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.     

Chemical modification studies on Abrus agglutinin. Involvement of tryptophan residues in sugar binding.

The galactose-binding lectin from the seeds of the jequirity plant (Abrus precatorius) was subjected to various chemical modifications in order to detect the amino acid residues involved in its binding activity. Modification of lysine, tyrosine, arginine, histidine, glutamic acid and aspartic acid residues did not affect the carbohydrate-binding activity of the agglutinin. However, modification of tryptophan residues carried out in native and denaturing conditions with N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide led to a complete loss of its carbohydrate-binding activity. Under denaturing conditions 30 tryptophan residues/molecule were modified by both reagents, whereas only 16 and 18 residues/molecule were available for modification by N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide respectively under native conditions. The relative loss in haemagglutinating activity after the modification of tryptophan residues indicates that two residues/molecule are required for the carbohydrate-binding activity of the agglutinin. A partial protection was observed in the presence of saturating concentrations of lactose (0.15 M). The decrease in fluorescence intensity of Abrus agglutinin on modification of tryptophan residues is linear in the absence of lactose and shows a biphasic pattern in the presence of lactose, indicating that tryptophan residues go from a similar to a different molecular environment on saccharide binding. The secondary structure of the protein remains practically unchanged upon modification of tryptophan residues, as indicated by c.d. and immunodiffusion studies, confirming that the loss in activity is due to modification only.     

Chemical modification studies on a lectin from Saccharomyces cerevisiae (baker''s yeast).

The effect of chemical modification on a galactose-specific lectin isolated from a fatty acid auxotroph of Saccharomyces cerevisiae was investigated in order to identify the type of amino acids involved in its agglutinating activity. Modification of 50 free amino groups with succinic anhydride or citraconic anhydride led to an almost complete loss of activity. This could not be protected by the inhibitory sugar methyl alpha-D-galactopyranoside. Treatment with N-bromosuccinimide and N-acetylimidazole, for the modification of tryptophan and tyrosine residues, did not affect lectin activity. Modification of carboxy groups with glycine ethyl ester greatly affected lectin activity, although sugars afford partial protection. Modification of four thiol groups with N-ethylmaleimide was accompanied by a loss of 85% of the agglutinating activity, and two thiol groups were found to be present at the sugar-binding site of the lectin. Modification of 18 arginine residues with cyclohexane-1,2-dione and 26 histidine residues with ethoxyformic anhydride led to a loss of lectin activity. However, in these cases, modification was not protected by the abovementioned inhibitory sugar, suggesting the absence of these groups at the sugar-binding site. In all the cases, immunodiffusion studies with modified lectin showed no gross structural changes which could disrupt antigenic sites of the lectin.     

Chemical modification studies of Rhizomucor miehei protease: evidence for the role of basic amino acids in enzyme catalysis.

The effect of chemical modification on milk clotting and proteolytic activities of aspartyl protease obtained from Rhizomucor miehei NRRL 3500 was examined in the absence and the presence of its specific inhibitor pepstatin A. The effect on the ratio of milk clotting activity (MC) to proteolytic activity (PA), an index of the quality of milk clotting proteases was also determined. Modification of the enzyme with trinitrobenzenesulfonic acid, diethylpyrocarbonate and phenylglyoxal produced an increase in the ratio of MC/PA, while modification with 2- hydroxy-5-nitrobenzyl bromide did not affect the ratio. Modification with N-acetylimidazole resulted in a marginal increase in MC/PA ratio. Protection using pepstatin A during modification with phenylglyoxal, N-acetylimidazole and 2-hydroxy-5-nitrobenzyl bromide, protected both MC and PA. In the case of modification by diethylpyrocarbonate, pepstatin A protected only MC. Pepstatin A did not protect both the activities on the modification of the enzyme by trinitrobenzene sulfonic acid. These observations indicate the presence of arginine, tyrosine and tryptophan at the catalytic site of the enzyme, for eliciting MC and PA of the enzyme. In general, modification of the positively charged residues increases the MC/PA ratio of the enzyme. In addition the modified lysine residues responsible for the inactivation of the enzyme were not involved in the active site of the enzyme. Thus the lysine residues might have a secondary role in enzyme catalysis. Further, histidine at the catalytic site was found to be exclusively involved in milk clotting activity. The enzyme with modified histidine residues were more susceptible to autocatalysis, indicating that histidine residues protect the enzyme against autolysis.     

