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1.
G L Rowley K E Rubenstein J Huisjen E F Ullman 《The Journal of biological chemistry》1975,250(10):3759-3766
Antimorphine antibodies inhibit the activity of morphine conjugates of mitochondrial malate dehydrogenase. Conjugation of malate dehydrogenase through tyrosine and amino groups resulted in only moderate losses of enzyme activity. On conjugation through disulfide bonds the enzyme activity first increased but dropped sharply with increasing substitution. Only the former conjugates were inhibited by excess antibodies. The degree of inhibition (up to 86%) was directly related to the number of morphine residues bonded directly to amino groups. The maximum number of antibody binding sites that bind to enzyme was nearly equal to the number of haptens provided there were 16 or less haptens/enzyme. However up to 26 haptens/enzyme became completely bound by antibody on long incubation. Inhibition of enzyme activity was detectably reduced by 2 times 10 minus 9 M morphine or 2 times 10 minus 10 M codeine, thus providing a sensitive assay for these drugs. The data suggest that enzyme inhibition occurs by conformational freezing of the enzyme when antibody binds to a morphine residue attached to one specific amino group. 相似文献
2.
Pulmonary angiotensin-converting enzyme antienzyme antibody. 总被引:1,自引:0,他引:1
A method has been developed for quantitating anticatalytic activity in antibody preparations made in goats against pure solubilized angiotensin-converting enzyme from rabbit pulmonary membranes. Anticatalytic activity was purified about 90-fold from a single batch of serum by a procedure including diethylaminoethylcellulose chromatography and elution from Sepharose columns containing covalently bound pure enzyme. Antiholoenzyme antibody was fractionated with respect to charge and binding affinity; however, these different populations each inhibited enzymatic hydrolysis of hippurylhistidylleucine, angiotensin I, and bradykinin. The inhibition dose-response curves were similar for hydrolysis of hippurylhistidylleucine and angiotensin I despite the difference in molecular weight of these substrates. Evidence is presented suggesting that a single molecule of antibody can bind two molecules of enzyme and that at least 18% of the total antiholoenzyme antibody population is directed against determinants which influence catalytic activity. A competitive immunoassay was developed with radioiodinated pulmonary enzyme as displaceable antigen. The anticatalytic and radioimmune assays were used to examine immunological properties of converting enzymes in various rabbit organs and fluids. Kidney, brain, and serum were found to contain converting enzymes which were immunologically identified with that in rabbit lung. Converting enzyme in seminal plasma was similar to the lung enzyme in the anticatalytic assay, but showed lower immunoreactivity in the radioimmune assay. 相似文献
3.
J M Conroy H Hoffman E S Kirk H O Hirzel E H Sonnenblick R L Soffer 《The Journal of biological chemistry》1976,251(16):4828-4832
Goat antibody against pure rabbit pulmonary angiotensin-converting enzyme was used to probe homology of converting enzymes from other species. Immunologically cross-reactive material was found in detergent-solubilized extracts of lung particles from rat, guinea pig, and dog by double immunodiffusion, radioimmunoassay, and inhibition of enzyme activity. No homology was demonstrable with bovine, frog, or chicken lung extracts. Antibodies from different individual goats yielded comparable estimates of homology by immunodiffusion and radioimmunoassay. In contrast, they varied greatly in extent and specificity of their inhibitory action on heterologous enzyme activity. The vasopressor effect of angiotensin I and the vasodepressor effect of bradykinin were diminished and potentiated, respectively, in rats treated with anti-rabbit enzyme antibody. A smaller but significant immune-dependent inhibition of the vasopressor response to angiotensin II was also observed. 相似文献
4.
