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1.
Nuclear glutamate dehydrogenase: unique antigenic determinants and determinants common to the mitochondrial enzyme 总被引:1,自引:0,他引:1
An antiserum against glutamate dehydrogenase from ox liver nuclei precipitates both the nuclear and the mitochondrial enzymes, with different equivalence zones. The antibodies of this serum have been fractionated by means of an immunoadsorbent to which mitochondrial glutamate dehydrogenase is covalently linked. After the affinity chromatography, the unretained antibodies had virtually lost the ability to precipitate the mitochondrial enzyme, whereas the retained portion, after elution, precipitated both glutamate dehydrogenases. These findings suggest that nuclear glutamate dehydrogenase contains specific antigenic determinants as well as determinants common to the mitochondrial enzyme, and that only the antibodies against the latter determinants have been selectively removed by the affinity chromatography. 相似文献
2.
R Myllyl? 《Biochimica et biophysica acta》1981,658(2):299-307
Antibodies were prepared against chick-embryo galactosylhydroxylysyl glucosyltransferase and further purified by immunoaffinity chromatography. The antibodies gave a single precipitation line of identity by double immunodiffusion against crude or pure chick-embryo glucosyltransferase. The ability of the antibody to precipitate the transferase was not altered by destroying the secondary structure of the enzyme. The antibody also inhibited the enzyme activity. The degree of inhibition was higher with denatured citrate-soluble rat skin collagen as the substrate than with gelatinized rat skin insoluble collagen or free galactosylhydroxylysine. The cross-reactivity of the glucosyltransferase between different species was low when studied by double immunodiffusion or inhibition kinetics. The antiserum showed no detectable cross-reactivity against other intracellular enzymes of collagen biosynthesis. A line of complete identity was found in double immunodiffusion between the transferases from whole chick embryos and chick embryo tendon, kidney and cartilage. Inhibition by the antiserum of the enzyme from chick embryo tissues synthesizing different collagen types was relatively similar. The data do not support the hypothesis that galactosylhydroxylysyl glucosyltransferase has isoenzymes with markedly different specific activities or immunological properties. 相似文献
3.
Immunological cross-reactivity between chicken slow skeletal and ventricular muscle myosin 总被引:2,自引:0,他引:2
S Sartore 《Biochimica et biophysica acta》1981,667(1):143-156
Antibodies elicited in rabbits against chicken slow skeletal anterior latissimus dorsi and ventricular myosin were analyzed by double immunodiffusion for their ability to react with homologous and heterologous antigen at different stages of immunization (1--12 months). Each anti myosin antiserum formed a single, strong precipitin line with its immunogen after short time of immunization. This reaction was specific for myosin heavy chains as determined by GEDELISA (gel electrophoresis derived enzyme lined immunosorbent assay) test. In rabbits injected with ventricular myosin after long time of immunization a second, fainter precipitin line has generally been observed. The antigenic determinants responsible for this precipitin line have been localized on the light myosin subunits. By comparing the two types of anti myosin antisera with heterologous antigen we have obtained evidence for partial immunological cross-reactivity between slow skeletal and ventricular muscle myosins. In particular, all anti ventricular myosin antisera displayed a marked immunological reactivity with anterior latissimus dorsi myosin whereas most of anti anterior latissimus dorsi myosin antisera showed absence of reciprocity. By means of immunofluorescence and immunoabsorption techniques both common and unique slow skeletal and ventricular antigenic determinants have been demonstrated. 相似文献
4.
5.
Immunological characterization of lysyl hydroxylase, an enzyme of collagen synthesis 总被引:2,自引:1,他引:1
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Taina M. Turpeenniemi-Hujanen 《The Biochemical journal》1981,195(3):669-676
Antibodies to pure lysyl hydroxylase from whole chick embryos were prepared in rabbits and used for immunological characterization of this enzyme of collagen biosynthesis. In double immunodiffusion a single precipitation line was seen between the antiserum and crude or pure chick-embryo lysyl hydroxylase. The antiserum effectively inhibited chick-embryo lysyl hydroxylase activity, whether measured with the biologically prepared protocollagen substrate or a synthetic peptide consisting of only 12 amino acids. This suggests that the antigenic determinant was located near the active site of the enzyme molecule. Essentially identical amounts of the antiserum were required for 40% inhibition of the same amount of lysyl hydroxylase activity units from different chick-embryo tissues synthesizing various genetically distinct collagen types. In double immunodiffusion a single precipitation line of complete identity was found between the antiserum and the purified enzyme from whole chick embryos and the crude enzymes from chick-embryo tendon, cartilage and kidneys. These results do not support the hypothesis that lysyl hydroxylase has collagen-type-specific or tissue-specific isoenzymes with markedly different specific activities or immunological properties. The antibodies to chick-embryo lysyl hydroxylase showed a considerable degree of species specificity when examined either by activity-inhibition assay or by double immuno-diffusion. Nevertheless, a distinct, although weak, cross-reactivity was found between the chick-embryo enzyme and those from all mammalian tissues tested. The antiserum showed no cross-reactivity against prolyl 3-hydroxylase, hydroxylysyl galactosyl-transferase or galactosylhydroxylysyl glucosyltransferase in activity-inhibition assays, whereas a distinct cross-reactivity was found against prolyl 4-hydroxylase. Furthermore, antiserum to pure prolyl 4-hydroxylase inhibited lysyl hydroxylase activity. These findings suggest that there are structural similarities between these two enzymes, possibly close to or at their active sites. 相似文献
6.
