Effect of L-leucine on p-aminohippurate accumulation by mammalian renal cortical slices

The effect of several amino acids (L-glutamate, L-phenylalanine, L-leucine, glycine, L-tryptophan, L-histidine, L-valine) on p-aminohippurate accumulation was evaluated in rat, mouse and rabbit kidneys. Only leucine was found to enhance p-aminohippurate accumulation in rat and mouse renal cortical slices but not in rabbit slices. Leucine had no effect on tetraethylammonium accumulation. In rat renal cortical slices, leucine increased the uptake and decreased the runout of p-aminohippurate, each effect contributing to the increase in p-aminohippurate accumulation. The apparent K_m of p-aminohippurate uptake was decreased by leucine with no change in the apparent V. Aminooxyacetate (an inhibitor of transamination of leucine) partially depressed the stimulating effect of leucine on p-aminohippurate accumulation, whereas α-ketoisocaproic acid (a metabolite of leucine formed by transamination) enhanced p-aminohippurate accumulation, suggesting that the metabolism of leucine in kidney slices may be necessary for the stimulating effect on p-aminohippurate transport.     

Studies on compartmentation of S-adenosyl-L-methionine in Saccharomyces cerevisiae and isolated rat hapatocytes

The existence of metabolically distinct pools of $\text{S-}\text{adenosyl-L-methionine}$ in Saccharomyces cerevisiae and isolated rat hepatocytes was investigated. Utilizing a relatively long labeling period with [methyl-¹⁴C]methionine, a metabolically ‘stable’ pool was labeled. A subsequent short labeling with [methyl-³H]methionine selectively labeled a putative metabolically ‘labile’ pool. The existence of these distinguishable pools was ascertained by following the ³H and ¹⁴C label disappearance in $\text{S-}\text{adenosyl-L-methionine}$ during the chase-period in label-free media containing cycloleycine to prevent futher synthesis of $\text{S-}\text{adenosyl-L-methionine}$. In both yeast and hepatocytes, the ${}^3\text{H}{}^{14}\text{C}$ ratio in $\text{S-}\text{adenosyl-L-methionine}$ decreased sharply. The individual ³H and ¹⁴C decrease in $\text{S-}\text{adenosyl-L-methionine}$ showed $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ values of 3 and 8 min for yeast and 4 and 18 min for hepatocytes. The results strongly indicate that at least two metabolically distinct $\text{S-}\text{adenosyl-L-methionine}$ pools actually do exist in both systems. Subcellular fractionation revealed that the ‘labile’ pool exist in the cytosol for both yeast and hepatocytes while the ‘stable’ pool exists in the vacuolar and the mitochondrial fraction for the yeast and hepatocytes respectively. The $\text{S-}\text{adenosyl-L-methionine}$ pools were also studied in normal yeast under anaerobic chase condition and petite mutant yeast. Sharply contrasting with aerobically chased normal yeast, both showed closely parallel ³H and ¹⁴C decreases in $\text{S-}\text{adenosyl-L-methionine}$.     

Aspartokinase of a lysine producing mutant ofMicrococcus glutamicus

Aspartokinase fromMicrococcus glutamicus AEC RN-13-6/1 [a homoserine requiring, S-(2-aminoethyl)-L-cysteine resistant, lysine producing strain] was purified 71 fold. The partially purified enzyme was inhibited by L-lysine. L-threonine, L-methionine, L-isoleucine, L-valine and L-phenylalanine activated the enzyme and reversed the inhibition by L-lysine. Aspartokinase activity was not derepressed by growth-limiting concentrations of L-threonine and/or L-methionine. It was not repressed by an excess of L-lysine (20 mM) and/or L-isoleucine (15.3 mM). The degree of activation or inhibition by amino acids was dependant on the composition of the growth medium. This observation is in contrast with the enzyme from the original (non-lysine-producing) strain which was inhibited by lysine or threonine and in a concerted manner by threonine plus lysine.     

A new NAD+-dependent opine dehydrogenase from Arthrobacter sp. strain 1C.

