Characterization of [14C]apiogalacturonans synthesized in a cell-free system from Lemna minor.

Radioactive polysaccharide was synthesized when uridine 5′-(α-d-[U-¹⁴C]apio-d-furanosyl pyrophosphate) (containing some uridine 5′-(α-d-[U-¹⁴C]xylopyranosyl pyrophosphate)) was incubated with a particulate enzyme preparation from Lemna minor. Characterization experiments established that the product: (i) was insoluble in methanol and water, (ii) contained d-[U-¹⁴C]apiose (75%) and d-[U-¹⁴C]xylose (25%), and (iii) was soluble in 1% ammonium oxalate. The material solubilized by ammonium oxalate (solubilized product): (i) was separated into five fractions by column chromatography with diethylaminoethyl-Sephadex (DEAE-Sephadex), (ii) contained [U-¹⁴C]apiobiose side chains that were removed by hydrolysis at pH 4, and (iii) was degraded by fungal pectinase. Both d-[U-¹⁴C]apiose residues of the [U-¹⁴C]apiobiose side chains were synthesized in vivo since radioactivity was distributed equally between the two residues. The presence of uridine 5′-(α-d-galactopyranosyluronic acid pyrophosphate) during synthesis of radioactive polysaccharide resulted in: (i) an increase in the incorporation of radioactive d-[U-¹⁴C]apiose into solubilized product, (ii) an increase in the ratio of d-[U-¹⁴C]apiose to d-[U-¹⁴C]xylose present in solubilized product, (iii) an increase in the amount of [U-¹⁴C]apiobiose plus d-[U-¹⁴C]apiose released from the solubilized product by hydrolysis at pH 4, and (iv) a tighter binding of the solubilized product to DEAE-Sephadex. These results show that apiogalacturonans similar to or the same as those synthesized by the intact plant were synthesized in the particulate enzyme preparation isolated from L. minor. [¹⁴C]Apiogalacturonans completely free of d-[U-^l4C]xylose were not isolated. The [¹⁴C]apiogalacturonan with the least d-[U-¹⁴C]xylose still had 4.8% of its radioactivity present in d-[U-¹⁴C]xylose. The possibility remains that d-xylose is a normal constituent of the apiogalacturonans of the cell wall of L. minor.     

A general procedure for the preparation of labeled l-ascorbic acid from labeled d-glucose

A simple, three-step conversion of 1,2-O-isopropylidene-α-d-glucofuranose into l-ascorbic acid, originally described by Bakke and Theander, was used to prepare l-[4-¹⁴C]ascorbic acid from milligram amounts of d-[3-¹⁴C]glucopyranose in 28% radioisotopic yield. In addition, l-[6-¹⁴C]- and l-[U-¹⁴C]-ascorbic acid were prepared from d-[1-¹⁴C]- and d-[U-¹⁴C]-glucopyranose, respectively. The procedure is useful for the synthesis of l-ascorbic acid bearing isotopic hydrogen, carbon, or oxygen atoms at specific positions, subject only to the availability of starting material.     

Characterization of particulate D-apiosyl-and D-xylosyltransferase from Lemna minor.

A particulate enzyme preparation capable of catalyzing the transfer of d-[U-¹⁴C]apiose and d-[U-¹⁴C]xylose from uridine 5′-(α-d-[U-¹⁴C]apio-d-furanosyl pyrophosphate) (UDP[U-¹⁴C]Api) and uridine 5′-(α-d-[U-¹⁴C]xylopyranosyl pyrophosphate) (UDP[U-¹⁴C]Xyl) to endogenous acceptor molecules was isolated from Lemna minor. The two enzymes were named UDP-d-apiose:acceptor d-apiosyltransferase and UDP-d-xylose:acceptor d-xylosyltransferase and were associated with particulate material sedimenting between 480 and 34,800g. The rate of d-[U-¹⁴C]apiose or d-[U-¹⁴C]xylose incorporation was proportional to the quantity of enzyme preparation used and was constant with time to 1.5 min. Both enzymes showed a pH optimum of 5.7 in citrate-phosphate buffer. The d-apiosyltransferase has a K_m for UDP[U-¹⁴C]Api of 4.9 μm. Bovine serum albumin and sucrose stimulated the rate of incorporation of both pentoses. Both enzymes rapidly lost activity; with our best conditions, approximately 50% of each enzyme activity was lost in 6 min at 25 °C or in 3 h at 4 °C. Incorporation of d-[U-¹⁴C]apiose was obtained in the absence of added uridine 5′-(α-d-galactopyranosyluronic acid pyrophosphate) (UDPGalUA); however, the addition of UDPGalUA not only almost doubled the rate of incorporation, but also increased the total incorporation of d-[U-^l4C]apiose and extended the proportional range of incorporation at 25 °C from 1.5 to 2 min.     

