Presence Of L-leucine Research Articles

Manipulation of Spray-Drying Conditions to Develop an Inhalable Ivermectin Dry Powder.

SARS-CoV-2, the causative agent of COVID-19, predominantly affects the respiratory tract. As a consequence, it seems intuitive to develop antiviral agents capable of targeting the virus right on its main anatomical site of replication. Ivermectin, a U.S. FDA-approved anti-parasitic drug, was originally shown to inhibit SARS-CoV-2 replication in vitro, albeit at relatively high concentrations, which is difficult to achieve in the lung. In this study, we tested the spray-drying conditions to develop an inhalable dry powder formulation that could ensure sufficient antiviral drug concentrations, which are difficult to achieve in the lungs based on the oral dosage used in clinical trials. Here, by using ivermectin as a proof-of-concept, we evaluated spray-drying conditions that could lead to the development of antivirals in an inhalable dry powder formulation, which could then be used to ensure sufficient drug concentrations in the lung. Thus, we used ivermectin in proof-of-principle experiments to evaluate our system, including physical characterization and in vitro aerosolization of prepared dry powder. The ivermectin dry powder was prepared with a mini spray-dryer (Buchi B-290), using a 23 factorial design and manipulating spray-drying conditions such as feed concentration (0.2% w/v and 0.8% w/v), inlet temperature (80 °C and 100 °C) and presence/absence of L-leucine (0% and 10%). The prepared dry powder was in the size range of 1–5 μm and amorphous in nature with wrinkle morphology. We observed a higher fine particle fraction (82.5 ± 1.4%) in high feed concentration (0.8% w/v), high inlet temperature (100 °C) and the presence of L-leucine (10% w/w). The stability study conducted for 28 days confirmed that the spray-dried powder was stable at 25 ± 2 °C/<15% RH and 25 ± 2 °C/ 53% RH. Interestingly, the ivermectin dry powder formulation inhibited SARS-CoV-2 replication in vitro with a potency similar to ivermectin solution (EC50 values of 15.8 µM and 14.1 µM, respectively), with a comparable cell toxicity profile in Calu-3 cells. In summary, we were able to manipulate the spray-drying conditions to develop an effective ivermectin inhalable dry powder. Ongoing studies based on this system will allow the development of novel formulations based on single or combinations of drugs that could be used to inhibit SARS-CoV-2 replication in the respiratory tract.

Improving the dynamic properties of silk particles by co-spray drying with L-leucine

In this work, we describe a novel approach to engineer silk into composite micro-sized particles by a combination of wet milling and homogenization followed by co-spray drying of the silk micro-particle suspension with L-leucine. We report the first example of the formation of a discrete L-leucine layer onto a composite micro-powder via co-spray drying of the milled suspension. We used milled silk suspension with or without subsequent high-pressure homogenisation. High pressure homogenization was done to further reduce particle size and to understand if the change in size could change the interaction with L-leucine during spray drying. Powder rheometric results revealed that the co-spray dried silk powder (both unhomogenised and homogenised samples) has substantially modified the dynamic bulk properties compared to untreated powder, and the bulk behaviour has varied with the level of L-leucine addition. The silk powder without L-leucine exhibited higher compressibility (35%) than the L-leucine-added samples (25%) at 1 kPa pressure. Overall, the outcomes from this study revealed that L-leucine co-spray drying has reduced the cohesiveness and improved the stability and flowability of the silk powders irrespective of the final step of homogenization was performed or not. There were no substantial changes in particle size in the presence of L-leucine, based on laser size distribution measurements. These improved properties are expected to aid and enable further formulation processing and applications of silk protein powder in diverse fields such as cosmetics, composites, biomedical, and others.

Influence of mutation in the regulatory domain of α-isopropylmalate synthase from Saccharomyces cerevisiae on its activity and feedback inhibition.

