Regeneration of catalytic activity of glutamine synthetase mutants by chemical activation: exploration of the role of arginines 339 and 359 in activity.

In order to understand the nature of ATP and L-glutamate binding to glutamine synthetase, and the involvement of Arg 339 and Arg 359 in catalysis, these amino acids were changed to cysteine via site-directed mutagenesis. Individual mutations (Arg-->Cys) at positions 339 and 359 led to a sharp drop in catalytic activity. Additionally, the Km values for the substrates ATP and glutamate were elevated substantially above the values for wild-type (WT) enzyme. Each cysteine was in turn chemically modified to an arginine "analog" to attempt to "rescue" catalytic activity by covalent modification; 2-chloroacetamidine (CA) (producing a thioether) and 2,2'-dithiobis (acetamidine)(DTBA) (producing a disulfide) were the reagents used to effect these chemical transformations. Upon reaction with CA, both R339C and R359C mutants showed a significant regain of catalytic activity (50% and 70% of WT, respectively) and a drop in Km value for ATP close to that for WT enzyme. With DTBA, chemically modified R339C had a greater kcat than WT glutamine synthetase, but chemically modified R359C only regained a small amount of activity. Modification with DTBA was quantitative for each mutant and each modified enzyme had similar Km values for both ATP and glutamate. The high catalytic activity of DTBA-modified R339C could be reversed to that of unmodified R339C by treatment with dithiothreitol, as expected for a modified enzyme containing a disulfide bond. Modification of each cysteine-containing mutant to a lysine "analog" was accomplished using 3-bromopropylamine (BPA).(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemical and physical properties of the disulfides of bovine neurophysin-II.

Bovine neurophysin-II is shown to be very susceptible to partial reduction in the absence of urea. Reduction of an average of one disulfide leads to major changes in conformation and disulfide optical activity, manifest in part by pronounced far-uv ellipticity changes, complete loss of the 248-nm ellipticity band, and a shift of the 278-nm ellipticity band to shorter wavelengths with loss of half its intensity; the reduction process generates a mixture of products and appears to be accompanied by disulfide interchange. The circular dichroism data indicate that the disulfide(s) most susceptible to reduction or interchange are either the principal contributors to the 248- and 278-nm ellipticity bands or that the optical activity of other disulfides is dependent on their integrity. Peptides that bind to the hormone-binding site of neurophysin-II protect against reduction. On reoxidation of partially reduced neurophysin-II there is only a partial return of the native circular dichroism spectrum and electrophoretic behavior. The percentage of native protein in samples reoxidized following different degrees of reduction was estimated by comparison of the circular dichroism spectra of these samples with those of the fractionated native and denatured components of monoreduced-reoxidized neurophysin. Under our reoxidation conditions, less than 50% native protein was found in monoreduced-reoxidized neurophysin and less than 10% native protein was found in completely reduced-reoxidized neurophysin. The results are interpreted with qualified reference to a model in which one or more disulfides are "strained" in the native state and in which the native protein is unstable relative to species in which the disulfides are differently paired.     

Disulfide between Cys392 and Cys438 of human serum albumin is redox-active, which is responsible for the thioredoxin-supported lipid peroxidase activity

Human serum albumin (HSA) is an abundant protein found in blood plasma and extracellular fluids. Previously, we found that HSA has a distinct thioredoxin (Trx)-dependent lipid peroxidase activity in the presence of palmitoyl-CoA. In this paper, we identified the redox-active disulfide, which can be specifically reduced by Trx, responsible for the Trx-dependent lipid peroxidase activity. The IIB-III fragment of HSA (Pro299-Leu585) sustained the Trx-dependent lipid peroxidase activity. Chemical modification of the Trx-reduced IIB-III with a thiol-specific modification agent resulted in a complete loss of the peroxidase activity. The analysis of tryptic-peptides derived from the inactivated HSA and IIB-III revealed that Cys392 and Cys438, which exist as an intramolecular disulfide bond in HSA, were preferentially modified in both HSA and IIB-III. Taken together, these results suggested that HSA has a capability to reduce lipid hydroperoxide with the use of Trx as an in vivo electron donor, and that the redox-active disulfide between Cys392 and Cys438 acts as a primary site of the catalysis for the Trx-linked lipid peroxidase activity.     

