Enzymatic production of the lignin precursor trans-[U-14C]cinnamic acid from l-[U-14C]phenylalanine using l-phenylalanine ammonia-lyase

An enzymatic method using phenylalanine ammonia-lyase (l-phenylalanine ammonia-lyase, EC 4.3.1.5) for the rapid conversion of l-[U-¹⁴C]phenylalanine to the deaminated lignin precursor trans-[U-¹⁴C]cinnamic acid is described. The method produces an experimentally useful ¹⁴C-labelled deaminated lignin precursor unavailable from radiochemical supply companies.     

Metabolism of L-(U-14C)valine, L-(U-14C)leucine, L-(U-14C)histidine and L-(U-14C) phenylalanine by the isolated perfused lactating guinea-pig mammary gland.

1. The incorporation of L-[U-14C]leucine, L[U-14C]histidine and L-[U-14C]phenylalanine into casein secreted during perfusion of isolated guinea-pig mammary glands was demonstrated. 2. The extent of incorporation of label into casein residues was consistent with their being derived from free amino acids of the perfusate plasma. 3. The mean transit time of the amino acids from perfusate into secreted casein was approx. 100 min. 4. Whereas radioactive histidine and phenylalanine were incorporated solely into milk protein, radioactivity from [U-14C]valine was also transferred to CO2 and to an unidentified plasma component, and from [U-14C]leucine to plasma glutamic acid. 5. Evidence from experiments with [U-14C]phenylalanine suggests that, as in rats, but in contrast with ruminant species, guinea-pig mammary tissue does not possess phenyl alanine hydroxylase activity. 6. The results are discussed in relation to the possible role of essential amino acid catabolism in the control of milk-protein synthesis.     

Rapid method for the assay of 4-aminobutyric acid (GABA), glutamic acid and aspartic acid in brain tissue and subcellular fractions

The thin-layer electrophoretic separation at pH 4.8 of brain extracts and a procedure for fluorescent staining of the plates with fluorescamine are described for the rapid routine determination of 4-aminobutyric acid (GABA), glutamic acid and aspartic acid in brain extracts and in particulate fractions of brain tissue. Automated sample application, electrophoretic separation using two chambers, and quantitation by in situ fluorescence scanning allows the assay of 280 samples within three working days. The method is reproducible (S.D. <8% of the mean) within the range of 0.2–2 nmole per spot. The staining procedure can be applied to a variety of related analytical problems. The method has proved useful for the determination of the specific radioactivities of GABA, glutamic acid and aspartic acid in metabolic studies.     

Utilization of U-14C amino acids or U-14C protein by adult Glossina morsitans during in utero development of larva

Administration of U¹⁴C protein hydrolysate in the diet of adult female Glossina morsitans at different times throughout the second reproductive cycle was followed by analysis of the distribution of radioactivity between the adult flies, their excreta, and the fully grown third instar larvae produced by these flies. A constant proportion of the total administered label was recoverable independently of the time lapse between administration and assay. Peak incorporation of labelled material occurred in the larva between the seventh and eighth day of a 9 or 10 day interlarval period, indicating that the larva feeds avidly on recently synthesized maternal uterine gland secretion at this time. Haemocoelic injection of U¹⁴C protein hydrolysate into similar adult females, between feeds, resulted in continued incorporation of labelled material by the larva to within 12 hr of parturition. Results are consistent with the hypothesis that uterine gland secretion and larval feeding continue throughout the intrauterine life of the larva.A constant and low proportion of detectable label remained in the adult fly while increased incorporation by the larva was paralleled by a reduction of detectable label in the adult excreta. This indicates direct competition between the uterine gland cells and those of the Malpighian tubules for free amino acids in the haemolymph.Administration of U¹⁴C protein in the adult diet did not result in incorporation of label by the developing larva, and the bulk was excreted as protein by the adult fly. Apparently the midgut trypsin of G. morsitans is incapable of splitting this labelled protein.Analysis of urine and haemolymph samples from flies in early pregnancy, recently fed on a diet containing U¹⁴C protein hydrolysate or U¹⁴C protein, shows that free labelled amino acids in the diet enter the adult haemolymph almost immediately after feeding, and are excreted along with dietary water during initial diuresis. The labelled protein used in these experiments was not taken up by the haemolymph and consequently did not appear in the urine.Implications are that the adult female G. morsitans possesses little storage capacity for substances in the diet which are destined to provide nutrients for the developing larva. Assuming a 48 hr digestion time, the digestive products of a blood meal ingested on day 5 or 6 of a 9 day interlarval period will provide the bulk of nutrients for larval growth. It is therefore significant that blood meals ingested at this time are larger than those ingested earlier or later in the cycle.     

[14C]acetylcholine synthesis and [14C]carbon dioxide production from [U-14C]glucose by tissue prisms from human neocortex.

