PHA stimulation of human lymphocytes during amino acid deprivation. Protein,RNA and DNA synthesis

Resting lymphocytes are in the G₀ phase of the cell cycle. Upon activation by PHA, they progress into G₁ with accompanying increased protein and RNA synthesis, initiate DNA synthesis and divide. We have studied the kinetics of inhibition of macromolecular synthesis during activation in the absence of single amino acids. Three types of kinetics are observed. In the absence of tryptophan or isoleucine, stimulated lymphocytes show a normal increase in protein and RNA synthesis during the first 30 hours of stimulation, initiate DNA synthesis but are subsequently inhibited. In phenylalanine-deficient medium, no DNA synthesis occurs in spite of a slight increase in protein synthesis. No increase in macromolecular synthesis is observed in medium lacking any one of the other essential amino acids (eg: lysine). Our results indicate that the kinetics of macromolecular synthesis in tryptophan-deficient medium is the result of a limited reserve of protein-bound tryptophan which becomes exhausted after 30 hours. On the other hand, delayed inhibition of synthesis in isoleucine-deficient medium probably reflects an initially low requirement for this amino acid followed by inhibition of the synthesis of isoleucine-rich proteins involved in some late event of stimulation. Partial deprivation of lysine results in kinetics of protein synthesis similar to that in tryptophan- or isoleucine-deficient media. The results indicate that the kinetics of macromolecular synthesis during activation of lymphocytes in the absence of an essential amino acid is a function of the quantitative requirement for that amino acid, at a given time during stimulation. Upon replacement of lysine, lymphocytes inhibited by lysine deficiency begin RNA and protein synthesis immediately and at a rate faster than that of unstimulated cultures to which PHA is added. They also initiate DNA synthesis earlier and therefore, are closer to the S phase than resting lymphocytes. It is concluded that lymphocytes stimulated in the absence of lysine are activated even though no overall increase in macromolecular synthesis is observed. Furthermore, the kinetics of DNA synthesis following reversal of inhibition by phenylalanine suggests that lymphocytes stimulated during phenylalanine deprivation become arrested at most six hours before S. These results indicate that amino acid deficiencies lead to arrest of activated lymphocytes at various stages of stimulation, depending on how stringent these deficiencies are.     

Engineering of the aspartate family biosynthetic pathway in barley (Hordeum vulgare L.) by transformation with heterologous genes encoding feed-back-insensitive aspartate kinase and dihydrodipicolinate synthase

In prokaryotes and plants the synthesis of the essential amino acids lysine and threonine is predominantly regulated by feed-back inhibition of aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS). In order to modify the flux through the aspartate family pathway in barley and enhance the accumulation of the corresponding amino acids, we have generated transgenic barley plants that constitutively express mutant Escherichia coli genes encoding lysine feed-back insensitive forms of AK and DHPS. As a result, leaves of primary transformants (T₀) exhibited a 14-fold increase of free lysine and an 8-fold increase in free methionine. In mature seeds of the DHPS transgenics, there was a 2-fold increase in free lysine, arginine and asparagine and a 50% reduction in free proline, while no changes were observed in the seeds of the two AK transgenic lines analysed. When compared to that of control seeds, no differences were observed in the composition of total amino acids. The introduced genes were inherited in the T₁ generation where enzymic activities revealed a 2.3-fold increase of AK activity and a 4.0–9.5-fold increase for DHPS. T₁ seeds of DHPS transformants showed the same changes in free amino acids as observed in T₀ seeds. It is concluded that the aspartate family pathway may be genetically engineered by the introduction of genes coding for feed-back-insensitive enzymes, preferentially giving elevated levels of lysine and methionine.     

Selection for efficient translation initiation biases codon usage at second amino acid position in secretory proteins

The definition of a typical sec-dependent bacterial signal peptide contains a positive charge at the N-terminus, thought to be required for membrane association. In this study the amino acid distribution of all Escherichia coli secretory proteins were analysed. This revealed that there was a statistically significant bias for lysine at the second codon position (P2), consistent with a role for the positive charge in secretion. Removal of the positively charged residue P2 in two different model systems revealed that a positive charge is not required for protein export. A well-characterized feature of large amino acids like lysine at P2 is inhibition of N-terminal methionine removal by methionyl amino-peptidase (MAP). Substitution of lysine at P2 for other large or small amino acids did not affect protein export. Analysis of codon usage revealed that there was a bias for the AAA lysine codon at P2, suggesting that a non-coding function for the AAA codon may be responsible for the strong bias for lysine at P2 of secretory signal sequences. We conclude that the selection for high translation initiation efficiency maybe the selective pressure that has led to codon and consequent amino acid usage at P2 of secretory proteins.     

