Purification and properties of L-alanine aminotransferase from Chlamydomonas reinhardtii.

An enzyme which catalyzes the transamination of L-alanine with 2-oxoglutarate has been purified 157-fold to electrophoretic homogeneity from the unicellular green alga Chlamydomonas reinhardtii 6145c. The enzyme showed maximal activity at pH 7.3 and 50 degrees C, has an apparent molecular mass of 105 kDa as estimated by gel filtration, and consists of two identical subunits of 45 kDa each as deduced from PAGE/SDS studies. A stoichiometry of two moles pyridoxal 5-phosphate/mole enzyme was calculated. The enzyme has an isoelectric point of 8.3 and its absorption spectrum exhibits a maximum at 412 nm which is shifted to 330 nm upon addition of L-alanine. Pyridoxal 5-phosphate protected activity against heat inactivation and, to a minor extent, L-alanine and 2-oxoglutarate, but not L-glutamate. Spectral data and activity inhibition and protection studies strongly support the involvement of pyridoxal 5-phosphate in enzyme catalysis through a Schiff's base formation. The purified enzyme was able to transaminate only L-alanine and L-glutamate with glyoxylate out of ten amino acids tested. L-Alanine aminotransferase exhibited hyperbolic kinetic for 2-oxoglutarate, pyruvate, and L-glutamate, and nonhyperbolic behaviour for L-alanine. Apparent Km values were 0.054 mM for 2-oxoglutarate, 0.52 for L-glutamate, 0.24 mM for pyruvate, and 2.7 mM for L-alanine. Transamination of L-alanine in C. reinhardtii is a bisubstrate reaction with a bi-bi ping-pong mechanism, and is not inhibited by substrates.     

Kinetics of the alanine aminotransferase reaction in the mitochondrial and cell sap fractions of rat brain.

1. A reversible transamination reaction between L-glutamate and pyruvate, or L-alanine and 2-oxoglutarate, takes place in the mitochondrial and cell sap fractions of rat brain. 2. The maximum rate of the transamination reaction in both subfractions was observed in the presence of a keto- substrate concentration of 2.5 mM only, but an amino- donor concentration of 20 mM. 3. The apparent Menten-Michaelis constants for pyruvate and 2-oxoglutarate were of a 10(-4) M and for L-glutamate and L-alanine of a 10(-3) M order and were approximately the same for both fractions. 4. The ratio of the initial rate of the L-alanine + 2-oxoglutarate to the L-glutamate + pyruvate transamination reaction in the cell sap and mitochondrial fractions amounted to up to 2. 5. The apparent equilibrium constant derived from the Haldane equation was 7.01 for cell sap alanine aminotransferase and 4 for the mitochondrial enzyme. 6. Increasing pyridoxal-5'-phosphate concentrations in the incubation medium were accompanied by only non-significant stimulation of alanine aminotransferase activity in the mitochondrial and cell sap fractions. 7. A comparison of the kinetic data obtained on mitochondrial and cell sap alanine aminotransferases in vitro with the actual substrate concentrations in the transamination reaction in nervous tissue in vivo indicates that the direction of the transamination reaction in situ seems to be determined simply by compartmentation and by dynamic changes in amino- and keto- substrates in the mitochondrial and cell sap spaces.     

Purification and properties of alanine aminotransferase from maize (Zea mays L.) leaves

Alanine aminotransferase (AlaAT, EC 2.6.1.2) from leaves of 14-day-old maize seedlings was purified over 1600-fold to electrophoretical homogeneity. Specific activity of the purified enzyme measured with L-alanine and 2-oxoglutarate as substrates was 2125 nkat·(mg protein)⁻¹ at 30 °C. The molecular weights of the native and sodium dodecyl sulfate — denatured AlaAT protein were 95 kDa and 50 kDa respectively, indicating that the native enzyme is probably a homodimer. AlaAT almost exclusively catalyzed amino group transfer from L-alanine to 2-oxoglutarate and the reverse reaction. The inhibitory experiments showed that pirydoxal phosphate is directly involved in the enzymatic catalysis and the enzyme molecule contains essential SH groups. The use of phenylglyoxal demonstrated the presence of arginine residue as anionic binding site in the active centre of AlaAT. This work was supported by the State Committee for Scientific Research, a grant No. 5PO6A00510     