Substrate utilization for energy production and lipid synthesis in oligodendrocyte-enriched cultures prepared from rat brain

The metabolism of oligodendrocytes has been studied using cultures of oligodendrocyte-enriched glial cells isolated from cerebra of 5–8-day old rats. Cultures containing 60–80% oligodendrocytes were incubated for 16h with [3-¹⁴C]acetoacetate, d-[3-¹⁴C]3-hydroxybutyrate, [U-¹⁴C]glucose, l-[U-¹⁴C]glutamine and [1-¹⁴C]pyruvate or [2-¹⁴C]pyruvate in the presence or absence of other oxidizable substrates. Labelled CO₂ was collected as an index of oxidative metabolism and the incorporation of label into total lipids, fatty acids and cholesterol was used as an index of the de novo synthesis of lipids. Glucose, acetoacetate, D-3-hydroxybutyrate, pyruvate and l-lactate were measured to determine substrate utilization and product formation under various conditions. Our results indicate that glucose is rapidly converted to lactate and is a relatively poor substrate for oxidative metabolism and lipid synthesis. Ketone bodies were used as an energy source and as precursors for the synthesis of fatty acids and cholesterol. Preferential incorporation of acetoacetate into cholesterol was not observed. Exogenous pyruvate was incorporated into both the glycerol skeleton of complex lipids and into cholesterol and fatty acids. l-Glutamine appeared to be an important substrate for the energy metabolism of these cells.     

Fluoride-Induced Inhibition of Starch Biosynthesis in Developing Potato, Solanum tuberosum L., Tubers Is Associated with Pyrophosphate Accumulation

Pretreatment of discs excised from developing tubers of potato (Solanum tuberosum L.) with 10 millimolar sodium fluoride induced a transient increase in 3-phosphoglycerate content. This was followed by increases in triose-phosphate, fructose 1,6-bisphosphate and hexose-phosphate (glucose 6-phosphate + fructose 6-phosphate + glucose 1-phosphate). The effect of fluoride is attributed to an inhibition of glycolysis and a stimulation of triose-phosphate recycling (the latter confirmed by the pattern of ¹³C-labeling [NMR] in sucrose when tissue was supplied with [2-¹³C]glucose). Fluoride inhibited the incorporation of [U-¹⁴C] glucose, [U-¹⁴C]sucrose, [U-¹⁴C]glucose 1-phosphate, and [U-¹⁴C] glycerol into starch. The incorporation of [U-¹⁴C]ADPglucose was unaffected. Inhibition of starch biosynthesis was accompanied by an almost proportional increase in the incorporation of ¹⁴C into sucrose. The inhibition of starch synthesis was accompanied by a 10-fold increase in tissue pyrophosphate (PPi) content. Although the subcellular localization of PPi was not determined, a hypothesis is presented that argues that the PPi accumulates in the amyloplast due to inhibition of alkaline inorganic pyrophosphatase by fluoride ions.     

The involvement of sucrose,glucose and other metabolites in the synthesis of triterpenes and dopa in the laticifers of Euphorbia lathyris