Comparative Kinetics of Arginine and Lysine Transport by Epimastigotes and Trypomastigotes from Two Strains of Trypanosoma cruzi*

SYNOPSIS. Three-day-old cultures of Y and MR strains of Trypanosoma cruzi had a higher rate of lysine and arginine uptake than 10-day cultures. Amino acid uptake by cells of the MR strain was consistently higher than that of the Y strain. Flagellates separated on DEAE-cellulose columns have normal structure, motility, and infectivity; they have higher rates of lysine and arginine uptake than the original 3- and 10-day cultures. In addition, passage through DEAE-cellulose columns modified the kinetic behavior of amino acid transport systems in the flagellate membranes. Methionine inhibited uncompetitively uptake of lysine and arginine by MR and Y strains. Lysine inhibited arginine uptake by both strains by an uncompetitive mechanism. Lysine, however, inhibited the uptake of arginine by 10-day culture cells of the Y strain by a mixed-type of inhibition. Arginine also inhibited the lysine uptake of both strains by an uncompetitive mechanism. In all experiments, beyond a certain level, a further increase in inhibitor concentration resulted in a decreased inhibition, which eventually disappeared altogether. Inhibition of amino-acid uptake by any of the substances tested was not observed after passage of flagellates through a DEAE-cellulose column. A model for amino acid transport was formulated which includes a recognition site amenable to modulation by effectors.     

Regulation of the synthesis of aspartokinase 3 of Escherichia coli by amino acids other than lysine

Summary When Escherichia coli B is grown in the presence of methionine, leucine and some other amino acids, lysine-sensitive aspartokinase (aspartokinase III) and aspartic semialdehyde dehydrogenase syntheses are derepressed. This can be explained by a synergistic inhibition between lysine and these amino acids on the lysine-sensitive aspartokinase, which leads to a decrease of the lysine intracellular pool.     

Selection and characterization of a feedback-insensitive tissue culture of maize

Tissue culture selection techniques were used to isolate a maize (Zea mays L.) variant D33, in which the aspartate family pathway was less sensitive to feedback inhibition by lysine. D33 was recovered by successively subculturing cultures originally derived from immature embryos on MS medium containing growth-inhibitory levels of lysine+threonine. The ability of D33 to grow vigorously on lysine+ threonine medium was retained after growth for 12 months on nonselection medium. New cultures initiated from shoot tissues of plants regenerated from D33 also were resistant to lysine+threonine inhibition. The K_i of aspartokinase for its feedback inhibitor, lysine, was about 9-fold higher in D33 than for the enzyme from unselected cultures. The free pools of lysine, threonine, isoleucine and methionine were increased 2–9-fold in D33 cultures. This was consistent with the observed change in feedback regulation of aspartokinase, the first enzyme common to the biosynthesis of these amino acids in the aspartate pathway. The accumulated evidence including the stability of resistance in the cultures, the resistance of cultures initiated from regenerated plants, the altered feedback regulation, and the increased free amino acids, indicates a mutational origin for these traits in line D33.Abbreviation LT lysine+threonine in equimolar concentration Paper No. 10880, Scientific Journal Series, Minnesota Agricultural Expertment Station     

Lysine excretion by Corynebacterium glutamicum. 1. Identification of a specific secretion carrier system

Corynebacterium glutamicum effectively excretes lysine when the internal lysine concentration is elevated. Lysine efflux was investigated using selected mutants which are not able to regulate lysine biosynthesis by feedback inhibition. Secretion of lysine is not the consequence of unspecific permeability of the plasma membrane but is mediated by a secretion carrier which is specific for lysine. Lysine export is characterized by high activation energy and follows Michaelis-Menten type kinetics with an internal Km of 20 mM and a Vmax of 12 nmol.min-1.mg dry cells-1. Excretion can proceed against a preexisting chemical gradient and against the electrical potential, which rules out a previously suggested pore model. Lysine excretion can also be observed in the wild-type strain especially under conditions of peptide uptake. Its possible physiological function may be related to regulation of internal amino acid concentrations under special growth conditions.     