A new NAD+-dependent opine dehydrogenase was purified to homogeneity from Arthrobacter sp. strain 1C isolated from soil by an enrichment culture technique. The enzyme has a molecular weight of about 70,000 and consists of two identical subunits with molecular weights of about 36,000. The enzyme catalyzed a reversible oxidation-reduction reaction of opine-type secondary amine dicarboxylic acids. In the oxidative deamination reaction, the enzyme was active toward unusual opines, such as N-[1-R-(carboxyl)ethyl]-S-methionine and N-[1-R-(carboxyl)ethyl]-S-phenylalanine. In the reductive secondary amine-forming reaction with NADH as a cofactor, the enzyme utilized L-amino acids such as L-methionine, L-isoleucine, L-valine, L-phenylalanine, L-leucine, L-alanine, and L-threonine as amino donors and alpha-keto acids such as pyruvate, oxaloacetate, glyoxylate, and alpha-ketobutyrate as amino acceptors. The product enzymatically synthesized from L-phenylalanine and pyruvate in the presence of NADH was identified as N-[1-R-(carboxyl)ethyl]-S-phenylalanine.     

Effect of L-ethionine the expression of the SOS system in Escherichia coli

The effect of L-ethionine, the ethyl analog of the essential amino acid methionine the SOS system of Escherichia coli was studied. This compound does not induce either inhibition of cell division nor cessation of cell respiration in a RecA⁺ Met⁺ RelA⁺ strain, nor in RecA⁺ Met⁻ RelA⁺ or RecA⁺ Met⁻ RelA⁻ mutants. Nevertheless, L-ethionine blocks the expression of both cited SOS functions in a recA441 mutant when it is growing at the restrictive temperature of 42°C. Furthermore, the inhibitory effect of the L-ethionine on the induction of the SOS system in this mutant is increased when the cells are preincubated for several hours in the presence of the analog, before the temperature shift. Moreover, cultures of the recA441 mutant incubated at 42°C in the presence of both L-ethionine and L-methionine present the same behaviour as the cultures of this mutant growing at the same temperature but without either amino acid. On the other hand, L-ethionine does not have any effect on the expression of the two mentioned SOS functions when these are induced by UV-irradiation in a RecA⁺ strain even if this compound is added to the cells several hours before irradiation.     

Postnatal amino acid uptake by the rat small intestine. Changes in membrane transport systems for amino acids associated with maturation of jejunal morphology.

The uptake of a number of amino acids by the developing small intestine of the rat was investigated in vitro. L-valine, L-leucine, L-methionine, L-phenylalanine, L-arginine and L-lysine were all taken up by active transport and concentrated within the jejunal mucosa. GABA was not actively transported by the jejunum. The kinetics of carrier transport of amino acids was determined from birth to maturity. The Michaelis constant (Km) of the L-leucine, L-methionine, L-arginine and l-lysine transport systems was found to be low postnatally and increased with age, particularly after the time of weaning. The rate of l-leucine, L-methionine, L-phenylalanine and L-lysine transport (Vmax) was high postnatally but decreased after weaning. Neutral amino acids were transported at higher rates than basic amino acids. l-arginine was poorly transported by the jejunum. The specificity of transport systems for amino acids was investigated in inhibition studies. Amino acid transport systems appeared to be polyfunctional in the postnatal period but were more specific in post-weaned animals. The changes in kinetics and specificity of amino acid transport in the small intestine are discussed with reference to their possible functional significance and to the maturational changes in the jejunum, particularly with the appearance of a functionally distinct absorptive cell lining the intestinal villi during the third postnatal week (the time of weaning).     

