Biosynthesis of L-[15N]aspartic acid and L-[15N]alanine by immobilized bacteria

Glycoproteins in nitrocellulose transfers of electrophoretically separated mixtures of cellular and viral proteins are rapidly and sensitively located by sequential incubation with the lectin concanavalin A and the enzymatically active glycoprotein horseradish peroxidase. The bound enzyme is located by incubation with a substrate which is converted to a highly insoluble colored product. The specificity of the method is demonstrated by the abolition of concanavalin A binding in the presence of α-methyl mannoside. The method is capable of detecting as little as 60 ng of a purified model glycoprotein after electrophoresis. It has been applied to the analysis of the glycoproteins of purified Lassa virus and of the virus-specific glycoproteins in Japanese encephalitis virus-infected cells.     

Respiration of 14CO2 by intact animals of various species given l-[1-14C]fucose or d-[1-14C]arabinose

The production of ¹⁴CO₂ from l-[1-¹⁴C]fucose and d-[1-¹⁴C]arabinose has been studied in five mammalian species.Cats, guinea pigs, mice, and rabbits respired about 22% of the label of l[1-¹⁴C]fucose or of d-[1-¹⁴C]arabinose within 6 h after intraperitoneal injection of the sugar. Rats respired only 1.5% of the l-fucose label and 5% of the d-arabinose label in the same time period.Liver homogenates from cat, guinea pig, and rabbit produced significantly more ¹⁴CO₂ from l-[1-¹⁴C]fucose or d-[1-¹⁴C]arabinose than mouse or rat liver homogenates. Unlike those of the other species, guinea pig liver homogenates had very low l-fucose dehydrogenase activity.The results suggest that substantial catabolism of l-fucose and d-arabinose occurs in the tissues of some animal species. Investigators wishing to employ l-fucose as a tracer of glycoprotein metabolism must, therefore, ensure that the species that they employ does not metabolize l-fucose to products interfering with their studies.     

The exchange reaction of peptides R-D-alanyl-D-alanine with D-[14C]alanine to R-D-alanyl-D-[14C]alanine and D-alanine, catalysed by the membranes of Streptococcus faecalis ATCC 9790.

Under alkaline conditions, the membrane-bound DD-carboxypeptidase of Streptococcus faecalis ATCC 9790 catalyses exchange reactions in which the X-L-R3-D-Ala moiety of peptides of the type X-L-R3-D-Ala-D-Ala is transferred to simple amino compounds such as D-alanine, glycine and glycyl-glycine. The enzyme system is unable, however, to catalyse complex reactions that would simulate the natural transpeptidation reaction.     

Transport of L-[14C]cystine and L-[14C]cysteine by subtypes of high affinity glutamate transporters over-expressed in HEK cells

Transport of L-cystine across the cell membrane is essential for synthesis of the major cellular antioxidant, glutathione (gamma-glutamylcysteinylglycine). In this study, uptake of L-[14C]cystine by three of the high affinity sodium-dependent mammalian glutamate transporters (GLT1, GLAST and EAAC1) individually expressed in HEK cells has been determined. All three transporters display saturable uptake of L-[14C]cystine with Michaelis affinity (K(m)) constants in the range of 20-110 microM. L-glutamate and L-homocysteate are potent inhibitors of sodium-dependent L-[14C]cystine uptake in HEK(GLAST), HEK(GLT1) and HEK(EAAC1) cells. Reduction of L-[14C]cystine to L-[14C]cysteine in the presence of 1mM cysteinylglycine increases the uptake rate in HEK(GLT1), HEK(GLAST) and HEK(EAAC1) cells, but only a small proportion (<10%) of L-[14C]cysteine uptake in HEK(GLT1) and HEK(GLAST) cells occurs by the high affinity glutamate transporters. The majority (>90%) of L-[14C]cysteine transport in these cells is mediated by the ASC transport system. In HEK(EAAC1) cells, on the other hand, L-[14C]cysteine is transported equally by the ASC and EAAC1 transporters. L-homocysteine inhibits L-[14C]cysteine transport in both HEK(GLAST) and HEK(GLT1) cells, but not in HEK(EAAC1) cells. It is concluded that the quantity of L-[14C]cyst(e)ine taken up by individual high affinity sodium-dependent glutamate transporters is determined both by the extracellular concentration of amino acids, such as glutamate and homocysteine, and by the extracellular redox potential, which will control the oxidation state of L-cystine.     