Isoamyl alcohol (i-AmOH) is produced from α-ketoisocaproate in the l-leucine biosynthetic pathway in yeast and controlled by the negative feedback regulation of α-isopropylmalate synthase (IPMS), which senses the accumulation of l-leucine. It is known that i-AmOH production increases when mutations in the regulatory domain reduce the susceptibility to feedback inhibition. However, the impact of mutations in this domain on the IPMS activity has not been examined. In this study, we obtained 5 IPMS mutants, encoding the LEU4 gene, N515D/S520P/S542F/A551D/A551V, that are tolerant to 5,5,5-trifluoro-dl-leucine. All mutant proteins were purified and examined for both IPMS activity and negative feedback activity by in vitro experiments. The results showed that not only the negative-feedback regulation by l-leucine was almost lost in all mutants, but also the IPMS activity was greatly decreased and the difference in IPMS activity among Leu4 mutants in the presence of l-leucine was significantly correlated with i-AmOH production.

Combination of Melatonin and Small Molecules Improved Reprogramming Neural Cell Fates via Autophagy Activation.

Reprogramming cell fates towards mature cell types are a promising cell supply for treating degenerative diseases. Recently, transcription factors and some small molecules have turned into impressive modulating elements for reprogramming cell fates. Melatonin, a pineal hormone, has neuroprotective functions including neural stem cell (NSC) proliferative and differentiative modulation in both embryonic and adult brain. We developed a protocol that could be implemented in the direct reprogramming of human skin fibroblast towards neural cells by using histone deacetylase (HDAC) inhibitor, glycogen synthase kinase-3 (GSK3) inhibitor (CHIR99021), c-Jun N-terminal kinase(JNK) inhibitor, rho-associated protein kinase inhibitor (Y-27632), cAMP activator, and melatonin treatment. We found that melatonin enhanced neural-transcription factor genes expressions, including brain-specific homeobox/POU domain protein 2 (BRN2), Achaete-Scute Family BHLH transcription Factor 1 (ASCL1), and Myelin Transcription Factor 1 Like (MYT1L). Melatonin also increased the expression of different neural-specific proteins such as doublecortin (DCX), Sex determining region Y-box 2 (Sox2), and neuronal nuclei (NeuN) compared with other five small molecules (valproic acid (VPA), CHIR99021, Forskolin, 1,9 pyrazoloanthrone (SP600125), and Y-27632) combination in the presence and absence of melatonin. A noticeable upregulation of autophagy proteins (microtubule-associated protein 1A/1B-light chain 3 (LC3) and Beclin-1) were seen in the melatonin treatment during the induction period while these were reverted in the presence of L-leucine, an autophagy inhibitor. In addition, the expression of NeuN was also significantly reduced by L-leucine. Collectively, our findings revealed an activation of autophagy during neural induction; melatonin enhanced reprogramming efficiency for neuron induction through the modulation of autophagy activation.

Use of leucine to improve aerodynamic properties of ciprofloxacin-loaded maltose microparticles for inhalation

Ciprofloxacin (CIP) apparent permeability and absorption rate across the pulmonary epithelium can be controlled by its complexation with copper (II) ion. The aim of the current study was to formulate CIP-Cu-loaded microparticles comprising three main excipients, calcium carbonate, maltose and L-leucine, and to process by spray drying so as to generate particles with suitable aerodynamic properties for pulmonary delivery using a dry powder inhaler. Different maltose:calcium carbonate ratios were used to prepare microparticles, and the role of the excipients on the particles’ physicochemical properties, stability, and aerosolization characteristics were investigated. All the formulations without L-leucine were fully X-ray amorphous. In the presence of L-leucine, diffraction peaks of low intensity were observed, which were attributed to the crystallization of the L-leucine at the particle surfaces. The addition of L-leucine modified the particle morphology and reduced the median geometric and aerodynamic diameters to 3.2 and 3.4 µm, respectively. The fine particle fraction of powder emitted from a Handihaler® device was increased up to 65.4%, predicting high total lung deposition. Stability studies showed that the powder X-ray diffraction pattern did not change over 21 months of storage in desiccated conditions, suggesting a good physical stability of the optimized formulation comprised of CIP-Cu, maltose and L-Leucine.