Interactions between the branched-chain amino acids in the growth of Spirodela polyrhiza

The joint action of L-valine and L-isoleucine, L-leucine and L-isoleucine, and L-valine and L-leucine on the growth of Spirodela polyrhiza was established. The effect of one branched-chain amino acid on growth inhibition by another one was compared with the non-specific antagonisms which glycine and L-alanine exert on growth inhibition by singly supplied branched-chain amino acids. In this way specific and non-specific interactions could be distinguished. It appeared that: (1) L-isoleucine was a specific antagonist of L-valine; (2) L-leucine was a specific antagonist of L-isoleucine; (3) L-valine and L-leucine were synergistic growth inhibitors. Further, it was found that: (4) growth inhibition by L-leucine was specifically antagonized by simultaneously supplied L-valine and L-isoleucine; (5) an excess of L-isoleucine strongly inhibited the conversion of exogenous valine into leucine; (6) accumulation of valine was typical of isoleucine-induced growth inhibition. The results are consistent with the view that growth inhibition by L-valine and L-leucine is due to the blocking of acetohydroxy acid synthetase, the first common enzyme in the valine-isoleucine biosynthetic pathway. Growth inhibition by L-isoleucine, however, seems to result from inhibition of leucine synthesis at a step after 2-oxoisovaleric acid. Some aspects of the regulation of branched-chain amino acid biosynthesis in higher plants are discussed.     

Repression and inhibition of transport systems for branched-chain amino acids in Salmonella typhimurium.

Kinetics of the transport systems common for entry of L-isoleucine, L-leucine, and L-valine in Salmonella typhimurium LT2 have been analyzed as a function of substrateconcentration in the range of 0.5 to 45 muM. The systems of transport mutants, KA203 (ilvT3) and KA204 (ilvT4), are composed of two components; apparent Km values for uptake of isoleucine, leucine, and valine by the low Km component are 2 muM, 2 to 3 muM, and 1 muM, respectively, and by the high Km component 30 muM, 20 to 40 muM, and 0.1 mM, respectively. The transport system(s) of the wild type has not been separated into components but rather displays single Km values of 9 muM for isoleucine, 10 muM for leucine, and 30 muM for valine. The transport activity of the wild type was repressed by L-leucine, alpha ketoisocaproate, glycyl-L-isoleucine, glycyl-L-leucine, and glycyl-L-methionine. That for the transport mutants was repressed by L-alanine, L-isoleucine, L-methionine, L-valine, alpha-ketoisovalerate, alpha-keto-beta-methylvalerate, glycyl-L-alanine, glycyl-L-threonine, and glycyl-L-valine, in addition to the compounds described above. Repression of the mutant transport systems resulted in disappearance of the low Km component for valine uptake, together with a decrease in Vmax of the high Km component; the kinetic analysis with isoleucine and leucine as substrates was not possible because of poor uptake. The maximum reduction of the transport activity for isoleucine was obtained after growing cells for two to three generations in a medium supplemented with repressor, and for the depression, protein synthesis was essential after removal of the repressor. The transport activity for labeled isoleucine in the transport mutant and wild-type strains was inhibited by unlabeled L-alanine, L-cysteine, L-isoleucine, L-leucine, L-methionine, L-threonine, and L-valine. D-Amino acids neither repressed nor inhibited the transport activity of cells for entry of isoleucine.     

Decrease in plasma tryptophan after tryptophan-free amino acid mixtures in man

Male healthy subjects, fasting 12 hours, ingested increasing amounts of a mixture containing a fixed proportion of seven essential amino acids (L-isoleucine 11.5%, L-leucine 18.0%, L-lysine 13.1%, L-methionine 18.0%, L-phenylalanine 18.0%, L-threonine 8.2%, L-valine 13.1%) and lacking tryptophan. The diets produced a rapid fall in plasma tryptophan which was proportional to the total amount of the amino acids ingested. Following the highest dose administered (36.6 g) plasma tryptophan fell to a minimum of about 35% the initial level and remained markedly reduced at 6 hours after treatment. The mechanism of this decrease and its potential clinical relevance are discussed.     

Construction and co-expression of polycistronic plasmid encoding d-hydantoinase and d-carbamoylase for the production of d-amino acids

d-Hydantoinase and d-carbamoylase genes from Agrobacterium radiobacter TH572 were cloned by polymerase chain reaction (PCR). The plasmid pUCCH3 with a polycistronic structure that is controlled by the native hydantoinase promoter was constructed to co-express the two genes and transformed into Escherichia coli strain JM105. To obtain the highest level of expression of the d-carbamoylase and avoid intermediate accumulation, the d-carbamoylase gene was cloned closer to the promoter and the RBS region in the upstream of it was optimized. This resulted in high active expression of soluble d-hydantoinase and d-carbamoylase that is obtained without any inducer. Thus, by the constitutive recombinant JM105/pUCCH3, d-p-hydroxyphenylglycine (d-HPG) was obtained directly with 95.2% production yield and 96.3% conversion yield.     

