Characterization of chitin synthase 2 of Saccharomyces cerevisiae. Implication of two highly conserved domains as possible catalytic sites

Chitin synthase 2 of Saccharomyces cerevisiae was characterized by means of site-directed mutagenesis and subsequent expression of the mutant enzymes in yeast cells. Chitin synthase 2 shares a region whose sequence is highly conserved in all chitin synthases. Substitutions of conserved amino acids in this region with alanine (alanine scanning) identified two domains in which any conserved amino acid could not be replaced by alanine to retain enzyme activity. These two domains contained unique sequences, Glu561-Asp562-Arg563 and Gln601-Arg602-Arg603-Arg604-Trp605, that were conserved in all types of chitin synthases. Glu561 or arginine at 563, 602, and 603 could be substituted by glutamic acid and lysine, respectively, without significant loss of enzyme activity. However, even conservative substitutions of Asp562 with glutamic acid, Gln601 with asparagine, Arg604 with lysine, or Trp605 with tyrosine drastically decreased the activity, but did not affect apparent Km values for the substrate significantly. In addition to these amino acids, Asp441 was also found in all chitin synthase. The mutant harboring a glutamic acid substitution for Asp441 severely lost activity, but it showed a similar apparent Km value for the substrate. Amounts of the mutant enzymes in total membranes were more or less the same as found in the wild type. Furthermore, Asp441, Asp562, Gln601, Arg604, and Trp605 are completely conserved in other proteins possessing N-acetylglucosaminyltransferase activity such as NodC proteins of Rhizobium bacterias. These results suggest that Asp441, Asp562, Gln601, Arg604, and Trp605 are located in the active pocket and that they function as the catalytic residues of the enzyme.     

Site-directed mutational analysis for the membrane binding of DnaA protein. Identification of amino acids involved in the functional interaction between DnaA protein and acidic phospholipids

DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli, interacts with acidic phospholipids, such as cardiolipin, and its activity seems to be regulated by membrane binding in cells. In this study we introduced site-directed mutations at the positions of hydrophobic or basic amino acids which are conserved among various bacteria species and which are located in the putative membrane-binding region of DnaA protein (from Asp357 to Val374). All mutant DnaA proteins showed much the same ATP and ADP binding activity as that of the wild-type protein. The release of ATP bound to the mutant DnaA protein, in which three hydrophobic amino acids were mutated to hydrophilic ones, was stimulated by cardiolipin, as in the case of the wild-type protein. On the other hand, the release of ATP bound to another mutant DnaA protein, in which three basic amino acids were mutated to acidic ones, was not stimulated by cardiolipin. These results suggest not only that the region is a membrane-binding domain of DnaA protein but also that these basic amino acids are important for the binding and the ionic interaction between the basic amino acids and acidic residues of cardiolipin and is involved in the interaction between DnaA protein and cardiolipin.     

Dissociation of fibronectin from gelatin-agarose by amino compounds

Soluble fibronectin of human plasma was specifically dissociated at neutral pH from gelatin-agarose by several cationic amino compounds, notably the polyamines spermine, spermidine and putrescine, the basic amino acid arginine, and amino sugars. The neutral and acidic amino acids and the N-acetylated derivatives of amino sugars tested were ineffective. Gel-filtration experiments demonstrated that [14C]spermidine bound to fibronectin but not to gelatin.     

Lysophosphatidylcholine acyltransferase activity in Saccharomyces cerevisiae: regulation by a high-affinity Zn2+ binding site

Variants of human pancreatic carboxypeptidase B (HCPB), with specificity for hydrolysis of C-terminal glutamic acid and aspartic acid, were prepared by site-directed mutagenesis of the human gene and expressed in the periplasm of Escherichia coli. By changing residues in the lining of the S1' pocket of the enzyme, it was possible to reverse the substrate specificity to give variants able to hydrolyse prior to C-terminal acidic amino acid residues instead of the normal C-terminal basic residues. This was achieved by mutating Asp253 at the base of the S1' specificity pocket, which normally interacts with the basic side-chain of the substrate, to either Lys or Arg. The resulting enzymes had the desired reversed polarity and enzyme activity was improved significantly with further mutations at residue 251. The [G251T,D253K]HCPB double mutant was 100 times more active against hippuryl-L-glutamic acid (hipp-Glu) as substrate than was the single mutant, [D253K]HCPB. Triple mutants, containing additional changes at Ala248, had improved activity against hipp-Glu substrate when position 251 was Asn. These reversed-polarity mutants of a human enzyme have the potential to be used in antibody-directed enzyme prodrug therapy of cancer.     