Modification of protein by disulfide S-monoxide and disulfide S-dioxide: distinctive effects on PKC

Disulfide S-monoxide (DSMO) and disulfide S-dioxide (DSDO) have been proposed as proximal mediators for the oxidant-mediated modification of proteins. These disulfide S-oxides (DSOs) derived from glutathione (GSH) and captopril (CPSH) were synthesized by iron- or methyltrioxorhenium (VII)-catalyzed oxidation of the thiols with H2O2. Treatment of mouse hippocampal extracts with [35S]GS-DSOs revealed that a large number of proteins were susceptible to thionylation; however, only a limited number of the them were detectable by the commonly used antibody against GS-associated proteins. Using protein kinase C (PKC) as a model, we found that DSOs derived from different thiols modified this kinase with different efficacy and specificity; for example, the inhibitory potency of the kinase was glutathione disulfide S-dioxide (GS-DSDO) (IC50, approximately 30 microM) > captopril disulfide S-dioxide (CPS-DSDO) (IC50, approximately 450 microM) > glutathione disulfide S-monoxide (GS-DSMO) and captopril disulfide S-monoxide (CPS-DSMO). The stoichiometries of thionylation of PKC beta mediated by [35S]GS-DSMO and [35S]GS-DSDO were approximately 1 and 5 mol/mol, respectively, and at least four glutathionylation sites were identified in the GS-DSDO-treated kinase. Modification of PKC by GS-DSDO and CPS-DSDO rendered the kinase very susceptible to limited proteolysis; the former preferentially caused the degradation of the catalytic and the latter the regulatory domain of the kinase. Furthermore, CPS-DSDO-mediated modification of PKC increased the autonomous kinase activity; this was not the case for GS-DSDO-mediated modification. Since DSOs of different oxidative states as well as those derived from different thiols exert different effects on a target protein, these molecules could cause distinct cellular responses if derived from endogenous cellular reactions or even if they arise from exogenous sources.     

红花菜豆(矮生红花变种)凝集素分子的色氨酸残基修饰与其活性的关系

用各种化学试剂修饰红花菜豆（Ｐｈａｓｅｏｌｕｓｃｏｃｃｉｎｅｕｓｖａｒｒｕｂｒｏｎａｎｕｓ,Ｂｅｒｒｙ）凝集素（简称ＰＣＬ）分子,测定与其活性相关的氨基酸残基．经ＮＢＳ修饰表明ＰＣＬ具有８个Ｔｒｐ残基,其中４个暴露于分子表面,此４个Ｔｒｐ残基被修饰后,ＰＣＬ的凝血活性完全丧失．比较ＰＣＬ修饰前后的ＣＤ光谱表明修饰不改变其二级结构。修饰Ｔｙｒ,Ａｒｇ,Ｈｉｓ残基和游离氨基及羧基不影响ＰＣＬ的血凝活性．巯基也不是血凝活性所必需,但是ＰＣＬ分子中的二硫键被还原,或被ＣＮＢｒ分解为两个片断则使蛋白质丧失血凝活性,提示分子的完整结构对ＰＣＬ的血凝活力是重要的     

