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1.
Acid phosphatase purified from maize scutellum did not dissociate into subunits upon acylation with succinic anhydride. The enzyme maintained its catalytic activity after succinylation of 52 free amino groups permolecule. The results also showed that free amino groups may play an important role in the maintenance of enzyme stability at pH values greater than 5.4. 相似文献
2.
The catalase succinylation by succinic anhydride excess results in an almost complete dissociation of the enzyme into subunits possessing no catalase activity. The catalase subunits show the peroxidatic activity on o-dianisidine oxidation. The oxidation kinetics of this substrate by the succinylated enzyme was studied at various temperatures. The activation energy for this process is 10.1 kcal/mole. Within the temperature range of 31-65.5 degrees, the succinylated enzyme thermostability was studied by monitoring the peroxidatic activity decrease upon o-dianisidine oxidation. The activation energy for the succinylated catalase thermoinactivation equals to 15.5 kcal/mole. The peroxidatic activity of catalase subunits obtained by enzyme succinylation and acidic solution treatment was compared to that of horseradish peroxidase in the oxidation of the same substrate, i.e., o-dianisidine. 相似文献
3.
R Freyer K Nissler G Kopperschl?ger E Hofmann 《Acta biologica et medica Germanica》1975,34(4):563-572
Modification of yeast phosphofructokinase by succinic and maleic anhydride influences the catalytic activity and the allosteric behaviour of the enzyme. Depending on the degree of succinylation and maleinylation a decrease of maximum activity, an increase of the apparent affinity for fructose-6-phosphate, a decrease of the Hill-coefficient and a diminution of ATP-inhibition are observed. Up to about 40% of the lysyl residues could be succinylated without dissociation of the hexameric protein, however with a decrease of the enzyme activity. More extensive succinylation or maleinylation causes a dissociation into subunits. The sedimentation coefficient is lowered from 20 S to about 3 S. The molecular weight of the smallest dissociation product was determined to 50 000 (+/- 10 000) by the sedimentation equilibrium method. The number of bound succinyl groups, as determined from radioactivity incorporation, exceeds the content of lysyl groups of the enzyme, indicating that the modifying reagent is also reacting with other amino acid residues. 相似文献
4.
Yeast hexokinase A (ATP:D-hexose 6-phosphotransferase, EC2.7.1.1) dissociates into its subunits upon reaction with succinic anhydride. The chemically modified subunits could be isolated in a catalytically active form. The Km values found for ATP and for glucose were of the some order as those found for the native enzyme. Of the 37 amino groups present per enzyme subunit, 2-3 of these groups might be located in the proximity of the region of subunit interactions. The 50% loss of the initial activity, which follows the succinylation of these more reactive amino groups, does not seem to be due to the modification of a residue on the enzyme active site or to a change of the tertiary structure of the protein. This 50%loss of the enzyme activity may be related to the dissociation of the dimer into monomers. Both native enzyme and the succinylated subunits have the same H-dependent denaturation rate profiles in response to 2 M urea. Moreover, the apparent pK of the group involved in the transition from a more stable conformation of the protein in the acid range to a less stable one at alkaline pH seems to be similar to the pK of the group implicated in the transition between the protonated inactive form of the enzyme and an active deprotonated form. The succinylated subunit presents 'negative co-operativity' with respect to ATP at slightly acid pH; however, the burst-type slow transient in the reaction progress curve and the activation effect induced by physiological polyanions, effects observed for the native enzyme, were not detected in the standard experimental conditions with the succinylated subunit. 相似文献
5.
Chemical modification studies on a lectin from winged-bean [Psophocarpus tetragonolobus (L.) DC] tubers.
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The effect of chemical modification on a D(+)-galactose-specific lectin isolated from winged-bean tubers was investigated to identify the type of amino acid involved in its haemagglutinating activity. Various anhydrides of dicarboxylic acids, such as acetic anhydride, succinic anhydride, maleic anhydride and citraconic anhydride, modified 57-68% of the amino groups of the winged-bean tuber lectin. Treatment with N-acetylimidazole modified only 45% of the total amino groups. Reductive methylation of free amino groups modified 57% of the amino groups. Modification of the amino groups of the lectin by acetic anhydride and succinic anhydride did not lead to any significant change in the haemagglutinating activity (greater than or equal to 75% active). However, citraconylation and maleylation of the lectin led to a significant decrease in the haemagglutinating activity (less than or equal to 20% active). Acetylation and succinylation (3-carboxypropionylation) of the lectin led to a decrease in the pI value of the native lectin from approx. 9.5 to approx. 4.5. Treatment of the lectin with N-bromosuccinimide led to the modification of two and four tryptophan residues per molecule in the absence and in the presence of 8 M-urea respectively. The immunological identity of all the modified lectin preparations showed no gross structural changes except the lectin modified with N-bromosuccinimide in the presence of urea at pH 4.0. 相似文献
6.