Effect of amino acid residue and oligosaccharide chain chemical modifications on spectral and hemagglutinating activity of Millettia dielsiana Harms. ex Diels. lectin

The effects of modifying the carbohydrate chain and amino acids on the conformation and activity of Millettia dielsiana Harms. ex Diels. lectin (MDL) were studied by hemagglutination, fluorescence and circular dichroism analysis. The modification of tryptophan residues led to a compete loss of hemagglutinating activity; however, the addition of mannose was able to prevent this loss of activity. The results indicate that two tryptophan residues are involved in the carbohydrate-binding site. Modifications of the carboxyl group residues produced an 80% loss of activity, but the presence of mannose protected against the modification. The results suggest that the carboxyl groups of aspartic and glutamic acids are involved in the carbohydrate-binding site of the lectin. However, oxidation of the carbohydrate chain and modification of the histidine and arginine residues did not affect the hemagglutinating activity of MDL. Fluorescence studies of MDL indicate that tryptophan residues are present in a relatively hydrophobic region, and the binding of mannose to MDL could quench tryptophan fluorescence without any change in λmax. The circular dichroism spectrum showed that all of these modifications affected the conformation of the MDL molecule to different extents, except the modification of arginine residues. Fluorescence quenching showed that acrylamide and iodoacetic acids are able to quench 77% and 98% of the fluorescence of tryptophan in MDL, respectively. However, KI produced a barely perceptible effect on the fluorescence of MDL, even when the concentration of I^- was 0.15M. This demonstrates that most of tryptophan residues are located in relatively hydrophobic or negatively charged areas near the surface of the MDL molecule.     

Studies on the active site of rat glutathione S-transferase isoenzyme 4-4. Chemical modification by tetrachloro-1,4-benzoquinone and its glutathione conjugate

The active site of glutathione S-transferase isoenzyme 4-4, purified from rat liver, was studied by chemical modification. Tetrachloro-1,4-benzoquinone, a compound previously shown to inactivate glutathione S-transferases very efficiently by covalent binding in or close to the active site, completely prevented the alkylation of the enzyme by iodoacetamide, indicating that the reaction had taken place with cysteine residues. Both from radioactive labeling and spectral quantification experiments, evidence was obtained for the covalent binding of three benzoquinone molecules per subunit, i.e. equivalent to the number of cysteine residues present. This threefold binding was achieved with a fourfold molar excess of the benzoquinone, illustrating the high reactivity of this compound. Comparison of the number of amino acid residues modified by tetrachloro-1,4-benzoquinone with the decrease of catalytic activity revealed an almost complete inhibition after modification of one cysteine residue. Chemical modification studies with diethylpyrocarbonate indicated that all four histidine residues of the subunit are ethoxyformylated in an at least partially sequential manner. Modification of the second histidine residue resulted in complete loss of catalytic activity. Preincubation of the transferase with the glutathione conjugate of tetrachloro-1,4-benzoquinone resulted in 78% protection against this modification. However, glutathione itself hardly protected against the reaction with diethylpyrocarbonate. The intrinsic fluorescence properties of the enzyme were affected by covalent binding of tetrachloro-1,4-benzoquinone. The concentration dependency of the fluorescence quenching is strongly correlated with the inactivation of the enzyme, indicating that covalent binding of the benzoquinone occurs in the vicinity of at least one tryptophan residue. Finally, the binding of bilirubin, as measured by means of circular dichroism, was inhibited by preincubation of the enzyme with tetrachloro-1,4-benzoquinone in a manner which strongly correlated with the loss of enzymatic activity, the protection against inactivation by diethylpyrocarbonate, and the fluorescence quenching. All processes showed a 70-80% decrease after incubation of the enzyme with an equimolar amount of the benzoquinone. Thus, evidence is presented for the presence of a cysteine, a histidine and a tryptophan residue in, or in the vicinity of, the active site of the glutathione S-transferase 4 subunit.     