Inhibition of hepatic gluconeogenesis by dichloroacetate

Gluconeogenesis from lactate by isolated hepatocytes suspended in a low bicarbonate medium is effectively inhibited by the hypoglycemic agent dichloroacetate. With this medium dichloroacetate suppresses the accumulation of the components of the malateaspartate shuttle, limits mitochondrial utilization of cytoplasmic reducing equivalents, and makes the availability of pyruvate and/or oxaloacetate limiting for gluconeogenesis. Much less inhibition is observed with hepatocytes suspended in a medium (Krebs?Henseleit saline) containing physiological concentrations of bicarbonate. No inhibition is observed with Krebs-Henseleit saline supplemented with lysine as a source of amino groups for the malate-aspartate shuttle. Thus, dichloroacetate inhibition of gluconeogenesis is observed only when hepatocytes are incubated in a medium deficient in bicarbonate and amino acids. This means that the action of dichloroacetate as a hypoglycemi agent is best explained by stimulation of peripheral tissue utilization of glucose and potential precursors for hepati gluconeogenesis rather than by direct inhibition of hepatic gluconeogenesis.     

Amino Acid Transport into Cultured Tobacco Cells: I. LYSINE TRANSPORT

Lysine transport into suspension-cultured Wisconsin-38 tobacco cells was observed. Uptake was linear (up to 90 minutes) with respect to time and amount of tissue only after 4 to 6 hours preincubation in calcium-containing medium. The observed cellular accumulation of lysine was against a concentration gradient and not due to exchange diffusion. Transport was stimulated by low pH and characterized by a biphasic uptake isotherm with two K(m) values for lysine. System I (K(m) approximately 5 x 10(-6) molar; V(max) approximately 180 nanomoles per gram fresh weight per hour) and system II (K(m) approximately 10(-4) molar; V(max) approximately 1900 nanomoles per gram fresh weight per hour) were inhibited by N-ethylmaleimide and a variety of respiratory inhibitors. This inhibition was not due to increased efflux. In antagonism experiments, system I was inhibited most effectively by basic amino acids, followed by the sulfur amino acids. System I was only slightly inhibited by the neutral and aromatic amino acids and was not inhibited by the acidic amino acids aspartic and glutamic acids. Transport by system II was inhibited by all of the tested amino acids (including aspartic and glutamic acids) and analogs; however, this system was not inhibited by d-arginine. Neither system was strongly inhibited by d-lysine or the lysine analog S-2-aminoethyl-l-cysteine. Arginine was shown to be a competitive inhibitor of both systems with values for K(i) similar to the respective K(m) values.These studies suggest the presence of at least two amino acid permeases in W-38 tobacco cells.     

Aspartate kinase and the synthesis of aspartate-derived amino acids in wheat

Aspartate kinase (EC 2.7.2.4.) has been purified from 7 day etiolated wheat (Triticum aestivum L. var. Maris Freeman) seedlings and from embryos imbibed for 8 h. The enzyme was 50% inhibited by 0.25 mM lysine. In this study wheat aspartate kinase was not inhibited by threonine alone or cooperatively with lysine; these results contrast with those published previously. In vivo regulation of the synthesis of aspartate-derived amino acids was examined by feeding [¹⁴C]acetate and [³⁵S]sulphate to 2–3 day germinating wheat embryos in culture in the presence of exogenous amino acids. Lysine (1 mM) inhibited lysine synthesis by 86%. Threonine (1 mM) inhibited threonine synthesis by 79%. Lysine (1 mM) plus threonine (1 mM) inhibited threonine synthesis by 97%. Methionine synthesis was relatively unaffected by these amino acids, suggesting that there are important regulatory sites other than aspartate kinase and homoserine dehydrogenase. [³⁵S]sulphate incorporation into methionine was inhibited 50% by lysine (2 mM) plus threonine (2 mM) correlating with the reported 50% inhibition of growth by these amino acids in this system. The synergistic inhibition of growth, methionine synthesis and threonine synthesis by lysine plus threonine is discussed in terms of lysine inhibition of aspartate kinase and threonine inhibition of homoserine dehydrogenase.Abbreviations AEC S-(2-aminoethyl) cysteine     

Lysine biosynthesis and nitrogen metabolism in quinoa (Chenopodium quinoa): study of enzymes and nitrogen-containing compounds.

