Chemical Modification of Tryptophan Residues in Castor Bean Hemagglutinin

Modification of tryptophan residues in castor bean hemagglutinin (CBH) with N-bromosuccinimide (NBS) was investigated in detail. Tryptophan residues accessible to NBS increased with lowering pH and six tryptophan residues/mol were oxidized at pH 3.0, while two tryptophan residues/mol were oxidized at pH 5.0. From the pH-dependence curve for tryptophan oxidation, we suggest that the extent of modification of tryptophan in CBH is influenced by an ionizable group with pK_a = 3.6. The saccharide-binding activity was decreased greatly by modification of tryptophan concomitantly with a loss of fluorescence. A loss of the saccharide-binding activity was found to be principally due to the modification of two tryptophan residues/mol located on the surface of the protein molecule. In the presence of raffinose, two tryptophan residues/mol remained unmodified with retention of fairly high saccharide-binding activity. The results suggest that one tryptophan residue is involved in each saccharide-binding site on each B-chain of CBH.     

Isoamylamine as the Possible Neuroactive Metabolite of l-Leucine

The states of tryptophan residues in Abrus precatorius agglutinin (APA) were analyzed by chemical modification and solvent perturbation UV-difference spectroscopy. The number of tryptophan residues available for N-bromosuccinimide (NBS) oxidation increased with lowering pH, and 20 out of the 24 tryptophans in APA were modified at pH 3.0, while 2 tryptophans were eventually oxidized at pH 5.0. Modification of tryptophan greatly decreased the binding of APA with saccharides, and only 4% of the hemagglutinating activity was retained after modification of 4 tryptophan residues/molecule. When the modification was done in the presence of lactose or galactose, 2 tryptophan residues/molecule remained unmodified with a retention of a fairly high hemagglutinating activity. The data from solvent perturbation UV-difference spectroscopy indicated that 6 tryptophans were on the surface of the APA molecule, and 4 tryptophan residues/molecule were shielded from the perturbing effect of the solvent upon binding with lactose.

Based on these results, we proposed that in the saccharide-binding site on each B-chain of APA there exists one tryptophan residue directly involved in saccharide binding, and near the binding site there is another tryptophan residue whose state is also changeable upon binding with saccharide.     

Significance of the Tryptophan Residue at the Low Affinity Saccharide-binding Site of Ricin E

Chemical modification of tryptophan residues in ricin E was investigated with regard to saccharide-binding. Two out of ten tryptophan residues in ricin E were modified with N- bromosuccinimide at pH 4.5 in the absence of specific saccharide accompanied by a marked decrease in the cytoagglutinating activity. Such a loss of the cytoagglutinating activity was found to be principally due to the oxidation of one tryptophan residue per B-chain. In the presence of lactose, one tryptophan residue/mol was protected from the modification with retention of a fairly high cytoagglutinating activity. However, G a IN Ac did not show such a protective effect. The binding of lactose to ricin E altered the environment of the tryptophan residue at the low affinity binding site of ricin E, leading to a blue shift of the fluorescence spectrum and an UV-difference spectrum with a maximum at 290 nm and a trough at 300 nm. The ability to generate such spectroscopic changes induced by lactose was retained in the derivative in which one tryptophan residue/mol was oxidized in the presence of lactose, but not in the derivative in which two tryptophan residues/mol were oxidized in the absence of lactose. Based on these results, it is suggested that one of the two surface-localized tryptophan residues is responsible for saccharide binding at the low affinity binding site of ricin E, which can bind lactose but lacks the ability to bind GalNAc.     