Inhaled powder formulation of naked siRNA using spray drying technology with l-leucine as dispersion enhancer

Pulmonary delivery of short interfering RNA (siRNA) has been widely studied in both animal and clinical studies to treat various respiratory diseases by gene silencing through RNA interference. Some of these studies showed that the administration of naked siRNA (without the use of any delivery vectors) could achieve satisfactory gene silencing effect, a unique feature to pulmonary delivery. Liquid aerosols were mostly used with very limited studies on the use of powder aerosols for siRNA. In this study, siRNA was co-spray dried with mannitol and l-leucine, the latter being a dispersion enhancer. To the best of our knowledge, this is the first time that siRNA in its naked form was formulated into an inhalable dry powder using spray drying technology. The aerosol performance of the powder was evaluated by Next Generation Impactor (NGI). The presence of l-leucine in the formulation could improve the aerosolization of siRNA-containing powders. Results from the X-ray photoelectron spectroscopy (XPS) suggested that l-leucine was enriched on the particle surface and promote powder dispersion. Among the different siRNA formulations being examined, the one that contained 50% w/w of l-leucine exhibited the best aerodynamic performance, with a high emitted fraction (EF) of around 80% and a modest fine particle fraction (FPF) of 45%. Importantly, the integrity of siRNA was successfully retained as evaluated by gel retardation assay and high performance liquid chromatography (HPLC).

Dry powder aerosols of curcumin-chitosan nanoparticle complex prepared by spray freeze drying and their antimicrobial efficacy against common respiratory bacterial pathogens

While the therapeutic benefits of curcumin delivery to the lung to treat various pulmonary disorders have been established, development of inhaled curcumin formulation that can address its inherently low aqueous solubility remains lacking. Although curcumin nanocapsules prepared by conventional encapsulation methods can improve the dissolution rate, their intricate preparation makes them less attractive for widespread implementation. Recently, our group developed a new class of curcumin nanoparticles in the form of curcumin-chitosan nanoparticle complex (or curcumin nanoplex in short) by a simple, cost-effective, and highly efficient method based on self-assembly drug-polysaccharide complexation. Owing to its nanosize and amorphous state, the curcumin nanoplex possessed high supersaturation generation capability upon dissolution that in turn produced high apparent solubility of curcumin.In the present work, we developed dry powder aerosol formulation of the curcumin nanoplex by spray freeze drying (SFD) using l-leucine and d-mannitol as adjuvants. The curcumin nanoplex aerosols were found to exhibit excellent aerosolization efficiency attributed to their large and low-density morphology, and the presence of l-leucine – a well-established aerosol dispersion enhancer – in their matrix. The aerosols, however, exhibited weaker supersaturation generation capability compared to the aqueous nanoplex suspension due to their slower dissolution rates caused by irreversible aggregations of the nanoplex during SFD. Nevertheless, the curcumin nanoplex aerosols still produced apparent solubility that was approximately 50% higher than the native curcumin’s solubility, thus signifying their dissolution enhancement capability. Despite their slower dissolution rate, the curcumin nanoplex aerosols maintained the same antimicrobial activity as the nanoplex suspension against four clinically-derived respiratory bacterial pathogens (i.e. Pseudomonas aeruginosa, Burkholderia cepacia, Klebsiella pneumoniae, and Stenotrophomonas maltophilia). Lastly, the aerosols exhibited minimal cytotoxicity towards the lung epithelium cells just like the native curcumin.

정신분열증 치료제에 의한 사람 글루탐산염 탈수소효소 동종효소의 억제효과

Glutamate is one of the major excitatory neurotransmitters in the central nervous system of vertebrates. Human GDH (hGDH) is the enzyme that regulates the glutamate metabolism and its expression is higher in the brains of schizophrenia patients than in normal subjects. This study examined the changes in the hGDH enzymatic activity caused by antipsychotic drugs (haloperidol, risperidone, (±)-sulpride, chlopromazine hydrochloride, melperone, (±)butaclamol, domperidone, clozapine) related to schizophrenia. First of all, hGDH isozymes (hGDH1, hGDH2) were synthesized by genetic recombination. As a result of the enzyme assay, haloperidol, (±)-sulpride, melperone and clozapine had an inhibitory effect on the hGDH isozymes. In addition, haloperidol showed a non-competitive inhibition against the substrate, 2-oxoglutarate. In contrast, it showed an uncompetitive inhibition against another substrate, NADH. The inhibitory effect of haloperidol on hGDH2 was abolished by the presence of L-leucine, an allosteric effector of hGDH, but by not other antipsychotic drugs. These results revealed the inhibition of enzyme activity by psychotropic drugs in hGDH isoenzymes (hGDH1 and hGDH2) and the possibility that haloperidol may be used to regulate the GDH activity and glutamate concentration in the central nervous system.