Aspartate kinase (AK, EC 2.7.2.4), homoserine dehydrogenase (HSDH, EC 1.1.1.3) and dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52) were isolated and partially purified from immature Chenopodium quinoa Willd seeds. Enzyme activities were studied in the presence of the aspartate-derived amino acids lysine, threonine and methionine and also the lysine analogue S-2-aminoethyl-l-cysteine (AEC), at 1 mM and 5 mM. The results confirmed the existence of, at least, two AK isoenzymes, one inhibited by lysine and the other inhibited by threonine, the latter being predominant in quinoa seeds. HSDH activity was also shown to be partially inhibited by threonine, whereas some of the activity was resistant to the inhibitory effect, indicating the presence of two isoenzymes, one resistant and another sensitive to threonine inhibition. Only one DHDPS isoenzyme highly sensitive to lysine inhibition was detected. The results suggest that the high concentration of lysine observed in quinoa seeds is possibly due to a combined effect of increased lysine synthesis and accumulation in the soluble form and/or as protein lysine. Nitrogen assimilation was also investigated and based on nitrate content, nitrate reductase activity, amino acid distribution and ureide content, the leaves were identified as the predominant site of nitrate reduction in this plant species. The amino acid profile analysis in leaves and roots also indicated an important role of soluble glutamine as a nitrogen transporting compound.     

Gender differences in the regulation of amino acid metabolism.

Exercising men, compared with women, have a greater increase in leucine oxidation but not lysine rate of appearance. The cause for this sexual dimorphism is unknown; however, an inhibition of beta-adrenoreceptor activity has previously been shown to mediate amino acid metabolism (Lamont LS, McCullough AJ, and Kalhan SC. Am J Physiol Endocrinol Metab 268: E910-E916, 1995; Lamont LS, Patel DG, and Kalhan SC. J Appl Physiol 67: 221-225, 1989). This study was a gender comparison of leucine and lysine kinetics during a beta-adrenoreceptor blockade (beta1,beta2-blockade) and a placebo control by using a double-blind crossover protocol. Subjects exercised at 50% of their trial-specific maximal O2 consumption (1 h) after 7 days of dietary control. During exercise with beta-blockade, men had an increased nonprotein respiratory exchange ratio (P < 0.001), whereas women had an increased circulation of free fatty acids (P < 0.001). The genders also displayed distinct differences in exercise amino acid kinetics. The men, but not the women, increased leucine oxidation (P < 0.005) and lysine rate of appearance (P < 0.009) when exercising during beta-adrenergic blockade. This study indicates that during beta-blockade, exercising men increase their need for amino acids (and carbohydrate) to fuel energy needs, whereas women increase their mobilization of fat, thereby requiring less alternative fuels such as carbohydrate and amino acids. Gender-specific fuel preferences during exercise are regulated by beta-adrenergic-receptor activity. Substrate availability during exercise appears to modulate the amino acid oxidation differences between genders.     

Characterization of system L and system y+ amino acid transport activity in cultured vascular smooth muscle cells

The uptake of L-leucine and L-lysine into vascular smooth muscle cells cultured from the aortas of rats has been investigated. Both amino acids are taken up by saturable systems that are independent of the presence of a ^·Na⁺ gradient and can be stimulated in trans by neutral bulky amino acids for leucine and cationic amino acids for lysine. Leucine uptake is inhibited competitively in cis by several neutral amino acids, whereas lysine uptake is inhibited strongly by other cationic amino acids but also significantly by neutral amino acids such as leucine. The leucine inhibition is noncompetitive. Cells preloaded with leucine and lysine could also export these amino acids and the rate of efflux was stimulated by the presence of appropriate amino acids in trans. These data are all consistent with leucine being transported largely if not entirely by System L and lysine by the System y⁺ transporter. © 1993 Wiley-Liss, Inc.     

