Biosynthesis of the Branched-Chain Amino Acids in Yeast: a Leucine-Binding Component and Regulation of Leucine Uptake

Use of an ion-exchange resin assay has shown that leucine is bound to a component of a dialyzed extract of yeast. Leucine binding may be related to in vivo uptake of the amino acid. A yeast strain with a 30-fold lower affinity for leucine uptake in vivo has a parallel reduction in affinity for in vitro leucine binding; the rate of leucine uptake in wild-type yeast can be increased four- to fivefold by growth on leucine as a sole nitrogen source. Under these conditions, the specific activity of the leucine-binding component also increases over threefold. Regulation of leucine uptake was studied by using wild-type strain 60615 and a mutant 60615/fl(2) with a constitutively elevated leucine uptake system. Leucine pool formation in the mutant was accompanied by an overshoot, leading to a loss of leucine from the pool. The phenomenon could be observed in the wild type under certain conditions. The mechanism of this process was examined. The leucine uptake system was found to be stable in the absence of protein synthesis. The rate of leucine uptake increased on reduction of the pool of amino acids, and in strain 60615/fl(2) the ability to overshoot was rapidly recovered on depletion of the leucine pool. The results suggest a control of leucine uptake by feedback inhibition, in which leucine or other amino acids, e.g., isoleucine, inhibit leucine uptake. The results do not exclude control by a rapidly activated-inactivated system.     

Microbial Decomposition in Aquatic Environments: Combined Process of Extracellular Enzyme Activity and Substrate Uptake

The aim of this study was to define a model for the coupling between extracellular enzyme activity and substrate uptake by bacterial populations in natural waters. The balance between uptake of leucine and extracellular hydrolytic production of leucine from a peptide model substrate was investigated in a combined fluorescence-radiotracer experiment with [H]leucine as a marker for the leucine pool and l-leucine-4 methyl-7-coumarinylamide (Leu-MCA) as a marker for the pool of dissolved peptide substrates. Results show that at low concentrations of the model substrate the input and uptake processes of leucine are nearly balanced, whereas at high concentrations of the model substrate much more leucine is liberated than taken up. In addition, samples from one polluted and one less polluted station in the Kiel Fjord were investigated for their extracellular enzymatic and uptake properties in an annual cycle. It was found that turnover rates of leucine (T(r), percent per hour) and hydrolysis rates of Leu-MCA (H(r), percent per hour), as well as the quotient T(r)/H(r), reflect the impact of environmental conditions on decomposition processes at both sampling sites. The quotient T(r)/H(r) is interpreted as an indirect measurement of the pool size ratio (polymers/monomers), which may serve as an index of hydrolysis-uptake coupling in bacterial utilization of dissolved protein. Calculated on an annual average basis, turnover rates are ca. nine times higher than hydrolysis rates at the polluted station and ca. five times higher at the less polluted station. From the described model, this would mean that the relative fraction of polymers within the total dissolved organic carbon pool (with regard to the substrate combination dissolved protein-leucine) is about twice that at the polluted than at the less polluted station.     

Functional homogeneity of leucine pool in retina cells

Data on leucine metabolism in isolated rabbit retina are examined for evidence, for or against, a common intracellular pool of free leucine. Data include values for: concentrations, transport rates, degradative metabolism and protein incorporation of labelled leucine measured over a wide range of concentrations; protein incorporation of labelled threonine, measured simultaneously; and an indirect measurement of protein breakdown. The fall in labelled leucine incorporation into protein, when medium leucine was reduced below 100 microM, corresponded closely with the fall in intracellular specific activity predicted from rate of influx of labelled leucine from medium and rate of release of unlabelled leucine from protein breakdown. Protein incorporation of labelled leucine competed with decarboxylation and outward transport and reduced the free intracellular leucine in about the amounts predicted for a common pool. Implications for measurements using labelled amino acid are discussed.     

