DeltapH-Dependent Amino Acid Transport into Plasma Membrane Vesicles Isolated from Sugar Beet Leaves: I. Evidence for Carrier-Mediated, Electrogenic Flux through Multiple Transport Systems

Amino acid transport into plasma membrane vesicles isolated from mature sugar beet (Beta vulgaris L. cv Great Western) leaves was investigated. The transport of alanine, leucine, glutamine, glutamate, isoleucine, and arginine was driven by a trans-membrane proton concentration difference. ΔpH-Dependent alanine, leucine, glutamine, and glutamate transport exhibited simple Michaelis-Menten kinetics, and double-reciprocal plots of the data were linear with apparent K_m values of 272, 346, 258, and 1981 micromolar, respectively. These results are consistent with carrier mediated transport. ΔpH-Dependent isoleucine and arginine transport exhibited biphasic kinetics, suggesting these amino acids may be transported by at least two transport systems. Symport mediated alanine transport was electrogenic as demonstrated by the effect of membrane potential (ΔΨ) on ΔpH-dependent flux. In the absence of significant charge compensation, a low rate of alanine transport was observed. When ΔΨ was held at 0 millivolt with symmetric potassium concentrations and valinomycin, the rate of flux was stimulated fourfold. In the presence of a negative ΔΨ, alanine transport increased sixfold. These results are consistent with an electrogenic transport process which results in a net flux of positive charge into the vesicles. The effect of changing ΔΨ on the kinetics of alanine transport altered V_max with no apparent change in K_m. Amino acid transport was inhibited by the protein modifier diethyl pyrocarbonate, but was insensitive to N-ethylmaleimide, 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid, p-chloromercuribenzenesulfonic acid, phenylglyoxal, and N,N′-dicyclohexylcarbodiimide. Four amino acid symport systems, two neutral, one acidic, and one basic, were resolved based on inter-amino acid competition experiments. One neutral system appears to be active for all neutral amino acids while the second exhibited a low affinity for isoleucine, threonine, valine, and proline. Although each symport was relatively specific for a given group of amino acids, each system exhibited some crossover specificity for amino acids in other groups.     

Amino Acid Metabolism of Lemna minor L. : I. Responses to Methionine Sulfoximine

When Lemna minor L. is supplied with the potent inhibitor of glutamine synthetase, methionine sulfoximine, rapid changes in free amino acid levels occur. Glutamine, glutamate, asparagine, aspartate, alanine, and serine levels decline concomitantly with ammonia accumulation. However, not all free amino acid pools deplete in response to this inhibitor. Several free amino acids including proline, valine, leucine, isoleucine, threonine, lysine, phenylalanine, tyrosine, histidine, and methionine exhibit severalfold accumulations within 24 hours of methionine sulfoximine treatment. To investigate whether these latter amino acid accumulations result from de novo synthesis via a methionine sulfoximine insensitive pathway of ammonia assimilation (e.g. glutamate dehydrogenase) or from protein turnover, fronds of Lemna minor were prelabeled with [¹⁵N]H₄⁺ prior to supplying the inhibitor. Analyses of the ¹⁵N abundance of free amino acids suggest that protein turnover is the major source of these methionine sulfoximine induced amino acid accumulations. Thus, the pools of valine, leucine, isoleucine, proline, and threonine accumulated in response to the inhibitor in the presence of [¹⁵N]H₄⁺, are ¹⁴N enriched and are not apparently derived from ¹⁵N-labeled precursors. To account for the selective accumulation of amino acids, such as valine, leucine, isoleucine, proline, and threonine, it is necessary to envisage that these free amino acids are relatively poorly catabolized in vivo. The amino acids which deplete in response to methionine sulfoximine (i.e. glutamate, glutamine, alanine, aspartate, asparagine, and serine) are all presumably rapidly catabolized to ammonia, either in the photorespiratory pathway or by alternative routes.     