A quantitative triterpene analysis was made of latex stem tissue of Euphorbia lathyris. Young plants seedlings of E. lathyris were incubated with various labelled precursors. Incorporation into triterpenes was obtained from [2-¹⁴C]mevalonic acid, [1-¹⁴C]acetate, [3-¹⁴C]pyruvate, [U-¹⁴C]sucrose, [U-¹⁴C]glucose, [U-¹⁴C]xylose, [U-¹⁴C]glyoxylate, [2,3-¹⁴C]succinic acid, [1-¹⁴C]glycerol [U-¹⁴C]serine. Both sugars tyrosine appeared to be effective precursors in DOPA synthesis inside the laticifers. Exogenously supplied mevalonic acid was only involved in triterpene synthesis outside the laticifers. GC-RC of triterpenes synthesized from [U-¹⁴C]glucose revealed the origin of these compounds in the latex. The labelled triterpenes obtained after incorporation of the other mentioned labelled precursors were only partly synthesized in the laticifers. For quantitative data on latex triterpene synthesis seedlings were incubated with [U-¹⁴C]sucrose, [U-¹⁴C]glucose, [U-¹⁴C]xylose [1-¹⁴C]acetate in the presence of increasing amounts of unlabelled substrate. From the amount of ¹⁴C incorporated into the triterpenes the amount of substrate directly involved in triterpene synthesis was calculated, as was the absolute triterpene yield. Sucrose showed the highest triterpene yield, equivalent to the daily increase of the triterpene content of growing seedlings. The possible significance of the other precursors in triterpene synthesis in the laticifers is discussed.     

beta-Carotene Synthesis in Isolated Spinach Chloroplasts : Its Tight Linkage to Photosynthetic Carbon Metabolism

Carefully isolated intact spinach chloroplasts virtually free of contamination of other organelles effectively form β-carotene from NaH¹⁴CO₃ or [U-¹⁴C]-3-phosphoglycerate (PGA) under photosynthetic conditions. The photosynthate pool formed in chloroplasts from 1 to 2 millimolar [U-¹⁴C]-3-PGA or 3 to 6 millimolar NaH¹⁴CO₃ was fully sufficient to supply β-carotene synthesis with intermediates for about 1 hour at maximal rates of about 20 nanomoles ¹⁴C incorporated per milligram chlorophyll per hour. Fatty acid synthesis remains, under these circumstances, in linear dependence to substrate concentrations with far lower activity. Isotopic dilution of the β-carotene synthesis by adding unlabeled glyceraldehyde 3-phosphate, dihydroxyacetone-P, 3-PGA, 2-PGA, phosphoenolpyruvate, pyruvate, respectively, may be interpreted as a direct substrate flow from photosynthetically fixed CO₂ to isopentenyl pyrophosphate synthesizing system. Unlabeled acetate did not dilute β-carotene synthesis. Fatty acid synthesis acted similarly with unlabeled substrates; but it also was diluted by unlabeled acetate. These results indicate a tight linkage of photosynthetic carbon fixation and plastid isoprenoid synthesis.     

Distribution of radiolabeled l-glutamate and d-aspartate from blood into peripheral tissues in naive rats: Significance for brain neuroprotection

Excess l-glutamate (glutamate) levels in brain interstitial and cerebrospinal fluids (ISF and CSF, respectively) are the hallmark of several neurodegenerative conditions such as stroke, traumatic brain injury or amyotrophic lateral sclerosis. Its removal could prevent the glutamate excitotoxicity that causes long-lasting neurological deficits. As in previous studies, we have established the role of blood glutamate levels in brain neuroprotection, we have now investigated the contribution of the peripheral organs to the homeostasis of glutamate in blood. We have administered naive rats with intravenous injections of either l-[1-¹⁴C] Glutamic acid (l-[1-¹⁴C] Glu), l-[G-³H] Glutamic acid (l-[G-³H] Glu) or d-[2,3-³H] Aspartic acid (d-[2,3-³H] Asp), a non-metabolized analog of glutamate, and have followed their distribution into peripheral organs. We have observed that the decay of the radioactivity associated with l-[1-¹⁴C] Glu and l-[G-³H] Glu was faster than that associated with glutamate non-metabolized analog, d-[2,3-³H] Asp. l-[1-¹⁴C] Glu was subjected in blood to a rapid decarboxylation with the loss of ¹⁴CO₂. The three major sequestrating organs, serving as depots for the eliminated glutamate and/or its metabolites were skeletal muscle, liver and gut, contributing together 92% or 87% of total l-[U-¹⁴C] Glu or d-[2,3-³H] Asp radioactivity capture. l-[U-¹⁴C] Glu and d-[2,3-³H] Asp showed a different organ sequestration pattern. We conclude that glutamate is rapidly eliminated from the blood into peripheral tissues, mainly in non-metabolized form. The liver plays a central role in glutamate metabolism and serves as an origin for glutamate metabolites that redistribute into skeletal muscle and gut. The findings of this study suggest now that pharmacological manipulations that reduce the liver glutamate release rate or cause a boosting of the skeletal muscle glutamate pumping rate are likely to cause brain neuroprotection.     