Transamination pathways influencing L-glutamine and L-glutamate oxidation by rat enterocyte mitochondria and the subcellular localization of L-alanine aminotransferase and L-aspartate aminotransferase

Using analytical subcellular fractionation techniques, 12% of the total L-alanine aminotransferase activity and 26% of the total L-aspartate aminotransferase activity was localized in enterocyte mitochondria. Alanine and aspartate were products from the oxidation of glutamine and glutamate by enterocyte mitochondria. At low concentrations, malate stimulated aspartate synthesis but was inhibitory at higher concentrations. The malate inhibition of aspartate synthesis, which increased in the presence of pyruvate, was accompanied by an increase in alanine synthesis. With glutamine as substrate in the presence of pyruvate and malate, alanine synthesis was increased by 127% on addition of purified L-alanine aminotransferase, in spite of large amounts of glutamate generated. It was concluded that when pyruvate is available the important route for glutamine or glutamate oxidation by transamination was via L-alanine:2-oxoglutarate aminotransferase and not via L-aspartate:2-oxoglutarate aminotransferase. Results suggested that mitochondria may account for 50% of alanine production from glutamine in the enterocyte despite the relatively low activity of L-alanine aminotransferase therein.     

Kinetics of the interaction between aspartic aminotransferase and anions.

The kinetics of the interaction between deionized supernatant aspartic aminotransferase and various anions (cacodylate, phosphate and chloride) were studied by the temperature-jump technique. The anion concentration in the range covered by our experiments does not affect the transamination rate. On the other hand the conformational transition, recently observed at the active site of the enzyme, is hindered by an excess of anions. A single relaxation effect was observed at the enzyme chromophore wavelength in systems containing the aldimine form of the enzyme and the above anions. It is shown that this effect corresponds to the protonation of the chromophore. The relaxation times were of about 10 mus with phosphate, 20-100 mus with cacodylate and 1-2 ms with chloride. The pH and concentration dependence of this effect were studied. The fits of experimental data to a rate equations for various models were tested by a chi2 analysis. The best fit was obtained with models where anions bind rapidly to a site close to the chromophore, so that the pK of the chromophore is affected by anions binding. The rate of the observed relaxation considerably increased when the anion has buffering capacities; this indicates, in the case of cacodylate and phosphate, that the acidic component of the buffer directly exchanges a proton with the enzyme chromophore.     

Purification and properties of L-aspartate aminotransferase of Chlamydomonas reinhardtii

An enzyme which catalyzes the transamination of L-aspartate with 2-oxoglutarate has been purified 400-fold to electrophoretic homogeneity from the unicellular green alga Chlamydomonas reinhardtii 6145c. An apparent relative molecular mass of 138,000 was estimated by gel filtration. The enzyme is a dimer consisting of two identical subunits of Mr 65,000 each as deduced from PAGE/SDS studies. A stoichiometry of two molecules pyridoxal 5-phosphate/enzyme molecule was calculated. The enzyme has an isoelectric point of 8.48 and its absorption spectrum exhibits a maximum at 412 nm which is shifted to 330 nm upon addition of L-aspartate. L-Aspartate or pyridoxal 5-phosphate, but not 2-oxoglutarate, protected the enzyme from heat inactivation. The purified enzyme was able to transaminate, although to a low extent, L-phenylalanine and L-tyrosine with 2-oxoglutarate, and L-serine, L-alanine and L-glutamine with oxaloacetate. L-Aspartate aminotransferase exhibited hyperbolic kinetics for 2-oxoglutarate and oxaloacetate, and nonhyperbolic behaviour for L-aspartate and L-glutamate. Apparent Km values were 0.55 mM for 2-oxoglutarate, 0.044 mM for oxaloacetate, 2.53 mM for L-aspartate and 3.88 mM for L-glutamate. Transamination of L-aspartate in C. reinhardtii is a bisubstrate reaction with a bi-bi ping-pong mechanism, and is not inhibited by substrates.     

L-alanine binding sites and Na+, K(+)-ATPase in cilia and other membrane fractions from olfactory rosettes of Atlantic salmon.