Membrane interaction of synthetic peptides related to the putative fusogenic region of PH-30 alpha, a protein in sperm-egg fusion

In order to investigate the relationship between structure and function of a putative fusogenic region of PH-30a, a protein active in sperm-egg fusion, two peptides, SFP22 and SFP23, whose sequences correspond to the residues 90-111 and 89-111 of PH-30 alpha, respectively, were chemically synthesized. An analog of SFP23, SFP23AA, which has an Ala-Ala sequence instead of the Pro-Pro sequence in SFP23, was also prepared. The CD study indicated that SFP22 and SFP23 mainly took a beta-structure in the presence of DPPC and DPPC/DPPG (3/1) vesicles, while SFP23AA showed an alpha-helical pattern though the alpha-helical content calculated was low (25-30%). alpha-Helical CD curve was observed for these peptides in trifluoroethanol. The membrane-perturbing activity of SFP22 and SFP23 was weaker than that of SFP23AA. On the other hand, the membrane-fusogenic activity of SFP22 and SFP23 to acidic phospholipid bilayers was much stronger than that of SFP23AA. All the peptides caused very weak cell lysis. These results are consistent with the reported speculation [Blobel, C. P. et al. (1992), Nature (London) 356, 248-252] that residues 90-111 of PH-30 alpha may be the fusogenic region and suggest that the Pro-Pro sequence is one of the important factors for holding the active secondary structure of the fusogenic region of PH-30 alpha in membranes.     

Mutagenesis and chemical rescue indicate residues involved in beta-aspartyl-AMP formation by Escherichia coli asparagine synthetase B

Site-directed mutagenesis and kinetic studies have been employed to identify amino acid residues involved in aspartate binding and transition state stabilization during the formation of beta-aspartyl-AMP in the reaction mechanism of Escherichia coli asparagine synthetase B (AS-B). Three conserved amino acids in the segment defined by residues 317-330 appear particularly crucial for enzymatic activity. For example, when Arg-325 is replaced by alanine or lysine, the resulting mutant enzymes possess no detectable asparagine synthetase activity. The catalytic activity of the R325A AS-B mutant can, however, be restored to about 1/6 of that of wild-type AS-B by the addition of guanidinium HCl (GdmHCl). Detailed kinetic analysis of the rescued activity suggests that Arg-325 is involved in stabilization of a pentacovalent intermediate leading to the formation beta-aspartyl-AMP. This rescue experiment is the second example in which the function of a critical arginine residue that has been substituted by mutagenesis is restored by GdmHCl. Mutation of Thr-322 and Thr-323 also produces enzymes with altered kinetic properties, suggesting that these threonines are involved in aspartate binding and/or stabilization of intermediates en route to beta-aspartyl-AMP. These experiments are the first to identify residues outside of the N-terminal glutamine amide transfer domain that have any functional role in asparagine synthesis.     

Intramolecular assistance of cis/trans isomerization of the histidine-proline moiety

Peptidyl-prolyl cis/trans isomerization is a slow conformational interconversion in the polypeptide backbone that is frequently rate-limiting in refolding of proteins and is thought to play a role in cellular restructuring of proteins. In order to probe the influence of positively charged amino acids located in sequence segments adjacent to proline, the rotational barriers of Arg-Pro- and His-Pro-containing peptides were determined by isomer-specific proteolysis and dynamic NMR spectroscopy for Suc-Ala-His-Pro-Phe-NH-Np, Ac-Ala-Arg-Pro-Ala-Lys-NH2, Ac-Ala-His-Pro-Ala-Lys-NH2, angiotensin III, thyrotropin-releasing hormone (TRH), and [His(3-Me)2]TRH in aqueous solution. In contrast to the guanidinium group of arginine, the protonated side chain of histidine preceding proline led to an acceleration of the prolyl isomerization up to 10-fold relative to the unprotonated state. Both arginine and histidine residues succeeding proline in an amino acid sequence proved to be ineffective. Under basic and acidic conditions the kinetic solvent deuterium isotope effects Kc-->tH20/Kc-->tD20 for angiotensin III were 1.0 +/- 0.1 and 2.0 +/- 0.1, respectively. The results are interpreted in terms of intramolecular general acid catalysis of prolyl bond rotation by the imidazolium group that is without precedent in intermolecular catalysis.     