Lipid synthesis in the laticifers of Euphorbia lathyris L. seedlings

Various solutions of labeled precursors were absorbed by the cotyledons of etiolated Euphorbia lathyris L. seedlings. Incorporation of ¹⁴C into triterpenes from [2-¹⁴C]mevalonic acid, [1-¹⁴C]acetate, [3-¹⁴C]pyruvate, [U-¹⁴C]glyoxylate, [U-¹⁴C]glycerol, [U-¹⁴C]serine, [U-¹⁴C]xylose, [U-¹⁴C]glucose, and [U-¹⁴C]sucrose was obtained. The [¹⁴] triterpenes synthesized from [¹⁴C] sugars were mainly of latex origin. [¹⁴C]mevalonic acid was only involved in terpenoid synthesis outside the laticifers. Exogenously supplied glyoxylate, serine, and glycerol were hardly involved in lipid synthesis at all. The ¹⁴C-distribution over the various triterpenols was consistent with the mass distribution of these constituents in gas liquid chromatography when [¹⁴C]sugars, [¹⁴C]acetate, and [¹⁴C]pyruvate were used. These precursors were supplied to the seedlings in the presence of increasing amounts of unlabeled substrates. The amount of substrate directly involved in lipid synthesis as well as the absolute triterpenol yield was calculated from the obtained [¹⁴C]triterpenols. The highest yield was obtained in the sucrose incorporated seedlings, being 25% of the daily increase of latex triterpenes in growing seedlings.     

Ontogenetic Study of the Effects of Energetic Nutrients on Amino Acid Metabolism of Rat Cerebral Cortex

We studied the effect of various energetic nutrients on metabolism of l-[U-¹⁴C]leucine and [1–¹⁴C]glycine in cerebral cortex of rats at different ages. At gestational age, glucose and lactate stimulated protein synthesis from l-[U-¹⁴C]leucine and [1–¹⁴C]glycine and from l-[U-¹⁴C]leucine, respectively; glucose, -OH-butyrate and lactate stimulated lipid synthesis from l-[U-¹⁴C]leucine. At 10 days of age, glucose, mannose, and fructose stimulated protein synthesis, and glucose and mannose stimulated oxidation to CO₂ as well as lipid synthesis from l-[U-¹⁴C]leucine. In adult rats, glucose, mannose, and fructose stimulated protein synthesis from l-[U-¹⁴C]leucine and [1–¹⁴C]glycine; glutamine also markedly decreased the oxidation of l-[U-¹⁴C]leucine and [1–¹⁴C]glycine in 10–day-old and adult rats.     

Studies in lipogenesis in vivo: Fatty acid and cholesterol synthesis during starvation and re-feeding

1. Lipogenesis in vivo has been studied in mice given a 250mg. meal of [U-¹⁴C]glucose (2·5μc) or given an intraperitoneal injection of 25μg. of [U-¹⁴C]glucose (2·0μc). 2. The ability to convert a [U-¹⁴C]glucose meal into fatty acid was not significantly depressed by 6–7hr. of starvation. In contrast, incorporation of ¹⁴C into fatty acid in the liver after the intraperitoneal dose of [¹⁴C]glucose was depressed by 80% and by more than 90% by 1 and 2hr. of starvation respectively. Carcass fatty acid synthesis from the [U-¹⁴C]glucose meal was not depressed by 12hr. of starvation, whereas from the tracer dose of [U-¹⁴C]glucose the depression in incorporation was 80% after 6hr. of starvation. 3. Re-feeding for 3 days, after 3 days' starvation, raised fatty acid synthesis and cholesterol synthesis in the liver fivefold and tenfold respectively above the levels in non-starved control mice. These increases were associated with an increased amount of both fatty acid and cholesterol in the liver. 4. After 18hr. of starvation incorporation of a [U-¹⁴C]glucose meal into carcass and liver glycogen were both increased threefold.     