Amino Acid Metabolism of Lemna minor L. : III. Responses to Aminooxyacetate

Aminooxyacetate, a known inhibitor of transaminase reactions and glycine decarboxylase, promotes rapid depletion of the free pools of serine and aspartate in nitrate grown Lemna minor L. This compound markedly inhibits the methionine sulfoximine-induced accumulation of free ammonium ions and greatly restricts the methionine sulfoximine-induced depletion of amino acids such as glutamate, alanine, and asparagine. These results suggest that glutamate, alanine, and asparagine are normally catabolized to ammonia by transaminase-dependent pathways rather than via dehydrogenase or amidohydrolase reactions. Aminooxyacetate does not inhibit the methionine sulfoximine-induced irreversible deactivation of glutamine synthetase in vivo, indicating that these effects cannot be simply ascribed to inhibition of methionine sulfoximine uptake by amino-oxyacetate. This transaminase inhibitor promotes extensive accumulation of several amino acids including valine, leucine, isoleucine, alanine, glycine, threonine, proline, phenylalanine, lysine, and tyrosine. Since the aminooxyacetate induced accumulations of valine, leucine, and isoleucine are not inhibited by the branched-chain amino acid biosynthesis inhibitor, chlorsulfuron, these amino acid accumulations most probably involve protein turnover. Depletions of soluble protein bound amino acids are shown to be approximately stoichiometric with the free amino acid pool accumulations induced by aminooxyacetate. Aminooxyacetate is demonstrated to inhibit the chlorsulfuron-induced accumulation of α-amino-n-butyrate in L. minor, supporting the notion that this amino acid is derived from transamination of 2-oxobutyrate.     

Studies on Amino Acid Sequence Determination from N-Terminal of Peptide by Methylthiohydantoin

Methylisothiocyanate reacts with the amino group of amino acid in alkaline solution to give methylthiocarbamyl amino acid. This is converted to the methylthiohydantoin derivative (MTH-amino acid) by subsequent acidification.

Gas chromatographical identification of 20 amino acids were examined by making MTH-amino acids. Among them, ten amino acids (glycine, alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, serine and threonine) were successfully identified. Aspartic acid and glutamic acid could be identified as the methyl esters of the MTH-amino acids.     

Amino Acid Transport across the Tonoplast of Vacuoles Isolated from Barley Mesophyll Protoplasts : Uptake of Alanine, Leucine, and Glutamine

Mesophyll protoplasts from leaves of well-fertilized barley (Hordeum vulgare L.) plants contained amino acids at concentrations as high as 120 millimoles per liter. With the exception of glutamic acid, which is predominantly localized in the cytoplasm, a major part of all other amino acids was contained inside the large central vacuole. Alanine, leucine, and glutamine are the dominant vacuolar amino acids in barley. Their transport into isolated vacuoles was studied using ¹⁴C-labeled amino acids. Uptake was slow in the absence of ATP. A three- to sixfold stimulation of uptake was observed after addition of ATP or adenylyl imidodiphosphate an ATP analogue not being hydrolyzed by ATPases. Other nucleotides were ineffective in increasing the rate of uptake. ATP-Stimulated amino acid transport was not dependent on the transtonoplast pH or membrane potential. p-Chloromercuriphenylsulfonic acid and n-ethyl maleimide increased transport independently of ATP. Neutral amino acids such as valine or leucine effectively decreased the rate of alanine transport. Glutamine and glycine were less effective or not effective as competitive inhibitors of alanine transport. The results indicate the existence of a uniport translocator specific for neutral or basic amino acids that is under control of metabolic effectors.     

Amino acid synthesis from glucose-U-14C in Glossina morsitans

Amino acid synthesis from glucose-U-¹⁴C was investigated in 2 day post-emergent and pregnant females of Glossina morsitans. This insect can synthesize alanine, aspartic acid, cystine, glutamic acid, glycine, proline, and serine from glucose. Arginine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, taurine, threonine, and valine showed no radioactivity and hence may be classified as nutritionally indispensable amino acids. Although tyrosine and hydroxyproline were not synthesized from glucose, they are at least partially dispensable nutrients for this insect because their synthesis from phenylalanine has been demonstrated. After the labelled glucose injection the highest radioactivity was recovered in the proline fraction. This is probably related to its rôle as an important energy reserve for flight. The radioactive amino acids recovered from females and from their offspring following glucose-U-¹⁴C injection were similar to those recovered from younger females. Radioactivity was also detected in the expired CO₂ and the excreta. The amino acids alanine, arginine, cystine, glycine, histidine, leucine/isoleucine, lysine, methionine, proline, and valine were identified in the excreta, of which arginine and histidine were in the largest amounts. Only excreted alanine, glycine, and proline showed radioactivity.     

