Presence Of L-lysine Research Articles

Enhanced stabilization of collagen-based dermal regeneration scaffolds through the combination of physical and chemical crosslinking

The use of collagen in the biomedical device industry has led to major advances in soft tissue repair. This is attributed largely to the favourable biological and physiochemical properties of collagen. Regenerative medicine and tissue engineering favoured the use of this biomaterial and various commercial products have become available in the past few decades. This study aims to develop a collagen and chondroitin-6-sulphate dermal regeneration scaffold with enhanced resistance against enzymatic degradation. Frozen slurries (0.5% collagen) were dried under vacuum, coated with silicone, crosslinked and then thoroughly rinsed. The scaffolds were subjected to a range of quantitative and qualitative tests that included: scanning electron microscopy analysis, collagenase enzymatic degradation, and cytotoxicity assessment. Scaffold resistance to enzymatic degradation was manipulated after dehydrothermal treatment by employing combinations of crosslinking agents, such as glutaraldehyde and/or carbodiimide, with or without the presence of L-lysine. Results indicate that highly porous (mean pore diameter of 87.3 µm), bioactive, non-cytotoxic tissue engineering matrices were obtained. Enhanced stability of these scaffolds was achieved through extensive crosslinking and suggests the potential to prevent in vivo wound contraction sufficiently.

Inhibition of Human Dimethylarginine Dimethylaminohydrolase-1 by S-Nitroso-L-homocysteine and Hydrogen Peroxide

The plasma concentrations of two cardiovascular risk factors, total homocysteine (tHcy) and asymmetric dimethylarginine (ADMA), correlate with decreased levels of endothelium-derived nitric oxide and subsequent endothelial dysfunction. Homocysteine has been proposed to inhibit the catabolic enzyme of ADMA, dimethylarginine dimethylaminohydrolase (DDAH), but the mechanism of this inhibition has not been fully elucidated. Here, the human DDAH isoform-1 (DDAH-1) is heterologously expressed and purified. Cys(274) and His(173) are identified as active site residues and the pH rate dependence is described. Because oxidation of the active site Cys has been suggested as an inhibitory mechanism in patients with hyperhomocysteinemia, the sensitivity of DDAH-1 to inhibition by L-homocysteine, H(2)O(2), and S-nitroso-L-homocysteine is quantified. DDAH-1 is surprisingly insensitive to inactivation by the powerful oxidant, H(2)O(2) (0.088 M(-1) s(-1)), possibly because of a substrate-assisted mechanism that allows the active site cysteine to remain predominantly protonated and less reactive in the resting enzyme. In contrast, DDAH-1 is sensitive to inactivation by S-nitroso-L-homocysteine (3.79 M(-1) s(-1)). This work illustrates how a particular catalytic mechanism can result in selective redox regulation and has possible implications for hyperhomocysteinemia.

Binding Energy in the One-Electron Reductive Cleavage of S-Adenosylmethionine in Lysine 2,3-Aminomutase, a Radical SAM Enzyme

The common step in the actions of members of the radical SAM superfamily of enzymes is the one-electron reductive cleavage of S-adenosyl-l-methionine (SAM) into methionine and the 5'-deoxyadenosyl radical. The source of the electron is the [4Fe-4S]1+ cluster characterizing the radical SAM superfamily, to which SAM is directly ligated through its methionyl carboxylate and amino groups. The energetics of the reductive cleavage of SAM is an outstanding question in the actions of radical SAM enzymes. The energetics is here reported for the action of lysine 2,3-aminomutase (LAM), which catalyzes the interconversion of l-lysine and l-beta-lysine. From earlier work, the reduction potential of the [4Fe-4S]2+/1+ cluster in LAM is -0.43 V with SAM bound to the cluster (Hinckley, G. T., and Frey, P. A. (2006) Biochemistry 45, 3219-3225), 1.4 V higher than the reported value for trialkylsulfonium ions in solution. The midpoint reduction potential upon binding l-lysine has been estimated to be -0.6 V from the values of midpoint potentials measured with SAM bound to the cluster and l-alanine in place of l-lysine, with S-adenosyl-l-homocysteine (SAH) bound to the cluster in the presence of l-lysine, and with SAH bound to the cluster in the presence of l-alanine or of l-alanine and ethylamine in place of l-lysine. The reduction potential for SAM has been estimated to be -0.99 V from the measured value for S-3',4'-anhydroadenosyl-l-methionine. The reduction potential for the [4Fe-4S] cluster is lowered 0.17 V by the binding of lysine to LAM, and the binding of SAM to the [4Fe-4S] cluster in LAM elevates its reduction potential by 0.81 V. Thus, the binding of l-lysine to LAM contributes 4 kcal mol-1, and the binding of SAM to the [4Fe-4S] cluster in LAM contributes 19 kcal mol-1 toward lowering the barrier for reductive cleavage of SAM from 32 kcal mol-1 in solution to 9 kcal mol-1 at the active site of LAM.