1. Membrane fractions were obtained from homogenates of olfactory rosettes from Atlantic salmon (Salmo salar) or from isolated olfactory cilia and homogenates of deciliated olfactory rosettes. 2. Specific binding of L-[3H]alanine was saturable, high-affinity, and effectively inhibited by L-threonine, L-serine and L-alanine but not by L-lysine or L-glutamic acid. Comparable results were obtained with L-[3H]serine except for the presence of a second, lower affinity, binding site for L-alanine but not L-serine. 3. Specific binding of L-[3H]alanine was inhibited by low concentrations of mercury ion, acidic pH, and high concentrations of cadmium, copper or zinc ions. Aluminum had no effect. 4. Specific binding sites for L-alanine were present in membranes from isolated cilia at a level 2-fold that of membranes prepared from the deciliated rosette. 5. Ouabain sensitive Na+, K(+)-ATPase activity was also determined in cilia preparations. This enzyme was present in cilia at a level approximately 3-fold that of membranes prepared from the deciliated rosette. 6. The results are consistent with the presence of an olfactory alanine receptor in S. salar with binding characteristics similar to those of a variety of other fish species and with a localization on olfactory cilia as well as non-ciliated receptor cell membranes.     

The route of lysine breakdown in Candida tropicalis

Candida tropicalis was found to contain high levels of the following enzymes after growth in defined medium on L-lysine as sole nitrogen source: L-lysine N6-acetyltransferase, N6-acetyl-lysine aminotransferase, and aminotransferase activity for 5-aminovalerate and 4-aminobutyrate. Extracts were also capable of converting 5-acetamidovalerate (and 4-acetamidobutyrate) to acetate. N6-Acetyllysine however, only gave rise to acetate in the presence of 2-oxoglutarate, NAD+ and thiamine pyrophosphate. These activities were undetectable or present in much lower concentrations in cells that had been grown on ammonium sulphate as sole nitrogen source. It is concluded that L-lysine is degraded in this organism via N6-acetyllysine, 5-acetamidovalerate and 5-aminovalerate, both nitrogen atoms being removed by transamination.     

Alanine aminotransferase and glycine aminotransferase from maize (Zea mays L.) leaves.

Alanine aminotransferase (AlaAT, EC 2.6.1.2) and glycine aminotransferase (GlyAT, EC 2.6.1.4), two different enzymes catalyzing transamination reactions with L-alanine as the amino-acid substrate, were examined in maize in which alanine participates substantially in nitrogen transport. Preparative PAGE of a partially purified preparation of aminotransferases from maize leaves gave 6 fractions differing in electrophoretic mobility. The fastest migrating fraction I represents AlaAT specific for L-alanine as amino donor and 2-oxoglutarate as amino acceptor. The remaining fractions showed three aminotransferase activities: L-alanine-2-oxoglutarate, L-alanine-glyoxylate and L-glutamate-glyoxylate. By means of molecular sieving on Zorbax SE-250 two groups of enzymes were distinguished in the PAGE fractions: of about 100 kDa and 50 kDa. Molecular mass of 104 kDa was ascribed to AlaAT in fraction I, while the molecular mass of the three enzymatic activities in 3 fractions of the low electrophoretic mobility was about 50 kDa. The response of these fractions to: aminooxyacetate, 3-chloro-L-alanine and competing amino acids prompted us to suggest that five out of the six preparative PAGE fractions represented GlyAT isoforms, differing from each other by the L-glutamate-glyoxylate:L-alanine-glyoxylate:L-alanine-2-oxoglutarate activity ratio.     

Role of 4-aminobutyrate aminotransferase in the arginine metabolism of Pseudomonas aeruginosa.

4-Aminobutyrate aminotransferase (GABAT) from Pseudomonas aeruginosa was purified 64-fold to apparent electrophoretic homogeneity from cells grown with 4-aminobutyrate as the only source of carbon and nitrogen. Purified GABAT catalyzed the transamination of 4-aminobutyrate, N2-acetyl-L-ornithine, L-ornithine, putrescine, L-lysine, and cadaverine with 2-oxoglutarate (listed in order of decreasing activity). The enzyme is induced in cells grown on 4-guanidinobutyrate, 4-aminobutyrate, or putrescine as the only carbon and nitrogen source. Cells grown on arginine or on glutamate contained low levels of the enzyme. The regulation of the synthesis of GABAT as well as the properties of the mutant with an inactive N2-acetyl-L-ornithin 5-aminotransferase suggest that GABAT functions in the biosynthesis of arginine by convertine N2-acetyl-L-glutamate 5-semialdehyde to N2-acetyl-Lornithine as well as in catabolic reactions during growth on putrescine or 4-guanidinobutyrate but not during growth on arginine.     