Identification of amino acids in the leader peptide of Methanococcus voltae preflagellin that are important in posttranslational processing

Archaeal flagellins are made initially as preproteins with short, positively charged leader peptides. Analysis of all available archaeal preflagellin sequences indicates that the -1 position is always held by a glycine while the -2 and -3 positions are almost always held by charged amino acids. To evaluate the importance of these and other amino acids in the leader peptides of archaeal flagellins for processing by a peptidase, Methanococcus voltae mutant FlaB2 preflagellin genes were generated by PCR and the proteins tested in a methanogen preflagellin peptidase assay that detects the removal of the leader peptide from preflagellin. When the -1 position was changed from glycine to other amino acids tested, no cleavage was observed by the peptidase, with the exception of a change to alanine at which poor, partial processing was observed. Amino acid substitutions at the -2 lysine position resulted in a complete loss of processing by the peptidase, while changes at the -3 lysine resulted in partial processing. A mutant preflagellin with a leader peptide shortened from 12 amino acids to 6 amino acids was not processed. When the invariant glycine residue present at position +3 was changed to a valine, no processing of this mutant preflagellin was observed. The identification of critical amino acids in FlaB2 required for proper processing suggests that a specific preflagellin peptidase may cleave archaeal flagellins by recognition of a conserved sequence of amino acids.     

Lysine uptake and exchange in Corynebacterium glutamicum.

Resting cells of Corynebacterium glutamicum (ATCC 13032) accumulate [14C]lysine by a transport system with a relatively high affinity (10 microMs) and a low maximum velocity (0.15 nmol/min per mg [dry weight]). Uptake of lysine was not inhibited by uncouplers or by ionophores affecting the ion gradients and the energetic state of the cell. Analysis of intracellular amino acid concentrations during the transport reaction as well as kinetic studies revealed that the observed uptake of lysine in fact represents a homologous antiport between extracellular [14C]lysine and intracellular unlabeled lysine. Intracellular [14C]lysine could only be released by the addition of unlabeled lysine to the bacterial suspension. In contrast to this homologous antiport reaction, we observed net uptake of lysine in lysine-depleted cells of a lysine auxotrophic strain. This net uptake was found to be electrogenic and could also be observed as a heterologous antiport reaction in wild-type cells under particular conditions. In this case exchange was mediated between internal lysine and external alanine, isoleucine, or valine. This antiport was electrogenic, since the substrates differ in charge. The cells can switch between electroneutral homologous exchange and electrogenic heterologous antiport mode during fermentation because of changing metabolic conditions.     

Control of lysine biosynthesis in Bacillus subtilis: inhibition of diaminopimelate decarboxylase by lysine.

Diaminopimelate decarboxylase has been characterized in extracts of Bacillus subtilis and resolved from aspartokinases I and II. Under certain conditions, the enzyme is specifically inhibited by physiological concentrations of L-lysine, but less specificity and altered kinetics of inhibition are observed if lower ionic strengths are employed in the assay procedure. Diaminopimelate decarboxylase can be desensitized to lysine inhibition by either lowering the pH or diluting the enzyme in Tris buffer in the absence of pyridoxal phosphate. Evidence is presented to incidate that, under proper conditions, lysine inhibition involves an interaction of the amino acid with the enzyme rather than competition for available pyridoxal phosphate in the assay. Lysine, by affecting the level of meso-diaminopimelate, may thus regulate its biosynthesis through sequential feedback inhibition. Analysis of the diaminopimelate decarboxylase of 15 revertants of mutants that had originally lacked diaminopimelate decarboxylase activity indicates that as little as 5% of the specific activity of enzyme observed in the wild-type strain is sufficient to permit normal growth rates. In the growing cell, diaminopimelate decarboxylase may therefore exist largely in an inhibited state.     

Lysine accumulation in maize cell cultures transformed with a lysine-insensitive form of maize dihydrodipicolinate synthase

Lysine is one of the nutritionally limiting amino acids in food and feed products made from maize (Zea mays L.). Two enzymes in the lysine biosynthesis pathway, aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS), have primary roles in regulating the level of lysine accumulation in plant cells because both enzymes are feedback-inhibited by lysine. An isolated cDNA clone for maize DHPS was modified to encode a DHPS much less sensitive to lysine inhibition. The altered DHPS cDNA was transformed into maize cell suspension cultures to determine the effect on DHPS activity and lysine accumulation. Partially purified DHPS (wildtype plus mutant) from transformed cultures was less sensitive to lysine inhibition than wild-type DHPS from nontransformed cultures. Transformed cultures had cellular free lysine levels as much as four times higher than those of nontransformed controls. Thus, we have shown that reducing the feedback inhibition of DHPS by lysine can lead to increased lysine accumulation in maize cells. Increasing the capacity for lysine synthesis may be an important step in improving the nutritional quality of food and feed products made from maize.     