L-lysine as a recognition molecule for the VAP-1 function of SSAO

Semicarbazide-sensitive amine oxidase (EC 1.4.3.6) acts as a vascular-adhesion protein (VAP-1), mediating the adhesion of lymphocytes to vascular endothelial cells under inflammatory conditions. The relationship between the adhesive and the enzymatic functions of SSAO have not yet been fully defined. Previous studies from this laboratory showed aminohexoses, which were neither substrates nor direct inhibitors of SSAO, bound to the enzyme as reversible inhibitors in the presence of H(2)O(2) generated during substrate oxidation. The possibility that surface L-lysine could act similarly has been investigated in the present study. The presence of L-lysine during the oxidation of benzylamine resulted in time- and dose-dependent inhibition of SSAO activity, in a process that was dependent on the H(2)O(2) formed during benzylamine oxidation. The possible implications of this in terms of the therapeutic uses of lysine are discussed.

Separation of l-lysine from dilute aqueous solution using molecular imprinting technique

In the present study, separation of l-lysine from dilute aqueous solution by solid-phase extraction based on molecular imprinting technique using a polar porogen was investigated. l-Lysine imprinted polymer (LLIP) was prepared by free radical solution polymerization of methacrylic acid and ethylene glycol dimethacrylate as functional and cross-linking monomers, in the presence of l-lysine as an imprint molecule, mixture of water and methanol as solvent and AIBN as an initiator. Non-imprinted polymer (NIP) as control was also prepared by the same procedure in the absence of template molecules. LLIP particles were applied to determine the optimum operational condition for l-lysine separation from dilute aqueous solution. In adsorption step, optimum pH and retention time were 7.8 and 90 min, while corresponding values in extraction step were 12 and 50 min, respectively. l and d-Lysine recovery by LLIP at optimum condition were found to be 96 and 58% with corresponding distribution coefficients of 8000 and 460, respectively. The retention capacity of LLIP was 27.26 mg l-lys/g of polymer at optimum condition.

Microbacterium deminutum sp. nov., Microbacterium pumilum sp. nov. and Microbacterium aoyamense sp. nov.

Three novel bacterial strains were isolated from a soil sample collected in Japan by culture on a GPM agar plate supplemented with superoxide dismutase and catalase. The strains were Gram-positive, catalase-positive, non-motile bacteria with L-ornithine as a diagnostic diamino acid of the peptidoglycan. The acyl type of the peptidoglycan was N-glycolyl. The major menaquinones were MK-12, 13 and 14. Mycolic acids were not detected. G+C contents of the DNA were in the range 69-71 mol%. Comparative 16S rRNA gene sequence analysis revealed that the isolates belonged to the genus Microbacterium and were closely related to Microbacterium terregens, Microbacterium aurum, Microbacterium koreense, Microbacterium schleiferi and Microbacterium lacticum. However, M. aurum, M. koreense and M. lacticum clearly differed from the isolated strains based on the presence of L-lysine as the cell-wall diamino acid and various other chemotaxonomic characteristics. Levels of DNA-DNA relatedness showed that the isolated strains represented three separate genomic species. Based on both phenotypic and genotypic data, the following novel species of the genus Microbacterium are proposed: Microbacterium deminutum sp. nov. (type strain KV-483(T)=NRRL B-24453(T)=NBRC 101278(T)), Microbacterium pumilum sp. nov. (type strain KV-488(T)=NRRL B-24452(T)=NBRC 101279(T)) and Microbacterium aoyamense sp. nov. (type strain KV-492(T)=NRRL B-24451(T)=NBRC 101280(T)).