Two residues that may ligate Ca2+ in transmembrane domain six of the plasma membrane Ca(2+)-ATPase

In order to identify Ca2+ ligands in the putative transmembrane domain 6 of the plasma membrane Ca2+ pump, amino acids Asn879, Met882, Asp883, and Ser887 were singly altered. Asn879, Met882, and Asp883 were chosen because the corresponding amino acids have been proposed as Ca2+ ligands in the sarcoplasmic reticulum Ca2+ pump (Clarke, D. M., Loo, T. W., and MacLennan, D. H. (1990) J. Biol. Chem. 265, 6262-6267). For the alterations, a fully active truncated version of the pump was used, because the interaction of Ca2+ with the pump could be studied without interference from calmodulin binding. The mutants at Asn and Asp did not carry out ATP-supported Ca2+ uptake and formed no acylphosphate from [gamma-32P]ATP, suggesting that, like the corresponding amino acids in the sarcoplasmic reticulum Ca2+ pump, these two are Ca2+ ligands. However, all the mutants at the position of Met882 showed some activity. Indeed, the Met882--> Ile mutant was fully active at a saturating Ca2+ concentration and only the K1/2 for Ca2+ activation was shifted slightly upward. Converting the Met to Thr (which is the corresponding residue in the sarcoplasmic reticulum Ca2+ pump) reduced the activity to 20% of the wild type, further emphasizing the differences between the two Ca2+ pumps. The mutant Ser887--> Ala was expressed in greater amounts than, and had a specific activity about 50% higher than, the wild type, indicating that this serine also could not be a Ca2+ ligand and could not replace the missing Thr at position Met882.     

Identification of a polar region in transmembrane domain 6 that regulates the function of the G protein-coupled alpha-factor receptor

The alpha-factor pheromone receptor (Ste2p) of the yeast Saccharomyces cerevisiae belongs to the family of G protein-coupled receptors that contain seven transmembrane domains (TMDs). Because polar residues can influence receptor structure by forming intramolecular contacts between TMDs, we tested the role of the five polar amino acids in TMD6 of the alpha-factor receptor by mutating these residues to nonpolar leucine. Interestingly, a subset of these mutants showed increased affinity for ligand and constitutive receptor activity. The mutation of the most polar residue, Q253L, resulted in 25-fold increased affinity and a 5-fold-higher basal level of signaling that was equal to about 19% of the alpha-factor induced maximum signal. Mutation of the adjacent residue, S254L, caused weaker constitutive activity and a 5-fold increase in affinity. Comparison of nine different mutations affecting Ser254 showed that an S254F mutation caused higher constitutive activity, suggesting that a large hydrophobic amino acid residue at position 254 alters transmembrane helix packing. Thus, these studies indicate that Gln253 and Ser254 are likely to be involved in intramolecular interactions with other TMDs. Furthermore, Gln253 and Ser254 fall on one side of the transmembrane helix that is on the opposite side from residues that do not cause constitutive activity when mutated. These results suggest that Gln253 and Ser254 face inward toward the other TMDs and thus provide the first experimental evidence to suggest the orientation of a TMD in this receptor. Consistent with this, we identified two residues in TMD7 (Ser288 and Ser292) that are potential contact residues for Gln253 because mutations affecting these residues also cause constitutive activity. Altogether, these results identify a new domain of the alpha-factor receptor that regulates its ability to enter the activated conformation.     

Amino acid metabolism in the piglet. 2. Influence of fasting on plasma free amino acid concentration and in vivo oxidation of methionine, isoleucine and threonine

1. The influence of a 24 h fast on the concentrations of free amino acids in the plasma, and upon the oxidation rates of methionine, isoleucine and threonine was studied (using early weaned, 4-week-old piglets which were receiving a semi-purified diet. 2. There was no change in the total concentration of the essential amino acids as a result of the 24 h fast: the concentration of the branched-chain amino acids increased, but the effect of this was offset by decreases in the concentrations of arginine, histidine, lysine, methionine and phenylalanine. There was a reduction in the concentration of the non-essential amino acids. 3. The piglets received infusions of L-[I-14C]methionine, L-[U-14C]isoleucine and L-[U-14C]-threonine, and the recovery of the label in carbon dioxide was determined. Less than 5% of the activity from methionine was recovered in the CO2 from the fed piglets, whereas 12% was recovered from the fasted piglets. The corresponding values with threonine were 11 and 19% but there was no effect of fasting on the recovery of the label from isoleucine in CO2. 4. The initial dilution of a single dose of a labelled amino acid infused into the bloodstream depends on the plasma concentration of the amino acid. Nutritional regimens may effect the free amino acid concentration in the plasma. Thus comparisons based upon direct determination of activity recovered in CO2 from the labelled dose of an amino acid with animals on different nutritional regimens could not misleading, unless the differences in the concentrations of the amino acid in the plasma are considered.     