Functional rescue of a bacterial dapA auxotroph with a plant cDNA library selects for mutant clones encoding a feedback-insensitive dihydrodipicolinate synthase

Dihydrodipicolinate synthase (DHDPS; EC4.2.1.52) catalyses the first reaction of lysine biosynthesis in plants and bacteria. Plant DHDPS enzymes are strongly inhibited by lysine (I0.5 approximately 10 microM), whereas the bacterial enzymes are less (50-fold) or insensitive to lysine inhibition. We found that plant dhdps sequences expressing lysine-sensitive DHDPS enzymes are unable to complement a bacterial auxotroph, although a functional plant DHDPS enzyme is formed. As a consequence of this, plant dhdps cDNA clones which have been isolated through functional complementation using the DHDPS-deficient Escherichia coli strain encode mutated DHDPS enzymes impaired in lysine inhibition. The experiments outlined in this article emphasize that heterologous complementation can select for mutant clones when altered protein properties are requisite for functional rescue. In addition, the mutants rescued by heterologous complementation revealed a new critical amino acid substitution which renders lysine insensitivity to the plant DHDPS enzyme. An interpretation is given for the impaired inhibition mechanism of the mutant DHDPS enzyme by integrating the identified amino acid substitution in the DHDPS protein structure.     

Chinese hamster ovary mRNA-dependent, Na(+)-independent L-leucine transport in Xenopus laevis oocytes.

In freshly prepared uninjected folliculated oocytes, Na(+)-independent leucine uptake is mediated predominantly by a system L-like transport system. Removal of follicular cells, however, results in an irreversible loss of this transport activity. When total poly(A)+ mRNA derived from Chinese hamster ovary (CHO) cells was injected into prophase-arrested stage V or VI Xenopus laevis oocytes, enhanced expression of Na(+)-independent leucine transport was observed. The injected mRNAs associated with increased levels of leucine uptake were between 2 and 3 kb in length. The newly expressed leucine transport activity exhibited important differences from the known characteristics of system L, which is the dominant Na(+)-independent leucine transporter in CHO cells as well as in freshly isolated folliculated oocytes. The CHO mRNA-dependent leucine uptake in oocytes was highly sensitive to the cationic amino acids lysine, arginine, and and ornithine (> 95% inhibition). As with the leucine uptake, an enhanced lysine uptake was also observed in size-fractionated CHO mRNA-injected oocytes. The uptakes of leucine and lysine were mutually inhibitable, suggesting that the newly expressed transporter was responsible for uptakes of both leucine and lysine. The inhibition of uptake of lysine by leucine was Na+ independent, thus clearly distinguishing it from the previously reported endogenous system y+ activity. Furthermore, the high sensitivity to tryptophan of the CHO mRNA-dependent leucine transport was in sharp contrast to the properties of the recently cloned leucine transport-associated gene from rat kidney tissue, although leucine transport from both sources was sensitive to cationic amino acids. Our results suggest that there may be a family of leucine transporters operative in different tissues and possibly under different conditions.     

Relationship of low lysine and high arginine concentrations to efficient ethanolic fermentation of wheat mash.

Very high gravity wheat mashes containing 20 or more grams of carbohydrates per 100 mL were fermented completely by Saccharomyces cerevisiae, even though these mashes contained low amounts of assimilable nitrogen. Supplementation of wheat mashes with various amino acids or with yeast extract, urea, or ammonium sulfate reduced the fermentation time. However, lysine or glycine added as single supplements, inhibited yeast growth and fermentation. With lysine, yeast growth was severely inhibited, and a loss of cell viability as high as 80% was seen. Partial or complete reversal of lysine-induced inhibition was achieved by the addition of a number of nitrogen sources. All nitrogen sources that relieved lysine-induced inhibition of yeast growth also promoted uptake of lysine and restored cell viability to the level observed in the control. They also increased the rate of fermentation. Experiments with minimal media showed that for lysine to be inhibitory to yeast growth, assimilable nitrogen in the medium must be in growth-limiting concentrations or totally absent. In the presence of excess nitrogen, lysine stimulated yeast growth and fermentation. Results indicate that supplementing wheat mash with other nitrogen sources increases the rate of fermentation not only by providing extra nitrogen but also by reducing or eliminating the inhibitory effect of lysine on yeast growth.