Die Bildung höherer Alkohole bei Aminosäuremangelmutanten von Saccharomyces cerevisiae

1. The influence of varying amounts of amino acids on the uptake of threonine, isoleucine, valine and leucine and their degradation to higher alcohols was investigated using a mutant strain of Saccharomyces cerevisiae, mating type a, genetic markers ade2, hom2, thr4, ilv2, leu1.
2. The cell mass is increased by increasing concentrations of threonine, isoleucine, valine and leucine, the latter two resulting in a higher dry weight. The amino acids are completely utilised at low concentrations. At higher contents up to 20% of the amino acids remain in the medium. The uptake of threonine, isoleucine, valine and leucine depends on the relative amounts of the concentrations of these amino acids in the medium. A greater amount of an amino acid is taken up if its concentration is comparatively higher than those of the other amino acids. There is a competition between the amino acids for the uptake into the cells.

Higher amounts of intracellular isoleucine and leucine are converted to 2-and 3-methylbutanol when compared with the degradation of valine and threonine to isobutanol and n-propanol-1, isoleucine and leucine up to 90%, valine up to 24% and threonine up to 20%. There is a competition between the four amino acids for their degradation to the corresponding higher alcohols. This behaviour confirms the earlier assumption of a degradation of the four amino acids by unspecific enzymes.     

Amino Acids from Heterodera glycines

Amino acids emitted and extracted from surface-sterilized larvae and adults of Heterodera glycines were identified by paper chromatography and quantitatively analyzed by column chromatography. Five amino acids (alanine, aspartic acid, glutamic acid, glycine and serine) were emitted by H. glycines larvae and eight others (asparagine, glutamine, leucine/isoleucine, lysine, methionine sulfoxide, threonine, tyrosine, valine/methionine) were found in extracts from crushed larvae.In addition to the amino acids emitted or extracted from larvae, four others were emitted by adults (γ-aminobutyric acid, histidine, phenylalanine, and proline). Four different amino acids (arginine, cystathionine, hydroxyproline, and ornithine) were found only in the extract from crushed adults. Greater quantities of alanine, aspartic acid and glycine were emitted than could be detected in nematode extracts suggesting selective emission.Subsamples of nematode populations were taken from growing plants 19, 26, 33, and 40 days after inoculation and extracted to determine whether changes in specific amino acid content correlated with aging. Proline content shifted most, increasing from 4.1% to 21.5% of the total amino acid complement from the 19th to the 40th days.     

Amino Acid interactions in the regulation of nitrate reductase induction in cotton root tips

Glycine, asparagine, and glutamine inhibited the induction by nitrate of nitrate reductase activity in root tips of cotton (Gossypium hirsutum L.). This inhibition was partially or entirely prevented when the inhibitor was applied in combination with any of several other amino acids. Studies of ¹⁴C-labeled amino acid uptake showed that, in most cases, the apparent antagonism resulted simply from competition for uptake. However, certain antagonists did not curtail uptake. The most effective of these were leucine (against all three inhibitors), and isoleucine and valine (against asparagine or glutamine, but not glycine). These results show that interactions among amino acids in the regulation of nitrate reductase induction result from at least two mechanisms, one acting on uptake of inhibitory amino acids, and the other involving true antagonism.     

Uptake of Branched-Chain Amino Acids by Streptococcus thermophilus

The transport of branched-chain amino acids in Streptococcus thermophilus was energy dependent. The metabolic inhibitors of glycolysis and ATPase enzymes were active, but the proton-conducting uncouplers were not. Transport was optimal at temperatures of between 30 and 45°C and at pH 7.0 for the three amino acids leucine, valine, and isoleucine; a second peak existed at pH 5.0 with valine and isoleucine. By competition and kinetics studies, the branched-chain amino acids were found to share at least a common transport system.     