Convenient preparative synthesis of [14C]trehalose from [14C]glucose by intact Escherichia coli cells.

At high osmolarity, Escherichia coli synthesizes trehalose intracellularly, irrespective of the nature of the carbon source. Synthesis proceeds via the transfer of UDP-glucose to glucose 6-phosphate, yielding trehalose 6-phosphate, followed by its dephosphorylation to trehalose (H.M. Giaeyer, B.O. Styrvold, I. Kaasen, and A.R. Strøm, J. Bacteriol. 170:2841-2849, 1988). This reaction was exploited to preparatively synthesize [14C]trehalose from exogenous [14C]glucose by using intact bacteria of a mutant (DF214) that could not metabolize glucose. The total yield of radiochemically pure trehalose from glucose was routinely more than 50%.     

In vitro [14C]-labelled 6-benzyladenine uptake and14CO2 evolution in two Japanese plum cultivars

Japanese plum cultivars Obilnaga and Santa Rosa showed different proliferation responses when grown in similar conditions in vitro. This led us to investigate BA uptake by shoots of both cultivars grown for different times (15 hrs, 1, 3, 6, 9, 13, 16 and 20 days) on an incubation medium enriched with 10Ci (370 kBq) 8-[¹⁴C]BA/250 ml. The decrease of radioactivity in the medium and its increase in the EtOH-soluble and-insoluble fractions of shoots of both cultivars grown for different times (15 h, 1, 3, 6, 9, 13, 16 and 20 days) on cultivars. Increasing amounts of¹⁴CO₂ were detected in the culture atmosphere when shoots were grown in gas-tight vials. The type of container closure strongly affected proliferation. From the results reported here it is not possible to state whether different optimal subculture intervals and different proliferation responses of Santa Rosa and Obilnaja cultures are due to different tissue sensitivity to the cytokinin and/or to different metabolic activities. Nevertheless the highest proliferation rates of both plums are clearly related to a drop of EtOH-soluble radioactivity of shoots by the end of subculture.This paper is No. 620 of the Istituto di Coltivazioni Arboree. Part of the results were presented as a poster at the 15th International Symposium on Growth Regulators in Fruit Production, Rimini (Italy) 2–6 September 1985. The research was partially supported by funds of the Ministry of Education, Roma (M.P.I. 60%).     

Relationship between synaptosomal uptake and release of [14C]gaba, [14C]diaminobutyric acid and [14C]beta-alanine.

[¹⁴C]GABA is taken up by rat brain synaptosomes via a high affinity, Na⁺-dependent process. Subsequent addition of depolarizing levels of potassium (56.2 MM) or veratridine (100 μM) stimulates the release of synaptosomal [¹⁴C]GABA by a process which is sensitive to the external concentration of divalent cations such as Ca²⁺, Mg²⁺, and Mn²⁺. However, the relatively smaller amount of [¹⁴C]GABA taken up by synaptosomes in the absence of Na⁺ is not released from synaptosomes by Ca²⁺ -dependent, K ⁺-stimulation. [¹⁴C]DABA, a competitive inhibitor of synaptosomal uptake of GABA (Iversen & Johnson , 1971) is also taken up by synaptosomal fractions via a Na ⁺ -dependent process; and is subsequently released by Ca²⁺ -dependent, K⁺-stimulation. On the other hand, [¹⁴C]β-alanine, a purported blocker of glial uptake systems for GABA (Schon & Kelly , 1974) is a poor competitor of GABA uptake into synaptosomes. Comparatively small amounts of [¹⁴C] β-alanine are taken up by synaptosomes and no significant amount is released by Ca²⁺ -dependent, K⁺-stimulation. These data suggest that entry of [¹⁴C]GABA into a releasable pool requires external Na⁺ ions and maximal evoked release of [¹⁴C]GABA from the synaptosomal pool requires external Ca²⁺ ions. The GABA analogue, DABA, is apparently successful in entering the same or similar synaptosomal pool. The GABA analogue, β-alanine, is not. None of the compounds or conditions studied were found to simultaneously affect both uptake and release processes. Compounds which stimulated release (veratridine) or inhibited release (magnesium) were found to have minimal effect on synaptosomal uptake. Likewise compounds (DABA) or conditions (Na⁺-free medium) which inhibited uptake, had little effect on release.     