Enzymatic synthesis of beta-[U-14C]alanine and D-[1,2,3-14C]pantothenate of high specific radioactivity

β-[U-¹⁴C]Alanine can be synthesized in >95% yield from l-[U-¹⁴C]aspartic acid using the aspartate 1-decarboxylase of Escherichia coli and converted to d-[1,2,3-¹⁴C]pantothenate in a 10–20% yield using the pantothenate synthetase of E. coli. Sufficiently pure preparations of both enzymes are readily obtained.     

LABELLING OF BRAIN PROTEINS AT EARLY PERIODS AFTER SUBCUTANEOUS INJECTION OF A MIXTURE OF [U-14C]GLUCOSE AND [3H]GLUTAMATE

To obtain evidence of the site of conversion of [U-¹⁴C]glucose into glutamate and related amino acids of the brain, a mixture of [U-¹⁴C]glucose and [³H]glutamate was injected subcutaneously into rats. [³H]Glutamate gave rise to several ³H-labelled amino acids in rat liver and blood; only ³H-labelled glutamate, glutamine or γ-aminobutyrate were found in the brain. The specific radioactivity of [³H]glutamine in the brain was higher than that of [³H]glutamate indicating the entry of [³H]glutamate mainly in the ‘small glutamate compartment’. The ¹⁴C-labelling pattern of amino acids in the brain and liver after injection of [U-¹⁴C]glucose was similar to that previously reported (Gaitonde et al., 1965). The specific radioactivity of [¹⁴C]glutamine in the blood and liver after injection of both precursors was greater than that of glutamate between 10 and 60 min after the injection of the precursors. The extent of labelling of alanine and aspartate was greater than that of other amino acids in the blood after injection of [U-¹⁴C]glucose. There was no labelling of brain protein with [³H]glutamate during the 10 min period, but significant label was found at 30 and 60 min. The highest relative incorporation of [¹⁴C]glutamate and [¹⁴C]aspartate in rat brain protein was observed at 5 min after the injection of [U-¹⁴C]glucose. The results have been discussed in the context of transport of glutamine synthesized in the brain and the site of metabolism of [U-¹⁴C]glucose in the brain.     

DEVELOPMENT OF LIPOGENESIS IN RAT BRAIN CORTEX: THE DIFFERENTIAL INCORPORATION OF GLUCOSE AND ACETATE INTO BRAIN LIPIDS IN VITRO

—The oxidation to CO₂ and the incorporation of [U-¹⁴C]glucose and [U-¹⁴C]acetate into lipids by cortex slices from rat brain during the postnatal period were investigated. The oxidation of [U-¹⁴C]glucose was low in 2-day-old rat brain, and increased by about two-fold during the 2nd and 3rd postnatal weeks. The oxidation of [U-¹⁴C]acetate was increased markedly in the second postnatal week, but decreased to rates observed in 2-day-old rat brain at the time of weaning. Both labeled substrates were readily incorporated into non-saponifiable lipids and fatty acids by brain slices from 2-day-old rat. Their rates of incorporation and the days on which maximum rates occurred were different, however, maximum incorporation of [U-¹⁴C]glucose and [U-¹⁴]acetate into lipid fractions being observed on about the 7th and 12th postanatal days, respectively. The metabolic compartmentation in the utilization of these substrates for lipogenesis is suggested. The activities of glucose-6-phosphate dehydrogenase, cytosolic NADP-malate dehydrogenase, cytosolic NADP-isocitrate dehydrogenase, ATP-citrate lyase and acetyl CoA carboxylase were measured in rat brain during the postnatal period. All enzymes followed somewhat different courses of development; the activity of acetyl CoA carboxylase was, however, the lowest among other key enzymes in the biosynthetic pathway, and its developmental pattern paralleled closely the fatty acid synthesis from [U-¹⁴C]glucose. It is suggested that acetyl CoA carboxylase is a rate-limiting step in the synthesis de novo of fatty acids in developing rat brain.     

The metabolism of l[3-3H]lactate by isolated hamster liver cells

The rate of tritium removal from l[3-³H]lactate by hamster liver cells is faster than the analytical rate of lactate utilization, or the rate of ¹⁴C disappearance from l[U-¹⁴C]lactate, with the result that the ³H/¹⁴C ratio in residual lactate from l-[U-¹⁴C,3-³H]lactate decreases. However, addition of low concentrations (0.1 to 1.0 mM) of l-cycloserine, a glutamate pyruvate transaminase inhibitor, nearly equalizes the rates of isotope utilization from l-[3-³H]lactate and l-[U-¹⁴C]lactate. The results suggest a very limited rate of recycling of phosphoenolpyruvate back to pyruvate during gluconeogenesis from lactate in fasted hamster liver cells.     