Intestinal absorption of amino acids by rainbow trout,Salmo gairdneri (Richardson)

Summary A technique for the in vitro maintenance of isolated portions of rainbow trout intestine is described. Uptake of¹⁴C-L-leucine occurs by an active mechanism which is stereospecific, sodium-dependent and susceptible to inhibition by other neutral amino acids.K _t for leucine uptake is 2.72 mM with aV _max of 19.61 moles/g ethanol extracted dry wt.·10 min. L-valine and L-methionine are competitive inhibitors of L-leucine uptake withK _i values of 24.30 mM and 2.56 mM, respectively. Evidence suggests that at least two uptake sites for the transport of neutral amino acids are present in the intestine of this species.     

Dose-response decrease in plasma tryptophan and in brain tryptophan and serotonin after tryptophan-free amino acid mixtures in rats

Rats fasted 15 hours were treated p.o. with increasing amounts (660 and 1320 mg/kg body weight) of a mixture containing a fixed proportion of seven essential amino acids (L-phenylalanine 13.6%, L-leucine 6.0%, L-isoleucine 12.1%, L-methionine 12.1%, L-lysine 30.3%, L-threonine 10.6%, L-valine 15.2%) and lacking tryptophan. The mixtures produced a dose-response decrease of free (by 34% after the lower dose and by 58% after the higher dose of the mixture) and total (by 10 and 31%) plasma tryptophan and of brain tryptophan (by 38 and 65%), serotonin (by 17 and 41%) and 5-hydroxyindole acetic acid (by 21 and 49%). The mechanisms of these changes are discussed.     

Aspartate Kinase from Some Higher Plants and Algae

Regulation of aspartate kinase activity was examined in some higher plant seedlings (Helianthus annuus, Raphanus sativus, Cucutnis sativus, Sinapis alba), a green (Chlorellapyrenoidosa), and a blue-green alga (Anacystis nidulans). In Cucumis sativus a concerted feedback regulation by L-lysine and L-threonine was indicated. In all the other species examined, aspartate kinase was inhibited by both L-lysine and L-threonine and it depended upon the species which of them was the strongest inhibitor. Only slight effects of L-leucine, L-isoIeucine and L-valine were observed, except in the Sinapis alba enzyme.     

Evidence of multiple operational affinities for d-glucose inside the human erythrocyte membrane

1. 1. The Michaelis-Menten parameters of labelled d-glucose exit from human erythrocytes at 2°C into external solution containing 50 mM d-galactose were obtained. The K_m is 3.4 ± 0.4 mM, V 17.3 ± 1.4 mmol · 1⁻¹ cell water · min⁻¹ for this infinite-trans exit procedure.

2. 2. The kinetic parameters of equilibrium exchange of d-glucose at 2°C are K_m = 25 ± 3.4 mM, V 30 ± 4.1 mmol · 1⁻¹ cell water · min⁻¹.

3. 3. The K_m for net exit of d-glucose into solutions containing zero sugar is 15.8 ± 1.7 mM, V 9.3 ± 3.3 mol 9.3 ± 3.3 mol · 1⁻¹ cell water · min⁻¹.

4. 4. This experimental evidence corroborates the previous finding of Hankin, B.L., Lieb, W.R. and Stein, W.D. [(1972) Biochim. Biophys. Acta 255, 126–132] that there are sites with both high and low operational affinities for d-glucose at the inner surface of the human erythrocyte membrane. This result is inconsistent with current asymmetric carrier models of sugar transport.