Leucine treatment enhances oxidative capacity through complete carbohydrate oxidation and increased mitochondrial density in skeletal muscle cells

Leucine has been largely implicated for increasing muscle protein synthesis in addition to stimulating mitochondrial biosynthesis. Limited evidence is currently available on the effects and potential benefits of leucine treatment on skeletal muscle cell glycolytic and oxidative metabolism. This work identified the effects of leucine treatment on oxidative and glycolytic metabolism as well as metabolic rate of human and murine skeletal muscle cells. Human rhabdomyosarcoma cells (RD) and mouse myoblast cells (C2C12) were treated with leucine at either 100 or 500 μM for 24 or 48 h. Glycolytic metabolism was quantified by measuring extracellular acidification rate (ECAR) and oxidative metabolism was quantified by measuring oxygen consumption rate. Peroxisome proliferator-activated receptor coactivator 1 alpha (PGC-1α), an important stimulator of mitochondrial biosynthesis, was quantified using flow cytometry and verified by immunofluorescent confocal microscopy. Mitochondrial content was quantified using mitochondrial and cytochrome C staining measured by flow cytometry and confirmed with confocal microscopy. Treatment with leucine significantly increased both basal and peak oxidative metabolism in both cell models. Leucine treated cells also exhibited significantly greater mitochondrial proton leak, which is associated with heightened energy expenditure. Basal ECAR was significantly reduced in both cell models following leucine treatment, evidence of reduced lactate export and more complete carbohydrate oxidation. In addition, both PGC-1α and cytochrome C expression were significantly elevated in addition to mitochondrial content following 48 h of leucine treatment. Our observations demonstrated few dose-dependent responses induced by leucine; however, leucine treatment did induce a significant dose-dependent expression of PGC-1α in both cell models. Interestingly, C2C12 cells treated with leucine exhibited dose-dependently reduced ATP content, while RD ATP content remain unchanged. Leucine presents a potent dietary constituent with low lethality with numerous beneficial effects for increasing oxidative preference and capacity in skeletal muscle. Our observations demonstrate that leucine can enhance oxidative capacity and carbohydrate oxidation efficiency, as well as verify previous observations of increased mitochondrial content.     

Biosynthesis of branched-chain amino acids in yeast: effect of carbon source on leucine biosynthetic enzymes.

The three enzymes in the leucine biosynthetic pathway of yeast do not exhibit coordinate repression and derepression in response to the carbon source available in the culture medium. Growth in an acetate medium results in derepression of the first enzyme in the pathway, alpha-isopropylmalate synthase, and repression of the second two enzymes, alpha-isopropylmalate isomerase and beta-isopropylmalate dehydrogenase, relative to the levels found in glucose-grown cells. The role of endogenous leucine pools as a mediator of these differences was investigated. The leucine pools did not differ significantly between acetate-grown and glucose-grown cells. However, an elevated endogenous leucine pool, caused by exogenous leucine in the growth medium, did decrease the rate of decay of alpha-isopropylmalate synthase activity observed when acetate-grown cells were shifted to glucose. Evidence is provided suggesting that an elevated endogenous leucine pool may increase the in vivo stability of alpha-isopropylmalate synthase under several different conditions. Studies on the kinetics of alpha-isopropylmalate synthase decay in vivo and sensitivity to leucine inhibition indicate that there are two classes of the enzyme in acetate-grown yeast cells.     

Ethanol and leucine oxidation--I. Leucine oxidation by the rat in vivo

The effect of ethanol upon the oxidation of leucine by the rat in vivo was determined. The rate of leucine oxidation was not significantly altered by chronic administration of ethanol (20% v/v solution as drinking water for 28 days). Ethanol administered acutely (8 g kg 0.73) significantly decreased leucine oxidation by the rat in vivo. This decrease appeared to be independent of a more general depression of oxidation metabolism. Decrease in leucine oxidation by ethanol is discussed in relation to the regulation of tissue leucine pool sizes in vivo.     

