Chemical modification of the active site of the NADP-linked glutamate dehydrogenase from Trypanosoma cruzi

The NADP-linked glutamate dehydrogenase (NADP-gluDH) purified from epimastigotes of the Tulahuén strain, Tul 2 stock, of Trypanosoma cruzi, was inhibited by Cibacron Blue FG3A, and inactivated by preincubation with phenylglyoxal or Woodward's Reagent K. The inhibition by Cibracron Blue FG3A, competitive towards NADPH with an apparent Ki of 20 microM, suggests that the enzyme presents the "dinucleotide fold" characteristic of most dehydrogenases and kinases. The inactivation of the NADP-gluDH by preincubation with phenylglyoxal, with a reaction order of 1, and the partial protection afforded by alpha-oxoglutarate, suggest the presence of one arginine residue in the active site of the enzyme, which might participate in the binding of alpha-oxoglutarate through interaction with one of the carboxyl groups of the substrate. The inactivation of the NADP-gluDH by preincubation with Woodward's Reagent K suggests the presence of a carboxyl group, from an aspartic or glutamic acid residue, at the active site, which might participate in the binding of the cationic substrate NH+4. The presence of NADPH during preincubation with the reagent increased the inactivation rate, which suggests that binding of the coenzyme increases the exposure of the reactive carboxyl group.     

Evidence for the presence of one carboxyl group in the catalytic center of D-beta-hydroxybutyrate dehydrogenase. Inactivation and binding studies with carbodiimide reagents

D-beta-Hydroxybutyrate dehydrogenase D-3-hydroxybutyrate: NAD+ oxidoreductase, EC 1.1.1.30), a phosphatidylcholine-requiring enzyme, was irreversibly inactivated by a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) or a hydrophobic carbodiimide, N,N'-dicyclohexylcarbodiimide (DCCD). The inactivation is pseudo-first-order with a kinetic stoichiometry of about 1. Phospholipid-free apoenzyme was more sensitive towards these reagents than reconstituted phospholipid-enzyme or membrane-bound enzyme forms. Reduced coenzyme (NADH) protected the enzyme against the inactivation, while oxidized coenzyme (NAD+) in presence of 2-methylmalonate (a pseudo-substrate) gave a better protection. It was found that the phospholipid-free apoenzyme bound about 1 mol [14C]DCCD. This incorporation was prevented by EDAC, indicating that both reagents react at the same site. [14C]Glycine ethyl ester, a nucleophilic compound which reacts specifically with the carboxylcarbodiimide derivative was incorporated to the enzyme (1 mol [14C]glycine ethyl ester per polypeptide chain), whatever its form, in the presence of DCCD or EDAC. These results indicate the presence of one carboxyl group probably located at or near the coenzyme-binding site and near the interacting domain of the enzyme with phospholipid.     

Role of protein dissociation in the transport of acidic amino acids by the Ehrlich ascites tumor cell.

The pH profile for the uptake of L-glutamic acid by the Ehrlich ascites tumor cell arises largely as a sum of the decline with falling pH of a slow, Na+-dependent uptake by System A, and an increasing uptake by Na+-independent System L. The latter maximizes at about pH 4.5, following approximately the titration curve of the distal carboxyl group. This shift in route of uptake was verified by (a) a declining Na+-dependent component, (b) an almost corresponding decline in the 2-(methylamino)-isobutyric acid-inhibitable component, (c) a rising component inhibited by 2-aminonorbornane-2-carboxylic acid. Other amino acids recognized as principally reactive with Systems A or L yielded corresponding inhibitory effects with some conspicious exceptions: 2-Aminoisobutyric acid and even glycine become better substrates of System L as the pH is lowered; hence their inhibitory action on glutamic acid uptake is not lost. The above results were characterized by generally consistent relations among the half-saturation concentrations of the interacting amino acids with respect to: their own uptake, their inhibition of the uptake, one by another, and their trans stimulation of exodus, one by another. A small Na+-dependent component of uptake retained by L-glutamic acid but not by D-glutamic acid at pH 4.5 is inhibitable by methionine but by neither 2-(methylamino)-isobutyric acid nor the norbornane amino acid. We provisionally identified this component with System ASC, which transports L-glutamine throughout the pH range studied. No transport activity specific to the anionic amino acids was detected, and the unequivocally anionic cysteic acid showed neither significant mediated uptake nor inhibition of the uptake of glutamic aic or of the norbornane amino acid. The dicarboxylic amino acids take the sequence, aspartic acid less than glutamic acid less than alpha-aminoadipic acid less than S-carboxymethylcysteine, in their rate of mediated, Na+-independent uptake at low pH. Diiodotyrosine and two dissimilas isomers of nitrotyrosine also show acceleration of uptake as the phenolate group on the sidechain is protonated, a result indicating that the acidic group need not be a carboxyl group and need not take a specific position in space to be accepted at the receptor site L. The presence of the carboxyl group does not upset the normal stereospecificity of System L until it falls on the beta-carbon in aspartic acid; even then it is the presence of the carbonyl group and not of the intact carboxyl group nor of its hydroxyl group that cancels out the stereospecificity, as was shown by the absence of normal stereospecificity for aspartic acid and asparagine and its presence in glutamic acid, homoserine and glutamine. In agreement, the uptak of aspartic acid is peculiarly sensitive to the presence of an alpha-methyl group or of other structures that modify the orientation of the sidechain.     