Identification of the tryptophan residue located at the low-affinity saccharide binding site of ricin D

The saccharide binding ability of the low affinity (LA-) binding site of ricin D was abrogated by N-bromosuccinimide (NBS)-oxidation, while in the presence of lactose the number of tryptophan residues eventually oxidized decreased by 1 mol/mol and the saccharide binding ability was retained (Hatakeyama et al., (1986) J. Biochem. 99, 1049-1056). Based on these findings, the tryptophan residue located at the LA-binding site of ricin D was identified. Two derivatives of ricin D which were modified with NBS in the presence and absence of lactose were separated into their constituent polypeptide chains (A- and B-chains), respectively. The modified tryptophan residue or residues was/were found to be contained in the B-chain, but not in the A-chain. From lysylendopeptidase and chymotryptic digests, peptides containing oxidized tryptophan residues were isolated by gel filtration on Bio-Gel P-30 and HPLC. Analysis of the peptides containing oxidized tryptophan revealed that three tryptophan residues at positions 37, 93, and 160 on the B-chain were oxidized in the inactive derivative of ricin D, in which the saccharide binding ability of the LA-binding site was abrogated by NBS-oxidation. On the other hand, the modified residues were determined to be tryptophans at positions 93 and 160 in the active derivative of ricin D which was modified in the presence of lactose, indicating that upon binding with lactose, the tryptophan residue at position 37 of the B-chain was protected from NBS-oxidation. From these results, it is suggested that tryptophan at position 37 on the B-chain is the essential residue for saccharide binding at the LA-binding site of ricin D.     

Evidence for involvement of tryptophan residue in the low-affinity saccharide binding site of ricin D

The nature of the saccharide-binding site of ricin D, which is a galactose- and N-acetylgalactosamine-specific lectin, was studied by chemical modification and spectroscopy. With excitation at 290 nm, ricin D displayed a fluorescence spectrum with a maximum at 335 nm. Upon binding of the specific saccharides, the spectrum shifted to shorter wavelength by 3 nm. However, binding of galactosamine and N-acetylgalactosamine failed to induce such a change in the fluorescence spectrum. The interaction of ricin D with its specific saccharides was analyzed in terms of the variation of the intensity at 320 nm as a function of saccharide concentration. The results indicate that the change in the fluorescence spectrum induced by saccharide binding is attributable to the binding of saccharide to the low-affinity (LA-) binding site of ricin D. The cytoagglutinating activity of ricin D decreased to 2% upon modification of two tryptophan residues/mol with N-bromosuccinimide at pH 4.0, but in the presence of galactose or lactose one tryptophan residue/mol remained unmodified, and a fairly high cytoagglutinating activity was retained. Galactosamine and N-acetylgalactosamine did not show such a protective effect. Spectroscopic analyses indicate that the decrease in the cytoagglutinating activity of ricin D upon tryptophan modification is principally due to the loss of the saccharide binding activity of the LA-binding site. The results suggest that one tryptophan residue is essential for saccharide binding at the LA-binding site, which can bind galactose and lactose but lacks the ability to bind N-acetylgalactosamine and galactosamine.     

Identification of the Tryptophan Residue Located at the Saccharide Binding Site of Castor Bean Hemagglutinin

The tryptophan residue present at the saccharide-binding site of castor bean hemagglutinin (CBH) was identified. A peptide containing a modified tryptophan residue was isolated from the tryptic digest of S-car boxy methylated B-chain obtained from an inactive derivative of CBH (2-Oxa-CBH), in which two tryptophan residues/mol were oxidized with Af-bromosuccinimide, by gel filtration on a Sephadex G-50 followed by high performance liquid chromatography. Analytical data for the isolated peptide indicated that the tryptophan residue at position 131 on the B-chain was modified in 2-Oxa-CBH.

From these and earlier results, it is suggested that the tryptophan residue at 131 on each B-chain is closely associated with the saccharide-binding activity of CBH. The specific role of tryptophan residue at 131 in the saccharide-binding site of CBH is also discussed.     

