Two additional polypeptides of molecular masses 23 and 12 kDa, not normally resolved by gel-electrophoresis, were detected. Reduction of the complex resulted in a significant change of¹⁴C/³H labeling ratio of core protein only, whereas treatment of the complex with antimycin resulted in decreases in¹⁴C/³H labeling ratios of core proteins I and II, cytochromec ₁, and a polypeptide of molecular mass 13 kDa identified as an antimycin-binding protein.     

Cell-free labeling in thyroid rough microsomes of lipid-linked and protein-linked oligosaccharides: I. Mannosylated units

In order to evaluate the possibility in a pig thyroid rough microsomal system of a transfer of pre-assembled sugar cores from sugar-lipids to protein, we have examined after incubation with GDP-[¹⁴C]Man the compounds bearing labeled saccharides and have determined some properties of their released saccharide moieties. The [¹⁴C]Man material specifically soluble in CHCl₃/CH₃OH/H₂O, 10 : 10 : 3, behaved on DEAE-cellulose and when treated with hot alkali and alkaline phosphatase as a lipid pyrophosphate (sometimes accompanied by some $\text{dolichol}\text{-P-[}{}^{14}\text{C}\text{]}\text{Man}$). Its saccharide moiety, released by mild acid, exhibited properties (molecular size, sensitivity to α-mannosidase, affinity for concanavalin A and charge modification introduced by a strong reductive alkaline treatment) pointing to a polymannosylated N,N′-diacetylchitobiose containing an average of nine monosaccharide units (from six to twelve). The [¹⁴C]mannosylated glycoproteins have represented all the polymeric label remaining after lipid extraction. From the susceptibility of their pronase glycopeptides to a differential reductive alkaline hydrolysis, it was concluded that their label belonged mainly to N-glycosidically linked units. Released saccharides exhibited the same properties as those from lipids, a result substantiating the possibility raised from previous studies of a transfer of pre-assembled moieties.     

Oxalyltransferase,a plant cell‐wall acyltransferase activity,transfers oxalate groups from ascorbate metabolites to carbohydrates

In the plant apoplast, ascorbate is oxidised, via dehydroascorbic acid, to O‐oxalyl esters [oxalyl‐l ‐threonate (OxT) and cyclic oxalyl‐l ‐threonate (cOxT)]. We tested whether OxT and cOxT can donate the oxalyl group in transacylation reactions to form oxalyl‐polysaccharides, potentially modifying the cell wall. [oxalyl‐¹⁴C]OxT was incubated with living spinach (Spinacia oleracea) and Arabidopsis cell‐suspension cultures in the presence or absence of proposed acceptor substrates (carbohydrates). In addition, [¹⁴C]OxT and [¹⁴C]cOxT were incubated in vitro with cell‐wall enzyme preparations plus proposed acceptor substrates. Radioactive products were monitored electrophoretically. Oxalyltransferase activity was detected. Living cells incorporated oxalate groups from OxT into cell‐wall polymers via ester bonds. When sugars were added, [¹⁴C]oxalyl‐sugars were formed, in competition with OxT hydrolysis. Preferred acceptor substrates were carbohydrates possessing primary alcohols e.g. glucose. A model transacylation product, [¹⁴C]oxalyl‐glucose, was relatively stable in vivo (half‐life >24 h), whereas [¹⁴C]OxT underwent rapid turnover (half‐life ~6 h). Ionically wall‐bound enzymes catalysed similar transacylation reactions in vitro with OxT or cOxT as oxalyl donor substrates and any of a range of sugars or hemicelluloses as acceptor substrates. Glucosamine was O‐oxalylated, not N‐oxalylated. We conclude that plants possess apoplastic acyltransferase (oxalyltransferase) activity that transfers oxalyl groups from ascorbate catabolites to carbohydrates, forming relatively long‐lived O‐oxalyl‐carbohydrates. The findings increase the range of known metabolites whose accumulation in vivo indicates vitamin C catabolism. Possible signalling roles of the resulting oxalyl‐sugars can now be investigated, as can the potential ability of polysaccharide oxalylation to modify the wall's physical properties.