Carboxymethylation of a minor ribonuclease from Aspergillus saitoi.

(1) RNase Ms was inactivated by iodoacetate. The inactivation was most rapid at pH 6.0, and was inhibited in the presence of a denaturant such as 8 m urea or 6 m guanidine-HCL. (2) Competitive inhibitors protected RNase Ms from inactivation by iodoacetate; the effect was in the order 2',(3')-GTP greater than 2',(3')-AMP, 2',(3')-UMP greater than or equal to 2',(3')-CMP. The order is not consistent with that of the binding constants of the 4 nucleotides towards RNase Ms (A is greater than C greater than G greater than U). (3) RNase Ms was inactivated with the concomitant incorporation of one molar equivalent of carboxymethly group. The following evidence indicated that the carboxymethyl group was incorporated into the carboxyl group of an aspartic acid or glutamic acid residue. (i) The carboxymethyl group incorporated into RNase Ms was liberated by treatment with 0.1 n NaOH or 1 m hydroxylamine. (ii) The amino acid composition of carboxymethylated RNase Ms (CM RNase Ms) after acid hydrolysis is similar to that of RNase Ms. (4) 14C-Labeled CM RNase Ms was digested successively with alkaline protease and amino-peptidase M. The radioactive amino acid released was eluted just before aspartate on an amino acid analyzer. After hydrolysis with 6 n HCL, glutamic acid was produced exclusively from the radioactive amino acid. The specific radioactivity of this amino acid calculated from the radioactivity and glutamic acid formed was practctically the same as that of CM RNase Ms. Thus, it was concluded that a carboxymethyl group was incorporated at the carboxyl group of a glutamic acid residue of RNnase Ms. (5) CM RNase Ms bound with 2'-AMP to the same extent as native RNase Ms, but bound to a lesser extent with 2',(3')-GMP. (6) Although the conformation of CM RNase Ms as judged from the CD spectrum was practically the same as that of native RNase Ms, the reactivity of CM RNase Ms towards dinitrofluorobenzene was different from that of native RNase Ms, indicating some difference in the conformation. (7) These results indicate that one glutamic acid residue is involved in the active of RNase Ms.     

Synthesis of some long-chain acylamidoalkyl glucosides

4-O-β-D-Galactopyranosyl-α,β-D-glucopyranosylamine (lactosylamine), β-D-gluco-, α- and β-D-manno-pyranosylamines were bound to the carbodiimide-activated groups of lysozyme. Of the 11 free carboxyl groups of the protein, ≈3 were substituted by α,β-6-lactosylamine, and ≈2 by the monohexo-sylamines. One of the 4 glycopeptides isolated from the tryptic digest of the lysozyme-lactosylamine conjugate was identical to synthetic l-N-L-leucinoyl-4-O-β-D-galactopyranosyl-β-D-glucopyranosylamine, indicating the substitution of the carboxyl group of the C-terminal leucine residue. The isolation of a glycopeptide containing the aspartic acid residue in position 117 indicates that the second α,β-lactosylamine residue is linked to the carboxyl group of this amino acid. Both of the 2 other glycopeptides contain the same free carboxyl groups (one glutamic and two aspartic acid residues in positions 35, 48, and 52, respectively). The third α,β-lactosylamine residue seems to be linked to one of these carboxyl groups.     