Nonexistence of Exo-cellobiohydrolase (CBH) in the Cellulase System of Trichoderma viride

Chemical modification of histidine residues in ricin E was studied with regard to saccharide binding. The analytical data indicate that 6 out of 7 histidine residues in ricin E are eventually modified with diethylpyrocarbonate (DEP) at pH 6.0 and 25°C in the absence of specific saccharides. Modification of histidine residues greatly decreased the cytoagglutinating activity of ricin E, and only 10% of the residual activity was found after modification of 6 histidine residues/mol. The data of affinity chromatography using lactamyl- and galactosamine-cellulofine columns suggest that modification of histidine residues does not have much effect on the binding ability at the low affinity saccharide-binding site of ricin E but abolishes the binding ability at the high affinity saccharide-binding site. In the presence of lactose, one histidine residue/mol was protected from the DEP modification with retention of a fairly high cytoagglutinating activity. Such a protective effect was also observed for specific saccharides such as galactose and A^-acetylgalactosamine, but not for glucose, a non-specific saccharide. On treatment with hydroxylamine, the modified ricin E restored 67 % of the cytoagglutinating activity. Based on these findings, it is suggested that in the high affinity saccharide- binding site of ricin E there exists one histidine residue responsible for saccharide binding.     

Effect of N-bromosuccinimide modification on dihydrofolate reductase from a methotrexate-resistant strain of Escherichia coli. Activity, spectrophotometric, fluorescence and circular dichroism studies.

When dihydrofolate reductase from a methotrexate-resistant strain of Escherichia coli B, MB 1428, is treated with approximately a 5 mol ratio of N-bromosuccinimide (NBS) to enzyme at pH 7.2 and assayed at the same pH, there is a 40% loss of activity due to the modification of 1 histidine residue and possibly 1 methionine residue before oxidation of tryptophan occurs. The initial modification is accompanied by a shift of the pH for maximal enzymatic activity from pH 7.2 to pH 5.5 Upon further treatment with N-bromosuccinimide, the activity is gradually reduced from 60 to 0% as tryptophan residues become oxidized. An NBS to enzyme mole ratio of approximately 20 results in 90% inactivation of the enzyme. When the enzyme is titrated with NBS in 6 M guanidine HCl, 5 mol of tryptophan react per mol of enzyme, a result in agreement with the total tryptophan content as determined by magnetic circular dichroism. The 40% NBS-inactivated sample posses full binding capacity for methotrexate and reduced triphosphopyridine nucleotide, and the Km values for dihydrofolate and TPNH are the same as for the native enzyme. After 90% inactivation, only half of the enzyme molecules bind methotrexate, and the dissociation constant for methotrexate is 40 nM as compared to 4 nM for native enzyme in solutions of 0.1 M ionic strength, pH 7.2 Also, TPNH is not bound as tightly to the modified enzyme-methotrexate complex as to the unmodified enzyme-methotrexate complex. Circular dichroism studies indicate the 90% NBS-inactivated enzyme has the same alpha helix content as the native enzyme but less beta structure, while the 40% inactivated enzyme is essentially the same as the native enzyme. Protection experiments were complicated by the fact that NBS reacts with the substrates and cofactors of the enzyme. Although protection of specific residues was not determined, it was clear that TPNH was partially protected from NBS reaction when bound to the enzyme, and the enzyme, and the enzyme was not inactivated by NBS until the TPNH had reacted.     

Identification of the Tryptophan Residue Located at the Substrate-binding Site of Rye Seed Chitinase-c

Chemical modifications of rye seed chitinase-c (RSC-c) with various reagents suggested the involvements of tryptophan and glutamic/aspartic acid residues in the activity. Of these, the modification of tryptophan residues with N-bromosuccinimide (NBS) was investigated in detail.

In the NBS-oxidation at pH 4.0, two of the six tryptophan residues in RSC-c were rapidly oxidized and the chitinase activity was almost completely lost. On the other hand, in the NBS-oxidation at pH 5.9, only one tryptophan residue was oxidized and the activity was greatly reduced. Analyses of the oxidized tryptophan-containing peptides from the tryptic and chymotryptic digests of the modified RSC-c showed that two tryptophan residues oxidized at pH 4.0 are Trp72 and Trp82, and that oxidized at pH 5.9 is Trp72.