C E Olson V Chhajlani J T August E D Schmell 《Archives of biochemistry and biophysics》1990,277(2):361-367
Monoclonal antibodies against human erythrocyte acetylcholinesterase (acetylcholine acetylhydrolase EC 3.1.1.7) have been examined for inhibition of enzyme activity. Of sixteen antibodies analyzed, only one (C1B7) inhibited enzyme activity, indicating selection of an unusual susceptible site. The inhibitory activity of C1B7 was characterized and compared to another inhibitory antibody, AE-2, previously described by Fambrough et al. (Proc. Natl. Acad. Sci. USA 79, 1078, 1982). Maximal demonstrated inhibition was 84% for C1B7 and 72% for AE-2 and antibody inhibition of enzyme activity was equivalent for the reduced and alkylated acetylcholinesterase monomer and the intact dimer. The Ki (stoichiometry of the enzyme-antibody reaction estimated from enzyme kinetics) was 1.0 for C1B7 and 4.8 molecules of antibody per monomer of acetylcholinesterase for AE-2. The antibodies did not compete with one another for binding to acetylcholinesterase, indicating that they have different target epitopes on the enzyme. Antibody binding to the enzyme was not specifically affected by any of the anticholinesterase agents tested: (a) the irreversible esteratic site-directed inhibitor diisopropylfluorophosphate; (b) the reversible active site-directed inhibitors edrophonium, neostigmine, BW284c51, and carbachol; and (c) allosteric site-directed compounds propidium and gallamine. Kinetic analysis of their effects provide evidence that both antibodies decrease the catalytic rate of enzyme activity and have little or no effect on substrate binding. 相似文献
5.
Human alpha-L-iduronidase. 1. Purification, monoclonal antibody production, native and subunit molecular mass 总被引:6,自引:0,他引:6
Human alpha-L-iduronidase from liver was purified about 20 000-fold with a new rapid three-step, five-column procedure which consisted of a Concanavalin-A-Sepharose/Blue-A-Agarose coupled step, a CM-Sepharose/Bio-Gel HT coupled step followed by a cupric-ion-chelating Sepharose 6B step. The behaviour of alpha-L-iduronidase on gel permeation chromatography was dependent upon both pH and ionic strength of the eluting buffer. The formation of species with enzyme activity which behaved as large-molecular-mass aggregates was favoured under conditions of low ionic strength and neutral pH. The amount of high-Mr species diminished as the pH decreased or the ionic strength increased to favour a single active species of Mr 65 000. A specific monoclonal antibody was generated against liver alpha-L-iduronidase. The antibody specifically immunoprecipitated enzyme activity from both crude and purified sources. The subunit Mr of liver alpha-L-iduronidase was estimated to be 65 000 using SDS-PAGE. Monoclonal antibody immunoprecipitation of radiolabelled enzyme was used to provide definitive confirmation of this subunit size. 相似文献
6.
A monoclonal antibody to bovine brain inositol monophosphatase. Immunoaffinity purification of the brain and kidney enzymes and evidence for their structural identity. 总被引:1,自引:1,他引:0
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A monoclonal IgG2b(K) antibody, G-2A4, has been generated against bovine brain myo-inositol monophosphatase (EC 3.1.3.25). The identity of the antigen recognized by the antibody was established by using e.l.i.s.a. and Western blotting procedures, and by immunoprecipitation of enzyme activity from crude brain supernatant. In addition, the hydrolysis of Ins1P by crude brain extract was inhibited by up to 83% by the pure antibody. Under identical conditions, the hydrolysis of Ins(1,4)P2 was unaffected. An immunoadsorbent column containing monoclonal antibody G-2A4 covalently attached to CNBr-activated Sepharose 4B has been used for rapid purification of the brain enzyme. Elution conditions have been optimized to allow isolation of the enzyme in high yield (54%) with full retention of column-binding capacity. The enzyme was electrophoretically homogeneous, Mr 30,000 and of higher specific activity than that purified conventionally. Chromatography of the pure enzyme on high resolution ion-exchange columns revealed some charge heterogeneity, possibly indicative of some type of post-translational modification. The immunoadsorbent column has also been used to purify the bovine kidney cortex enzyme to homogeneity. Partial proteolytic fragmentation patterns of the brain and kidney enzymes using endoprotease glu-C were identical, suggesting that they are almost certainly products of the same gene. 相似文献
7.