Studies on lipogenesis in vivo: Comparison of cholesterol and fatty acid synthesis in rats and mice

1. The importance of fatty acid synthesis as a pathway for the disposal of ingested glucose has been evaluated in rats and mice given a purified diet high in glucose and low in fat. [U-¹⁴C]Glucose was either added to the diet and fed for 24hr. or given by stomach tube as a 250mg. (mice) or 1000mg. (rats) meal. The two methods of isotope administration gave similar results. 2. Under the conditions employed fatty acid synthesis appeared to be a more important pathway for glucose disposal in mice than in rats. In mice 15·3% of ingested [U-¹⁴C]glucose was converted into fatty acid and in rats the corresponding value was 8·6%. In contrast, the conversion of [U-¹⁴C]glucose into cholesterol, as a percentage of dose, was twice as high in rats as in mice. 3. The effect of 20% of corn oil in the diet on the conversion of dietary [U-¹⁴C]glucose into fat was also investigated. Mice given diets containing 1% or 20% of corn oil converted 14·6% or 7·0% respectively of dietary [U-¹⁴C]glucose into fatty acid over a 24hr. period. There was no effect of fat on the incorporation of the isotope into cholesterol. 4. In mice given diets containing 1% or 20% of corn oil approx. 10% and 2% respectively of newly synthesized fatty acids were found in the liver. Hepatic fatty acid synthesis appears to be more sensitive to dietary fat than is extrahepatic synthesis.     

The inhibition of oxalate biosynthesis in isolated perfused rat liver by DL-phenyllactate and n-heptanoate

Glycolate oxidase was isolated and partially purified from human and rat liver. The enzyme preparation readily catalyzed the oxidation of glycolate, glyoxylate, lactate, hydroxyisocaproate and α-hydroxybutyrate. The oxidation of glycolate and glyoxylate by glycolate oxidase was completely inhibited by 0.02 m dl-phenyllactate or n-heptanoate. The oxidation of glyoxylate by lactic dehydrogenase or xanthine oxidase was not inhibited by 0.067 m dl-phenyllactate or n-heptanoate. The conversion of [U-¹⁴C] glyoxylate to [¹⁴C] oxalate by isolated perfused rat liver was completely inhibited by dl-phenyllactate and n-heptanoate confirming the major contribution of glycolate oxidase in oxalate synthesis. Since the inhibition of oxalate was 100%, lactic dehydrogenase and xanthine oxidase do not contribute to oxalate biosynthesis in isolated perfused rat liver. dl-Phenyllactate also inhibited [¹⁴C] oxalate synthesis from [1-¹⁴C] glycolate, [U-¹⁴C] ethylene glycol, [U-¹⁴C] glycine, [3-¹⁴C] serine, and [U-¹⁴C] ethanolamine in isolated perfused rat liver. Oxalate synthesis from ethylene glycol was inhibited by dl-phenyllactate in the intact male rat confirming the role of glycolate oxidase in oxalate synthesis in vivo and indicating the feasibility of regulating oxalate metabolism in primary hyperoxaluria, ethylene glycol poisoning, and kidney stone formation by enzyme inhibitors.     

Purification and characterisation of a novel starch synthase selective for uridine 5′-diphosphate glucose from the red alga Gracilaria tenuistipitata

Red algae (Rhodophyceae) are photosynthetic eukaryotes that accumulate starch granules in the cytosol. Starch synthase activity in crude extracts of Gracilaria tenuistipitata Chang et Xia was almost 9-fold higher with UDP[U-¹⁴C]glucose than with ADP[U-¹⁴C]glucose. The activity with UDP[U-¹⁴C]glucose was sensitive to proteolytic and oxidative inhibition during extraction whilst the activity with ADP[U-¹⁴C]glucose appeared unaffected. This indicates the presence of separate starch synthases with different substrate specificities in G. tenuistipitata. The UDPglucose: starch synthase was purified and characterised. The enzyme appears to be a homotetramer with a native M_r of 580 kDa and displays kinetic properties similar to other α-glucan synthases such as stimulation by citrate, product (UDP) inhibition and broad primer specificity. We propose that this enzyme is involved in cytosolic starch synthesis in red algae and thus is the first starch synthase described that utilises UDPglucose in vivo. The biochemical implications of the different compartmentalisation of starch synthesis in red algae and green algae/plants are also discussed. Received: 29 January 1999 / Accepted: 11 March 1999     