Sensitive determination of l-lysine with a new amperometric microbial biosensor based on Saccharomyces cerevisiae yeast cells

A new amperometric microbial biosensor based on Saccharomyces cerevisiae NRRL-12632 cells, which had been induced for lysine oxidase enzyme and immobilized in gelatin by a cross-linking agent was developed for the sensitive determination of L-lysine amino acid. To construct the microbial biosensor S. cerevisiae cells were activated and cultured in a suitable culture medium. By using gelatine (8.43 mg cm(-2)) and glutaraldehyde (0.25%), cells obtained in the logarithmic phase of the growth curve at the end of a 14 h period were immobilized and fixed on a pretreated oxygen sensitive Teflon membrane of a dissolved oxygen probe. The assay procedure of the microbial biosensor is based on the determination of the differences of the respiration activity of the cells on the oxygenmeter in the absence and the presence of L-lysine. According to the end point measurement technique used in the experiments it was determined that the microbial biosensor response depended linearly on L-lysine concentrations between 1.0 and 10.0 microM with a 1 min response time. In optimization studies of the microbial biosensor, the most suitable microorganism quantities were found to be 0.97x10(5)CFU cm(-2). In addition phosphate buffer (pH 7.5; 50 mM) and 30 degrees C were obtained as the optimum working conditions. In characterization studies of the microbial biosensor some parameters such as substrate specificity, interference effects of some substances on the microbial biosensor responses, reproducibility of the biosensor and operational and storage stability were investigated.

Pharmacokinetics and biodistribution of 177Lu-labeled multivalent single-chain Fv construct of the pancarcinoma monoclonal antibody CC49

Lutetium-177 (177Lu) is a radionuclide of interest for radioimmunoimaging (RII) and radioimmunotherapy (RIT) on account of its short half-life (161 h) and the ability to emit both beta and gamma radiation. Single-chain Fv (scFv) constructs have shown advancement in cancer diagnosis and therapy due to the pharmacokinetics advantage and seem to be intriguing tools in oncology. The objective of this study was to evaluate the pharmacokinetics and biodistribution characteristics of the 177Lu-labeled tetravalent scFv of CC49 MAb and intact CC49 IgG in vivo. Conjugation and labeling conditions of multivalent scFv with 177Lu were optimized without affecting integrity and immunoreactivity. For this purpose, multivalent scFv constructs {dimer, sc(Fv)2; tetramer, [sc(Fv)2]2} of the MAb CC49 were expressed as secretory proteins in Pichia pastoris. The purified scFv constructs and IgG form of CC49 were conjugated with a bifunctional chelating agent, ITCB-DTPA, and labeled with 177Lu. The comparative biodistribution, blood clearance, and tumor-targeting characteristics of 177Lu-labeled tetravalent [sc(Fv)2]2 construct of CC49 MAb and intact CC49 IgG were investigated in the athymic mice bearing LS-174T xenografts. Approximately, 90% of 177Lu incorporation was achieved using ITCB-DTPA chelator, and the labeled immunoconjugates maintained integrity and immunoreactivity. Blood clearance studies demonstrated an alpha half-life (t1/2alpha) of 177Lu-labeled [sc(Fv)2]2 and IgG of CC49 at 4.40 and 9.50 min and a beta half-life (t1/2beta) at 375 and 2,193 min, respectively. At 8 h post administration, the percent of the injected dose accumulated/gram (%ID/g) of the LS-174T tumor was 6.4+/-1.3 and 8.9+/-0.6 for 177Lu-labeled [sc(Fv)2]2 and IgG of CC49, respectively, in the absence of L-lysine. The corresponding values were 8.0+/-0.6 and 8.4+/-1.2 in the presence of L-lysine. Renal accumulation of [sc(Fv)2]2 was significantly (p<0.005) reduced in the presence of L-lysine. The results of this study demonstrate that the ITCB-DTPA conjugation and 177Lu-labeling of scFvs are feasible without influencing the antibody characteristics. 177Lu-labeled [sc(Fv)2]2 showed faster clearance and equivalent tumor uptake at 8 h compared with its IgG form, with a markedly reduced renal uptake in the presence of L-lysine. Therefore, 177Lu-labeled [sc(Fv)2]2 may be a potential radiopharmaceutical for the treatment of cancer.