The inhibition by anion binding of reactions of inorganic radical anions with bovine carbonic anhydrase B

Reactions of the inorganic radical anions, Br(2) and (SCN)2, with bovine carbonic anhydrase (carbonate hydrolyase, EC 4.2.1.1) have been studied by pulse radiolysis. Reaction is almost completely inhibited by the binding of Br-, SCN- and ClO4- to an electrophilic site at the active centre of the enzyme. Dissociation constants for anion binding calculated from the reduction in free radical reactivity agree well with inhibition constants for these anions. The anions OCN- and CN-, although potent inhibitors of carbonic anhydrase activity, have relatively little effect on the reactivity of radical anions with the enzyme. Reaction of radical anions occurs mainly with tryptophan and tyrosine residues in the hydrophobic core of the enzyme, through a channel at the active site. This channel is closed by the anions in accord with their position in the lyotropic series.     

Anion binding to yeast phosphoglycerate kinase.

The single thiol of yeast phosphoglycerate kinase was labelled with the chromophoric sulfhydryl reagent, 2-chloromercuri-4-nitrophenol. Sequential additions of individual anions to this modified enzyme brought about a decrease in absorbance at 410 nm that reflected the degree of saturation of the enzyme with anion. The binding curves were analyzed to determine the dissociation constants of a number of anions with charges varying from--1 to--4.1. A linear relationship was found between the charge of the anion and the negative logarithm of the dissociation constant for the labelled enzyme-anion complex. The highly charged anions, such as ATP, bound more tightly than did anions with less charge, such as Cl-. The average number of binding sites for those anions for which accurate results could be obtained was 1.06 mol per 47000 g of enzyme. Several lines of evidence suggested that titration of the active center was not being monitored. Anions bound to phosphoglycerate kinase decreased the rate of reaction between the enzyme thiol and 5,5'-dithiobis(2-nitrobenzoic acid). The relationship between the degree of saturation of the anion binding site and the reaction rate constant was used to calculate the dissociation constant between anion and enzyme. Dissociation constants determined in this manner were in good agreement with those determined by titration of the enzyme-mercurial complex.     

Dibasic amino acid interactions with Na+-independent transport system asc in horse erythrocytes. Kinetic evidence of functional and structural homology with Na+-dependent system ASC

Amino acid transport in horse erythrocytes is regulated by three co-dominant allelomorphic genes coding for high-affinity transport activity (system asc1), low-affinity transport activity (system asc2) and transport-deficiency, respectively. The asc systems are selective for neutral amino acids of intermediate size, but unlike conventional system ASC, do not require Na+ for activity. In the present series of experiments we have used a combined kinetic and genetic approach to establish that dibasic amino acids are also asc substrates, systems asc1 and asc2 representing the only mediated routes of cationic amino acid transport in horse erythrocytes. Both transporters were found to exhibit a strong preference for dibasic amino acids compared with neutral amino acids of similar size. Apparent Km values (mM) for influx via system asc1 were L-lysine (9), L-ornithine (27), L-arginine (27), L-alanine (0.35). Corresponding Vmax estimates (mmol/l cells per h, 37 degrees C) were L-lysine (1.65), L-ornithine (2.15), L-arginine (0.54), L-alanine (1.69). Apparent Km values for L-lysine and L-ornithine influx via system asc2 were approximately 90 and greater than 100 mM, respectively, with Vmax values greater than 2 and greater than 1 mmol/l cells per h, respectively. Apparent Km and Vmax values for L-alanine uptake by system asc2 were 14 mM and 6.90 mmol/l cells per h. In contrast, L-arginine was transported by system asc2 with the same apparent Km as L-alanine (14 mM), but with a 77-fold lower Vmax. This dibasic amino acid was shown to cause cis- and trans-inhibition of system asc2 in a manner analogous to its interaction with system ASC, where the side-chain guanidinium group is considered to occupy the Na+-binding site on the transporter. Concentrations of extracellular L-arginine causing 50% inhibition of zero-trans L-alanine influx and half-maximum inhibition of L-alanine zero-trans efflux were 14 mM (extracellular L-alanine concentration 15 mM) and 3 mM (intracellular L-alanine concentration 15.5 mM), respectively. We interpret these observations as evidence of structural homology between the horse erythrocyte asc transporters and system ASC. Physiologically, intracellular L-arginine may function as an endogenous inhibitor of system asc2 activity.     