The functional role of cysteine residues for c-Abl kinase activity

S-glutathionylation, the formation of mixed disulfides of glutathione with cysteine residues of proteins, is a broadly observed physiological modification that occurs in response to oxidative stress. Since cysteine residues are particularly susceptible to oxidative modification by reactive oxygen species, S-glutathionylation can protect proteins from irreversible oxidation. In this study, we show that the kinase activity of the non-receptor tyrosine kinase c-Abl is inhibited by in vitro thiol modification; specifically, the cysteine residues of c-Abl are modified by S-glutathionylation and by thiol alkylating agents such as 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid and N-ethylmaleimide. Modification of cysteine residues of c-Abl tyrosine kinase using glutathione disulfide and thiol alkylating agents corresponds to a concomitant loss of kinase activity. We also demonstrate that S-glutathionylation of c-Abl can be reversed using a physiological system involving glutaredoxin and this reversal restores c-Abl kinase activity. To our knowledge, these are the first data to show S-glutathionylation of c-Abl, and this modification may represent a mechanism of regulation of c-Abl kinase activity in cells under oxidative stress.     

Secretion in yeast of mutant human lysozymes with and without glutathione bound to cysteine 95

A mutant human lysozyme C77A, in which Cys-77 is replaced with Ala, was secreted by Saccharomyces cerevisiae as two proteins (C77A-a and C77A-b) with different specific activities. A peptide fragment from Val93 to Ala108 was obtained from C77A-a by pepsin digestion, and examined by fast atom bombardment mass spectrometry and amino acid analysis. The results showed that glutathione was attached to the thiol group of Cys95 of the fragment through a disulfide linkage. This observation was confirmed by quantitative formation of free glutathionesulfonic acid from C77A-a by performic acid treatment. In contrast, there was no modification in the case of C77A-b. These results indicate that C77A-a contained a mixed disulfide with glutathione attached to cysteine residue 95. In C77A-b, there appears to be a free thiol of Cys95 surrounded by many side chains, which was not modified by iodoacetic acid under native conditions, suggesting that the attachment of glutathione occurs during folding. These findings further suggest that in the oxidation step of disulfide bond formation in human lysozyme secreted by yeast, mixed disulfides are formed with glutathione and that posttranslational modification with glutathione can occur even in a protein secreted by yeast.     

Bovine brain acetylcholinesterase primary sequence involved in intersubunit disulfide linkages

Three distinct classes of membrane-bound acetylcholinesterases (AChEs) have been identified. A12 AChE is composed of 12 catalytic subunits that are linked to noncatalytic collagen-like subunits through intersubunit disulfide bonds. G2 AChE is localized in membranes by a glycoinositol phospholipid covalently linked to the C-terminal amino acid. Brain G4 AChE involves two catalytic subunits linked by a direct intersubunit disulfide bond while the other two are disulfide-linked to a membrane-binding 20-kDa noncatalytic subunit. Molecular cloning studies have so far failed to find evidence of more than one AChE gene in any organism although alternative splicing of torpedo AChE mRNA results in different C-terminal sequences for the A12 and G2 AChE forms. Support for a single bovine AChE gene is provided in this report by amino acid sequencing of the N-terminal domains from the G2 erythrocyte, G4 fetal serum, and G4 brain AChE. Comparison of the 38-amino acid sequences reveals virtually complete identity among the three AChE forms. Additional extensive identity between the fetal serum and brain AChEs was demonstrated by sequencing several brain AChE peptides isolated by high performance liquid chromatography after trypsin digestion of nitrocellulose blots of brain AChE catalytic subunits. Cysteines involved in intersubunit disulfide linkages in brain AChE were reduced selectively with dithiothreitol in the absence of denaturants and radioalkylated with iodoacetamide. The observed sequence of the major radiolabeled tryptic peptide was C*SDL, where C* was the radioalkylated cysteine residue. This sequence is precisely the same as that observed at the C terminus of fetal bovine serum AChE and shows close homology to the C-terminal sequence of torpedo A12 AChE. We conclude that the mammalian brain G4 AChEs utilize the same exon splicing pattern as the A12 AChEs and that factors other than the primary sequence of the AChE catalytic subunits dictate assembly with either the collagen-like or the 20-kDa noncatalytic subunits.     