Convenient preparative synthesis of [14C]trehalose from [14C]glucose by intact Escherichia coli cells

At high osmolarity, Escherichia coli synthesizes trehalose intracellularly, irrespective of the nature of the carbon source. Synthesis proceeds via the transfer of UDP-glucose to glucose 6-phosphate, yielding trehalose 6-phosphate, followed by its dephosphorylation to trehalose (H.M. Giaeyer, B.O. Styrvold, I. Kaasen, and A.R. Str?m, J. Bacteriol. 170:2841-2849, 1988). This reaction was exploited to preparatively synthesize [14C]trehalose from exogenous [14C]glucose by using intact bacteria of a mutant (DF214) that could not metabolize glucose. The total yield of radiochemically pure trehalose from glucose was routinely more than 50%.     

Detritiation of L-[3-3H]alanine in the glutamate-pyruvate transaminase reaction.

The detritiation of L-[3-3H]alanine in the reaction catalyzed by pig heart glutamate-pyruvate transaminase was monitored in the absence or presence of lactate dehydrogenase. The results indicated that each monodirectional conversion of L-[3-3H]alanine to [3-3H]pyruvate resulted in the generation of 3HOH at a rate representing one-third of the total 3H flux. No isotopic discrimination in reaction velocity between tritiated and 14C-labelled L-alanine was observed. The mathematical modelling of the reaction revealed that, as a consequence of the detritiation process, the steady-state ratio in L-[3-3H]alanine/[3-3H]pyruvate does not inform on either the absolute or relative size of the amino acid and 2-keto acid pools.     

Uptake of L-[propyl-2,3-3H]dihydroalprenolol by intact HeLa cells

This report describes the uptake of L-[propyl-2,3-3H]dihydroalprenolol, a beta-adrenergic antagonist, by HeLa (human adenocarcinoma) cells. [3H]Dihydroalprenolol binds to sites of high capacity and low affinity in intact HeLa cells. The binding achieves equilibrium rapidly and is rapidly reversible. Bound [3H]dihydroalprenolol is displaceable by beta-adrenergic antagonists in a nonstereoselective fashion, but is not displaceable by isoproterenol, an adrenergic agonist. Phentolamine, an alpha-adrenergic antagonist, and chloroquine, a lysosomotropic amine, also compete for [3H]dihydroalprenolol binding sites. [3H]Dihydroalprenolol binding is inhibited by metabolic inhibitors, but not by cytoskeletal blocking agents. The binding is sensitive to extracellular pH (less binding at lower pH) and is temperature-sensitive (less binding at lower temperatures). The bound radioligand is rapidly reversed following hypotonic lysis of the cells. These [3H]dihydroalprenolol binding sites in intact HeLa cells therefore do not have the characteristics expected for beta-adrenergic receptors. Further studies showed that beta-adrenergic receptors could be detected in a HeLa membrane preparation using [125I]iodohydroxybenzylpindolol, and that chloroquine had very low affinity for these receptors. We conclude that [3H]dihydroalprenolol diffuses across the plasma membrane of intact HeLa cells and accumulates in acidic intracellular compartments.