Candida L-norleucine,leucine:2-oxoglutarate aminotransferase. Purification and properties

A new enzyme which catalyzes the transamination of L-norleucine (2-aminohexanoic acid) and L-leucine with 2-oxoglutarate was purified to homogeneity from cells of Candida guilliermondii var. membranaefaciens. The relative molecular mass determined by gel filtration was estimated to be close to 100,000. The transaminase behaved as a dimer which consists of two subunits identical in molecular mass (Mr 51,000). The enzyme has a maximum activity in the pH range of 8.0-8.5 and at 55 degrees C. 2-Oxoglutarate, and to a lesser extent pyridoxal 5'-phosphate, were effective protecting agents against increasing temperature. The enzyme exhibits absorption maximum at 330 nm and 410 nm. L-Norleucine, and L-leucine to a lesser extent, are the best amino donors with 2-oxoglutarate as amino acceptor. The Km values for L-norleucine, L-leucine and 2-oxoglutarate determined from the Lineweaver-Burk plot were 1.8 mM, 6.6 mM and 2.0 mM respectively. A ping-pong bi-bi mechanism of inhibition with alternative substrates is found when the enzyme is in the presence of both L-norleucine and L-leucine. The inhibitory effect of various amino acid analogs on the transamination reaction between L-norleucine and 2-oxoglutarate was studied and Ki values were determined.     

Glutamate-glyoxylate aminotransferase in rat liver cytosol. Purification, properties and identity with alanine-2-oxoglutarate aminotransferase

After cortisone injection, virtually identical increases in rat liver cytosol alanine-2-oxoglutarate aminotransferase and glutamate-glyoxylate aminotransferase activities were observed. The two activities were co-purified to homogeneity from rat liver cytosol. The purified enzyme was specific for L-alanine with 2-oxoglutarate as amino acceptor. With glyoxylate, however, the enzyme utilized various L-amino acids as amino donors in the following order of activity: glutamate greater than alanine greater than glutamine greater than methionine. The ratio of alanine-2-oxoglutarate aminotransferase activity to glutamate-glyoxylate aminotransferase activity remained constant during purification and was unchanged by a variety of treatments of the purified enzyme. These results suggest that glutamate-glyoxylate aminotransferase is identical with alanine-2-oxoglutarate aminotransferase. Evidence was obtained that the two enzyme activities in the cytosol of dog, cat and human liver are also properties of the same protein.     

Characterization of a novel enzyme,N6-acetyl-L-lysine: 2-oxoglutarate aminotransferase,which catalyses the second step of lysine catabolism inCandida maltosa

A novel aminotransferase catalyzing the second step of lysine catabolism, the oxidative transamination of the -group of N⁶-acetyllysine, was identified and characterized in the yeastCandida maltosa. The enzyme was strongly induced in cells grown on L-lysine as sole carbon source. Its activity was specific for both N⁶-acetyllysine and 2-oxoglutarate. The K_m values were 14 mM for the donor, 4 mM for the acceptor and 1.7 M for pyridoxal-5-phosphate. The enzyme had a maximum activity at pH 8.1 and 32°C. Its molecular mass estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis was 55 kDa. Since the native molecular mass determined by gel filtration was 120 kDa, the enzyme is probably a homodimer.     

Effect of chemical modification and carboxylate anions on transamination of phenylalanine and alanine in the active center of chicken cytosol aspartate transaminase

Photooxidation of a histidine residue in aspartate transaminase leads to proportionate loss of the enzyme activity in reactions with L-aspartate and L-phenylalanine. Modification of two arginine residues by 1,2-cyclohexanedione strongly inhibits transamination of aspartate but, in contrast, slightly increases the rate of phenylalanine transamination. A stimulatory effect of a number of aromatic and aliphatic monocarboxylate anions on the rate of alanine transamination in the active site was observed. Indolylbutyrate was the most effective compound among those tested. Indolylbutyrate and indolylacetate act as competitive inhibitors in the case of transamination of phenylalanine or aspartate. The results were interpreted as indicating the presence in the active center of transaminase of a hydrophobic subsite participating in the binding of aromatic aminoacids.     