Structure and biological activity of chemically modified nisin A species

Nisin, a 34-residue peptide bacteriocin, contains the less common amino acids lanthionine, beta-methyl-lanthionine, dehydroalanine (Dha), and dehydrobutyrine (Dhb). Several chemically modified nisin A species were purified by reverse-phase HPLC and characterized by two-dimensional NMR and electrospray mass spectrometry. Five constituents, [2-hydroxy-Ala5]nisin, [Ile4-amide,pyruvyl-Leu6]des-Dha5-nisin, [Met(O)21]nisin, [Ser33]nisin, and nisin-(1-32)-peptide amide, were found in a commercial nisin sample. A further species, [2-hydroxy-Ala5]nisin-(1-32)-peptide amide, was obtained by freeze drying an acidic nisin solution. These compounds are formed by chemical modification of nisin: the addition of a water molecule to the dehydroalanine residues, which can lead to the cleavage of the polypeptide chain, or the oxidation of methionine residues. The 2-hydroxyalanine-containing products have a limited stability; they are spontaneously converted into the corresponding des-dehydroalanine derivatives. The growth-inhibiting activity of the modified nisins towards different bacteria was determined. The 2-hydroxyalanine-containing species and the des-dehydroalanine derivative show a strong reduction in biological activity as compared to native nisin. [Met(O)21]nisin and [Ser33]nisin show moderate or no reduction in biological activity.     

Extensive random mutagenesis analysis of the Na+/K+-ATPase alpha subunit identifies known and previously unidentified amino acid residues that alter ouabain sensitivity--implications for ouabain binding

Random mutagenesis with ouabain selection has been used to comprehensively scan the extracellular and transmembrane domains of the alpha1 subunit of the sheep Na+/K+-ATPase for amino acid residues that alter ouabain sensitivity. The four random mutant libraries used in this study include all of the transmembrane and extracellular regions of the molecule as well as 75% of the cytoplasmic domains. Through an extensive number of HeLa cell transfections of these libraries and subsequent ouabain selection, 24 ouabain-resistant clones have been identified. All previously described amino acids that confer ouabain resistance were identified, confirming the completeness of this random mutagenesis screen. The amino acid substitutions that confer the greatest ouabain resistance, such as Gln111-->Arg, Asp121-->Gly, Asp121-->Glu, Asn122-->Asp, and Thr797-->Ala were identified more than once in this study. This extensive survey of the extracellular and transmembrane regions of the Na+/K+-ATPase molecule has identified two new regions of the molecule that affect ouabain sensitivity: the H4 and the H10 transmembrane regions. The new substitutions identified in this study are Leu330-->Gln, Ala331-->Gly, Thr338-->Ala, and Thr338-->Asn in the H4 transmembrane domain and Phe982-->Ser in the H10 transmembrane domain. These substitutions confer modest increases in the concentration of cardiac glycoside needed to produce 50% inhibition of activity (IC50 values), 3.1-7.9-fold difference. The results of this extensive screening of the Na+/K+-ATPase alpha1 subunit to identify amino acids residues that are important in ouabain sensitivity further supports our hypothesis that the H1-H2 and H4-H8 regions represent the major binding sites for the cardiac glycoside class of drugs.     

Percutaneous biopsy of the thoracic spine

The aspartate aminotransferase gene (AspAT, EC 2.6.1.1) of an extremely thermophilic bacterium, Thermus thermophilus HB8, was cloned and sequenced, and its gene product was overproduced. The purified T. thermophilus AspAT was stable up to about 80 degrees C at neutral pH. T. thermophilus AspAT was strictly specific for acidic amino acid substrates, such as aspartate, glutamate, and the respective keto acids. The gene coding for T. thermophilus AspAT showed that it comprised 1,155 bp with a high G+C content (70 mol%), and encoded a 385-residue protein with a molecular weight of 42,050. The amino acid sequence of T. thermophilus AspAT deduced from its gene showed about 15, 46, and 29% homology with those from Escherichia coli, Bacillus sp. YM-2, and Sulfolobus solfataricus, respectively. When the amino acid sequence of T. thermophilus AspAT was compared with that of E. coli AspAT, the number of Cys was found to have decreased from 5 to 1, that of Asn from 23 to 9, that of Gln from 16 to 8, and that of Asp from 20 to 13, all of which are known to be relatively labile at high temperatures. Conversely, the number of Pro was increased from 15 to 25, Arg from 22 to 32, and Glu 27 to 37. As shown by the E. coli AspAT structure, there was a marked tendency for the extra prolyl residues to be located around the surface of the molecule. This was quite different from that in the case of RecA protein, which shows an increased number of prolyl residues in the interior of its molecule. Different strategies of different proteins as to prolyl contribution to thermostability have been suggested. Despite the high degree of conservation of active-site residues, Arg292 in E. coli AspAT, which interacts with the distal carboxylate of the substrate, was not found in T. thermophilus AspAT. Arg89 may complement the function of Arg292.     