The Hydrazine Derivative Aminoguanidine Inhibits the Reaction of Tetrahydrofolic Acid with Hydroxymethylarginine Biomolecule

Aminoguanidine (AG), a hydrazine derivative is known to inhibit the formation of Advanced Glycosylation Endproducts (AGE) and AG has been proposed as an agent in prophylaxis of diabetic complications. However, treatment with hydrazine produced liver and lung tumors by formation of N7- and O6-methylguanine in the DNA of rodents. The hydrazine derivative, isonicotinic acid hydrazide induced pulmonary tumors in mice. N(G)-hydroxymethyl-arginine (HMA) was synthesized by our research group and it showed anticancer effect against experimental tumors. HMA was found earlier in human blood and urine, and recently in many plants (in fruits and vegetables). We could demonstrate a reaction (pH = 7.5, 37 degrees C, 1h) between HMA and tetrahydrofolate (THF) producing N5,N10-methylene-tetrahydrofolate (CH2-THF), the coenzyme of thymidylate synthase (TS). In model experiments AG proved to react with formaldehyde (HCHO) and to eliminate the C1-fragment of HMA, but not that of CH2-THF. In the presence of AG burst chemiluminescence and a higher speed of the formylation and methylation reactions were found in the AG, HCHO, hydrogen peroxide (H2O2) and L-lysine system than without AG. HMA as a biomolecule is one of the compounds which are responsible for the endogenous HCHO level. The biochemical function of HMA may be the direct supply of C1-fragment for the folate cycle. AG can disturb the above function of HMA. The reaction between AG and HCHO seems to be dangerous for biological systems because of the possible presence of L-lysine and H2O2. The burst chemiluminescence indicates excited molecules with extreme high energy producing uncontrolled formylation and methylation reactions. Considering the results of the experiments with AG its use as a medicament seems to be questionable.

Preferential production of rapamycin vs prolylrapamycin by Streptomyces hygroscopicus

A trace of prolylrapamycin is often produced in rapamycin fermentations carried out by strains of Streptomyces hygroscopicus. Prolylrapamycin was produced as the major rapamycin when L-proline was added to the fermentation medium. Addition of proline plus thiazolidine-2-carboxylic acid (T2CA), a sulfur-containing proline analog, prevented rapamycin production and stimulated prolylrapamycin production, thereby resulting in an even greater selective production of prolylrapamycin. T2CA addition inhibited rapamycin production even in the presence of Llysine which is converted into pipecolic acid intracellularly and normally stimulates rapamycin formation. Addition of the rapamycin precursor, DL-pipecolic acid, surprisingly failed to stimulate rapamycin production. However, when DL-pipecolic acid was added with L-proline, it reduced the formation of prolylrapamycin and stimulated rapamycin production; this was evident especially in the presence of T2CA. The evidence suggests that T2CA suppresses rapamycin production by inhibiting intracellular conversion of L-lysine into pipecolate. Furthermore, the data suggest that uptake of pipecolate into the cell is stimulated or induced by growth in the presence of L-proline and/or T2CA.

Distribution and identification of actinomycetes lysing cyanobacteria in a eutrophic lake

Among 83 actinomycete strains isolated from lake sediments, about half were found to lyse cyanobacteria. One (S-9 strain), identified as Streptomyces phaeofaciens, grew well on lawns of living cyanobacteria and rapidly lysed the cyanobacterial cells. The amino acid, L-lysine, secreted by this isolate, was found to be one cause of lysis. Scanning electron microscopy of cyanobacterial cells incubated in the presence of L-lysine revealed that L-lysine caused severe damage to the cell wall.

Arginine uptake and metabolism in cultured murine macrophages.

Transport of L-arginine and nitrite production were examined in the murine macrophage cell line J774. Lipopolysaccharide (LPS) induced a dose- and time-dependent stimulation of nitrite production which was further increased in the presence of interferon-gamma (IFN-gamma). Nitrite synthesis was dependent on extracellular arginine and inhibited in the presence of L-lysine. Treatment of cells with LPS and IFN-gamma caused a reduction in intracellular L-arginine concentration which was accompanied by increases in the levels of L-citrulline in both the cells and culture medium. These findings indicate that activation of J774 cells with LPS produces an increase in both L-arginine transport and nitrite synthesis. The elevated rate of L-arginine transport in activated J774 cells may provide a mechanism for sustained substrate supply during enhanced utilization of L-arginine for the generation of nitric oxide.

L-arginine transport is increased in macrophages generating nitric oxide.