A A ten Have-Opbroek 《Developmental biology》1975,46(2):390-403
After preparation of an antiserum specifically recognizing the antigenic determinants of adult mouse lung tissue, the existence of a lung-specific antigen could be demonstrated by immunodiffusion studies in agar. This specific anti-adult lung serum was used in immunofluorescence studies to localize the corresponding antigen in adult mouse lung tissue. Comparison of the results of immunofluorescent staining with those of histochemical enzyme staining showed that the lung-specific antigen is localized in the great alveolar cell.Subsequently, the same antiserum was used in immunodiffusion studies to follow the development of the lung-specific antigen in the mouse embryo. The results indicate that the lung-specific antigen first appears in embryos with a weight of between 760 and 900 mg, which corresponds with a developmental age of between 16.76 and 17.23 days. 相似文献
7.
Nuclear glutamate dehydrogenase (EC 1.4.1.3) activity has been demonstrated in Chinese hamster ovary cells. Some characteristics of this enzyme have been examined and compared with those of the mitochondrial glutamate dehydrogenase from the same source. Differences were detected in the extent of the activation by inorganic phosphate, in the pH versus activity curves, in the affinity of the two enzymes for the cofactor NAD+ and in the electrophosretic mobility. A different rate of decay of the two enzymes has been observed in cells grown in the presence of chloramphenicol. Immunological studies show that, as in ox liver, the nuclear enzyme has specific antigenic determinants besides those in common with mitochondrial glutamate dehydrogenase. Finally, experiments of thermal inactivation indicate a higher stability of the mitochondrial enzyme. 相似文献
8.
Cecilia Gotor Eloísa Pajuelo Luís C. Romero Antonio J. Márquez José M. Vega 《Archives of microbiology》1990,153(3):230-234
Polyclonal antiserum specific for ferredoxin-nitrite reductase (EC 1.7.7.1) from the green alga Chlamydomonas reinhardii recognized the nitrite reductase from other green algae, but did not cross-react with the corresponding enzyme from different cyanobacteria or higher plant leaves. An analogous situation was also found for ferredoxin-glutamate synthase (EC 1.4.7.1), using its specific antiserum. Besides, the antibodies raised against C. reinhardii ferredoxin-glutamate synthase were able to inactivate the ferredoxin-dependent activity of nitrite reductase from green algae.These results suggest that there exist similar domains in ferredoxin-nitrite reductases and ferredoxin-glutamate synthases from green algae. In addition, both types of enzymes share common antigenic determinants, probably located at the ferredoxin-binding domain. In spite of their physicochemical resemblances, no apparent antigenic correlation exists between the corresponding enzymes from green algae and those from higher plant leaves or cyanobacteria.Abbreviations Fd
ferredoxin
- GOGAT
glutamate synthase
- MV+
reduced methyl viologen (radical cation)
- NiR
nitrite reductase
- PMSF
phenylmethylsulphonyl fluoride
- SDS
sodium dodecyl sulfate 相似文献
9.