The NBS-oxidation of Trp72 at pH 5.9 was protected by a tetramer of N-acetylglucosamine (NAG₄), a very slowly reactive substrate for RSC-c, and the activity was almost fully retained. In the presence of NAG4, RSC-c exhibited an UV -difference spectrum with maxima at 284 nm and 293 nm, attributed to the red shift of the tryptophan residue, as well as a small trough around 300 nm probably due to an alteration of the environment of the tryptophan residue. From these results, it was suggested that Trp72 is exposed on the surface of the RSC-c molecule and involved in the binding to substrate.     

States of tryptophan residues in castor bean hemagglutinin: the perturbing effects of solvents on tryptophans in hemagglutinin and its complexes as analyzed by UV-difference spectroscopy

The states of tryptophan residues in castor bean hemagglutinin (CBH) were analyzed by solvent perturbation studies employing ultraviolet difference spectroscopy. Eight out of 22 tryptophan residues in CBH were exposed to ethylene glycol and glycerol, suggesting that the remaining 14 tryptophan residues are buried in the interior of the CBH molecule. The fraction of tryptophan residues accessible to the perturbant decreased with increase in the molecular size of the perturbant, and only 2 tryptophan residues were exposed to polyethylene glycol 600. Upon binding with raffinose, 2 tryptophan residues were shielded from the perturbing effect of the solvent, and binding of lactose reduced the number of tryptophan residues accessible to the perturbant by 1 mol per mol of protein. Binding of galactose, however, did not change the accessibility of tryptophan to the perturbant. On the other hand, the accessibility of tyrosine to the perturbant remained unchanged after binding with raffinose and lactose, suggesting that tyrosine is not directly involved in the saccharide binding of CBH. Based on these results, it is proposed that one tryptophan residue at the saccharide-binding site on each B-chain of CBH lies on the surface of the protein molecule and is located at a subsite which is accessible to a glucopyranoside moiety in the lactose molecule or a glycopyranosyl-fructofuranosyl moiety in the raffinose molecule, whereas such a residue is not present at the galactopyranoside-recognition site.     

葡萄糖淀粉酶色氨酸残基及底物结合位点的研究

 本文用N-溴代琥珀酰亚胺(NBS)对葡萄糖淀粉酶进行特异性修饰,当酶分子表面有3个色氨酸残基被修饰后,酶活力完全丧失。用邹氏图解法测得酶活性中心有一个色氨酸残基是必需的。如果在酶液中加入不同的底物再用NBS氧化,用荧光发射和荧光猝灭光谱检测表明,底物对酶分子有不同程度的保护作用。在被测试的三种底物中,这种保护能力依为糊精>淀粉>麦芽糖。     

Evidence for the involvement of tryptophan 38 in the active site of glutathione S-transferase P.

Glutathione S-transferase P (GST-P) exists as a homodimeric form and has two tryptophan residues, Trp28 and Trp38, in each subunit. In order to elucidate the role of the two tryptophan residues in catalytic function, we examined intrinsic fluorescence of tryptophan residues and effect of chemical modification by N-bromosuccinimide (NBS). The quenching of intrinsic fluorescence was observed by the addition of S-hexylglutathione, a substrate analogue, and the enzymatic activity was totally lost when single tryptophan residue was oxidized by NBS. To identify which tryptophan residue is involved in the catalytic function, each tryptophan was changed to histidine by site-directed mutagenesis. Trp28His GST-P mutant enzyme showed a comparable enzymatic activity with that of the wild type one. Trp38His mutant neither was bound to S-hexylglutathione-linked Sepharose nor exhibited any GST activity. These findings indicate that Trp38 is important for the catalytic function and substrate binding of GST-P.     