Transport of L-arginine and nitrite production were examined in the murine macrophage cell line J774. Bacterial lipopolysaccharide (LPS) induced a dose- and time-dependent stimulation of nitrite production, which was further increased in the presence of interferon-gamma. Nitrite synthesis was absolutely dependent on extracellular L-arginine and inhibited in the presence of L-lysine or L-ornithine. In unactivated J774 cells L-arginine transport was saturable, with an apparent Km of 0.14 +/- 0.04 mM and Vmax. of 15 +/- 2 nmol/h per 10(6) cells. LPS (1 microgram/ml) induced a time-dependent stimulation of L-arginine transport, and after 24 h the Vmax. increased to 34 +/- 2 nmol/h per 10(6) cells. These findings indicate that activation of J774 cells with LPS produces an increase in both L-arginine transport and nitrite synthesis. The elevated rate of L-arginine transport in activated J774 cells may provide a mechanism for sustained substrate supply during enhanced utilization of L-arginine for the generation of NO.

Enzymatic production of alpha-aminoadipate-delta-semialdehyde and related compounds by lysine epsilon-dehydrogenase from Candida albicans.

L-Lysine epsilon-dehydrogenase [L-lysine:NADP+ oxidoreductase (epsilon-deaminating), EC 1.4.1.15] of Candida albicans was studied emphasizing its application for the production of alpha-aminoadipate-delta-semialdehyde and related compounds. A high enzyme level (240 pkat/mg of protein in the crude extract) could be attained during growth in the presence of L-lysine as sole nitrogen source. After optimization of the reaction conditions a partial purified enzyme (1.5 nkat/mg of protein) was used to produce alpha-aminoadipate-delta-semialdehyde, S-(beta-acetaldehyde)-cysteine, alpha-amino-delta-hydroxyadipate-semialdehyde and alpha-amino-gamma-hydroxyadipate-semialdehyde from the corresponding substrates lysine, S-(beta-aminoethyl)-cysteine, 5-hydroxylysine and 4-hydroxylysine, respectively. After purification of the compounds using Dowex 50 x 4 chromatography a yield of the products between 4.6 and 6.8% was achieved.

Purification and characterization of an inducible L-lysine: 2-oxoglutarate 6-aminotransferase from Candida utilis.

L-Lysine:2-oxoglutarate 6-aminotransferase (EC 2.1.6.36) was purified 202-fold from the yeast Candida utilis. The subunit Mr estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis was 40 kDa. The Mr of the native enzyme was estimated to be 83 kDa by gel filtration, suggesting a dimeric structure. The enzyme exhibits absorption maxima at 280, 340 and 420 nm, and binds 2 mol pyridoxal-5-phosphate/mol of the native enzyme. The aminotransferase has a maximum activity at pH 8.5 and at 4 degrees C. 2-Oxoglutarate is the best amino acceptor with L-lysine as amino donor. Lower activity is observed with oxaloacetate (38%), pyruvate (19%) and 2-oxoadipate (7%) as acceptor or with L-thialysine (13%) as donor. The Km values are 2.5 mM for L-lysine, 3.8 mM for 2-oxoglutarate and 0.015 mM for pyridoxal-5-phosphate. The enzyme activity is induced in cells grown in the presence of L-lysine.

Formation of excited formaldehyde in model reactions simulating real biological systems

By studying the oxidation of CH 2O by H 2O 2 in the presence of l-lysine under real biological circumstances (pH 7.4, T 298 K) we have found excited CH 2O molecules and singlet oxygen. Analysis of the chemiluminescence spectral distribution allows singlet oxygen lines and a band at 23 000 cm −1 characterizing the 3 A 2→ 1 A 1 transition of formaldehyde to be identified. According to ab initio SCF calculations, in our model reaction excited triplet formaldehyde could be liberated besides the ground state formaldehyde. The lowest 3 A 2 CH 2O state has an extremely long lifetime (about 0.025 s) because of the strongly forbidden triplet→singlet transition. We recognise the biological importance of this excited formaldehyde: because of its biradical character the molecule shows extreme reactivity, and may attack even the smallest reactive regions of biomolecules causing toxicity and carcinogenesis.

Variations of intestinal calcium absorption in adult frogs (Rana esculenta). Effect of lysine.