Effect of coordinated addition of specific amino acids on the synthesis of recombinant glucose isomerase

The amplified expression of a recombinant protein is known to lead to an intracellular depletion of specific amino acid pools which in turn may affect the production of the desired protein. In order to counteract and overcome such a situation during the fermentation of the recombinant Escherichia coli (PMSG27) containing the glucose isomerase (GI) gene from Streptomyces sp. NCIM 2730, the effect of addition of different amino acids on the specific activity of GI was studied. The amino acid composition of GI from Streptomyces sp. NCIM 2730 reveals predominantly aspartic acid, glutamic acid, and glycine; therefore, in the present paper, the effect of coordinated addition of the assorted combinations of these three amino acids on the synthesis of recombinant GI was studied. The results were analyzed using a 2³ factorial design. The following conclusions were derived from the analysis of two-factor interactions of the three amino acids: (i) The interaction between the aspartic and glutamic acid is independent of aspartic acid concentration but is affected by the increasing concentrations of glutamic acid, (ii) The effect of aspartic acid concentration is more than that of glycine, and (iii) During the interaction of glutamic acid and glycine, the effect of glutamic acid is more prominent than that of glycine. The three-factor interaction analyses reveal that the effect of the three amino acids is in the order aspartic acid > glutamic acid > glycine.     

Identification of catalytically active groups of pea (Pisum sativum L.) beta-glucosidase

Functional groups ofcytoplasmic pea beta-glucosidase pretreated to an electrophoretically homogeneous state were identified. Data on the pH dependence of the enzyme activity, calculated heat of ionization, photoinactivation of the enzyme in the presence of methylene blue, and inactivation of the enzyme with diethyl pyrocarbonate suggest that the catalytic site of beta-glucosidase contains the carboxyl group of glutamic or aspartic acids and the imidazole group of histidine.     

The 18O isotope effect in 13C nuclear magnetic resonance spectroscopy: mechanistic studies on asparaginase from Escherichia coli

The mechanism of the enzyme asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) from Escherichia coli was examined using 13C NMR spectroscopy. The pH-dependent oxygen exchange reactions between water and aspartic acid were followed by use of the 18O isotope-induced shift of the resonance positions of directly bonded 13C nuclei. Both L-1- and L-1,4-[13C]aspartic acid were used in experiments with previously 18O-labeled aspartic acid, or in experiments involving the use of 18O-labeled solvent water. Asparaginase catalyzes a relatively efficient exchange between the oxygens of water and those on one carboxyl group of aspartic acid. Exchange at C-4 occurs rapidly but, within experimental error, no exchange at C-1 could be detected. These and related experiments involving the position of 18O incorporation during hydrolysis of aspartic acid beta-methyl ester are all consistent with possible acyl-enzyme mechanisms involving C-4, but do not support a free aspartic acid anhydride mechanism.     

Restoration of enzymic activity and cytotoxicity of mutant, E553C, Pseudomonas aeruginosa exotoxin A by reaction with iodoacetic acid

Pseudomonas aeruginosa exotoxin A (ETA) is inactivated greater than 1,000-fold when an active site glutamic acid, E553, is mutated to aspartic acid (Douglas, C.M., and Collier, R. J. (1987) J. Bacteriol. 169, 4967-4971). To test the effect of creating a carboxyl-containing side chain at position 553 longer than that of glutamic acid, we first replaced Glu-553 with cysteine by site-directed mutagenesis of cloned ETA and then carboxymethylated the cysteine side chain with iodoacetic acid. The E553C mutation reduced ADP-ribosyltransferase and cytotoxic activities greater than 10,000-fold. Reaction of the mutant with iodoacetic acid enhanced enzymic activity 2,500-fold, to a level approximately one-sixth that of wild type toxin, and restored cytotoxicity to a slightly lesser extent. Iodoacetamide did not activate the mutant, and neither iodoacetic acid nor iodoacetamide affected the activity of wild type toxin. These results show that the carboxyl group of Glu-553 is important for ADP-ribosylation activity and imply flexibility in the enzyme-substrate complex in accommodating the slightly longer S-carboxymethylcysteine side chain. This general approach may have applications in protein engineering as well as in studying carboxyl side chain functions in enzymes.     