Isolation,purification, and physicochemical characterization of a D-galactose-binding lectin from seeds of Erythrina speciosa

A lectin was isolated from the saline extract of Erythrina speciosa seeds by affinity chromatography on lactose-Sepharose. The lectin content was about 265 mg/100g dry flour. E. speciosa seed lectin (EspecL) agglutinated all human RBC types, showing no human blood group specificity; however a slight preference toward the O blood group was evident. The lectin also agglutinated rabbit, sheep, and mouse blood cells and showed no effect on horse erythrocytes. Lactose was the most potent inhibitor of EspecL hemagglutinating activity (minimal inhibitory concentration (MIC)=0.25 mM) followed by N-acetyllactosamine, MIC=0.5mM, and then p-nitrophenyl alpha-galactopyranoside, MIC=2 mM. The lectin was a glycoprotein with a neutral carbohydrate content of 5.5% and had two pI values of 5.8 and 6.1 and E(1%)(1 cm) of 14.5. The native molecular mass of the lectin detected by hydrodynamic light scattering was 58 kDa and when examined by mass spectroscopy and SDS-PAGE it was found to be composed of two identical subunits of molecular mass of 27.6 kDa. The amino acid composition of the lectin revealed that it was rich in acidic and hydroxyl amino acids, contained a lesser amount of methionine, and totally lacked cysteine. The N-terminal of the lectin shared major similarities with other reported Erythrina lectins. The lectin was a metaloprotein that needed both Ca(2+) and Mn(2+) ions for its activity. Removal of these metals by EDTA rendered the lectin inactive whereas their addition restored the activity. EspecL was acidic pH sensitive and totally lost its activity when incubated with all pH values between pH 3 and pH 6. Above pH 6 and to pH 9.6 there was no effect on the lectin activity. At 65 degrees C for more than 90 min the lectin was fairly stable; however, when heated at 70 degrees C for 10 min it lost more than 80% of its original activity and was totally inactivated at 80 degrees C for less than 10 min. Fluorescence studies of EspecL indicated that tryptophan residues were present in a highly hydrophobic environment, and binding of lactose to EspecL neither quenched tryptophan fluorescence nor altered lambda(max) position. Treating purified EspecL with NBS an affinity-modifying reagent specific for tryptophan totally inactivated the lectin with total modification of three tryptophan residues. Of these residues only the third modified residue seemed to play a crucial role in the lectin activity. Addition of lactose to the assay medium did not provide protection against NBS modification which indicated that tryptophan might not be directly involved in the binding of haptenic sugar D-galactose. Modification of tyrosine with N-acetylimidazole led to a 50% drop in EspecL activity with concomitant acetylation of six tyrosine residues. The secondary structure of EspecL as studied by circular dichroism was found to be a typical beta-pleated-sheet structure which is comparable to the CD structure of Erythrina corallodendron lectin. Binding of lactose did not alter the EspecL secondary structure as revealed by CD examination.     

Hemagglutinating activity and conformation of a lactose-binding lectin from mushroom Agrocybe cylindracea

A lactose-binding lectin (Agrocybe cylindracea Lectin, ACL) purified from fruiting bodies of the mushroom A. cylindracea was investigated to determine the hemagglutinating activity and conformation changes after chemical modification, removal of metal ion and treatment at different temperatures and pH. ACL agglutinated both rabbit and human erythrocytes and its hemagglutinating activity could be inhibited by lactose. This lectin was stable in the pH range of 6-9 and temperature up to 60 degrees C. Fluorescence quenching and modification of tryptophan residues indicated that there were about two tryptophan residues in ACL molecule and one of them might be located on the surface, while the other was buried in the hydrophobic shallow groove near the surface. Chemical modification of serine/threonine and histidine showed that the partial necessity of these residues for the hemagglutinating activity of ACL. However, modifications of arginine, tyrosine and cysteine residues had no effect on its agglutinating activity.     