R Di Fraia V Wilquet M A Ciardiello V Carratore A Antignani L Camardella N Glansdorff G Di Prisco 《European journal of biochemistry》2000,267(1):121-131
The Antarctic psychrotolerant bacterium Psychrobacter sp. TAD1 contains two distinct glutamate dehydrogenases (GDH), each specific for either NADP+ or NAD+. This feature is quite unusual in bacteria, which generally have a single GDH. NADP+-dependent GDH has been purified to homogeneity and the gene encoding GDH has been cloned and expressed. The enzyme has a hexameric structure. The amino acid sequence determined by peptide and gene analyses comprises 447 residues, yielding a protein with a molecular mass of 49 285 Da. The sequence shows homology with hexameric GDHs, with identity levels of 52% and 49% with Escherichia coli and Clostridium symbiosum GDH, respectively. The coenzyme-binding fingerprint motif GXGXXG/A (common to all GDHs) has Ser at the last position in this enzyme. The overall hydrophilic character is increased and a five-residue insertion in a loop between two alpha-helices may contribute to the increase in protein flexibility. Psychrobacter sp. TAD1 GDH apparent temperature optimum is shifted towards low temperatures, whereas irreversible heat inactivation occurs at temperatures similar to those of E. coli GDH. The catalytic efficiency in the temperature range 10-30 degrees C is similar or lower than that of E. coli GDH. Unlike E. coli GDH the enzyme exhibits marked positive cooperativity towards 2-oxoglutarate and NADPH. This feature is generally absent in prokaryotic GDHs. These observations suggest a regulatory role for this GDH, the most crucial feature being the structural/functional properties required for fine regulation of activity, rather than the high catalytic efficiency and thermolability encountered in several cold-active enzymes. 相似文献
10.
Ferredoxin-nitrite reductase (EC 1.7.7.1), an enzyme which catalyzes the 6-electron reduction of nitrite to ammonia, has been isolated from green shoots of bean (Phaseolus angularis). The isolated enzyme (GR-NiR), having a molecular mass of 68 000, showed 1.4 times higher ferredoxin-dependent activity than methyl viologen-linked activity. The enzyme was homogeneous by polyacrylamide gel electrophoresis (PAGE). In the oxidized form, the enzyme had absorption maxima at 275, 393 (Soret band), 535 and 571 (α band) nm, indicating that siroheme is involved in the catalysis of nitrite reduction. The absorbance ratios, A393 : A275 and A571 : A393 were 0.26 and 0.32, respectively. Antibody against the isolated enzyme was raised in rabbits. Analysis of the antiserum by immunodiffusion and immunoelectrophoresis suggested that it was a specific antiserum against GR-NiR. Using the antiserum, immunodiffusion and immunoprecipitation procedures were employed to compare the immunological similarity of NiR from green shoots, etiolated shoots and roots of bean. These tests revealed that the three forms of assimilatory NiR have antigenic determinants in common. 相似文献
11.
Liver mitochondrial and microsomal DT-diaphorase have been purified from 3-methylcholanthrene-treated rats. A 1150-fold and 3500-fold purification of mitochondrial and microsomal DT-diaphorase, respectively, is achieved after solubilization of the membranes with deoxycholate followed by affinity chromatography on azodicoumarol Sepharose 6B and subsequent gel filtration on Sephadex G-100. From this purification procedure, 65–70% of mitochondrial DT-diaphorase is recovered and the purified enzyme has a specific activity comparable to that of cytosolic DT-diaphorase; i.e., 50.4 kat/kg protein. Microsomal DT-diaphorase is obtained with a yield of 45% and a specific activity of 15.5 kat/kg protein.Purified mitochondrial DT-diaphorase exhibits an absorption spectrum characteristic of a flavoprotein and very similar to that of the cytosolic enzyme. Purification of both mitochondrial and microsomal DT-diaphorase results in fractions enriched in a polypeptide with a molecular weight of 28,000 which comigrates with purified cytosolic DT-diaphorase on SDS-polyacrylamide gel electrophoresis. Employing antiserum raised against cytosolic DT-diaphorase, immunological identity between DT-diaphorase isolated from the three cell fractions is observed with both the Ouchterlony immunodiffusion technique and fused rocket immunoelectrophoresis. The latter method also reveals that DT-diaphorase isolated from mitochondria and microsomes contains several antigenic forms identical to those observed in purified cytosolic DT-diaphorase. Furthermore, this antiserum inhibits DT-diaphorase to about the same extent whether the enzyme is isolated from mitochondria, microsomes, or cytosol. In addition, this antiserum efficiently inhibits membrane-bound microsomal DT-diaphorase. 相似文献
12.
The relatedness of antigenic determinants of purified major core proteins of the murine, feline, RD 114/baboon, and woolly monkey/gibbon ape groups of RNA tumor viruses was examined by competition radioimmunoassay. In assay systems of a homologous antigen and antiserum, high affinity competition for binding to all of the antibodies was observed only with the homologous unlabeled protein; the core proteins of other groups of viruses showed only low affinity binding of a small fraction of antibodies, presumably those reactive with the interspecies determinants, at concentrations of competing protein 10- to 100-fold greater than that of the labeled antigen. The cross-reactive (interspecies) antigens of every two viruses were selectively examined by precipitating the purified 125-I-labeled protein with antiserum against each of the other proteins. The extent to which these shared determinants were common to the other viruses was then tested by the effectiveness of the proteins of each virus to compete for antibody binding. Several classes of interspecies determinants were distinguished: those common to two of the groups of viruses, others to three, and some to all four. Moreover, an even greater variety of interspecies determinants was indicated by differences in the affinity of the individual proteins for antibody binding, supporting the hypothesis that there are at least several, if not many, different interspecies determinants with a broad spectrum of antigenic cross-reactivity. These studies suggest that the murine and feline viruses are closely related as they contain cross-reactive antigenic determinants not shared with the other viruses, that the feline virus is more closely related to the woolly monkey virus than to RD 114, and that the RD 114 and woolly monkey viruses retain interspecies determinants shared relatively equally with each of the other viruses. 相似文献
13.