1. [14C]Acetylcholine synthesis and 14CO2 production from [U-14C]glucose has been measured in tissue prism preparations from human neocortex. 2. Electron micrographs of prisms from human and rat neocortex show that both contain intact synaptic endings with evenly-distributed vesicles and normal-appearing mitochondria, but only poorly preserved cell body structure. 3. Synthesis of [14C]acetylcholine in prisms from rat neocortex is similar to estimates for turnover in vivo. Synthesis in prisms from human neocortex is 18% of that in rat tissue and 64% of that in tissue from baboon neocortex for incubations performed in 31 mM-K+. 4. Investigations of prisms prepared from rat brains stored at 37 degrees C after death revealed that synthesis of [14C]acetylcholine in the presence of 31 mM-K+ was greatly decreased within 30 min of post-mortem incubation, whereas synthesis at 5 mM-K+ and production of 14CO2 at both K+ concentrations were only significantly affected after longer periods. Changes were similar in neocortex and striatum. Thus human autopsy material is unlikely to be suitable for use with this system. 5. Investigations using animal models suggest that [14C]acetylcholine synthesis and 14CO2 production are not affected by surgical or anaesthetic procedures. 6. Neither [14C]acetylcholine synthesis nor 14CO2 production in human prisms was significantly changed with age between 15 and 68 years. 7. Samples from patients with the dementing condition Alzheimer's disease showed a significant decrease in [14C]acetylcholine synthesis to 47% of normal samples and a significant increase of 39% in production of 14CO2.     

Intermediates of peroxisomal beta-oxidation of [U-14C]hexadecanedionoate. A study of the acyl-CoA esters which accumulate during peroxisomal beta-oxidation of [U-14C]hexadecanedionate and [U-14C]hexadecanedionoyl-mono-CoA.

1. The oxidation of [U-14C]hexadecanedionoyl-mono-CoA was stimulated by CoA, by carnitine in the absence of CoA and by the presence of an NAD(+)-regenerating system. 2. Substrate inhibition was observed with respect to [U-14C]hexadecanedionoyl-mono-CoA at concentrations greater than 35 microM. 3. Acetyl-CoA and the dicarboxyl-CoA esters of chain length C6-16 were detected by HPLC under standard incubation conditions. 4. In the absence of the NAD(+)-regenerating system, 2-enoyl-CoA and 3-hydroxacyl-CoA esters were detected. 5. In general, the peroxisomal beta-oxidation of dicarboxylates is very similar to that of monocarboxylates [Bartlett, K., Hovik, R., Eaton, S., Watmough, N. J. & Osmundsen, H. (1990) Biochem. J. 270, 175-180] except that chain shortening does not proceed beyond C6. 6. We conclude that the peroxisomal beta-oxidation of dicarboxylates is regulated by the redox state of the peroxisomal matrix and CoA availability.     

Enhanced mineralization of [U-(14)C]2,4-dichlorophenoxyacetic acid in soil from the rhizosphere of Trifolium pratense

Enhanced biodegradation in the rhizosphere has been reported for many organic xenobiotic compounds, although the mechanisms are not fully understood. The purpose of this study was to discover whether rhizosphere-enhanced biodegradation is due to selective enrichment of degraders through growth on compounds produced by rhizodeposition. We monitored the mineralization of [U-(14)C]2,4-dichlorophenoxyacetic acid (2,4-D) in rhizosphere soil with no history of herbicide application collected over a period of 0 to 116 days after sowing of Lolium perenne and Trifolium pratense. The relationships between the mineralization kinetics, the number of 2,4-D degraders, and the diversity of genes encoding 2,4-D/alpha-ketoglutarate dioxygenase (tfdA) were investigated. The rhizosphere effect on [(14)C]2,4-D mineralization (50 microg g(-1)) was shown to be plant species and plant age specific. In comparison with nonplanted soil, there were significant (P < 0.05) reductions in the lag phase and enhancements of the maximum mineralization rate for 25- and 60-day T. pratense soil but not for 116-day T. pratense rhizosphere soil or for L. perenne rhizosphere soil of any age. Numbers of 2,4-D degraders in planted and nonplanted soil were low (most probable number, <100 g(-1)) and were not related to plant species or age. Single-strand conformational polymorphism analysis showed that plant species had no impact on the diversity of alpha-Proteobacteria tfdA-like genes, although an impact of 2,4-D application was recorded. Our results indicate that enhanced mineralization in T. pratense rhizosphere soil is not due to enrichment of 2,4-D-degrading microorganisms by rhizodeposits. We suggest an alternative mechanism in which one or more components of the rhizodeposits induce the 2,4-D pathway.     

Oxidation of D-[U-(14)C] glucose and [1,12-(14)C] dodecanedioic acid by pancreatic islets from Goto-Kakizaki rats.

In pancreatic islets from hereditarily diabetic GK rats, [1,12 -(14)C] dodecanedioic acid (5.0 mM) was oxidized at a rate representing about 5 % of that of D-[U - (14)C] glucose (8.3 mM). Dioic acid and hexose failed to exert any significant reciprocal effects on their respective oxidation. The production of (14)CO(2) from [1,12 -(14)C] dodecanedioic acid was proportional to its concentration in the 0.2 - 5.0 mM range. These results were essentially comparable to those obtained in islets from control rats. They extend, therefore, to GK rats the knowledge that dodecanedioic acid acts as a nutrient in pancreatic islet cells.     