Interamino Acid Inhibition of Transport in Higher Plants : EVIDENCE FOR TWO TRANSPORT CHANNELS WITH ASCERTAINABLE AFFINITIES FOR AMINO ACIDS

Data from published experiments were analyzed to determine the number and specificities of amino acid transport channels in cells of higher plants. Each experiment measured the uptake of a labeled amino acid in the presence of unlabeled amino acids, used one at a time, in the incubating medium. The observed interamino acid inhibitions can be accounted for by two transport channels, each with characteristic affinities that were computed from the observed interamino acid inhibitions. The first channel is a general transport system with the following relative affinities for the amino acids: methionine 75, alanine 75, phenylalanine 64, tyrosine 64, leucine 63, cysteine 58, serine 57, glycine 56, tryptophan 54, glutamine 51, threonine 49, valine 44, isoleucine 44, glutamic acid 44, proline 43, histidine 33, lysine 32, asparagine 22, arginine 22, aspartic acid 18. The second channel is a basic amino acid tranport system with relative affinities for arginine, lysine, and histidine of 66, 39, and 21, respectively. The affinities for the other acids in the second channel are lower. Despite considerable diversity in the species, tissues, and solute concentrations employed in the experiments, multiple regression equations (Y = α + β₁X₁ + βX₂, in which Y is the observed transport inhibition and X₁ and X₂ are the relative transport affinities of the two channels) account for 50 to 99% of the variance in all but six experiments, five of which employed unusually high solute concentrations.     

Effect of spermine on the uptake of amino acids in Micrococcus lysodeikticus

Spermine inhibited the transport of neutral aliphatic amino acids (valine, leucine, isoleucine, alanine, and glycine) into cells of Micrococcus lysodeikticus. On the other hand, spermine did not affect the uptake of basic (arginine and histidine), acidic (glutamic acid), or aromatic (phenylalanine and tyrosine) amino acids. Inhibition of uptake of the neutral amino acids by spermine is apparently of a noncompetitive nature; the V(max) decreased, whereas the apparent K(m) remained unaltered. The inhibition is most likely due to a specific binding of spermine to the carrier(s) of these amino acids. Related polyamines, spermidine and cadaverine, also caused inhibition of valine uptake, though to a lesser extent; spermidine was less active than spermine, and cadaverine showed the weakest effect of all. Valine, leucine, and isoleucine were transported into M. lysodeikticus cells by a common carrier as evidenced from competition experiments. The uptake of these amino acids is an active process; it was temperature-dependent and inhibited by azide (10(-1)m to 2.5 x 10(-2)m) and dinitrophenol (10(-3)m). The intracellular concentration of valine was 100-fold higher than in the medium.     

Amino Acid Metabolism of Lemna minor L. : II. Responses to Chlorsulfuron

Chlorsulfuron, an inhibitor of acetolactate synthase (EC 4.1.3.18) (TB Ray 1984 Plant Physiol 75: 827-831), markedly inhibited the growth of Lemna minor at concentrations of 10⁻⁸ molar and above, but had no inhibitory effects on growth at 10⁻⁹ molar. At growth inhibitory concentrations, chlorsulfuron caused a pronounced increase in total free amino acid levels within 24 hours. Valine, leucine, and isoleucine, however, became smaller percentages of the total free amino acid pool as the concentration of chlorsulfuron was increased. At concentrations of chlorsulfuron of 10⁻⁸ molar and above, a new amino acid was accumulated in the free pool. This amino acid was identified as α-amino-n-butyrate by chemical ionization and electron impact gas chromatography-mass spectrometry. The amount of α-amino-n-butyrate increased from undetectable levels in untreated plants, to as high as 840 nanomoles per gram fresh weight (2.44% of the total free pool) in plants treated with 10⁻⁴ molar chlorsulfuron for 24 hours. The accumulation of this amino acid was completely inhibited by methionine sulfoximine. Chlorsulfuron did not inhibit the methionine sulfoximine induced accumulations of valine, leucine, and isoleucine, supporting the idea that the accumulation of the branched-chain amino acids in methionine sulfoximine treated plants is the result of protein turnover rather than enhanced synthesis. Protein turnover may be primarily responsible for the failure to achieve complete depletion of valine, leucine, and isoleucine even at concentrations of chlorsulfuron some 10⁴ times greater than that required to inhibit growth. Tracer studies with ¹⁵N demonstrate that chlorsulfuron inhibits the incorporation of ¹⁵N into valine, leucine, and isoleucine. The α-amino-n-butyrate accumulated in the presence of chlorsulfuron and [¹⁵N]H₄⁺ was heavily labeled with ¹⁵N at early time points and appeared to be derived by transamination from a rapidly labeled amino acid such as glutamate or alanine. We propose that chlorsulfuron inhibition of acetolactate synthase may lead to accumulation of 2-oxobutyrate in the isoleucine branch of the pathway, and transamination of 2-oxobutyrate to α-amino-n-butyrate by a constitutive transaminase utilizing either glutamate or alanine as α-amino-N donors.     