ENHANCED CEREBRAL PROTEIN SYNTHESIS IN DEVELOPING HYPOTHYROID RATS: EVIDENCE FOR DELAYED MATURATION

(1) Neonatal hypothyroidism resulted in a 40% increase in the incorporation of [¹⁴C]leucine into protein by cerebral cortical slices from 25-day-old rats. The uptake of the [¹⁴C]-labelled amino acid into the acid-soluble free amino acid pool was similar in hypothyroid and control groups which excluded the possibility that transport differences contributed to the observed differences in incorporation. (2) The conversion of [¹⁴C]leucine in the free amino acid pool to other metabolites was substantially greater in the hypothyroid state compared to euthyroid controls. (3) The correction of the incorporation data for radioactivity associated with [¹⁴C]leucine in the precursor pool, provided an estimate of cerebral protein synthetic rate which was markedly higher in thyroid hormone-deficient-rats compared to litter mate controls. (4) The administration of L-thyroxine to hypothyroid animals for two successive days essentially returned the accelerated metabolism of the precursor pool leucine to normal but failed to ameliorate the increased incorporation into protein. (5) Incubations conducted in the presence of high exogenous leucine levels, to eliminate possible differences in intracellular free amino acid pool size, provided additional evidence for an increased rate of cerebral protein synthesis in 25-day-old hypothyroid rats compared to controls. (6) The results are compatible with a retardation in the normal developmental decline in the rate of cerebral protein synthesis associated with hypothyroidism.     

Interpretation of Tracer Data: Significance of the Number of Terms in Specific Activity Functions

It has already been shown that the number of pools in an open system in the steady state cannot be determined from the number of exponential terms in the specific activity function of a pool, even if the data were free from experimental error. However, some information is conveyed by the number of exponential terms. The information is different depending upon whether the data are obtained from the pool into which the tracer is introduced or from another pool. In the latter case, the number of exponential terms is shown to indicate the maximum number of intermediate pools involved in the shortest path of transfer of material from the injected pool to the pool in question. With regard to the former case, this paper is restricted to functions with two exponential terms and shows which systems of n pools (n >/= 2) are consistent with such data. Consequently, biexponential experimental curves can be interpreted in terms of models consisting of an unrestricted number of pools in which each pool is defined in terms of fast mixing. The generalization to cases of functions with more than two exponential terms can be carried out in a similar manner.     

Changes in leucine uptake in the retina of the hamster after traumatic detachment

Protein metabolism was investigated in detached hamster retinas. By sucking off 0.2 ml of aqueous humor from the anterior chamber through limbic insertion of a 27-gauge needle, a tractional force pulled off the neural retina from the retinal pigment epithelium and created a simple detachment without retinal breaks in the right eyes of the hamsters. The left eyes were left untouched as normal controls and sham controls were induced by simple limbic insertion without suction. The animals were sacrificed at selected intervals of 1, 3, 6, 9, 16, 24, 32 days after the operation. Subsequently, scintillation counting and autoradiography were employed to study retinal protein metabolism using leucine uptake as an index. After tritiated leucine uptake, scintillation counting of radioactive substance indicated that detached retinas had taken in less tritiated leucine than normal controls from day 1 to 6 after the operation, but this change had normalized by day 9. For autoradiography, the change in leucine uptake rate was shown to be different in different layers. All the retinal cells seemed to show a decreased leucine uptake with the exception of the outer nuclear layer, in which leucine appeared to be significantly upregulated. This paper illustrates the patterns of protein metabolism and their change after traumatic detachment as well as their possible recovery.     

Protein synthesis rates of human PBMC and PMN can be determined simultaneously in vivo by using small blood samples