Intestinal calcium absorption was investigated in an adult frog (Rana esculenta) by injecting a CaCl2 solution containing 45Ca into the lumen. The 45Ca absorption coefficient in the proximal loop was higher than in the distal loop, only when the CaCl2 solution was left for 4 h. This coefficient increased both in the proximal and distal loops when a 4-h treatment was substituted for a 1-h treatment. The coefficient increased in the whole intestine during the first 2 h of treatment (1 h: 21%; 2 h: 55%) and remained stable afterwards in our experimental conditions. The intestinal calcium absorption increase occurred early in the presence of L-lysine (100 mM), since the coefficient already reached its maximum value (52%) after a 1-h treatment.

Phosphorylated proteins from anuran intestinal microvilli membranes—I. Relations with alkaline phosphatase

1. 1. The degree of phosphorylation of intestinal microvilli membrane proteins in an adult amphibian, Rana esculenta, was investigated under various experimental conditions. 2. 2. The microvilli protein phosphorylation rate rapidly increases during the first 4 min of incubation in a medium containing [γ- 32P]ATP. This increase is slower afterwards. 3. 3. Cyclic nucleotides (cyclic AMP, cyclic GMP) and sorbitol do not modify the microvilli protein phosphorylation rate. 4. 4. On the contrary, this phosphorylation rate significantly decreases in the presence of l-lysine, when its concentration in the incubation medium is greater than 25 mM. The time course of phosphorylation confirms the inhibitory effects of l-lysine (100 mM). 5. 5. The microvilli membrane proteins were distinguished by polyacrylamide gel electrophoresis. In heated samples, electrophoresis followed by an radioautograph systematically reveals the existence of a very phosphorylated protein with a mol. wt of 86 kDa. 6. 6. The phosphorylation of this protein is partially inhibited by l-lysine (100 mM). 7. 7. The very phosphorylated protein could be the monomer of alkaline phosphatase. The dimer (170 kDa) is visualized on electrophoretograms by its catalytic activity. 8. 8. In mammals, several authors have established a correlation between phosphorylation of the microvilli membrane proteins and the intensity of intestinal calcium absorption. Such a control is presently being investigated in adult Rana esculenta.

A method for the two-dimensional electrophoresis of leaf proteins

Proteins extracted from green leaves of tobacco were subjected to analysis by two-dimensional electrophoresis. It was found that electrophoretic separations were unsatisfactory when leaf extracts were analyzed directly without prior extraction of pigments, phenols, and lipids by acetone treatment. The plant pigments and several phenolic compounds present in leaves presumably interacted with the proteins and created charge heterogeneity, streaking, and other artifacts. It was found that these problems could be overcome by treatment of leaf extracts with acetone followed by solubilization and electrophoresis of the acetone-treated proteins. Leaf extracts were prepared by grinding deribbed leaf disks in a buffer containing 5 m m potassium carbonate, 9.5 m urea, 0.5 dithiothreitol, 2% Nonidet P-40 detergent, 500 μg/ml l-lysine, and 2% Ampholines. The extracts, after centrifugation to remove cell debris and insoluble material, were treated several times with ice-cold acetone. The acetone-precipitated proteins were treated with nucleases, reprecipitated with cold acetone, and then resuspended in the above grinding buffer. The presence of l-lysine and Ampholines were required for good electrophoretic separations. The resuspended proteins were subjected to two-dimensional electrophoresis and proteins detected by staining and or fluorography. At least 300 distinct proteins could be recognized in radioactive samples. The method gives reproducible patterns even after repeated freezing and thawing of the samples.

Inhibition of gas vesicle production in Microcyclus aquaticus by L-lysine.

The timing and degree of gas vesicle production in Microcyclus aquaticus was affected by nutritional conditions. If 50 microng L-lysine/ml was added to a glucose-mineral salts medium (DM), the organism did not form gas vesicles. This effect was specific for L-lysine, as neither D-lysine nor meso-diaminopimelic acid prevented gas vesicle production. Cells grown in the presence of L-lysine did not contain any immunologically detectable gas vesicle protein, which indicates that L-lysine affects expression of the structural gene for the gas vesicle protein rather than assembly of the protein into gas vesicles. The addition of L-lysine to cultures in DM did not immediately decrease the rate of gas vesicle assembly, nor did the removal of cells from DM plus L-lysine to DM result in immediate gas vesicle production. Gas vesicle production was also affected by the addition of L-threonine or L-cysteine to culture media or by an increase in the medium's ionic strength. These results are discussed in relation to the aspartic acid pathway of amino acid biosynthesis and effects upon the intracellular L-lysine concentration.