Kinetics and molecular characteristics of arginine transport by Leishmania donovani promastigotes

Characteristics of transport of L-arginine were studied in Leishmania donovani promastigotes grown in vitro in a defined medium. The promastigotes exhibited a time-dependent, temperature-sensitive, pH-dependent and saturable uptake of arginine. Metabolic inhibitors caused 81-92% inhibition, indicating that arginine influx in promastigotes is an energy requiring process. The presence of Na+ ions was necessary for full activity. Considerable inhibition was also noticed with valinomycin, gramicidin and amiloride. The transporter seems to involve an -SH group at the active site. The most distinctive feature of the leishmanial transporter was that lysine and ornithine did not show significant competition with arginine transport. Other neutral and acidic amino acids, as well as polyamines were also ineffective. The arginine analogues, viz., nitro-L-arginine methyl ester, N-nitro-L-arginine, aminoguanidine, agmatine and D-arginine were not recognised by the transporter, while N-methyl-L-arginine acetate and phospho-L-arginine showed competition, indicating stereo-specificity of the transporter and recognition of both the guanidino group, as well as the arginine side chain by the transporter. No exchange of intracellular [14C]arginine taken up by the promastigotes was noticed during incubation with 2 or 5 mM arginine in the extracellular medium. Eighty percent of the arginine taken up remained in the trichloroacetic acid-soluble fraction. Pentamidine caused competitive inhibition of arginine transport, exhibiting an IC50 value of 40 microM. Results indicate the presence of a novel distinct arginine transporter in Leishmania promastigotes.     

A conserved motif in the tail domain of vinculin mediates association with and insertion into acidic phospholipid bilayers

The tail domain of vinculin (Vt) contains a salt-insensitive binding site for acidic phospholipids which is masked by the intramolecular head-tail interaction in native vinculin [Johnson, R. P., and Craig, S. W. (1995) Biochem. Biophys. Res. Commun. 210, 159-164]. To characterize further this phospholipid binding site, we have used hydrophobic photolabeling with a photoactivatable phosphatidylcholine analogue to detect insertion of protein into the lipid bilayer. We show here that, although the properties of binding to acidic phospholipid vesicles and spontaneous insertion into the bilayer are cryptic and inactive in vinculin at physiologic ionic strength, these activities of the purified tail domain can be activated by physical and chemical disruption of the intramolecular interaction between the head and tail domains. By analyzing the lipid binding and insertion activity of a series of GST-Vt fusion proteins, we defined 55 amino acids, comprising vinculin residues 916-970, that mimic the lipid-binding and insertion activity of Vt. Predictions of secondary structure suggest that these 55 amino acids form a basic, amphipathic helical hairpin. This prediction is supported by circular dichroism analysis, which indicates that at least 80% of the residues in residues 916-970 are in a helical conformation. This predicted helical hairpin motif, which is conserved in all vinculins and is present in an acidic phospholipid-binding region of alpha-catenin, is distinct from C2 and PH domains, and likely represents a third type of acidic phospholipid-binding structure.     

The use of quantitative bacteriologic assessment of bone

A new sub-class of binding protein-dependent transporter with specificity for a broad range of polar amino acids has been identified by sequence comparison, in Rhizobium leguminosarum, Rhodobacter capsulatus, Escherichia coli and Pseudomonas fluorescens. Southern blotting and PCR analysis has shown that transporters from this new sub-class are widely distributed in Gram-negative bacteria, including, in addition to the above, Citrobacter freundii, Erwinia carotovorum and Rhizobium meliloti. ABC transporters of polar amino acids can be divided into two groups: those with narrow solute specificity and the newly identified sub-class with broad solute specificity. The binding and inner membrane proteins from transporters with a broad solute specificity are larger by approximately 30% than those with a narrow solute specificity. Multiple alignment of the inner membrane proteins from all sequenced polar amino acid transporters indicates there is an N-terminal conserved region that may be involved in solute specificity. A conserved arginine or lysine at residue 30 of this region is changed to glutamate in arginine transporters. Residue 53 also has a strong correlation with the charge on the transported solute, with basic amino acid transporters replacing an aliphatic amino acid at this position with a negatively charged amino acid. The general amino acid permease from R. leguminosarum, which will transport aliphatic as well as basic and acidic amino acids, juxtaposes two prolines at residues 52 and 53 of the N-terminal conserved region.     