Immune cell functions can be evaluated in vivo by measuring their specific protein fractional synthesis rates (FSR). Using stable isotope dilution techniques, we describe a new method allowing simultaneous in vivo assessment of FSR in two leukocyte populations in healthy human subjects, using small blood samples. Peripheral blood mononuclear cell (PBMC) and polymorphonuclear neutrophil (PMN) FSR were measured during primed continuous intravenous infusion of L-[1-¹³C]leucine. Immune cells from 6 ml of whole blood were isolated by density gradient centrifugation. In a first study, we calculated the FSR using plasma [¹³C]leucine or -[¹³C]ketoisocaproate (KIC) enrichments as precursor pools. In a second study, we compared protein FSR in leukocytes, using enrichments of either intracellular or plasma free [¹³C]leucine as immediate precursor pools. The present approach showed a steady-state enrichment of plasma and circulating immune cell free [¹³C]leucine precursor pools. The linearity of labeled amino acid incorporation rate within mixed PBMC and PMN proteins also was verified. Postabsorptive protein FSR was 4.09 ± 0.39%/day in PBMC and 1.44 ± 0.08%/day in PMN when plasma [¹³C]KIC was the precursor pool. The difference between PBMC and PMN FSR was statistically significant, whatever the precursor pool used, suggesting large differences in their synthetic activities and functions. Use of the plasma [¹³C]KIC pool led to an underestimation of leukocyte FSR compared with the intracellular pool (PBMC: 6.04 ± 0.94%/day; PMN: 2.98 ± 0.30%/day). Hence, the intracellular free amino acid pool must be used as precursor to obtain reliable results. In conclusion, it is possible to assess immune cell metabolism in vivo in humans by using small blood samples to directly indicate their metabolic activity in various clinical situations and in response to regulating factors. peripheral blood mononuclear cells; polymorphonuclear neutrophils; protein metabolism; stable isotopes; leucine     

Metabolism of Phosphate and Sulfate Groups Modifying the P0 Protein of Peripheral Nervous System Myelin

We have examined the metabolism of phosphate and sulfate groups modifying the P0 protein, the major protein of peripheral nervous system myelin, using an in vitro incubation system. Incorporation of [3H]leucine into the P0 peptide backbone decreased approximately 25-fold between 10 and 90 days of age, a finding reflecting a decreased rate of myelin synthesis in the older animals. In contrast, incorporation of [32P]phosphate into P0 decreased only four- to fivefold, a result indicating that phosphate groups are metabolized independently of the peptide backbone. Developmental decreases in the incorporation of sulfate groups into P0 were similar to those seen for leucine, an observation suggesting that this modifying group is metabolized together with the peptide backbone as a single metabolic entity. The time course of labeling of P0 isolated from the starting homogenate and from myelin was also compared. Results are consistent with sulfation of P0 protein taking place before insertion of newly synthesized P0 into myelin. In contrast, incorporation of phosphate into P0 appears to involve both the newly synthesized pool and the preexisting pool of P0 in myelin. Presumably, entry of phosphate into P0 in myelin involves turnover of preexisting phosphate groups and rephosphorylation by myelin protein kinases. Developmental decreases in the specific activity of P0 phosphate groups in myelin are consistent with the presence of a small, rapidly turning-over pool of phosphorylated P0 (perhaps associated with the axon-myelin interface), which does not increase to the same extent as the marked increase in bulk myelin that occurs during development.     

Construction of kinetic models to understand metabolism in vivo

This review describes increasingly complex kinetic models that simulate carbohydrate metabolism in a simple eucaryotic system which undergoes differentiation. Dynamic models of complex metabolic networks serve to organize and analyze the many interdependent variables involves and to define the rate-limiting events controlling metabolism in vivo. Since the ultimate justification for and test of any model are its predictive values, a series of predictions and related experiments will be described.     

Determination of leucine flux in vivo by gas chromatography—mass spectrometry utilizing stable isotopes for trace and internal standard

A simple and reliable method is described for the determination of leucine flux in vivo using two stable isotopes of leucine and gas chromatography—mass spectrometry (GC---MS). [6,6,6-²H₃]Leucine is administered as a primed-dose constant infusion in vivo and -[²H₇]leucine is added to plasma as an internal standard. Plasma leucine concentration and moles per cent enrichment of [²H₃]leucine can be determined simultaneously by GC---MS and selected ion monitoring. Leucine flux calculated from the [6,6,6-²H₃]leucine data was nearly identical to that obtained with -[U-¹⁴C]leucine in dogs. This method is readily applicable to the study of leucine metabolism in humans of all ages and laboratory animals.     