Effect of organic solvents on the molten globule state of procerain: beta-sheet to alpha-helix switchover in presence of trifluoroethanol

The effect of methanol and trifluoroethanol (TFE) on the structure and folding of molten globule state of procerain, a cysteine protease from Calotropis procera, was studied by circular dichroism spectroscopy. The magnitude of ellipticity at 215 nm, as a measure of beta-sheet content, is dependent on the concentration of the TFE. Interestingly, a switch over from the beta-sheet structure of the molten globule state to alpha-helix was observed at 60% TFE and the ellipticity at 222 nm increased as a function of TFE concentration beyond this critical TFE concentration. Temperature induced unfolding of the molten globule state of procerain in 10% methanol showed stabilization of alpha-rich domain with concomitant destabilization of beta-rich domain. Using higher concentration of methanol (20-40 %) had no stabilizing effect on the alpha-rich domain however, the beta-rich domain was destabilized, indicating that the stability of the domains were not interdependent and that a low concentration of methanol induced stabilization in alpha-rich domain.     

Interchain disulfide bonds promote protein cross-linking during protein folding

We provide evidence that in vitro protein cross-linking can be accomplished in three concerted steps: (i) a change in protein conformation; (ii) formation of interchain disulfide bonds; and (iii) formation of interchain isopeptide cross-links. Oxidative refolding and thermal unfolding of ribonuclease A, lysozyme, and protein disulfide isomerase led to the formation of cross-linked dimers/oligomers as revealed by SDS-polyacrylamide gel electrophoresis. Chemical modification of free amino groups in these proteins or unfolding at pH < 7.0 resulted in a loss of interchain isopeptide cross-linking without affecting interchain disulfide bond cross-linking. Furthermore, preformed interchain disulfide bonds were pivotal for promoting subsequent interchain isopeptide cross-links; no dimers/oligomers were detected when the refolding and unfolding solution contained the reducing agent dithiothreitol. Similarly, the Cys326Ser point mutation in protein disulfide isomerase abrogated its ability to cross-link into homodimers. Heterogeneous proteins become cross-linked following the formation of heteromolecular interchain disulfide bonds during thermal unfolding of a mixture of of ribonuclease A and lysozyme. The absence of glutathione and glutathione disulfide during the unfolding process attenuated both the interchain disulfide bond cross-links and interchain isopeptide cross-links. No dimers/oligomers were detected when the thermal unfolding temperature was lower than the midpoint of thermal denaturation temperature.     

Nitric oxide-induced S-glutathionylation and inactivation of glyceraldehyde-3-phosphate dehydrogenase

S-Nitrosylation of protein thiol groups by nitric oxide (NO) is a widely recognized protein modification. In this study we show that nitrosonium tetrafluoroborate (BF4NO), a NO+ donor, modified the thiol groups of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by S-nitrosylation and caused enzyme inhibition. The resultant protein-S-nitrosothiol was found to be unstable and to decompose spontaneously, thereby restoring enzyme activity. In contrast, the NO-releasing compound S-nitrosoglutathione (GSNO) promoted S-glutathionylation of a thiol group of GAPDH both in vitro and under cellular conditions. The GSH-mixed protein disulfide formed led to a permanent enzyme inhibition, but upon dithiothreitol addition a functional active GAPDH was recovered. This S-glutathionylation is specific for GSNO because GSH itself was unable to produce protein-mixed disulfides. During cellular nitrosative stress, the production of intracellular GSNO might channel signaling responses to form protein-mixed disulfide that can regulate intracellular function.     