Evolutionarily distinct versions of the multidomain enzyme α-isopropylmalate synthase share discrete mechanisms of V-type allosteric regulation.

Understanding the evolution of allostery in multidomain enzymes remains an important step in improving our ability to identify and exploit structure-function relationships in allosteric mechanisms. A recent protein similarity network for the DRE-TIM metallolyase superfamily indicated there are two evolutionarily distinct forms of the enzyme α-isopropylmalate synthase (IPMS) sharing approximately 20% sequence identity. IPMS from Mycobacterium tuberculosis has been extensively characterized with respect to catalysis and the mechanism of feedback regulation by l-leucine. Here, IPMS from Methanococcus jannaschii (MjIPMS) is used as a representative of the second form of the enzyme, and its catalytic and regulatory mechanism is compared with that of MtIPMS to identify any functional differences between the two forms. MjIPMS exhibits kinetic parameters similar to those of other reported IPMS enzymes and is partially inhibited by l-leucine in a V-type manner. Identical values of (D2O)kcat (3.1) were determined in the presence and absence of l-leucine, indicating the hydrolytic step is rate-determining in the absence of l-leucine and remains so in the inhibited form of the enzyme. This mechanism is identical to the mechanism identified for MtIPMS ((D2O)kcat = 3.3 ± 0.3 in the presence of l-leucine) despite product release being rate-determining in the uninhibited MtIPMS enzyme. The identification of identical regulatory mechanisms in enzymes with low sequence identity raises important evolutionary questions concerning the acquisition and divergence of multidomain allosteric enzymes and highlights the need for caution when comparing regulatory mechanisms for homologous enzymes.

Conversion of L-leucine to isovaleric acid by Propionibacterium freudenreichii TL 34 and ITGP23.

Several branched-chain volatile compounds are involved in the flavor of Swiss cheese. These compounds are probably produced by enzymatic conversion of branched-chain amino acids, but the flora and the pathways involved remain hypothetical. Our aim was to determine the ability of Propionibacterium freudenreichii, which is one of the main components of the secondary flora of Swiss cheese, to produce flavor compounds during leucine catabolism. Cell extracts and resting cells of two strains were incubated in the presence of L-leucine, alpha-ketoglutaric acid, and cofactors, and the metabolites produced were determined by high-performance liquid chromatography and gas chromatography. The first step of leucine catabolism was a transamination that produced alpha-ketoisocaproic acid, which was enzymatically converted to isovaleric acid. Both reactions were faster at pH 8.0 than at acidic pHs. Cell extracts catalyzed only the transamination step under our experimental conditions. Small amounts of 3-methylbutanol were also produced by resting cells, but neither 3-methylbutanal noralpha-hydroxyisocaproic acid was detected. L-Isoleucine and L-valine were also converted to the corresponding acids and alcohols. Isovaleric acid was produced by both strains during growth in a complex medium, even under conditions simulating Swiss cheese conditions (2.1% NaCl, pH 5.4, 24 degrees C). Our results show that P. frendenreichii could play a significant role in the formation of isovaleric acid during ripening.