Montes T Grazu V López-Gallego F Hermoso JA Guisan JM Fernandez-Lafuente R 《Biomacromolecules》2006,7(11):3052-3058
The enzyme penicillin G acylase (PGA) is not adsorbed at pH 7 on DEAE- or PEI-coated supports, neither is it adsorbed on carboxymethyl (CM)- or dextran sulfate (DS)-coated supports. The surface of the enzyme was chemically modified under controlled conditions: chemical amination of the protein surface of carboxylic groups (using soluble carbodiimide and ethylendiamine) and chemical succinylation (using succinic anhydride) of amino groups. The full chemical modification produced some negative effects on enzyme stability and activity, although partial modification (mainly succinylation) presented negligible effects on both enzyme features. The chemical amination of the protein surface permitted the immobilization of the enzyme on CM- and DS-coated support, while the chemical succinylation permitted the enzyme immobilization on DEAE- and PEI-coated supports. Immobilization was very strong on these supports, mainly in the polymeric ones, and dependent on the degree of modification, although the enzymes still can be desorbed after inactivation by incubation under drastic conditions. Moreover, the immobilization on ionic polymeric beds allowed a significant increase in enzyme stability against the inactivation and inhibitory effects of organic solvents, very likely by the promotion of a certain partition of the organic solvent out of the enzyme environment. These results suggest that the enrichment of the surface of proteins with ionic groups may be a good strategy to take advantage of the immobilization of industrial enzymes via ionic exchange on ionic polymeric beds. 相似文献
7.
Three enzymes used in cancer chemotherapy (asparaginases from Escherichia coli and Erwinia carotovora and glutaminase from Achromobacter) were each reacted with four amino specific reagents (ethyl acetimidate, O-methylisourea, succinic anhydride, and formaldehyde/sodium borohydride). The half-lives of the modified enzymes measured in the blood of rats showed that guanidation, acetimidation and reductive alkylation were more likely to increase the persistence of the native enzymes than succinylation. However, the improvement in the persistence of any one enzyme after any one modification could not be predicted from the results with the others. It was concluded that changes in persistence caused by each modification were due to the different effects on the tertiary structure of each native enzyme. The advantages of chemical modification for increasing the persistence of enzymes over other methods such as encapsulation or aggregation are discussed. 相似文献
8.
Role of lysines in mediating interaction of modified low density lipoproteins with the scavenger receptor of human monocyte macrophages 总被引:10,自引:0,他引:10
The ability of the scavenger receptor of human monocyte macrophages to recognize human low density lipoproteins (LDL) progressively modified by three lysine-specific reagents, malondialdehyde, acetic anhydride, or succinic anhydride, has been investigated. Regardless of the reagent utilized, receptor-mediated uptake was dependent upon modification of greater than 16% of the peptidyl lysines rather than upon the net negative charge of derivatized LDL. Rates of lysosomal hydrolysis of acetyl-LDL and succinyl-LDL increased as a function of progressive modification and reflected the amount of derivatized LDL binding to the receptor. Succinylation or acetylation of greater than 60% of the lysines was necessary to attain maximal ligand binding, internalization, and degradation. In contrast, modification of only 16% of the peptidyl lysines by malondialdehyde resulted in maximal levels of binding, uptake, and hydrolysis. The expression of receptor recognition site(s) appears to depend upon the charge modification of critical lysine residues of the LDL protein rather than the net negative charge of the lipoprotein complex. Malondialdehyde, a bifunctional reactant, may modify surface and sequestered lysines concomitantly and thus promote efficient formation of the recognition site(s). 相似文献
9.
Using succinic anhydride, a succinylated derivative of anti-urease IgG having 49 ± 6% modification was prepared and its physicochemical
and immunological properties were studied. IgG undergoes substantial changes in its native conformation on succinylation,
which was mainly attributed to electrostatic destabilization of the native protein conformation. The modified IgG exhibited
a decrease in its cross-reactivity with urease. This decrease is attributed to the conformational change in IgG upon succinylation
and/or is due to the disruption of the lysine residues in the antigen-binding site of IgG upon succinylation, which may be
involved in binding the antigen. IgG was able to bind to the specific antigen although its conformation was partially modified.
Therefore, partial modification of the conformation of the antigen-binding site of IgG is permissible in order to bind to
the antigen.