Characterization of an arginine:pyruvate transaminase in arginine catabolism of Pseudomonas aeruginosa PAO1

The arginine transaminase (ATA) pathway represents one of the multiple pathways for L-arginine catabolism in Pseudomonas aeruginosa. The AruH protein was proposed to catalyze the first step in the ATA pathway, converting the substrates L-arginine and pyruvate into 2-ketoarginine and L-alanine. Here we report the initial biochemical characterization of this enzyme. The aruH gene was overexpressed in Escherichia coli, and its product was purified to homogeneity. High-performance liquid chromatography and mass spectrometry (MS) analyses were employed to detect the presence of the transamination products 2-ketoarginine and L-alanine, thus demonstrating the proposed biochemical reaction catalyzed by AruH. The enzymatic properties and kinetic parameters of dimeric recombinant AruH were determined by a coupled reaction with NAD(+) and L-alanine dehydrogenase. The optimal activity of AruH was found at pH 9.0, and it has a novel substrate specificity with an order of preference of Arg > Lys > Met > Leu > Orn > Gln. With L-arginine and pyruvate as the substrates, Lineweaver-Burk plots of the data revealed a series of parallel lines characteristic of a ping-pong kinetic mechanism with calculated V(max) and k(cat) values of 54.6 +/- 2.5 micrromol/min/mg and 38.6 +/- 1.8 s(-1). The apparent K(m) and catalytic efficiency (k(cat)/K(m)) were 1.6 +/- 0.1 mM and 24.1 mM(-1) s(-1) for pyruvate and 13.9 +/- 0.8 mM and 2.8 mM(-1) s(-1) for l-arginine. When L-lysine was used as the substrate, MS analysis suggested Delta(1)-piperideine-2-carboxylate as its transamination product. These results implied that AruH may have a broader physiological function in amino acid catabolism.     

An NMR study of anion binding to yeast phosphoglycerate kinase

Anion binding to yeast phosphoglycerate kinase has been investigated using 1H-NMR spectroscopy. The use of anionic paramagnetic probes. [Cr(CN)6]3- and [Fe(CN)6]3-, has enabled the location of the primary anion binding site in the 'basic-patch' region of the amino-terminal domain. The anions interact most closely with Arg-65 and Arg-168. The binding of these and a variety of other anions to this site is directly competitive with the binding of the substrate, 3-phosphoglycerate. Binding of 3-phosphoglycerate and 1.3-bisphosphoglycerate is, however, stronger than expected on the basis of anionic charge and causes conformational changes in the protein not seen with any of the other simple spherical anions investigated. This must be part, at least, of the substrate specificity. Evidence for a secondary anion binding site involving the side chains of surface lysine residues is also presented. It is suggested that the primary anion site is responsible for the observed activation by anions at low concentrations while the secondary site leads to inhibition at higher anion concentrations. The kinetics fit these deductions and a scheme for kinase activity is presented.     

Two sites of interaction of anions with cytochrome a in oxidized bovine cytochrome c oxidase

An interaction between cytochrome a in oxidized cytochrome c oxidase (CcO) and anions has been characterized by EPR spectroscopy. Those anions that affect the EPR g = 3 signal of cytochrome a can be divided into two groups. One group consists of halides (Cl-, Br-, and I-) and induces an upfield shift of the g = 3 signal. Nitrogen-containing anions (CN-, NO2-, N3-, NO3-) are in the second group and shift the g = 3 signal downfield. The shifts in the EPR spectrum of CcO are unrelated to ligand binding to the binuclear center. The binding properties of one representative from each group, azide and chloride, were characterized in detail. The dependence of the shift on chloride concentration is consistent with a single binding site in the isolated oxidized enzyme with a Kd of approximately 3 mm. In mitochondria, the apparent Kd was found to be about four times larger than that of the isolated enzyme. The data indicate it is the chloride anion that is bound to CcO, and there is a hydrophilic size-selective access channel to this site from the cytosolic side of the mitochondrial membrane. An observed competition between azide and chloride is interpreted by azide binding to three sites: two that are apparent in the x-ray structure plus the chloride-binding site. It is suggested that either Mg2+ or Arg-438/Arg-439 is the chloride-binding site, and a mechanism for the ligand-induced shift of the g = 3 signal is proposed.