Chemical modification studies on Ricinus communis (Castor Bean) agglutinin

Ricinus communis agglutinin was subjected to various chemical treatments and the effect on its hemagglutinating and saccharide-binding properties was studied. Acetylation, succinylation and citraconylation led to a complete loss in the activity of the agglutinin, whereas reductive methylation had no effect on the activity, showing that charged amino groups were involved in the hemagglutinating and saccharide-binding activity of Ricinus agglutinin. Modification of tryptophyl, arginyl and carboxyl-group-containing residues did not lead to any loss in the activity of the agglutinin. Acetylation of tyrosyl groups with N-acetylimidazole strongly reduced the hemagglutinating and saccharide-binding property of Ricinus agglutinin. The loss in activity was restored on deacetylation of the tyrosyl groups. Modification of tyrosyl residues also led to a change in the immunological properties of the agglutinin. The initial rate of modification of tyrosyl and amino groups and the concomitant loss of activity was reduced in the presence of lactose.     

N-bromosuccinimide oxidation of a glucoamylase from Aspergillus saitoi

1. In order to elucidate the structure-function relation of a glucoamylase [EC 3.2.1.3, alpha-D-(1 leads to 4) glucan glucohydrolase] from Aspergillus saitoi (Gluc M1), the reaction of Gluc M1 with NBS was studied. 2. The tryptophan residues in Glu M1 were oxidized at various NBS/Gluc M1 ratios. The enzymatic activity decreased to about 80% of that of the native Gluc M1 with the oxidation of the first 2 tryptophan residues. The oxidation of these 2 tryptophan residues occurred within 0.2-0.5 s. On further oxidation of ca. 4-5 more tryptophan residues of Glu M1, the enzymatic activity of Gluc M1 decreased to almost zero (NBS/Gluc M1 = 20). Thus, the most essential tryptophan residue(s) is amongst these 4-5 tryptophan residues. 3. 7.5 tryptophan residues were found to be eventually oxidized with increasing concentrations of NBS up to NBS/Gluc M1 = 50. This value is comparable to the number of tryptophan residues which are located on the surface of the enzyme as judged from the solvent perturbation difference spectrum with ethylene glycol as perturbant. 4. In the presence of 10% soluble starch, about 5 tryptophan residues in Gluc M1 were oxidized at an NBS/Gluc M1 ratio of 20. The remaining activity of Glu M1 at this stage of oxidation was about 76%. On further oxidation, after removal of soluble starch, the enzymatic activity decreased to zero with the concomitant oxidation of 2 tryptophan residues. The results indicated that the essential tryptophan residue(s) is amongst these 2 tryptophans. 5. The UV difference spectrum induced by addition of maltose and maltitol to Gluc M1 showed 4 troughs at 281, 289, 297, and 303 nm. The latter 3 troughs were probably due to tryptophan residues of Gluc M1 and decreased with NBS oxidation.     

Tryptophan residue essential for activity of Naja naja atra phospholipase A2

When Naja naja atra phospholipase A2, which contains three tryptophan residues at the 18th, 19th, and 61st positions, was oxidized with N-bromosuccinimide at pH 4.0, its activity decreased in a convex manner with increase in the extent of oxidation of tryptophan residues. The curve shape showed that the tryptophan residue oxidized last is most responsible for the activity. The order of accessibilities of the three tryptophan residues, which was analyzed according to the method reported previously (Mohri et al. (1876) J. Biochem. 100, 883-893), was Trp-61 greater than Trp-19 greater than Trp-18. Thus, Trp-18 was evaluated to be essential for activity. Difference spectra of phospholipase A2 produced by titrating with laurylphosphorylcholine in the presence of Ca2+, which are due in large part to perturbation of the tryptophan residue(s), were retained with phospholipase A2 derivatives containing 1.2 and 2.0 mol of tryptophan residues oxidized but not with the derivative containing 3.0 mol of tryptophan residues oxidized. Such observations led us to assume that Trp-18 is involved in the specific site that interacts with phospholipid.     