Molecular characterization of l-amino acid oxidase from Agkistrodon halys blomhoffii with special reference to platelet aggregation

l-Amino acid oxidase (LAO, EC 1.4.3.2) is widely distributed in snake venom, and induces apoptosis in vascular endothelial cells, causing prolonged bleeding from vessel walls at bite sites. The effect of snake venom LAOs on platelet function is controversial. Further, we have little information on their structural characterization. We purified M (mamushi)-LAO, a single-chain glycoprotein with a molecular mass of 60 kDa and a p I of 4.9, from Agkistrodon halys blomhoffii (Japanese mamushi) venom, and determined the N-terminal and several internal amino acid sequences of this enzyme. Molecular cloning based on these data was conducted to elucidate its full-length cDNA structure (2192 nucleotides), which includes a putative 18 amino acid residue signal peptide and a 504 residue mature subunit. The predicted M-LAO translation product shares 87.3% identity with that of Crotalus adamanteus (Southeastern diamondback rattlesnake) LAO. M-LAO, up to a final concentration of 2.6 μM, inhibited both agonist- and shear stress-induced platelet aggregation (SIPA) dose-dependently. In agonist-induced platelet aggregation, M-LAO predominantly inhibited the second aggregation, but with a marginal inhibition of the first. In SIPA, the inhibition was more dramatic under low-shear stress than high-shear stress, and was enhanced by the presence of l-leucine, a substrate of this enzyme. Catalase, a H 2O 2 scavenger, totally quenched such enhancement. These results suggest that M-LAO inhibits the interaction between activated platelet integrin αIIb/β3 and fibrinogen through the continuous generation of H 2O 2, and may contribute to prolonged bleeding from the vessels at snake bite sites.

Purification and characterization of Sa-lrp, a DNA-binding protein from the extreme thermoacidophilic archaeon Sulfolobus acidocaldarius homologous to the bacterial global transcriptional regulator Lrp.

Archaea, constituting the third primary domain of life, harbor a basal transcription apparatus of the eukaryotic type, whereas curiously, a large fraction of the potential transcription regulation factors appear to be of the bacterial type. To date, little information is available on these predicted regulators and on the intriguing interplay that necessarily has to occur with the transcription machinery. Here, we focus on Sa-lrp of the extremely thermoacidophilic crenarchaeote Sulfolobus acidocaldarius, encoding an archaeal homologue of the Escherichia coli leucine-responsive regulatory protein Lrp, a global transcriptional regulator and genome organizer. Sa-lrp was shown to produce a monocistronic mRNA that was more abundant in the stationary-growth phase and produced in smaller amounts in complex medium, this down regulation being leucine independent. We report on Sa-Lrp protein purification from S. acidocaldarius and from recombinant E. coli, both identified by N-terminal amino acid sequence determination. Recombinant Sa-Lrp was shown to be homotetrameric and to bind to its own control region; this binding proved to be leucine independent and was stimulated at high temperatures. Interference binding experiments suggested an important role for minor groove recognition in the Sa-Lrp-DNA complex formation, and mutant analysis indicated the importance for DNA binding of the potential helix-turn-helix motif present at the N terminus of Sa-Lrp. The DNA-binding capacity of purified Sa-Lrp was found to be more resistant to irreversible heat inactivation in the presence of L-leucine, suggesting a potential physiological role of the amino acid as a cofactor.

Acetone formation in the Vibrio family: a new pathway for bacterial leucine catabolism.

There is current interest in biological sources of acetone, a volatile organic compound that impacts atmospheric chemistry. Here, we determined that leucine-dependent acetone formation is widespread in the Vibrionaceae. Sixteen Vibrio isolates, two Listonella species, and two Photobacterium angustum isolates produced acetone in the presence of L-leucine. Shewanella isolates produced much less acetone. Growth of Vibrio splendidus and P. angustum in a fermentor with controlled aeration revealed that acetone was produced after a lag in late logarithmic or stationary phase of growth, depending on the medium, and was not derived from acetoacetate by nonenzymatic decarboxylation in the medium. L-Leucine, but not D-leucine, was converted to acetone with a stoichiometry of approximately 0.61 mol of acetone per mol of L-leucine. Testing various potential leucine catabolites as precursors of acetone showed that only alpha-ketoisocaproate was efficiently converted by whole cells to acetone. Acetone production was blocked by a nitrogen atmosphere but not by electron transport inhibitors, suggesting that an oxygen-dependent reaction is required for leucine catabolism. Metabolic labeling with deuterated (isopropyl-d(7))-L-leucine revealed that the isopropyl carbons give rise to acetone with full retention of deuterium in each methyl group. These results suggest the operation of a new catabolic pathway for leucine in vibrios that is distinct from the 3-hydroxy-3-methylglutaryl-coenzyme A pathway seen in pseudomonads.