Published in Russian in Biokhimiya, 2006, Vol. 71, No. 12, pp. 1642–1647. 相似文献
10.
L N Pshenichnaia S E Tukachinski? 《Zhurnal mikrobiologii, epidemiologii, i immunobiologii》1977,(3):135-139
In using a series of the succinic anhydride concentration the authors obtained preparations of succinylated albumin with a different degree of modification. The effect of succinylation on the immunochemical and some physico-chemical properties of albumin were investigated; depending on the concentration of the modifying reagent preparations of succinylated albumin proved to differ by the antigenic properties, electrophoretic mobility in agar gel and the complex-forming activity in interaction with rivanol. Conditions of partial modification permitting to increase the complex-forming properties of albumin 3-fold with the minimal changes in its structure were determined. 相似文献
11.
A method for the succinylation of yeast Proteins during their isolation is described. Some factor affecting the extent of succinylation are described, e.g., heating of protein (80°C for 5 min) prior to dervatization reduced maximum succinylation of available ε-amino groups from 88 to 54%. Succinylation of amino groups progressively increased as the concentration of succinic anhydride was increased and there was a concomitant decrease in nucleic acid content of the precipitated protein concentrate from 12.5 to 5.3 for 0 and 88% succinylation, respectively, succinylation enhanced the solubility of the protein concentrates particularly below pH 7 and it decreased the isoelectric point pH. 4.5 to 4.0. 相似文献
12.
Wilbur DS Hamlin DK Meyer DL Mallett RW Quinn J Vessella RL Press OW 《Bioconjugate chemistry》2002,13(3):611-620
Recombinant streptavidin (rSAv) is of interest as a carrier of alpha-emitting radionuclides in pretargeting protocols for cancer therapy. Due to the inherently high kidney localization of rSAv, modification of this protein is required before it can be useful in pretargeting. Previous studies (Wilbur, D. S., Hamlin, D. K. et al. (1998) Bioconjugate Chem. 9, 322-330) have shown that succinylation of rSAv using succinic anhydride decreases the kidney localization appreciably. In continuing studies, the biotin binding characteristics and biodistribution in mice of rSAv modified by reaction with succinic anhydride (amine modification) or 1,2-cyclohexanedione (arginine modification) have been compared. Modification of rSAv was conducted using 5-50 mol equiv of succinic anhydride and 60-200 mol equiv of 1,2-cyclohexanedione. Most studies were conducted using rSAv modified with the highest quantities of reagents. Succinylation of rSAv did not alter binding with biotin derivatives, but a small increase in the biotin derivative dissociation rate was noted for arginine-modified rSAv. Amino acid analysis of 1,2-cyclohexanedione-treated rSAv indicated about 40% of the arginine residues, or an average of 1.6 residues per subunit, were modified, whereas none of the lysine residues were modified. IEF analyses showed that the pI of the arginine-modified rSAv was 5.3-6, whereas the pI for the succinylated rSAv was approximately 4. Electrospray mass spectral analyses indicated that one to three conjugates of 1,2-cyclohexanedione, and two to three conjugates of succinic anhydride, were obtained per subunit. Both modification reactions resulted in greatly decreasing the kidney localization of rSAv (normally 20-25% ID/g at 4, 24, and 48 h pi). However, the kidney concentration for the succinylated rSAv continued to decrease (5% ID/g to 1.5% ID/g) from 4 to 48 h pi, whereas the concentration (5% ID/g) remained constant over that period of time for the arginine-modified rSAv. In contrast to this, the liver concentration appeared to be slightly higher (3% ID/g vs 2% ID/g) at the later time points for the succinylated rSAv. When less than 50 mol equiv of succinic anhydride were employed in the modification of rSAv, a correlation between increasing kidney localization with decreasing equivalents reacted was observed. Although the differences in the two modified rSAv are not substantial, succinylated rSAv appears to have more favorable properties for pretargeting studies. 相似文献
13.