Antibiotic selection by the promiscuous aminoglycoside acetyltransferase-(3)-IIIb is thermodynamically achieved through the control of solvent rearrangement

The results presented here show the first known observation of opposite signs of change in heat capacity (ΔC(p)) of two structurally similar ligands binding to the same protein site. Neomycin and paromomycin are aminoglycoside antibiotics that are substrates for the resistance-conferring enzyme, the aminoglycoside acetyltransferase-(3)-IIIb (AAC). These antibiotics are identical to one another except at the 6' position where neomycin has an amine and paromomycin has a hydroxyl. The opposite trends in ΔC(p) of binding of these two drugs to AAC suggest a differential exposure of nonpolar amino acid side chains. Nuclear magnetic resonance experiments further demonstrate significantly different changes in AAC upon interaction with neomycin and paromomycin. Experiments in H(2)O and D(2)O reveal the first observed temperature dependence of solvent and vibrational contributions to ΔC(p). Coenzyme A significantly influences these effects. Together, the data suggest that AAC exploits solvent properties to facilitate favorable thermodynamic selection of antibiotics.     

Synthesis of beta- and gamma-fluorenylmethyl esters of respectively N alpha-Boc-L-aspartic acid and N alpha-Boc-L-glutamic acid

The orthogonal synthesis of N alpha-Boc-L-aspartic acid-gamma-fluorenylmethyl ester and N alpha-Boc-L-glutamic acid-delta-fluorenylmethyl ester is reported. This is a four-step synthesis that relies on the selective esterification of the side-chain carboxyl groups on N alpha-CBZ-L-aspartic acid and N alpha-CBZ-L-glutamic acid. Such selectivity is accomplished by initially protecting the alpha-carboxyl group through the formation of the corresponding 5-oxo-4-oxazolidinone ring. Following side-chain esterification, the alpha-carboxyl and alpha-amino groups are deprotected with acidolysis. Finally, the alpha-amino group is reprotected with the t-butyl-oxycarbonyl (Boc) group. Thus aspartic acid and glutamic acid have their side-chain carboxyl groups protected with the base-labile fluorenylmethyl ester (OFm) and their alpha-amino groups protected with the acid-labile Boc group. These residues, when used in conjunction with N alpha-Boc-N epsilon-Fmoc-L-lysine, are important in the formation of side-chain to side-chain cyclizations, via an amide bridge, during solid-phase peptide synthesis.     

Identification of catalytically active groups of pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.) β-glucosidase

Functional groups of cytoplasmic pea β-glucosidase pretreated to an electrophoretically homogeneous state were identified. Data on the pH dependence of the enzyme activity, calculated heat of ionization, photoinactivation of the enzyme in the presence of methylene blue, and inactivation of the enzyme with diethyl pyrocarbonate suggest that the catalytic site of β-glucosidase contains the carboxyl group of glutamic or aspartic acids and the imidazole group of histidine.     

五步蛇蛇毒磷脂酶A_2的纯化及部分性质

经Sephadex G-75和QAE-Sephadex A-50离子交换层析等方法,从湖南产五步蛇(Agkistrodon acutus)蛇毒中纯化一种均一的酸性磷脂酶A_2。SDS-PAGE测得分子量为15.8kD,按氨基酸残基计算其分子量为14.352kD,IEF-PAGE测得等电点为5.32。氨基酸组份分析表明磷脂酶A_2分子由128个氨基酸残基组成,富含Asp和Glu,不含中性糖。PLA_2酶活性的最适温度为45℃,最适pH为8.5左右,没有抗胰蛋白酶的活性,具一定的热稳定性。K~+、Ca~(++)和Na~+离子激活,而Cd~(++)、Sn~(++)、Cu~(++)、Li~+、Hg(++)、Zn~(++)、Fe~(++)和Co~(++)离子可抑制或完全丧失酶活力。手工微量顺序分析测得PLA_2分子N-末端氨基酸为Leu。此酶对小白鼠的LD_(50)至少大于10mg/kg(ip)。     

A new approach for detecting C‐terminal amidation of proteins and peptides by mass spectrometry in conjunction with chemical derivatization