Regulation of the activity of glyceraldehyde 3-phosphate dehydrogenase by glutathione and H2O2

The activity lost during storage of a solution of muscle glyceraldehyde 3-phosphate dehydrogenase was rapidly restored on adding a thiol compound, but not arsenite or azide. On treatment with H₂O₂, the enzyme was partially inactivated and complete loss of activity occurred in the presence of glutathione. Samples of the enzyme pretreated with glutathione followed by removal of the thiol compound by filtration on a Sephadex column showed both full activity and its complete loss on adding H₂O₂, in the absence of added glutathione. Most of the activity was restored when the H₂O₂-inactivated enzyme was incubated with glutathione (25mM) or dithiothreitol (5mM) whereas arsenite or azide were partly effective and ascorbate was ineffective. The need for incubation for a long time with a strong reducing agent for restoration of activity suggests that the oxidized group (disulfide or sulfenate) must be in a masked state in the H₂O₂-inactivated enzyme. Analysis by SDS-PAGE gave evidence for the formation of a small quantity of glutathione-reversible disulfide-form of the enzyme. Circular dichroic spectra indicated a decrease in -helical content in the inactivated form of the enzyme. The evidence suggest that glutathione and H₂O₂ can regulate the active state of this enzyme.     

Replacement of the interchain disulfide bridge-forming amino acids A7 and B7 by glutamate impairs the structure and activity of insulin

Insulin contains three disulfide bonds, one intrachain bond, A6-A11, and two interchain bonds, A7-B7 and A20-B19. Site-directed mutagenesis results (the two cysteine residues of disulfide A7-B7 were replaced by serine) showed that disulfide A7-B7 is crucial to both the structure and activity of insulin. However, chemical modification results showed that the insulin analogs still retained relatively high biological activity when A7Cys and B7Cys were modified by chemical groups with a negative charge. Did the negative charge of the modification groups restore the loss of activity and/or the disturbance of structure of these insulin analogs caused by deletion of disulfide A7-B7? To answer this question, an insulin analog with both A7Cys and B7Cys replaced by Glu, which has a long side-chain and a negative charge, was prepared by protein engineering, and its structure and activity were analyzed. Both the structure and activity of the present analog are very similar to that of the mutant with disulfide A7-B7 replaced by Ser, but significantly different from that of wild-type insulin. The present results suggest that removal of disulfide A7-B7 will result in serious loss of biological activity and the native conformation of insulin, even if the disulfide is replaced by residues with a negative charge.     

Glutathione disulfide production during arachidonic acid oxygenation in human platelets

Washed human platelets stimulated with 50 microM sodium arachidonate rapidly accumulated glutathione disulfide to a peak concentration of 0.620 nmole per 10(9) cells, 200% of control (unstimulated) levels. Total glutathione remained unchanged. The rise in glutathione disulfide was transitory, returning to control values within 30 seconds in aggregating platelets. Similar findings were observed in washed platelets aggregated with 5 U/ml thrombin. Platelet aggregation was not necessary for the generation of glutathione disulfide. However, cyclooxygenase activity was necessary for the generation of glutathione disulfide. Aspirin treated platelets aggregated with thrombin demonstrated no thromboxane B2 production and no glutathione disulfide generation. Dose response studies with both agonists demonstrated a direct relationship between the amount of thromboxane B2 produced and the amount of glutathione disulfide generated by stimulated platelets. During the conversion of arachidonic acid to thromboxane B2, unesterified arachidonic acid is oxygenated to prostaglandin G2 which is subsequently reduced to prostaglandin H2. Both reactions are catalyzed by the enzyme prostaglandin H synthase. Our data support the hypothesis that glutathione is an important supplier of reducing equivalents to prostaglandin H synthase during the production of prostaglandin H2 in human platelets.     

Chemical Modification of Catalytically Essential Functional Groups of NAD-Dependent Hydrogenase from Ralstonia eutropha H16

Amino acid residues His and Cys of the NAD-dependent hydrogenase from the hydrogen-oxidizing bacterium Ralstonia eutropha H16 were chemically modified with specific reagents. The modification of His residues of the nonactivated hydrogenase resulted in decrease in both hydrogenase and diaphorase activities of the enzyme. Activation of NADH hydrogenase under anaerobic conditions additionally modified a His residue (or residues) significant only for the hydrogenase activity. The rate of decrease in the diaphorase activity was unchanged. The modification of thiol groups of the nonactivated enzyme did not affect the hydrogenase activity. The effect of thiol-modifying agents on the activated hydrogenase was accompanied by inactivation of both diaphorase and hydrogenase activities. The modification degree and changes in the corresponding catalytic activities depended on conditions of the enzyme activation. Data on the modification of cysteine and histidine residues of the hydrogenase suggested that the enzyme activation should be associated with significant conformational changes in the protein globule.     