A redox‐active isopropylmalate dehydrogenase functions in the biosynthesis of glucosinolates and leucine in Arabidopsis

We report a detailed functional characterization of an Arabidopsis isopropylmalate dehydrogenase (AtIPMDH1) that is involved in both glucosinolate biosynthesis and leucine biosynthesis. AtIPMDH1 shares high homology with enzymes from bacteria and yeast that are known to function in leucine biosynthesis. In plants, AtIPMDH1 is co‐expressed with nearly all the genes known to be involved in aliphatic glucosinolate biosynthesis. Mutation of AtIPMDH1 leads to a significant reduction in the levels of free leucine and of glucosinolates with side chains of four or more carbons. Complementation of the mutant phenotype by ectopic expression of AtIPMDH1, together with the enzyme’s substrate specificity, implicates AtIPMDH1 in both glucosinolate and leucine biosynthesis. This functional assignment is substantiated by subcellular localization of the protein in the chloroplast stroma, and the gene expression patterns in various tissues. Interestingly, AtIPMDH1 activity is regulated by a thiol‐based redox modification. This work characterized an enzyme in plants that catalyzes the oxidative decarboxylation step in both leucine biosynthesis (primary metabolism) and methionine chain elongation of glucosinolates (specialized metabolism). It provides evidence for the hypothesis that the two pathways share a common origin, and suggests a role for redox regulation of glucosinolate and leucine synthesis in plants.     

Immunocytochemical localization of beta-methylcrotonyl-CoA carboxylase in astroglial cells and neurons in culture

Astroglia-rich primary cultures and brain slices rapidly metabolize branched-chain amino acids (BCAAs), in particular leucine, as energy substrates. To allocate the capacity to degrade leucine oxidatively in neural cells, we have purified beta-methylcrotonyl-CoA carboxylase (beta-MCC) from rat liver as one of the enzymes unique for the irreversible catabolic pathway of leucine. Polyclonal antibodies raised against beta-MCC specifically cross-reacted with both enzyme subunits in liver and brain homogenates. Immunocytochemical examination of astroglia-rich rat primary cultures demonstrated the presence of beta-MCC in astroglial cells, where the enzyme was found to be located in the mitochondria, the same organelle that the mitochondrial isoform of the BCA(A) aminotransferase (BCAT) is located in. This colocalization of the two enzymes supports the hypothesis that mitochondrial BCAT is the isoenzyme that in brain energy metabolism prepares the carbon skeleton of leucine for irreversible degradation in astrocytes. Analysis of neuron-rich primary cultures revealed also that the majority of neurons contained beta-MCC. The presence of beta-MCC in most neurons demonstrates their ability to degrade the alpha-ketoisocaproate that could be provided by neighboring astrocytes or could be generated locally from leucine by the action of the cytosolic isoform of BCAT that is known to occur in neurons.     

Role of transport systems in amino acid metabolism: leucine toxicity and the branched-chain amino acid transport systems.

The livR locus, which leads to a trans-recessive derepression of branched-chain amino acid transport and periplasmic branched-chain amino acid-binding proteins, is responsible for greatly increased sensitivity toward growth inhibition by leucine, valine, and serine and, as shown previously, for increased sensitivity toward toxicity by branched-chain amino acid analogues, such as 4-azaleucine or 5',5',5'-trifluoroleucine. These phenotypes are similar to those of relA mutants; however, the livR mutants retain the stringent response of ribonucleic acid synthesis. However, an increase in the rate of transport or in the steady-state intracellular level of amino acids in the livR strain cannot completely account for this sensitivity. The ability of the LIV-I transport system to carry out exchange of pool amino acids for extracellular leucine is a major factor in leucine sensitivity. The previous finding that inhibition of threonine deaminase by leucine contributes to growth inhibition is confirmed by simulating the in vivo conditions using a toluene-treated cell preparation with added amino acids at levels corresponding to the internal pool. The relationship between transport systems and corresponding biosynthetic pathways is discussed and the general principle of a coordination in the regulation of transport and biosynthetic pathways is forwarded. The finding that the LIV-I transport system functions well for amino acid exchange in contrast to the LIV-II system provides another feature that distinguishes these systems in addition to previously described differences in regulation and energetics.     