The effect of highly inhibitory rabbit antibodies on the enzymic activity of lobster-muscle arginine kinase has been studied. 100% inhibition was obtained at a 30-fold molar excess of antibody. The guanidine substrate L-arginine did not protect the enzyme from subsequent inhibition by its antibodies. On the other hand, the metal-nucleotide substrate Mg-ATP, or the substrate analog Mg-AMP, protected about 50% the enzyme activity, the extent of hte protection being irrespective of hte presence or of the absence of L-arginine and of the value of the molar ration Ab/Ag/ An antibody population directed against the antigenic determinant containing the essential tyrosine residue of the enzyme has been isolated by the affinity chromatography. No inhibitory acitivity was found in that fraction. Most of the inhibitory activity was found in the remaining antibody populations... 相似文献
8.
The immunization of rabbits with purified guinea pig liver transglutaminase resulted in the appearance of two antibody populations against the enzyme: one which reacted only with the Ca2+-enzyme complex and another which reacted with the intact as well as the Ca2+-enzyme. The Ca2+-induced confomrational change of the enzyme molecule exposes a new antigenic determinant which initiates the production of a specific antibody population. When the glutamine substrate of the enzyme was a dipeptide, the result of the interaction of the Ca2+-enzyme and its isolated specific antibody was an apparent activation of the catalytic activity. However, when protein substrates were used, an inhibition was observed. The characterization of the mechanism of the activation and the inhibition has led to the conclusion that the consequence of the interaction of Ca 2+-enzyme and its specific antibody is not only a limited steric hindrance of the active center but, besides that, a stabilization of the otherwise labile Ca2+-enzyme. The other antibody population reacts with both forms of the enzyme and its inhibitory effect, which has been observed in each assay, could be due to a prevention of the Ca2+-induced formation of the active enzyme. 相似文献
9.
The ascites form of a chemically induced guinea pig hepatoma, line-10, was resistant to killing in vitro by xenogeneic antibody and guinea pig complement. Pretreatment of line-10 cells with certain proteolytic enzymes rendered tham susceptible to the killing action of antibody and guinea pig complement. The effects of enzyme pretreatment were dependent on enzyme concentration, temperature, and could be blocked by addition of competitive or non-competitive inhibitors. The effect of the enzyme treatment could reversed by incubating the treated cells at 37 degrees C (but not at 0 degrees C), in the absence of the enzyme. Effective enzymes included ficin, bromelain, pronase, elastase, papain, trypsin, collagenase, lipases type I and type VI, and the neuraminidase preparation isolated from Clostridium perfringens. The activity of the lipase preparations and the neuraminidase preparation isolated from Clostridium perfringens appeared to be caused by proteolytic enzyme contamination. Enzyme preparations that proved ineffecitve in rendering the line-10 cells sensitive to killing by antibody and guinea pig complement included DNase, RNase, beta-glucuronidase type 6A or type B10, hyaluronidase type V or type VI, and pectinesterase. 相似文献
10.
E. D. Wachsmuth 《The Histochemical journal》1976,8(3):253-270
Synopsis Methods for detecting enzymes in tissue sections by antibody techniques are reviewed. In all these techniques, sections are first incubated with antibody. The bound antibody is visualized in one of four ways: identifying a label such as fluorescein linked to the antibody; using a labelled anti-antibody; employing complement and labelled anti-complement; or making use of a mixed aggregation immuno-cytochemical method.The last technique consists of three steps. A section is first incubated with antiserum, and secondly with the soluble enzyme under investigation. Thirdly the desired enzyme is stained using a conventional cytochemical method. The method is specific since, for example, the soluble enzyme used in the second step can bind only to antigenic determinants which are identical to those of the enzyme localized in the tissue. Thus purification of antigen and antibody sources is simplified, and chemical modifications of the antigen and antibody are avoided.Antibody also acts as a selective fixative for tissue antigen. It will inhibit the catalytic activity of its antigen and, in this way, permit the enzyme activity arising after the reaction of tissue enzyme-antibody complex with soluble enzyme to be amplified selectively. The mixed aggregation immuno-cytochemical technique has been used successfully with membrane-bound enzymes and cytoplasmic enzymes and for the demonstration of catalytically inactive enzyme precursors. 相似文献
11.