Effects of Sulphur Dioxide on Sugar and Free Amino Acid Content of Pine Seedlings

Treatment of jack pine (Pinus banksiana Lamb.) seedlings with gaseous SO₂ resulted in a shift between the reducing and non-reducing sugars. Increasing concentrations of gaseous SO₂ caused an increase in reducing sugars and a decline in the non-reducing sugars, suggesting a conversion from the latter to the former at high SO₂ concentrations. The total amino acid content of the intact tissues also increased with increasing concentrations of gaseous SO₂. Gas-liquid chromatographic analyses of the amino acids indicated that SO₂ (1. 34 mg · m^-3 for 96 h) resulted in an increase in the content of alanine, valine, glycine, isoleucine, leucine, threonine, aspartic acid tyrosine, lysine, and arginine, and a decrease in the content of serine and glutamic acid. The enzymatic and other implications of such changes are discussed.     

Quantitative analysis of free amino acids and carbohydrates in spring barley anthers at different stages of maturity

The content of the carbohydrates glucose, fructose and sucrose was determined in spring barley anthers at different stages of maturity. During maturation the sucrose content of the anthers increased markedly. The following 17 free amino acids were detected in anthers of different stages of maturity: aspartic acid, glutamic acid, serine, alanine, arginine, leucine, isoleucine, lysine, α-aminobutyric acid, glutamine, proline, tyrosine, phenylalanine, valine, threonine, cystine and glycine. Quantitative analysis was only carried out in amino acids present in higher concentrations in the analysed samples. These were: aspartic acid, glutamic acid, α-aminobutyric acid, proline, serine, valine and glutamine, and a mixture of amino acids (leucine, isoleucine, valine and phenylalanine). The total content of free amino acids increased with increasing maturity of the anthers. However, not all amino acids followed contributed to this increase, but only proline, glutamic acid, aspartic acid and glutamine. A small difference was found in the variety Gopal in which the aspartic acid content did not increase significantly, but the content of the mixture of amino acids and serine did. With the exception of green anthers of the variety Firlbecks Union, proline was present in the highest concentration in all samples analysed.     

Pyruvate-Derived Amino Acids in Spinach Chloroplasts : Synthesis and Regulation during Photosynthetic Carbon Metabolism

A probable carbon flow from the Calvin cycle to branched chain amino acids and lipids via phosphoenolpyruvate (PEP) and pyruvate was examined in spinach (Spinacia oleracea) chloroplasts. The interpendence of metabolic pathways in and outside chloroplasts as well as product and feedback inhibition were studied. It was shown that alanine, aromatic, and small amounts of branched chain amino acids were formed from bicarbonate in purified intact chloroplasts. Addition of PEP only favored formation of aromatic amino acids. Mechanisms of regulation remained unclear. Concentrations of PEP and pyruvate within the chloroplast impermeable space during photosynthetic carbon fixation were 15 times higher than in the reaction medium. A direct carbon flow to pyruvate was identified (0.1 micromoles per milligram chlorophyll per hour). Pyruvate was taken up by intact chloroplasts slowly, leading to the formation of lysine, alanine, valine, and leucine plus isoleucine (approximate ratios, 100-500:60-100:40-100:2-10). The K_m for the formation of valine and leucine plus isoleucine was estimated to be 0.1 millimolar. Ten micromolar glutamate optimized the transamination reaction regardless of whether bicarbonate or pyruvate was being applied. Alanine and valine formation was enhanced by the addition of acetate to the reaction mixture. The enhancement probably resulted from an inhibition of pyruvate dehydrogenase by acetyl-S-coenzyme A formed from acetate, and resulting accumulation of hydroxyethylthiamine diphosphate and pyruvate. High concentrations of valine and isoleucine inhibited their own and each others synthesis and enhanced alanine formation. When pyruvate was applied, only amino acids were formed; when complemented with bicarbonate, fatty acids were formed as well. This is probably the result of a requirement of acetyl-S-coenzyme A-carboxylase for bicarbonate.     