The involvement of carboxylate groups of putidaredoxin in the reaction with putidaredoxin reductase

Modification of carboxyl groups on putidaredoxin with 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC) resulted in loss of putidaredoxin reductase activity. The modification did not affect the visible absorption spectrum of putidaredoxin, indicating that the iron-sulfur center was not perturbed. In order to identify the carboxyl groups labeled by EDC, native and EDC-treated putidaredoxin were digested with a combination of trypsin and Staphylococcus aureus protease, and the resulting peptides were separated by high pressure liquid chromatography. The most heavily modified carboxyl groups were found to be those at residues 58, 65, 67, 72, and 77. These carboxyl groups are located in the same general region of the protein as those on adrenodoxin that have been shown to be involved in binding to both adrenodoxin reductase and cytochrome P-450scc. Chemical modification was also used to compare the role of lysine, arginine, and histidine residues on putidaredoxin and adrenodoxin. Modification of lysine and arginine residues had no effect on the reductase activity of either protein. The reductase activity of adrenodoxin was unaffected by labeling with 1 eq of diethyl pyrocarbonate/histidine residue, but labeling with a second equivalent completely abolished both activity and the iron-sulfur center spectrum. In contrast, modification of the 2 histidines in putidaredoxin with 1 eq each resulted in nearly complete loss of reductase activity. There was no significant activity for adrenodoxin in the putidaredoxin reductase assay or for putidaredoxin in the adrenodoxin reductase assay, demonstrating that, in spite of the structural similarity between the two proteins, they are not interchangeable functionally.     

On the reaction of papain and succinylpapin with diazo-1-H-tetrazole.

1. The reaction of papain and succinylpapain with diazo-1-H-tetrazole was investigated under different conditions. The extent of modification of the amino acids histidine, tyrosine, tryptophan and lysine was determined spectrophotometrically and/or by amino acid analysis. 2. Only one of the two histidine residues present in the enzyme reacts with diazo-1-H-tetrazole forming a monoazo derivative. The pH dependence of the coupling reaction reveals a normal pK of this reactive histidine. There are several arguments suggesting that this may be histidine 159 near the essential SH-group of papain. 3. All five tryptophan residues of the protein react with the diazonium ion below pH 7 forming a monoazo derivative with an absorption maximum at 370 nm, above pH 7 only four residues couple with diazo-1-H-tetrazole. The reaction of one tryptophan and one histidine are correlated as can be concluded from the pH dependence of the coupling rate of both amino acids and the parallel impairment of the catalytic acitivity. 4. 10-11 tyrosine residues out of 19 react with diazo-1-H-tetrazole to give bisazo compounds. 5 residues involved in hydrogen bridges form monoazo compounds. Only 12 tyrosines can be acylated by acetylimidazole. A relationship between the extent of modification of tyrosine and the activity of the enzyme could not be found.     

Chemical modification and complex formation studies with jack bean proteinase inhibitor.

Pretreatment of the purified jack bean inhibitor with enterokinase activated human pancreatic preparation for 1 hr decreased its inhibitory capacity against crystalline bovine alpha-chymotrypsin by 30% but did not affect its trypsin inhibitory activity. Preincubation of the inhibitor with bovine chymotrypsin for 60 min resulted in partial loss of the inhibitory potency. Complex formation studies by gel chromatography on Sephadex G-100 indicated that the trypsin-inhibitor and chymotrypsin-inhibitor complexes dissociated to release inactivated inhibitor and active proteinases. Gel chromatography of the inhibitor in presence of 1.5 M ammonium sulphate indicated that the inhibitor showed a tendency to aggregate without loss of biological activity. However, in 4.2 M salt medium after 3 hr, antichymotryptic activity was lost completely without any effect on antitryptic activity. Treatment with methylamine, a nucleophile, caused a greater loss of antichymotryptic activity. Trinitrobenzene sulphonate and ethylacetamidate, the amino group modifiers, affected only the antichymotryptic activity. Treatment with ninhydrin, a specific arginine modifier, at pH 9.0 abolished the antitryptic activity whereas only 50% of the antichymotryptic activity was lost. Diethylpyrocarbonate, a histidine reagent, also decreased only the antitryptic activity. Modification of tryptophan and cysteine residues of the inhibitor had no effect on its inhibitory potency. Treatment with mercaptoethanol and sodium borohydride caused nearly 50% loss of antitryptic and antichymotryptic activities. Chloramine-T, a reagent that modifies methionine residues, inactivated the inhibitor.     