Accuracy of femur reconstruction from sparse geometric data using a statistical shape model

Sparse geometric information from limited field-of-view medical images is often used to reconstruct the femur in biomechanical models of the hip and knee. However, the full femur geometry is needed to establish boundary conditions such as muscle attachment sites and joint axes which define the orientation of joint loads. Statistical shape models have been used to estimate the geometry of the full femur from varying amounts of sparse geometric information. However, the effect that different amounts of sparse data have on reconstruction accuracy has not been systematically assessed. In this study, we compared shape model and linear scaling reconstruction of the full femur surface from varying proportions of proximal and distal partial femur geometry in combination with morphometric and landmark data. We quantified reconstruction error in terms of surface-to-surface error as well as deviations in the reconstructed femur’s anatomical coordinate system which is important for biomechanical models. Using a partial proximal femur surface, mean shape model-based reconstruction surface error was 1.8 mm with 0.15° or less anatomic axis error, compared to 19.1 mm and 2.7–5.6° for linear scaling. Similar results were found when using a partial distal surface. However, varying amounts of proximal or distal partial surface data had a negligible effect on reconstruction accuracy. Our results show that given an appropriate set of sparse geometric data, a shape model can reconstruct full femur geometry with far greater accuracy than simple scaling.     

Dynamics of mixed populations having complementary metabolism

The dynamics of a system of two microbial populations having complementary metabolism are investigated by means of simple mathematical models of growth. Complementary metabolism as used here means that each population produces a substance—not present in the initial or feed medium—required by the other for growth. The simple models indicate that (1) something other than lack of the substrate or growth factor produced by its partner must limit the growth of at least one population and (2) the coexistence steady state of such populations in continuous culture is not stable with respect to large perturbations, though it is stable with respect to a wide range of perturbations.     

Effect of Herbivory and Plant Species Replacement on Primary Production

Grazing optimization occurs when herbivory increases primary production at low grazing intensities. In the case of simple plant-herbivore interactions, such an effect can result from recycling of a limiting nutrient. However, in more complex cases, herbivory can also lead to species replacement in plant communities, which in turn alters how primary production is affected by herbivory. Here we explore this issue using a model of a limiting nutrient cycle in an ecosystem with two plant species. We show that two major plant traits determine primary production at equilibrium: plant recycling efficiency (i.e., the fraction of the plant nutrient stock that stays within the ecosystem until it is returned to the nutrient pool in mineral form) and plant ability to deplete the soil mineral nutrient pool through consumption of this resource. In cases where sufficient time has occurred, grazing optimization requires that herbivory improve nutrient conservation in the system sufficiently. This condition sets a minimum threshold for herbivore nutrient recycling efficiency, the fraction of nutrient consumed by herbivores that is recycled within the ecosystem to the mineral nutrient pool. This threshold changes with plant community composition and herbivore preference and is, therefore, strongly affected by plant species replacement. The quantitative effects of these processes on grazing optimization are determined by both the recycling efficiencies and depletion abilities of the plant species. However, grazing optimization remains qualitatively possible even with plant species replacement.     

Sampling of the leucine pool from the growing peptide chain: difference in leucine specific activity of peptidyl-transfer RNA from free and membrane-bound polysomes.

The specific activity of leucine in newly synthesized protein was determined by isolating the nascent polypeptides of the growing polypeptide chains. The newt, Triturus viridescens, was labeled in vivo with [³H]leucine. Polysomes were prepared from the livers. Peptidyl-tRNA was released from the polysomes by EDTA, isolated by sucrose gradient and purified on hydroxylapatite. It was then hydrolyzed with HCl and the amino acids were reacted with ¹⁴C-labeled 1fluoro-2,4-dinitrobenzene. The specific activity of [³H]leucine was determined from the [¹⁴C]dinitrophenyl-[³H]leucine after purification by two-dimensional thin layer chromatography. By this approach we found twofold differences between leucine specific activity in the growing polypeptide chain of free polysomes and that of membrane-bound polysomes. Moreover, we recorded eight to tenfold differences between the specific activity of leucine in peptidyl-tRNA and that in the acid-soluble pool. Our results indicate and define the intracellular compartmentalization of the leucine pool available for protein synthesis.