We describe a mass spectrometric method for distinguishing between free and modified forms of the C‐terminal carboxyl group of peptides and proteins, in combination with chemical approaches for the isolation of C‐terminal peptides and site‐specific derivatization of the C‐terminal carboxyl group. This method could most advantageously be exploited to discriminate between peptides having C‐terminal carboxyl groups in the free (COOH) and amide (CONH₂) forms by increasing their mass difference from 1 to 14 Da by selectively converting the free carboxyl group into methylamide (CONHCH₃). This method has been proven to be applicable to peptides containing aspartic and glutamic acids, because all the carboxyl groups except the C‐terminal one are inert to derivatization, according to oxazolone chemistry. The efficiency of the method is illustrated by a comparison of the peaks of processed peptides obtained from a mixture of adrenomedullin, calcitonin, and BSA. Among these components of the mixture, only the C‐terminal peptide of BSA exhibited the mass shift of 13 Da upon treatment, eventually unambiguously validating the C‐terminal amide structures of adrenomedullin and calcitonin. The possibility of extending this method for the analysis of C‐terminal PTMs is also discussed.     

Tracer studies on the biosynthesis of amino acids from lactate by Peptostreptococcus elsdenii

Peptostreptococcus elsdenii, a strict anaerobe from the rumen, was grown on a medium containing yeast extract and [1-(14)C]- or [2-(14)C]-lactate. Radioisotope from lactate was found in all cell fractions, but mainly in the protein. The label in the protein fraction was largely confined to a few amino acids: alanine, serine, aspartic acid, glutamic acid and diaminopimelic acid. The alanine, serine, aspartic acid and glutamic acid were separated, purified and degraded to establish the distribution of (14)C from lactate within the amino acid molecules. The labelling patterns in alanine and serine suggested their formation from lactate without cleavage of the carbon chain. The pattern in aspartic acid suggested formation by condensation of a C(3) unit derived directly from lactate with a C(1) unit, probably carbon dioxide. The distribution in glutamic acid was consistent with two possible pathways of formation: (a) by the reactions of the tricarboxylic acid cycle leading from oxaloacetate to 2-oxoglutarate, followed by transamination; (b) by a pathway involving the reaction sequence 2 acetyl-CoA-->crotonyl-CoA-->glutaconate-->glutamate.     

Mechanism of Proline Production by Kurthia catenaforma

The fate of aspartic acid used for proline fermentation by Kurthia catenaforma was traced by using aspartic acid-U-(14)C. The radioactivities of proline and glutamic acid increased with the disappearance of aspartic acid. After 40 hr, aspartic acid disappeared from the medium and radioactive alpha-ketoglutaric acid was detected. The radioactivity of proline reached 44% of aspartic acid radioactivity at 40 hr. The specific radioactivities of these amino acids and of alpha-ketoglutaric acid supported the notion that proline is produced mainly from aspartic acid via alpha-ketoglutaric acid and glutamic acid. Since the levels of glutamic acid dehydrogenases (EC 1.4.1.2 and EC 1.4.1.4) were low in this organism, it appears that the nitrogen atom of aspartic acid enters proline by the action of aspartate aminotransferase (EC 2.6.1.1). The mechanism of proline production is discussed on the basis of the role of aspartic acid in this fermentation.     

Unusual amidation reaction of asparagine-containing glycopeptide antibiotics in the presence of (benzotriazole-1-yl)oxy-tris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP)

The coupling reagent (benzotriazole-1-yl)oxy-tris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP) is widely used for the synthesis of different peptides and their amides, particularly carboxamides of glycopeptide antibiotics of the vancomycin or teicoplanin groups. The amidation reaction of the carboxyl group of the seventh amino acid residue (AA7) in antibiotics in the presence of PyBOP is not usually accompanied by the formation of significant amounts of byproducts. However, the amidation of eremomycin (I) with bulky amines (e.g., decyl amine and adamantyl amine) in the presence of PyBOP at pH ～8.5 (Et₃N or (i-Pr)₂EtN) yielded N-unsubstituted carboxamide of eremomycin (Ia) as an admixture. The interaction of asparagine-containing antibiotics (eremomycin or vancomycin) with the excess of PyBOP and Et₃N (pH ～8.5) in the absence of amine or ammonia led to the formation of still larger amounts of corresponding unsubstituted AA7-amides (～20%). Their structure was determined by ¹H NMR and ESI MS methods and confirmed by comparing with authentic samples. It is assumed that the amide group of the asparagine residue (AA3) is the source of ammonia in the unususal amidation reaction of Asn-containing antibiotics.     