Limiting Role of Protein Disulfide Isomerase in the Expression of Collagen-tailed Acetylcholinesterase Forms in Muscle

The expression of acetylcholinesterase (AChE) in skeletal muscle is regulated by muscle activity; however, the underlying molecular mechanisms are incompletely understood. We show here that the expression of the synaptic collagen-tailed AChE form (ColQ-AChE) in quail muscle cultures can be regulated by muscle activity post-translationally. Inhibition of thiol oxidoreductase activity decreases expression of all active AChE forms. Likewise, primary quail myotubes transfected with protein disulfide isomerase (PDI) short hairpin RNAs showed a significant decrease of both the intracellular pool of all collagen-tailed AChE forms and cell surface AChE clusters. Conversely, overexpression of PDI, endoplasmic reticulum protein 72, or calnexin in muscle cells enhanced expression of all collagen-tailed AChE forms. Overexpression of PDI had the most dramatic effect with a 100% increase in the intracellular ColQ-AChE pool and cell surface enzyme activity. Moreover, the levels of PDI are regulated by muscle activity and correlate with the levels of ColQ-AChE and AChE tetramers. Finally, we demonstrate that PDI interacts directly with AChE intracellularly. These results show that collagen-tailed AChE form levels induced by muscle activity can be regulated by molecular chaperones and suggest that newly synthesized exportable proteins may compete for chaperone assistance during the folding process.     

Thiol/disulfide redox equilibrium and kinetic behavior of chicken liver fatty acid synthase

Chicken liver fatty acid synthase is rapidly inactivated and cross-linked at pH 7.2 and 8.0 by incubation with low concentrations of common biological disulfides including glutathione disulfide, coenzyme A disulfide, and glutathione-coenzyme A-mixed disulfide. Glutathione disulfide inactivation of the enzyme is accompanied by the oxidation of a total of 4-5 enzyme thiols per monomer. Only one glutathione equivalent is incorporated per monomer as a protein-mixed disulfide, and its rate of incorporation is significantly slower than the rate of inactivation. The formation of protein-SS-protein disulfides results in significant cross-linking of enzyme subunits. The inactive enzyme is rapidly and completely reactivated, and the cross-linking is completely reversed by incubation of the enzyme with thiols (10-20 mM) including dithiothreitol, mercaptoethanol, and glutathione. In a glutathione redox buffer (GSH + GSSG), disulfide bond formation comes to equilibrium. The enzyme activity at equilibrium is dependent both on the ratio of glutathione to glutathione disulfide and on the total glutathione concentration. The equilibrium constant for the redox equilibration of fatty acid synthase in a glutathione redox buffer is 15 mM (Ered + GSSG in equilibrium Eox + 2GSH). The formation of at least one protein-protein disulfide per monomer dominates the redox properties of the enzyme while the formation of one protein-mixed disulfide with glutathione (Kmixed = 0.45) has little effect on activity. The oxidation equilibrium constant suggests that there would be no significant cycling between the reduced and the oxidized enzyme in response to likely physiological variations in the hepatic glutathione status. The possibility that changes in the concentration of cellular glutathione may act as a mechanism for metabolic control of other enzymes is discussed.     