Cysteine-scanning mutagenesis of helix IV and the adjoining loops in the lactose permease of Escherichia coli: Glu126 and Arg144 are essential. off

Cys-scanning mutagenesis has been applied to the remaining 45 residues in lactose permease that have not been mutagenized previously (from Gln100 to Arg144 which comprise helix IV and adjoining loops). Of the 45 single-Cys mutants, 26 accumulate lactose to > 75% of the steady state observed with Cys-less permease, and 14 mutants exhibit lower but significant levels of accumulation (35-65% of Cys-less permease). Permease with Phe140-->Cys or Lys131-->Cys exhibits low activity (15-20% of Cys-less permease), while mutants Gly115-->Cys, Glu126-->Cys and Arg144-->Cys are completely unable to accumulate the dissacharide. However, Cys-less permease with Ala or Pro in place of Gly115 is highly active, and replacement of Lys131 or Phe140 with Cys in wild-type permease has a less deleterious effect on activity. In contrast, mutant Glu126-->Cys or Arg144-->Cys is inactive with respect to both uphill and downhill transport in either Cys-less or wild-type permease. Furthermore, mutants Glu126-->Ala or Gln and Arg144-->Ala or Gln are also inactive in both backgrounds, and activity is not rescued by double neutral replacements or inversion of the charged residues at these positions. Finally, a mutant with Lys in place of Arg144 accumulates lactose to about 25% of the steady state of wild-type, but at a slow rate. Replacement of Glu126 with Asp, in contrast, has relatively little effect on activity. None of the effects can be attributed to decreased expression of the mutants, as judged by immunoblot analysis. Although the activity of most of the single-Cys mutants is unaffected by N-ethylmaleimide, Cys replacement at three positions (Ala127, Val132, or Phe138) renders the permease highly sensitive to alkylation. The results indicate that the cytoplasmic loop between helices IV and V, where insertional mutagenesis has little effect on activity [McKenna, E., et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 11954-11958], contains residues that play an important role in permease activity and that a carboxyl group at position 126 and a positive charge at position 144 are absolutely required.     

NMDA-receptor antagonist requirements in conantokin-G

A series of variants of the neuroactive 17-residue gamma-carboxyglutamate-(Gla)-containing polypeptide, conantokin-G (con-G), were synthesized with the intention of determining those features that were important for its N-methyl-D-aspartate (NMDA) receptor-targeted antagonist activity and for adoption of its divalent cation-dependent alpha-helical conformation. Employing the binding of [3H]dizolcipine (MK-801) as an assay for open receptor ion channels in rat brain membranes, which displays inhibition by con-G (IC50 = 0.48 microM), it was found that replacement by an Ala residue of Gla4 led to complete inactivation of the peptide, whereas a similar replacement of Gla3 resulted in a 20-fold decreased potency. Ala substitutions for Gla10 and Gla14 did not substantially affect [3H]MK-801 binding. This same substitution at Gla7 appeared to slightly enhance binding. Ala replacements of non-Gla residues demonstrated that four of them, viz. Glu2, Leu5, Gln9, and Ile12, possessed at least 200-fold decreases in inhibitory potency, whereas similar replacements at Gly1, Leu11, and Arg13 resulted in peptides with 8- to 12-fold increases in the IC50 values. The remaining amino acid residues tested in the single Ala replacement series showed no significant changes in the inhibitory characteristics of wild-type con-G. Additional studies with carboxyl-terminal truncated peptides revealed that the carboxyl-terminal 4 amino acids were unimportant for this activity. There was no strict correlation of inhibition of [3H]MK-801 binding with the ability of these peptides to form cation-dependent alpha-helices. Peptides with notably low alpha-helical content in the presence of these cations were lacking at least one, or both, of Gla10 and Gla14. Con-G[Gla3,4,7,10,14E] and con-G[Gla7,10,14E] were the only peptides that remained in a completely random conformation upon metal ion addition.