Calcium binding by human erythrocyte membranes. Significance of carboxyl, amino and thiol groups

1. The role of the ionized carboxyl groups of proteins of the erythrocyte membrane as Ca(2+) receptor sites was investigated. A water-soluble carbodi-imide [1-cyclohexyl-3-(2-morpholinoethyl)carbodi-imide methotoluene-p-sulphonate], referred to as carbodi-imide reagent, and glycine methyl ester were used to modify the free carboxyl groups of the membrane. The degree of modification was estimated from amino acid analyses, which showed the amount of glycine incorporated. As the concentration of carbodi-imide reagent was raised (0.1-0.4m) incorporation of glycine increased and Ca(2+) binding decreased by about 77%. At 0.4m-carbodi-imide reagent all of the binding of Ca(2+) to protein was abolished and it was estimated that about 37% of the side-chain carboxyl groups of aspartic acid plus glutamic acid had been blocked by glycine. 2. Acetylation of all of the free amino groups was achieved by incubating the erythrocyte ;ghosts' at pH10.3 with acetic anhydride (10-15mg/10mg of ;ghost' protein). Acetylation increased by 1.5-fold the capacity of the ;ghost' to bind Ca(2+), indicating that the remaining carboxyl groups of aspartic acid and glutamic acid were made available for Ca(2+) binding by this procedure. These findings support the concept that in normal ;ghosts', at pH7.4, Ca(2+) binding to free carboxyl groups is partially hindered by the presence of charged amino groups. 3. Treatment of ;ghosts' with N-acetylneuraminidase, which removed 94% of sialic acid residues, and treatment with 1mm-p-chloromercuribenzoate did not alter Ca(2+) binding. The major effect of 5.8mm-p-chloromercuribenzoate upon ;ghosts' was to cause a solubilization of a calcium-membrane complex, which included about one-third of the ;ghost' protein. The molar ratio of Ca(2+): protein in the solubilized material was the same as that in the intact (untreated) ;ghosts'.     

Correlation between [U-14C]glucose metabolism and function in perfused cat brain

In brain perfusion experiments conducted with blood containing [U-14C]glucose the relative specific activity (RSA) of blood glucose carbon incorporated in brain intermediate metabolites was measured. It was demonstrated that the so-called metabolic pattern of Geiger is not constant, but it bears a close relation to the function of the brain. The results of the study may be summarized briefly as follows. (1) In a group (A) of cats with a high level of brain function, the RSA of lactic acid was 75 per cent; that of glutamic acid 80 per cent; aspartic acid 75 per cent; glutamine 61 per cent; GABA 43 per cent; and respiratory CO2 55 per cent. It was observed that the major part of the carbon of amino acids, such as glutamic acid and aspartic acid, which are directly associated with the tricarboxylic acid cycle are derived from blood glucose. (2) In a group (B) showing a low level of brain function, the RSA of each amino acid was considerably lowered. The RSA of glutamic acid and aspartic acid was about 50 per cent and that of respiratory CO2 was 27 per cent. (3) In a group (C) with a still lower level of brain function, each amino acid as well as the respiratory CO2 had still lower RSA values. (4) The metabolic pattern of Geiger corresponds to values obtained during low functional activity of the brain in our experiment.     

THE CONDUCTIVITIES OF AQUEOUS SOLUTIONS OF GLYCINE,d,l-VALINE,AND l-ASPARAGINE

1. The conductivities of aqueous solutions of glycine, d,l-valine, and l-asparagine have been determined, and comparisons have been made with similar data reported in the literature. 2. On the basis of certain theoretical considerations, calculations of the expected conductivities of aqueous solutions of glycine, asparagine, aspartic acid, and glutamic acid have been made and these data have been compared with similar data obtained experimentally. 3. The dissociation constants of the carboxyl groups of aspartic acid and glutamic acid have been calculated from conductivity data. 4. It is shown that alanine has no effect on the ionic atmosphere of solutions of potassium chloride.