Induction by 3, 5, 3′ l-triiodothyronine of l-ornithine decarboxylase in rat heart muscle

Injection of 3,5,3′ l-triiodothyronine (15 μg/100 g) induces a biphasic enhancement of rat heart ornithine decarboxylase (EC. 4.1.17) activity after 4 and 21 hours. This induction is observed after each daily injection, but to a lesser extent.The properties of partially purified basal enzyme and induced enzyme, at 21h, after single injections have been compared.

1) Affinity for ornithine is the same for both enzymes, but affinity for pyridoxal-phosphate is 40-fold higher for the induced one.

2) Thermostability studies suggest that basal and induced enzymes have different conformations.

3) The two enzymes have similar immunoreactivity.

4) The comparisons of the time-dependent activity curve after injection and of the antigen/activity ratio suggests that triiodothyronine induces the synthesis of new molecules of enzymes and that an inhibition of the enzyme activity also occurs which explains the biphasic induction.

Growth of Rhodopseudomonas capsulata on l- and d-malic acid

d-malate replaced l-malate in supporting both photosynthetic (anaerobic, light) and heterotrophic (aerobic, dark) growth of Rhodopseudomonas capsulata. Growth rates and cell yields were nearly equivalent with both enantiomorphs. Addition of glucose to malate culture media increased the growth rate and doubled the cell yield of heterotrophic cultures, but had little effect on photosynthetic cultures. Aerobically-grown cells showed a higher level of substrate-dependent oxygen uptake with l-malate than with d-malate. This preference for l-malate occured even in cells grown on d-malate. No malic racemase activity was detected in extracts of heterotrophically- or photosynthetically-grown cells.     

Biosynthese in vitro d'iodothyronines a partir du diiodo-3,5-L-tyrosyl-diiodo-3,5-L-tyrosine

In vitro biosynthesis of iodothyronines from diiodo-3,5-L-tyrosyl-diiodo-3,5-L-tyrosine A comparative study of two types of in vitro synthesis of iodothyronines has been done from 3,5-diiodotyrosine and from diiodo-3,5-L-tyrosyl-diiodo-3,5-L-tyrosine (Tyr(I)₂-Tyr(I)₂) (equimolecular in tyrosyl rings).Incubations are made with rat thyroid gland minces in Eagle's medium or with thyroid microsomal fraction.Synthesis of thyroid hormones from Tyr(I)₂-Tyr(I)₂ is faster and more important than from diiodo-3,5-L-tyrosine (Tyr(I)₂).A mechanism of iodothyronine formation via Tyr(I)₂ - Tyr(I)₂ and different from the one occuring for Tyr(I)₂ is suggested.

Résumé

Une étude comparative de deux types de synthèse in vitro d'iodothyronines a été faite à partir de la 3,5-diiodotyrosine Tyr(I)₂ et à partir d'un dipeptide iodé: le diiodo-3,5-L-tyrosyl-diiodo-3,5-L-tyrosine (Tyr(I)₂-Tyr(I)₂) dans des conditions équimoléculaires en noyaux tyrosyl.Les incubations sont effectuées en présence de coupes de thyroïdes de rat en milieu de survie ou en présence de fraction microsomale thyroïdienne.La synthèse d'hormones thyroïdienes à partir du Tyr(I)₂-Tyr(I)₂ est plus rapide et plus importante qu'à partir de la Tyr(I)₂.Un mécanisme de synthèse des iodothyronines à partir du Tyr(I)₂-Tyr(I)₂ différent de celui intervenant pour la Tyr(I)₂ est proposé.     

Effects of temperature on L-leucine transport in toadfish liver in vivo

The effect of body temperature in the 4–30°C range on L-leucine uptake by toadfish liver in vivo was examined by means of a single-injection pulse technique. The ratio of [¹⁴C]leucine to [³H]mannitol or [³H]inulin in blood leaving the liver was measured as a function of time after hepatic portal vein injection. Recoveries of the two isotopes in liver and [¹⁴C]leucine incorporation into protein were determined.The Q₁₀ value for influx was 3.8, that for efflux 2.8. At all temperatures, the leucine influx was 8–10-times higher than its incorporation into protein. The directly energy-linked reactions appear to be the main site of increased temperature sensitivity at low temperatures.