Rabbit antibodies against the Mn-dependent DNAse were obtained. The specificity of the anti-DNAse antibody was established by the enzyme inhibition in vitro. The enzyme activity was inhibited by more than 75 and 54% using rabbit antisera and affinity-purified IgG. Localization of the enzyme in the rat hepatocyte nuclei was studied by indirect immunofluorescence. 相似文献
12.
Goat antibody to swine kidney angiotensin I-converting enzyme was coupled to fluorescein isothiocyanate and was used to react with the converting enzyme in slices of swine kidney. Converting enzyme was present throughout the nephron and was concentrated in the convoluted tubules, especially in the proximal tubule. This finding is supported by high converting enzyme activity in the homogenate of Morris MK2 renal tumor, which is a transplanted adenocarcinoma of the proximal tubules of the rat kidney. 相似文献
13.
Induction of dopa (3,4-dihydroxyphenylalanine) decarboxylase in blowfly integument by ecdysone. A demonstration of synthesis of the enzyme de novo.
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The activity of the enzyme dopa (3,4-dihydroxyphenylalanine) decarboxylase, present in the epidermis cells of blowfly larvae, increases during the late third instar under the influence of the steroid hormone, ecdysone. By using the double-labelling technique and immune precipitation with univalent antibody to dopa decarboxylase, we demonstrated that the increase in enzyme activity was due to a stimulation of synthesis of enzyme molecules de novo. In this respect, the action of ecdysone is similar to the action of other steroid hormones. 相似文献
14.
Isolation of phenylalanine hydroxylase-stimulating monoclonal antibody by rat-myeloma--rat-spleen-cell fusion. 总被引:1,自引:1,他引:0
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A monoclonal antibody directed against monkey liver phenylalanine hydroxylase was produced by using a rat-myeloma--rat-spleen-cell-fusion system. This antibody showed the interesting property of increasing mammalian phenylalanine hydroxylase activity more than 2-fold. Perhaps monoclonal antibodies with this effect on other enzyme or proteins could be developed. 相似文献
15.
The inhibition of rat skeletal muscle glyceraldehyde-3-phosphate dehydrogenase by specific antibodies produced in rabbits has been studied. The results suggest that no influence on the enzyme active site is caused by the interaction with antibody, the inhibition being due entirely to the restricted accessibility for substrates of a part of dehydrogenase molecules included in the immune precipitate. Soluble complexes of the enzyme with monovalent Fab antibody fragments retain full catalytic activity. Modification of 8 -SH groups per mole of glyceraldehyde-3-phosphate dehydrogenase with p-chloromercuribenzoate results in no alterations in the quantitative precipitin curve, thus supporting the conclusion about the different localization of species-specific antigenic determinants of the enzyme and its active center. Interaction with monovalent Fab fragments of antibody stabilizes the structure of the dehydrogenase. Eight molar equivalents of Fab fragments almost completely protect the enzyme from cold inactivation in the presence of 0.15 M NaCl. Complex formation with Fab fragments does not prevent, however, the ADP-induced inactivation of the enzyme. 相似文献
16.
The level of acetyl-coenzyme-A carboxylase activity in Candida lipolytica undergoes large variations depending upon the carbon source on which the yeast is grown. Cells grown on n-alkanes or fatty acids exhibit a lower activity level than do cells grown on glucose. Among the n-alkanes and fatty acids tested, n-heptadecane, n-octadecane, oleic acid and linoleic acid reduce the enzyme activity to the lowest levels, which are 16-18% of the activity level in glucose-grown cells. Immunochemical titrations and Ouchterlony double-diffusion analysis with specific antibody as well as kinetic studies have indicated that the observed decrease in the level of acetyl-CoA carboxylase activity is due to a reduction in the cellular content of the enzyme. Furthermore, isotopic leucine incorporation studies with the use of the immunoprecipitation technique have demonstrated that the relative rate of synthesis of the enzyme in oleic-acid-grown cells is diminished to 12% of that in glucose-grown cells. Evidence has also been obtained to support the view that the enzyme in this yeast is not degraded at a rate high enough to contribute to the marked decrease in the cellular content of the enzyme. Thus, it is concluded that the reduction in acetyl-CoA carboxylase content in fatty-acid-grown cells is due to diminished synthesis of the enzyme. 相似文献
17.