Purification and properties of a fucoidan-binding protein from human placenta and its identification as immunoglobulin G.

1. A fucoidan-binding protein from human placenta was purified by affinity chromatography on immobilized fucoidan. 2. Characterization of molecular and immunological properties and peptide mapping indicated that the fucoidan-binding protein is an immunoglobulin G. 3. Cleavage with papain and transblot analysis with labelled fucoidan ascertained binding properties of the F(ab) fragments. 4. The specificity for fucoidan was furthermore substantiated by hapten inhibition of haemagglutination as well as by solid-phase assays with biotinylated fucoidan as ligand. The results emphasized the importance of structural features instead of simple ionic interactions. 5. Chemical modification with group-specific reagents to lysine, arginine, tryptophan, tyrosine and histidine resulted in substantial inactivation, their impact being markedly reduced by the presence of fucoidan in the cases of lysine, arginine and tryptophan.     

Chemical modification of acetylcholinesterase from eel and basal ganglia: effect on the acetylcholinesterase and aryl acylamidase activities

The effect of chemical modification on the acetylcholinesterase and the aryl acylamidase activities of purified acetylcholinesterase from electric eel and basal ganglia was investigated in the presence and absence of acetylcholine, the substrate of acetylcholinesterase, and 1,5-bis[4-(allyldimethylammonium)phenyl]pentan-3-one dibromide (BW284C51), a reversible competitive inhibitor of acetylcholinesterase. Trinitrobenzenesulfonic acid, pyridoxal phosphate, acetic anhydride, diethyl pyrocarbonate, and 2-hydroxy-5-nitrobenzyl bromide under specified conditions inactivated both acetylcholinesterase and aryl acylamidase in the absence of acetylcholine and BW284C51. Chemical modifications in the presence of acetylcholine and BW284C51 by all the above except diethyl pyrocarbonate selectively prevented the loss of acetylcholinesterase but not aryl acylamidase activity; modification by diethyl pyrocarbonate in the presence of acetylcholine and BW284C51 prevented the loss of both acetylcholinesterase and aryl acylamidase activities. Treatment with N-acetylimidazole resulted in the inactivation of acetylcholinesterase and the activation of aryl acylamidase. These changes in both the activities could be prevented by acetylcholine and BW284C51. Modification by phenylglyoxal, 2,4-pentanedione, or N-ethylmaleimide did not affect the enzyme activities. Indophenylacetate hydrolase activity followed a pattern similar to that of acetylcholinesterase in all the above modification studies. The results suggested essential lysine, tyrosine, tryptophan, and histidine residues for the active center of acetylcholinesterase and essential lysine, histidine, and tryptophan residues for the active center of aryl acylamidase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemical modification of acyl-CoA:cholesterol O-acyltransferase. 2. Identification of a coenzyme A regulatory site by p-mercuribenzoate modification