The abnormal human low-density lipoprotein class characteristic of biliary obstruction (LP-X) was reacted with [14C]succinic anhydride to an extent of 70–80 moles of succinyl groups incorporated per 105 g of LP-X protein. The modified lipoprotein retained the typical morphology and ultracentrifugal flotation and sedimentation properties of LP-X but failed to react with antiserum to the native lipoprotein. On agar and agarose gel electrophoresis the succinylated lipoprotein had an increased mobility toward the anode relative to LP-X, as a result of the increased negative charge on the protein component.Partial delipidation of succinylated LP-X and ultracentrifugal fractionation of the protein into a fraction containing phospholipids plus at least three relatively small proteins (Apo-X) and an essentially lipid-free protein, chemically similar and immunologically identical with albumin, permitted us to evaluate the extent of reaction of these two protein classes with succinic anhydride in intact LP-X. On the average, the Apo-X fraction had 72 moles of succinyl groups incorporated per 105 g of protein, whereas the albumin fraction incorporated 55 moles per 105 g of protein.Extensive reaction of susceptible amino acid residues (mostly lysines) with succinic anhydride, without disruption of the lipoprotein structure, indicates that these protein groups are accessible to the reagent and are not involved in critical protein-lipid interactions. Elimination of immunoreactivity upon succinylation of LP-X implies that, at least for the Apo-X component, lysine residues participate in the interaction with LP-X antibodies. Also, the present results strongly support the view that albumin is not merely adsorbed to LP-X, and suggest, furthermore, that protein-protein interactions are not directly responsible for the characteristic stacking of LP-X discs as seen in the electron microscope. 相似文献
14.
The activities of hybrid dimers of alkaline phosphatase containing two chemically modified subunits have been investigated. One hybrid species was prepared by dissociation and reconstitution of a mixture of two variants produced by chemical modification of the native enzyme with succinic anhydride and tetranitromethane, respectively. The succinyl-nitrotyrosyl hybrid was separated from the other members of the hybrid set by DEAE-Sephadex chromatography and then converted to a succinyl-aminotyrosyl hybrid by reduction of the modified tyrosine residues with sodium dithionite. A comparison of the activities of these two hybrids with the activities of the succinyl, nitrotyrosyl and aminotyrosyl derivatives has shown that either the subunits of alkaline phosphatase function independently or if the subunits turnover alternately in a reciprocating mechanism, then the intrinsic activity of each subunit must be strongly dependent on its partner subunit. 相似文献
15.
Effect of chemical modification of horseradish peroxidase lysine epsilon-amino groups by propionic, butyric, valeric, succinic anhydrides and trinitrobenzolsulfonic acid (TNBS) on catalytic properties of the enzyme is investigated. All the preparations of modified peroxidase have 100% peroxidase activity for o-dianizidine at pH 7.0, which indicates the absence of lysine epsilon-amino group in the enzyme active site. pH-dependencies of modified peroxidase relative activity are studied; modification by anhydrides of monobasic acids is not found to result in changes of the relative activity pH-profile, while modification by succinic anhydride widens it. Absorption and circular dichoism spectra of native and modified peroxidase within 260--270 nm are obtained, some changes in the enzyme tertiary structure after its epsilon-amino groups modification are observed. Modification of four epsilon-amino groups by buturic and succinic anhydrides and of three epsilon-amino groups by TNBS is found to increase the regidity of protein surrounding of heme, and modification of six epsilon-amino groups by TNBS results in more unwrapped enzyme structure as compared with its native molecule. 相似文献
16.
Chemical modification and composition of tetrameric isozyme K of alkaline phosphatase from harp seal intestinal mucosa 总被引:2,自引:0,他引:2
I E Sakharov IYuMakarova G A Ermolin 《Comparative biochemistry and physiology. B, Comparative biochemistry》1989,92(1):119-122
1. The carbohydrate content of isozyme K of alkaline phosphatase (EC 3.1.3.1) from harp seal intestinal mucosa was examined. The presence of N-acetylglucosamine, N-acetylgalactosamine and considerable amounts of mannose residues was shown. 2. The amino acid content of seal alkaline phosphatase was determined. A high extent of homology (85%) between bovine and seal alkaline phosphatases was demonstrated. 3. By chemical modification lysine, dicarboxylic acids, arginine and tyrosine residues of tetrameric seal alkaline phosphatase are located near or at the active site. By contrast, the modification of either thiol or imidazole groups resulted in no alterations of the enzyme activity. 4. It has been demonstrated that inorganic phosphate is an inhibitor of alkaline phosphatase and entirely prevents the enzyme inactivation with succinic anhydride. 相似文献
17.
Succinoylation of sugarcane bagasse under ultrasound irradiation 总被引:1,自引:0,他引:1
The chemical modification of sugarcane bagasse with succinic anhydride using pyridine as solvent after ultrasound irradiation was studied. The optimized parameters included ultrasound irradiating time 0-50 min, reaction time 30-120 min, succinic anhydride concentration by the ratio of dried sugarcane bagasse to succinic anhydride from 1:0.25 to 1:1.50, and reaction temperature 75-115 degrees C are required in the process. The extent of succinoylation was measured by the weight percent gain (WPG), which increased with increments of reaction time, succinic anhydride concentration, and reaction temperature. The ultrasound irradiation has a positive effect on bagasse succinoylation process. On the other hand, the ultrasonic pre-treatment application broke down the cell wall polymers, resulting in, therefore, a negative effect on the WPG. Evidences of succinoylation were also provided by FT-IR and CP MAS (13)C NMR and the results showed that the succinoylation at C-2 and C-3 occurred. The thermal stability of the succinylated bagasse decreased upon chemical modification. 相似文献
18.