大肠埃希菌Mn-SOD基因的克隆、表达及多克隆抗体制备 总被引:1,自引:0,他引:1
目的实现Mn-SOD基因在大肠埃希菌中的高可溶性表达,制备Mn-SOD的多克隆抗体。方法用PCR方法从一株野生型大肠埃希菌(E.coli)基因组中扩增Mn-SOD基因编码区.将它克隆到原核表达载体上进行大量表达和纯化,再用纯化的蛋白对新西兰大白兔进行背部多点注射,40d后取其血清,用Western-blot印迹实验测定抗体效果。结果SDS-PAGE分析表明SOD的表达量约为细菌总蛋白的50%;黄嘌呤氧化酶法测定表达蛋白活性,结果表明每毫克菌体可溶性总蛋白中表达产物酶比活为3921.77U/mg,是对照BL21的276.77倍;并制备了高效价的多克隆抗体。结论该研究成功地构建了大肠埃希菌Mn-SOD基因高效原核表达系统,所表达的Mn-SOD具有良好的免疫原性和免疫反应性。 相似文献
18.
Antibody to B. subtilis DNA polymerase III: use in enzyme purification and examination of homology among replication-specific DNA polymerases. 总被引:5,自引:4,他引:1
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Bacillus subtilis DNA polymerase III (pol III), an arylhydrazinopyrimidine-sensitive, replication-specific enzyme, was used to generate a non-precipitating rabbit antibody which specifically inhibited pol III activity in vitro. The antibody was used to examine structural relationships among several DNA polymerases, and it was linked covalently to agarose; the antibody:agarose was employed to develop a rapid, selective method of purification of catalytically active B. subtilis pol III. 相似文献
19.
Glutamine synthetase (L-glutamate : ammonia ligase, EC 6.3.1.2) fromPhaseolus aureus (mung bean) seedlings was purified to homogeneity by ammonium sulphate fractionation, DEAE-cellulose chromatography, Sephadex
G-200 gel filtration and affinity chromatography on histidine-Sepharose. The enzyme had a molecular weight of 775,000 ± 25,000.
The enzyme consisted of identical subunits with an approximate subunit molecular weight of 50,000. Hyperbolic saturation curves
were obtained with the substrates, glutamate, ATP and hydroxylamine.
Antibody, raised in the rabbit, against mung bean glutamine synthetase, completely inhibited the activity of the enzyme. Preincubation
of the enzyme with glutamate and ATP, prior to the addition of the antibody, partially protected the enzyme against inhibition.
TheK
m values of this enzyme-antibody complex and the native enzyme were identical (glutamate, 2.5mM; ATP, 1 mM; hydroxylamine,
0.5 mM). The Km values of the partially inhibited enzyme (the enzyme pretreated with antibody prior to the addition of substrates)
were 2-fold higher than those of the native enzyme. These results suggested that the substrate-induced conformational changes
in the enzyme were responsible for the protection against inhibition of the enzyme activity by the antibody. 相似文献
20.
Phosphopyruvate carboxylase induction by l-tryptophan. Effects on synthesis and degradation of the enzyme 总被引:12,自引:10,他引:2
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1. The administration of l-tryptophan to fed rats produces a twofold increase in hepatic phosphopyruvate carboxylase activity that represents a comparable increase in enzyme protein. With specific antibody against the enzyme we have shown that the increase in phosphopyruvate carboxylase is partially mediated via an actinomycin D-sensitive increase in enzyme synthesis. 2. In starved animals tryptophan increases the enzyme activity without any change in the relative rate of phosphopyruvate carboxylase synthesis. In this condition degradation of the enzyme is retarded by tryptophan by a mechanism that is not prevented by cycloheximide. 相似文献