Acyl-CoA:cholesterol O-acyltransferase (EC 2.3.1.26, ACAT) is the major intracellular cholesterol-esterifying activity in vascular tissue and is potentially a key regulator of intracellular cholesterol homeostasis during atherogenesis. We have previously reported inhibition of microsomal ACAT by histidine and sulfhydryl-selective chemical modification reagents and present here a more detailed analysis of the effect of sulfhydryl modification on ACAT activity. This analysis indicated two effects of sulfhydryl modification on ACAT activity. Modification of aortic microsomes with relatively low concentrations of p-mercuribenzoate (PMB) (100-200 microM) identified an inhibitory coenzyme A binding site on ACAT which contains a modifiable sulfhydryl group. This site binds CoA tightly (Ki = 20 microM), and PMB modification prevented subsequent ACAT inhibition by CoA without itself inhibiting enzyme activity. At higher concentrations (1-2 mM), PMB inhibited ACAT activity, indicating the presence of a modifiable sulfhydryl group necessary for cholesterol esterification by ACAT. Modification of both sites by PMB was reversible by thiols, and protection against modification was afforded in both cases by oleoyl-CoA, indicating that these sites may also bind oleoyl-CoA. Thus, at least two sulfhydryl groups influence ACAT activity: one is necessary for cholesterol esterification by ACAT, and one is at or near an inhibitory CoA binding site, which may be occupied at intracellular concentrations of CoA.     

Inactivation of the mitochondrial ATPase inhibitor protein by chemical modification with diethylpyrocarbonate

Modification of histidine residue(s) by diethylpyrocarbonate treatment of submitochondrial particles obtained by sonication results in inhibition of ATPase activity and stimulation of oligomycin-sensitive H+ conduction. The inhibition of the ATPase (EC 3.6.1.3) activity persisted in F1 isolated from diethylpyrocarbonate-treated submitochondrial particles, which exhibited the absorbance spectrum of modified histidine. Thus the inhibition of the ATPase activity results from histidine modification in F1 subunits. Removal of the natural inhibitor protein from submitochondrial particles resulted in stimulation of proton conduction. After removal of F1 inhibitor protein from the particles the stimulatory effect exerted by diethylpyrocarbonate treatment on proton conduction was lost. Reconstitution experiments showed that purified F1 inhibitor protein lost, after histidine modification, its capacity to inhibit the ATPase activity and proton conduction. These observations show that the stimulation of proton conduction by the ATPase complex effected by diethylpyrocarbonate treatment results from histidine modification in F1 inhibitor protein.     

Isolation of specific protease inhibitors from Neurospora crassa.

Four natural protease inhibitors have been partially purified by heat treatment, ion-exchange chromatography pand gel filtration from Neurospora crassa. The inhibitory activity has been estimated by measuring the inhibition of proteolysis of casein as well as by the protection of Neurospora tryptophan synthase from proteolytic inactivation. The inhibitors are all oligopeptides and possess molecular weights in the range 5000-24 000 and appear to be very specific to Neurospora proteases. They may be classified into two types. The first are specific to Neurospora alkaline protease and the second to acidic protease. None of them exhibited any effect on other proteases including trypsin, chymotrypsin, papain, pepsin, thermolysin, subtilisin and proteinase K. The possible physiological role of these inhibitors is discussed.     

Surface lysine and tyrosine residues are required for interaction of the major herpes simplex virus type 1 DNA-binding protein with single-stranded DNA.

Modification of the herpes simplex virus type 1 major DNA-binding protein (ICP8) with reagents and conditions specific for arginine, lysine, and tyrosine residues indicates that surface lysine and tyrosine residues are required for the interaction of this protein with single-stranded DNA. Modification of either of these two amino acids resulted in a loss and/or modification of binding activity as judged by nitrocellulose filter assays and gel shift. Modification specific for arginine residues did not affect binding within the limits of the assays used. Finally, quenching of the intrinsic tryptophan fluorescence of ICP8 in the presence of single-stranded DNA either suggests involvement of this amino acid in the binding reaction or reflects a conformational change in the protein upon binding.     

Cross-Pathway Regulation: Tryptophan-Mediated Control of Histidine and Arginine Biosynthetic Enzymes in Neurospora crassa

In Neurospora crassa, the starvation of tryptophan mutants for tryptophan resulted in the derepression of tryptophan, histidine, and arginine biosynthetic enzymes. This tryptophan-mediated derepression of histidine and arginine biosynthetic enzymes occurred despite the fact that the tryptophan-starved cells had a higher intracellular concentration of histidine and arginine than did nonstarved cells.     