Pseudomonas cytochrome oxidase (EC 1.9.3.2) is composed of two subunits. Each subunit has a molecular weight of approx. 63000 and, according to the iron determination, contains two hemes. Cytochrome oxidase was subjected to various dissociation procedures to determine the stability of the dimeric structure. Progressive succinylation of 14 to 68% of the lysine residues of the enzyme increases the amount of the protein appearing in the subunit form (S20,W approximately 4 S) from 18 to 92%. At a high degree of succinylation a component with a sedimentation coefficient of approx. 2 S appears. The subunits with sedimentation coefficients of approx. 4 S and 2 S are also formed when the pH is below 4 or above 11. The same molecular weight (63000) was found for these two components in sodium dodecylsulphate electrophoresis. No dissociation of cytochrome oxidase was observed in salt solutions like 3 M NaC1 and 1 M Na2SO4, or in 6 M urea. The slight decrease in the sedimentation coefficients in NaC1 solutions is partly explained by preferential hydratation of the protein. 相似文献
19.
Exposure of antimycin-treated Complex III (ubiquinol-cytochromec reductase) purified from bovine heart mitochondria to [3H]succinic anhydride plus [35S]p-diazobenzenesulfonate (DABS) resulted in somewhat uniform relative labeling of the eight measured subunits of the complex by [3H]succinic anhydride. In contrast, relative labeling by [35S]DABS was similar to [3H]succinic anhydride for the subunits of high molecular mass, i.e., core proteins, cytochromes, and the iron-sulfur protein, but greatly reduced for the polypeptides of molecular mass below 15 kDa. With Complex III depleted in the iron-sulfur protein the relative labeling of core protein I by exposure of the complex to [3H]succinic anhydride was significantly enhanced, whereas labeling of the polypeptides represented by SDS-PAGE bands 7 and 8 was significantly inhibited. Dual labeling of the subunits of Complex III by14C- and3H-labeled succinic anhydride before and after dissociation of the complex by sodium dodecyl sulfate, respectively, was measured with the complex in its oxidized, reduced, and antimycin-inhibited states. Subunits observed to be most accessible or reactive to succinic anhydride were core protein II, the iron-sulfur protein, and polypeptides of SDS-PAGE bands 7, 8, and 9. Two additional polypeptides of molecular masses 23 and 12 kDa, not normally resolved by gel-electrophoresis, were detected. Reduction of the complex resulted in a significant change of14C/3H labeling ratio of core protein only, whereas treatment of the complex with antimycin resulted in decreases in14C/3H labeling ratios of core proteins I and II, cytochromec
1, and a polypeptide of molecular mass 13 kDa identified as an antimycin-binding protein. 相似文献
20.
Effect of Phosphate Limitation on Synthesis of Periplasmic Cyclic (beta)-(1,2)-Glucans 总被引:1,自引:0,他引:1
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Rhizobium meliloti and Agrobacterium tumefaciens synthesize periplasmic cyclic (beta)-(1,2)-glucans during adaptation to hypoosmotic environments. It also appears that these glucans provide important functions during the interactions of these bacteria with plant hosts. A large fraction of these glucans may become modified with anionic substituents such as phosphoglycerol or succinic acid; however, the role(s) of these substituents is unknown. In this study, we show that growth of these bacteria in phosphate-limited media leads to a dramatic reduction in the levels of phosphoglycerol substituents present on the periplasmic cyclic (beta)-(1,2)-glucans. Under these growth conditions, R. meliloti 1021 was found to synthesize anionic cyclic (beta)-(1,2)-glucans containing only succinic acid substituents. Similar results were obtained with R. meliloti 7154 (an exoH mutant which lacks the ability to succinylate its high-molecular-weight exopolysaccharide), revealing that succinylation of the cyclic (beta)-(1,2)-glucans is mediated by an enzyme system distinct from that involved in the succinylation of exopolysaccharide. In contrast, when A. tumefaciens C58 was grown in a phosphate-limited medium, it was found to synthesize only neutral cyclic (beta)-(1,2)-glucans. 相似文献