Regulation of the Expression of Acetylcholinesterase by Muscular Activity in Avian Primary Cultures

Primary cultures of avian muscle cells express both globular and asymmetric molecular forms of acetylcholinesterase (AChE) when grown in a simple defined culture medium. Under these conditions, we analyzed the role of various agents interfering with muscular activity: tetrodotoxin (TTX) and veratridine, as well as a depolarizing concentration of KCl. These treatments caused the complete cessation of contractions in mature myotubes. We observed no influence on cellular AChE activity. The paralyzing treatments induced different effects on AChE secretion: TTX increased the secretion by approximately 25%, whereas KCl and veratridine reduced it by approximately 30%. The proportions of secreted molecular forms (mostly hydrophilic G4 and G2) were not modified significantly. TTX did not affect the pattern of molecular forms of cellular AChE (in particular, the proportion of A forms was not changed). Depolarization by veratridine or KCl induced an increase in the proportion of A forms in mature myotubes by a factor of 2-3. Similar results were obtained with quail myotubes cultured under the same conditions. This study shows that, in avian muscle cultures, the ionic balance across myotube membranes, rather than muscular activity per se, can regulate the level of A forms and the rate of AChE secretion. These results do not exclude the possible involvement of other factors, such as Ca2+ and/or peptidic factors. In addition, taking together our results and data from the literature. we conclude that the expression of AChE molecular forms depends both on the species and on the culture conditions used.     

Two partially unfolded states of Torpedo californica acetylcholinesterase.

Chemical modification with sulfhydryl reagents of the single, nonconserved cysteine residue Cys231 in each subunit of a disulfide-linked dimer of Torpedo californica acetylcholinesterase produces a partially unfolded inactive state. Another partially unfolded state can be obtained by exposure of the enzyme to 1-2 M guanidine hydrochloride. Both these states display several important features of a molten globule, but differ in their spectroscopic (CD, intrinsic fluorescence) and hydrodynamic (Stokes radii) characteristics. With reversal of chemical modification of the former state or removal of denaturant from the latter, both states retain their physiochemical characteristics. Thus, acetylcholinesterase can exist in two molten globule states, both of which are long-lived under physiologic conditions without aggregating, and without either intraconverting or reverting to the native state. Both states undergo spontaneous intramolecular thioldisulfide exchange, implying that they are flexible. As revealed by differential scanning calorimetry, the state produced by chemical modification lacks any heat capacity peak, presumably due to aggregation during scanning, whereas the state produced by guanidine hydrochloride unfolds as a single cooperative unit, thermal transition being completely reversible. Sucrose gradient centrifugation reveals that reduction of the interchain disulfide of the native acetylcholinesterase dimer converts it to monomers, whereas, after such reduction, the two subunits remain completely associated in the partially unfolded state generated by guanidine hydrochloride, and partially associated in that produced by chemical modification. It is suggested that a novel hydrophobic core, generated across the subunit interfaces, is responsible for this noncovalent association. Transition from the unfolded state generated by chemical modification to that produced by guanidine hydrochloride is observed only in the presence of the denaturant, yielding, on extrapolation to zero guanidine hydrochloride, a high free energy barrier (ca. 23.8 kcal/mol) separating these two flexible, partially unfolded states.     

Chemical modification studies of Artocarpus lakoocha lectin artocarpin.

The effect of chemical modification on an anti T-like lectin, artocarpin isolated from Artocarpus lakoocha seeds was investigated in order to identify the type of amino acids involved in its agglutinating activity. Modification of carboxyl groups, arginine and lysine residues, did not affect the lectin activity. However, modification of tryptophan, tyrosine and histidine residues led to a complete loss of its activity, indicating the involvement of these amino acids in the saccharide-binding ability. A protection was observed in the presence of inhibitory sugar. A marked decrease in the fluorescence emission was found when the tryptophan residues of lectin were modified. The circular dichroism spectra showed the presence of an identical pattern of conformation in the native and modified lectin, indicating that the loss in activity was due to modification only. The effect of pronase on artocarpin showed loss of activity whereas papain and trypsin had no effect. The specific activity of artocarpin remained unaltered on treatment with glycosidases but remarkable increase in the activity (of the same) was observed with xylanase treatment. Immunodiffusion studies with chemically modified lectin showed no gross structural changes, indicating that the group specific modifying agents did not alter the antigenic sites of the modified lectin.