Stimulation of Lysine Decarboxylase Production in Escherichia coli by Amino Acids and Peptides

A commercial hydrolysate of casein stimulated production of lysine decarboxylase (EC 4.1.1.18) by Escherichia coli B. Cellulose and gel chromatography of this hydrolysate yielded peptides which were variably effective in this stimulation. Replacement of individual, stimulatory peptides by equivalent amino acids duplicated the enzyme levels attained with those peptides. There was no indication of specific stimulation by any peptide. The peptides were probably taken up by the oligopeptide transport system of E. coli and hydrolyzed intracellularly by peptidases to their constituent amino acids for use in enzyme synthesis. Single omission of amino acids from mixtures was used to screen them for their relative lysine decarboxylase stimulating abilities. Over 100 different mixtures were evaluated in establishing the total amino acid requirements for maximal synthesis of lysine decarboxylase by E. coli B. A mixture containing all of the common amino acids except glutamic acid, aspartic acid, and alanine increased lysine decarboxylase threefold over an equivalent weight of casein hydrolysate. The nine most stimulatory amino acids were methionine, arginine, cystine, leucine, isoleucine, glutamine, threonine, tyrosine, and asparagine. Methionine and arginine quantitatively were the most important. A mixture of these nine was 87% as effective as the complete mixture. Several amino acids were inhibitory at moderate concentrations, and alanine (2.53 mM) was the most effective. Added pyridoxine increased lysine decarboxylase activity 30%, whereas other B vitamins and cyclic adenosine 5′-monophosphate had no effect.     

Rhipicephalus sanguineus: amino acid composition of egg shell

Acid hydrolysis of the protein fraction of a batch of egg shells of Rhipicephalus sanguineus was followed by determination of the amino acids in the hydrolysate. Using thin layer chromatography, the amino acids—lysine, arginine, aspartic acid, serine, glycine, glutamic acid, alanine, threonine, valine, tyrosine, isoleucine, and leucine were identified. Alkaline hydrolysis of the fraction followed by TLC revealed the presence of tryptophane. Chitin was revealed utilizing a chitosan test.     

Amino acid-selective isotope labeling of proteins for nuclear magnetic resonance study: Proteins secreted by Brevibacillus choshinensis

Here we report the first application of amino acid-type selective (AATS) isotope labeling of a recombinant protein secreted by Brevibacillus choshinensis for a nuclear magnetic resonance (NMR) study. To prepare the ¹⁵N-AATS-labeled protein, the transformed B. choshinensis was cultured in ¹⁵N-labeled amino acid-containing C.H.L. medium, which is commonly used in the Escherichia coli expression system. The analyses of the ¹H-¹⁵N heteronuclear single quantum coherence (HSQC) spectra of the secreted proteins with a ¹⁵N-labeled amino acid demonstrated that alanine, arginine, asparagine, cysteine, glutamine, histidine, lysine, methionine, and valine are suitable for selective labeling, although acidic and aromatic amino acids are not suitable. The ¹⁵N labeling for glycine, isoleucine, leucine, serine, and threonine resulted in scrambling to specific amino acids. These results indicate that the B. choshinensis expression system is an alternative tool for AATS labeling of recombinant proteins, especially secretory proteins, for NMR analyses.     

Some Effects of Protein Deficiency in Young Growing Pigs. II. Blood Plasma Free Amino Acids

The influence of protein deficiency, rehabilitation and total starvation on the free amino acid levels in the blood plasma of pigs has been investigated. It was found that the concentration of most amino acids was reduced during protein deficiency. The levels of leucine, isoleucine and valine were diminished by the greatest proportion, followed by threonine, tyrosine and citrulline. During the first few weeks of protein deficiency the levels of lysine, histidine and arginine were slightly increased, but later decreased below control values. Concentrations of glycine and alanine were altered in a similar way except that the initial increase was much more pronounced. The concentrations of most of these amino acids returned to control levels after rehabilitation. Total starvation led to an increase in concentration of leucine, isoleucine, valine, threonine and to a smaller extent phenylalanine, lysine, citrulline and arginine. The concentration of glycine, alanine and glutamic acid were very much reduced. The level of urea in the circulation dropped reversibly during protein deficiency and increased very much during total starvation.