Resistance of the insulinotropic action of alpha-D-glucose and beta-L-glucose pentaacetates to cholera and pertussis toxins.

The pentaacetate esters of alpha-D-glucose and beta-L-glucose were recently reported to stimulate insulin release. The possible participation of G-protein-coupled receptors to the insulinotropic action of these esters was investigated in rat pancreatic islets either preincubated with cholera toxin or obtained from animals injected with pertussis toxin. Neither procedure affected adversely the secretory response to the esters in islets incubated in the presence of L-leucine. Thus, in both situations, alpha-D-glucose pentaacetate and, to a lesser extent, beta-L-glucose pentaacetate augmented insulin release evoked by the branched-chain amino acid, whilst beta-L-galactose pentaacetate failed to do so. These findings suggest that G-proteins sensitive to either cholera or pertussis toxins are not involved in one of the two modalities by which these esters are thought to stimulate insulin secretion, namely that independent of the catabolic fate of their hexose moieties.

Functional Heterogeneity of Single Pancreatic β-Cells Stimulated by L-Leucine and the Methyl Ester of Succinic or Glutamic Acid

Single pancreatic β-cells exposed to D-glucose in the absence or presence of L-leucine and to the amino acid in the absence or presence of either the monomethyl ester of succinic acid (SME) or the dimethyl ester of glutamic acid (SME) were examined in a reverse hemolytic plaque assay for insulin release. At a D-glucose concentration of 11.1 mM, plaque-forming cells amounted to 84.8 ± 1.0% including 11.4 ± 4.1% of cells forming large plaques. These percentages were not increased by the incorporation of L-leucine (5 mM) into the incubation medium despite the fact that the amino acid, when tested in the absence of D-glucose, caused sizeable secretory activity. When the secretory response evoked by L-leucine was increased by either SME (10 mM) or GME (3 mM), marked heterogeneity of individual β-cells in terms of both the occurrence and magnitude of hemolytic plaques was again observed. These findings argue against the view that the heterogeneity in secretory behavior of isolated purified β-cells can be accounted for solely by differences in hexose metabolism as conceivably attributable to individual variations in glucokinase activity.

The influence of additives on the growth kinetics and mechanism of l-alanine crystals

Results for the characterization of the growth kinetics and mechanism of l-alanine crystals, grown from aqueous solution in the presence and absence of tailor-made additives are reported. l-Alanine crystals grown from water in the absence of additives are prismatic, elongated along the crystallographic c-axis bounded by the {120}, {010}, {110}, {210} and {011} faces. The growth rate of l-alanine crystals was measured by monitoring the change of size of individual crytals as a function of time. Examination of both the growth rate dependence on supersaturation and the structure of the crystal surface, reveals that the {120}, {010}, and {011} crystal faces of l-alanine grow following the spiral growth mechanism, both in the presence and absence of additives. This mechanism was confirmed by examining the surface topography of crystals by atomic force microscope. The growth rate of l-alanine crystals in the presence of l-leucine and l-phenylalanine at concentrations as low as 0.02 m (i.e., 0.3 g additive/100 g solvent or 0.18 g additive/100 g l-alanine) is drastically reduced and the crystals are bounded by the {120} and {011} faces. The inhibitory effect of these additives is explained by a Langmuir isotherm, assuming that the inhibition of the growth rate is proportional to the degree of surface coverage and that the crystal surface is homogeneous with respect to the energy of adsorption sites.

Leucine and serine induce mecillinam resistance in Escherichia coli.

We have previously shown that resistance to the beta-lactam mecillinam in Escherichia coli can be brought about by a high ppGpp pool, as observed under conditions of partial amino acid starvation and RelA-dependent induction of the stringent response. We show here that our E. coli wild-type strain, which is sensitive to mecillinam on minimal glucose plates, becomes resistant in the presence of L-leucine or L-serine (or cysteine, which inactivates the antibiotic). The resistance, which is not a transient effect and does not depend on the physiological state of the cells when plated, is specific for mecillinam and is reversed by the presence of isoleucine and valine in the medium. At least in the case of serine, the resistance is RelA-dependent. We conclude that the presence of leucine and serine in the growth medium cause partial starvation for isoleucine/valine, leading to induction of the stringent response and concomitant resistance to mecillinam.