Identification of essential amino acid residues of an alpha-amylase inhibitor from Phaseolus vulgaris white kidney beans.

Kidney bean (Phaseolus vulgaris) alpha-amylase inhibitors, which are bivalent inhibitors with the subunit stoichiometry of (alphabeta)(2) complex, have been inferred to contain unique arginine, tryptophan, and tyrosine residues essential for the inhibitory activity. To test the validity of this inference, an attempt was made to identify the essential amino acid residues of a white kidney bean (P. vulgaris) alpha-amylase inhibitor (PHA-I) by using the chemical modification technique combined with amino acid sequencing and mass spectrometry. Exhaustive modification of the arginine residues by phenylglyoxal did not lead to a marked loss of activity, suggesting that no arginine residue is directly associated with the inhibitory activity. N-Bromosuccinimide treatment of PHA-I in the presence or absence of a substrate alpha-amylase revealed the involvement of two tryptophan residues in alpha-amylase inhibition, and they were identified as Trp188 of the beta-subunit by amino acid sequencing and mass spectrometry of lysylendopeptidase peptides. Further, two tyrosine residues were preferentially modified either by N-acetylimidazole or by tetranitromethane, resulting in a concomitant loss of most of the PHA-I activity. Amino acid sequencing of the lysylendopeptidase peptides from a tetranitromethane-modified PHA-I identified Tyr186 of the beta-subunit as an essential residue.     

Chemical modification of dipeptidyl peptidase iv: involvement of an essential tryptophan residue at the substrate binding site

Inactivation of pig kidney dipeptidyl peptidase IV (EC 3.4.14.5) by photosensitization in the presence of methylene blue at pH 7.5 was observed to have pseudo-first-order kinetics. During the process, until over 95% inactivation was achieved, the histidine and tryptophan residues were decreased from 14.0 to 2.7 and 12.6 to 7.1, respectively, per 94,000-Da subunit, without any detectable changes in other photosensitive amino acids. Modification of four histidine residues per subunit using diethylpyrocarbonate resulted in only 30% inactivation of the enzyme, while N-bromosuccinimide almost completely inactivated the enzyme with the modification of only one tryptophan residue per subunit, as determined by absorption spectrophotometry at 280 nm. The protective action of the substrate and inhibitors such as Ala-Pro-Ala and Pro-Pro against the modification of tryptophan residues with N-bromosuccinimide was observed both fluorometrically and by measurement of activity. On the basis of these results it is suggested that one of the tryptophan residues in the enzyme subunit is essential for the functioning of the substrate binding site of pig kidney dipeptidyl peptidase IV.     

Interaction of the cysteine proteinase inhibitor chicken cystatin with papain

The two forms of chicken cystatin, with different isoelectric points, that have been described previously were indistinguishable in analyses of amino- and carboxy-terminal residues, amino acid composition, and peptide maps. The two forms thus are highly similar and most likely differ only in an amide group or in a small charged substituent. The binding of either cystatin form to highly purified, active papain was accompanied by the same pronounced changes in near-ultraviolet circular dichroism, ultraviolet absorption, and fluorescence emission. These changes were compatible with perturbations of the environment of aromatic residues in one or both proteins of the complex, arising from local interactions or from a conformational change. Modification of the single tryptophan residue of cystatin, at position 104, with N-bromosuccinimide resulted in considerably smaller spectroscopic changes on binding of the inhibitor to papain, indicating that the environment of this residue is affected by the binding. Analogous modification of Trp-69 and Trp-177 of papain markedly affected the fluorescence changes observed on binding of cystatin to the enzyme, similarly suggesting that these two residues of papain are involved in the interaction. The fluorescence increase of papain at alkaline pH, arising from Trp-177 and due to deprotonization of the adjacent His-159, was abolished on binding of cystatin to the enzyme, further supporting the proposal that this region of papain participates in the interaction with the inhibitor. A stoichiometry of binding of either cystatin form to papain of 1:1 and a lower limit for the binding constant of 10(9) M-1 were determined by titrations monitored by either the ultraviolet absorption or fluorescence changes induced by the interaction.