Periodate oxidation of sperm-whale myoglobin and the role of the methionine residues in the antigen–antibody reaction

1. Oxidation of sperm-whale metmyoglobin and its apoprotein with periodate has been investigated under various conditions of pH and temperature to find those under which the reagent acted with specificity. 2. At pH6.8 and 22 degrees consumption of periodate ceased in 3(1/2)hr. at 43 moles of periodate/mole of myoglobin. The two methionine residues, the two tryptophan residues, the three tyrosine residues and two histidine residues were oxidized; serine increased in the hydrolysates from 6 to 9 residues/mol. 3. At pH5.0 and 22 degrees , consumption levelled off in 4(1/2)hr. at 26 moles of periodate/mole of myoglobin and resulted in the modification of the two methionine residues, the two tryptophan residues, the three tyrosine residues and two histidine residues; serine increased from 6 to 7 residues/mol. and, also, ferrihaem suffered considerable oxidation. 4. Oxidation at pH5.0 and 0 degrees resulted at completion (4hr.) in the consumption of 22 moles of periodate/mole of myoglobin and in the modification of the methionine, tyrosine and tryptophan residues. Spectral studies indicated oxidation of the haem group. This derivative reacted very poorly with rabbit antisera to MbX (the major component no. 10 obtained by CM-cellulose chromatography; Atassi, 1964). 5. Oxidation of apomyoglobin at pH5.0 and 0 degrees was complete in 4hr. with the consumption of 7.23 moles of periodate/mole of apoprotein. The rate of oxidation in decreasing order was: methionine; tryptophan; tyrosine; and after 7hr. of reaction the following residues/mol. were oxidized: methionine, 2.0; tryptophan, 1.6; tyrosine, 0.99. No peptide bonds were cleaved. Metmyoglobin prepared from the 7hr.-oxidized apoprotein showed that the reactivity with antisera to MbX had diminished considerably. 6. Milder oxidation of apoprotein (2 molar excess of periodate, pH5.0, 0 degrees , 2hr.) resulted in the modification of 1.66 residues of methionine/mol. Metmyoglobin prepared from this apoprotein was identical with native MbX spectrally, electrophoretically and immunochemically. It was concluded that the methionine residues at positions 55 and 131 were not essential parts of the antigenic sites of metmyoglobin.     

Characterization of alpha-mannosidase from Erythrina indica seeds and influence of endogenous lectin on its activity

alpha-mannosidase from Erythrina indica seeds is a Zn(2+) dependent glycoprotein with 8.6% carbohydrate. The enzyme has a temperature optimum of 50 degrees C and energy of activation calculated from Arrhenius plot was found to be 23 kJ mol(-1). N-terminal sequence up to five amino acid residues was found to be DTQEN (Asp, Thr, Gln, Glu, and Asn). In chemical modification studies treatment of the enzyme with NBS led to total loss of enzyme activity and modification of a single tryptophan residue led to inactivation. Fluorescence studies over a pH range of 3-8 have shown tryptophan residue to be in highly hydrophobic environment and pH change did not bring about any appreciable change in its environment. Far-UV CD spectrum indicated predominance of alpha-helical structure in the enzyme. alpha-Mannosidase from E indica exhibits immunological identity with alpha-mannosidase from Canavalia ensiformis but not with the same enzyme from Glycine max and Cicer arietinum. Incubation of E. indica seed lectin with alpha-mannosidase resulted in 35% increase in its activity, while no such activation was observed for acid phosphatase from E. indica. Lectin induced activation of alpha-mannosidase could be completely abolished in presence of lactose, a sugar specific for lectin.     

Studies on the tryptophan residues of soybean agglutinin. Involvement in saccharide binding

Modification of tryptophan side chains of soybean agglutinin (SBA) with N-bromosuccinimide results in a loss of the hemagglutinating and carbohydrate binding activities of the protein. One residue/subunit is probably essential for the binding activity. Modification leads to a large decrease in the fluorescene of the protein accompained by a blue shift. Iodide ion quenching of the protein fluorescence shows that saccharide binding results in a decreased accessibility of some of the tryptophan side chains. These results strongly point towards the involvement of tryptophan residues in the active site of SBA.Abbreviations SBA soybean agglutinin - NBS N-bromosuccinimide - dansyl N-dimethyl 5-amino-naphthalene 1-sulphonyl - GalNAc N-acetyl D-galactosamine