Leucine dehydrogenase from Bacillus stearothermophilus: identification of active-site lysine by modification with pyridoxal phosphate.

We have constructed an efficient expression plasmid for the leucine dehydrogenase gene previously cloned from Bacillus stearothermophilus. The recombinant enzyme was overproduced in Escherichia coli cells to a level of more than 30% of the total soluble protein upon induction with isopropyl beta-D-thiogalactopyranoside. The enzyme could be readily purified to homogeneity by heat treatment and a single step of ion-exchange chromatography. The purified enzyme was inactivated in a time-dependent manner upon incubation with pyridoxal 5'-phosphate (PLP) followed by reduction with sodium borohydride. The inactivation was completely prevented in the copresence of L-leucine and NAD+. Concomitantly with the inactivation, several molecules of PLP were incorporated into each subunit of the hexameric enzyme. Sequence analysis of the fluorescent peptides isolated from a proteolytic digest of the modified protein revealed that Lys80, Lys91, Lys206, and Lys265 were labeled. Among these residues, Lys80 was predominantly labeled and, in the presence of L-leucine and NAD+, was specifically protected from the labeling. Furthermore, a linear relationship of about 1:1 was observed between the extent of inactivation and the amount of PLP incorporated into Lys80. A slightly active mutant enzyme, in which Lys80 is replaced by Ala, was not inactivated at all by incubation with PLP, showing that the inactivation is correlated with the labeling of only Lys80. Lys80is conserved in the corresponding regions of all the amino acid dehydrogenase sequences reported to date. These results suggest that Lys80 is located at the active site and plays an important role in the catalytic function of leucine dehydrogenase.

The leucine regulon of Escherichia coli K-12: a mutation in rblA alters expression of L-leucine-dependent metabolic operons

We have isolated and characterized a highly pleiotropic Escherichia coli mutant affected in the activity of a number of enzymes involved in different metabolic pathways, all of which are regulated by leucine. Selected for its ability to grow with L-serine as sole carbon source, the rbl-1::Tn10 mutant had high levels of L-serine deaminase activity (due to increased transcription of the structural gene) and of another amino acid-degrading enzyme, L-threonine dehydrogenase, and decreased transcription of the operons serA and ilvIH, coding for biosynthetic enzymes. The rbl mutation suppressed the slow growth of a metK mutant, deficient in S-adenosylmethionine synthetase. Furthermore, metK mutants spontaneously accumulated faster-growing rbl-like derivatives, and a commonly used metK strain, RG62, carries such a mutation. The rbl gene is located near 20 min on the E. coli genetic map. All phenotypes of the rbl mutant could be observed in rbl+ strains cultivated in the presence of L-leucine, and exogenous L-leucine had little further effect on the rbl strains. We propose that the rbl gene product is the regulator of a global response to leucine.

Anomeric specificity of the insulin and glucagon secretory responses to D-glucose in lean and obese Zucker rats.

The perfused pancreas of lean and obese Zucker rats was exposed, in the presence of L-leucine, to the anomers of D-glucose, which were administered on four successive occasions in the alpha/beta/alpha/beta or beta/alpha/beta/alpha sequence. In 5 lean and 6 obese rats, alpha-D-glucose was more efficient than beta-D-glucose in both stimulating insulin secretion and suppressing glucagon release. Although D-glucose evoked a greater release of insulin and, during prolonged exposure to the hexose, a less pronounced suppression of glucagon secretion in obese than lean rats, the anomeric specificity of these secretory responses was not different in the two groups of animals. In one lean rat, however, alpha-D-glucose, while efficiently stimulating insulin release, failed on two occasions to inhibit glucagon secretion. This isolated observation raises the possibility that the anomeric specificity of functional events evoked by D-glucose in the endocrine pancreas may occasionally be perturbed.