An Hg-sensitive channel mediates the diffusional component of glucose transport in olive cells

In several organisms solute transport is mediated by the simultaneous operation of saturable and non-saturable (diffusion-like) uptake, but often the nature of the diffusive component remains elusive. The present work investigates the nature of the diffusive glucose transport in Olea europaea cell cultures. In this system, glucose uptake is mediated by a glucose-repressible, H⁺-dependent active saturable transport system that is superimposed on a diffusional component. The latter represents the major mode of uptake when high external glucose concentrations are provided. In glucose-sufficient cells, initial velocities of d- and l-[U-¹⁴C]glucose uptake were equal and obeyed linear concentration dependence up to 100 mM sugar. In sugar starved cells, where glucose transport is mediated by the saturable system, countertransport of the sugar pairs 3-O-methyl-d-glucose/d-[U-¹⁴C]glucose and 3-O-methyl-d-glucose/3-O-methyl-d-[U-¹⁴C]glucose was demonstrated. This countertransport was completely absent in glucose-sufficient cells, indicating that linear glucose uptake is not mediated by a typical sugar permease. The endocytic inhibitors wortmannin-A and NH₄Cl inhibited neither the linear component of d- and l-glucose uptake nor the absorption of the nonmetabolizable glucose analog 3-O-methyl-d-[U-¹⁴C]glucose, thus excluding the involvement of endocytic mediated glucose uptake. Furthermore, the formation of endocytic vesicles assessed with the marker FM1-43 proceeded at a very slow rate. Activation energies for glucose transport in glucose sufficient cells and plasma membrane vesicles were 7 and 4 kcal mol^− 1, respectively, lower than the value estimated for diffusion of glucose through the lipid bilayer of phosphatidylethanolamine liposomes (12 kcal mol^− 1). Mercury chloride inhibited both the linear component of sugar uptake in sugar sufficient cells and plasma membrane vesicles, and the incorporation of the fluorescent glucose analog 2-NBDG, suggesting protein-mediated transport. Diffusive uptake of glucose was inhibited by a drop in cytosolic pH and stimulated by the protein kinase inhibitor staurosporine. The data demonstrate that the low-affinity, high-capacity, diffusional component of glucose uptake occurs through a channel-like structure whose transport capacity may be regulated by intracellular protonation and phosphorylation/dephosphorylation.     

Stimulatory effect of gamma-aminobutyric acid upon animo acid incorporation into protein by a ribosomal system from immature rat brain

Abstract—

• 1 GABAstimulated the incorporation of L-[U-¹⁴C]leucine, primarily into the particulate protein of a ribosomal system from immature rat brain, but not from immature rat liver.
• 2 The GABA effect required the presence of Na⁺ and occurred at GABA concentrations which are thought to be physiological (1–5 mM).
• 3 Of all other amino acids tested at tissue extract concentrations in the system, only glycine had a similar effect. No analogues of GABA tested had a significant stimulatory effect upon leucine incorporation into protein, with the exception of homocarnosine which was mildly stimulatory.
• 4 The effect of GABA upon the incorporation of L-[U-¹⁴C]leucine was examined in the presence of added amino acid substrates, both individually and as mixtures. Also, the incorporation of L-[U-¹⁴C]leucine was compared with incorporation of L-[U-¹⁴C]Iysine and L-[U-¹⁴C]phenylalanine. The results are discussed in terms of GABA interaction with activating, transfer and transport mechanisms of other amino acids, inhibition of proteinase activity, and the possibility that GABA is stimulating the synthesis or turnover of specific proteins in the brain ribosomal system.
• 5 The results illustrate the fact that studies of ‘protein synthesis’ in immature rat brain ribosomes, as measured by amino acid incorporation, will yield answers which depend heavily upon substrate conditions and upon the labelled amino acid used as the marker for protein synthesis or turnover.