Enzymatic synthesis of [U-14C] ribose-labeled inosine.

A very potent anticholinesterase compound, 7-(diethoxyphosphinyloxy)-N-methylquinolinium fluorosulfate, has been used to determine the normality of acetylcholinesterase solutions. The inhibitor reacts rapidly and completely with acetylcholinesterase. The bimolecular rate constant is 2.5 × 10⁸m^?1 min^?1 and the equilibrium constant is about 10⁶. The reaction produces an inactive diethylphosphoryl enzyme in which the active serine is phosphorylated. The reaction produces the highly fluorescent 1-methyl-7-hydroxyquinolinium dipolar ion as a leaving group. The inhibited enzyme is quite stable and hydrolyzes to produce active enzyme only at the rate of 0.04%/min. The inhibitor was used in two ways for measuring the normality of acetylcholinesterase solutions: (1) The very fast reaction of the inhibitor with cholinesterase makes it convenient to determine the normality of enzyme solutions by measuring the decrease in enzyme activity caused by the addition of an accurately known quantity of the inhibitor. (2) The highly fluorescent nature of the leaving group makes it possible to measure the low concentration that is produced by the reaction of excess inhibitor with the enzyme. The two methods yielded activities per site of 6.9 × 10⁵ min^?1 and 7.3 × 10⁵ min^?1 using enzyme normalities of 1–2 × 10^?8m and 1–5 × 10^?m, respectively, using a commercial 11 S enzyme preparation from electric eel and acetylthiocholine as the enzyme substrate.     

Hormonal control of [14C]glucose synthesis from [U-14C]dihydroxyacetone and glycerol in isolated rat hepatocytes.

The hormonal control of [14C]glucose synthesis from [U-14C-A1dihydroxyacetone was studied in hepatocytes from fed and starved rats. In cells from fed rats, glucagon lowered the concentration of substrate giving half-half-maximal rates of incorporation while it had little or no effect on the maximal rate. Inhibitors of gluconeogenesis from pyruvate had no effect on the ability of the hormone to stimulate the synthesis of [14C]glucose from dihydroxyacetone. The concentrations of glucagon and epinephrine giving half-maximal stimulation from dihydroxacetone were 0.3 to 0.4 mM and 0.3 to 0.5 muM, respectively. The meaximal catecholamine stimulation was much less than the maximal stimulation by glucagon and was mediated largely by the alpha receptor. Insulin had no effect on the basal rate of [14C]clucose synthesis but inhibited the effect of submaximal concentration of glucagon or of any concentration of catecholamine. Glucagon had no effect on the uptake of dihydroxyacetone but suppressed its conversion to lactate and pyruvate. This suppression accounted for most of the increase in glucose synthesis. In cells from gasted rats, where lactate production is greatly reduced and the rate of glucose synthesis is elevated, glucagon did not stimulate gluconeogenesis from dihydroxyacetone. Findings with glycerol as substrate were similar to those with dihyroxyacetone. Ethanol also stimulated glucose production from dihydroxyacetone while reducing proportionately the production of lactate. Ethanol is known to generate reducing equivalents fro clyceraldehyde-3-phosphate dehydrogenase and presumably thereby inhibits carbon flux to lactate at this site. Its effect was additive with that of glucagon. Estimates of the steady state levels of intermediary metabolites and flux rates suggested that glucagon activated conversion of fructose diphosphate to fructose 6-phosphate and suppressed conversion of phosphoenolpyruvate to pyruvate. More direct evidence for an inhibition of pyruvate kinase was the observation that brief exposure of cells to glucagon caused up to 70% inhibition of the enzyme activity in homogenates of these cells. The inhibition was not seen when the enzyme was assayed with 20 muM fructose diphosphate. The effect of glucagon to lower fructose diphosphate levels in intact cells may promote the inhibition of pyruvate kinase. The inhibition of pyruvate kinase may reduce recycling in the pathway of gluconeogenesis from major physiological substrates and probably accounts fromsome but not all the stimulatory effect of glucagon.     

Mechanism of the stereospectific irreversible inhibition of bacterial glutamic acid decarboxylase by (R)-(--)-4-aminohex-5-ynoic acid, an analogue of 4-aminobutyric acid.

4-Aminohex-5-ynoic acid inhibits bacterial glutamic acid decarboxylase in a time-dependent irreversible manner. The inhibition is stereospecific and requires the abstraction of the propargylic hydrogen from 4(R)-(--)-4-aminohex-5-ynoic acid. This leads to the generation of a reactive alkylating agent in the active site which can react with a nucleophilic residue. At complete inhibition, there is incorporation of one molecule of inhibitor per pyridoxal binding site. If the decarboxylation of glutamate occurs with retention of configuration, the irreversible inhibition of this enzyme by the 4-(R) isomer can be rationalized on the basis of reversibility of the protonation step in the normal catalytic mechanism.