A new purified antigen (E2) of Paracoccidioides brasiliensis mycelial growth phase was isolated by immunoadsorption from a crude metabolic soluble extract of the fungus. The antiserum prepared in a rabbit by inoculation of E2 antigen developed only one immunodiffusion line with the crude metabolic extract. Findings on immunological analysis showed that E2 antigen is the antigenic component of immunoelectrophoretic band E. The isolated antigens did not possess detectable alkaline phosphatase activity. It reacted in immunodiffusion tests with all the sera (14/14) from P. brasiliensis infected patients containing precipitating antibodies. 相似文献
14.
A highly specific rabbit antiserum against DNA polymerase alpha from regenerating rat liver (antigen AG 1) and an antiserum against the preparation of the enzyme proteolytic fragments possessing catalytic activity (antigen AG 2) were obtained. The enzyme neutralization test revealed that antibodies against AG 2 inhibit the DNA polymerase activity in a much stronger degree, than those against AG 1. Data from a kinetic analysis of the enzyme complexed with the antibodies against AG 1 suggest that the catalytic and binding sites for dNTP and free Mg2+ are altered. The value of apparent Km for activated DNA is unchanged in the DNA polymerase complexes with antibodies both against AG 1 and AG 2. 相似文献
15.
Highly specific antibodies against hemeprotein were obtained by immunizing rabbits with a highly purified cholesterol-hydroxylating cytochrome P-450scc from adrenocortical mitochondria. The antibodies do not specifically interact with other components of the adrenocortical electron transport chain, e. g., adrenodoxin reductase and adrenodoxin. Using double immunodiffusion technique (Ouchterlony method), it was shown that the antibodies did not precipitate the microsomal cytochromes P-450 LM2 and LM4, cytochrome b5 and 11 beta-hydroxylating cytochrome P-450 from adrenocortical mitochondria. Antibodies against cytochrome P-450scc inhibited the cholesterol side chain cleavage activity of cytochrome P-450scc in a reconstituted system. Limited proteolysis with trypsin and immunoelectrophoresis in the presence of specific antibodies revealed that antigenic determinants are present of the heme-containing catalytic domain of cytochrome P-450scc (F1) as well as on the domain responsible for the interaction with the phospholipid membrane (F2). 相似文献
16.
《Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology》1988,952(1):152-157
Polyclonal antisera were prepared against ferredoxin-nitrite reductase (EC 1.7.7.1) and ferredoxin-glutamate synthase (glutamate synthase (ferredoxin); EC 1.4.7.1) from the green algaChlamydomonas reinhardtii. The anti-glutamate synthase antibodies recognized both glutamate synthase and nitrite reductase, but inhibited only the ferredoxin-linked activity of the latter enzyme and not the activity dependent on methyl viologen. Analogously, the anti-nitrite reductase antibodies recognized glutamate synthase and nitrite reductase but the first enzyme was only poorly inhibited. Free ferredoxin protected the nitrite reductase against its inactivation by anti-glutamate synthase antibodies. These results indicate that the ferredoxin-dependent glutamate synthase and nitrite reductase from this alga share common antigenic determinants, and that these are located at the ferrodoxin-binding domains. 相似文献
17.
Antisera to purified gamma-glutamyltranspeptidase (gamma GTP) from human and rat kidney were prepared, and their reactivities toward purified gamma GTP from kidney, liver, and bile were tested. The following results were obtained: 1. On double immunodiffusion, Triton-solubilized gamma GTP, and papain-solubilized gamma GTP from rat kidney gave single precipitin lines which fused completely against antiserum to the purified enzyme from rat kidney. 2. An antigen-antibody complex of human kidney gamma GTP retained about 50% of the catalytic activity of the antigen. 3. Double immunodiffusion showed that the enzymes from human liver, kidney, and bile were immunologically identical. 4. Antiserum to rat kidney gamma GTP partially cross reacted with human gamma GTP, but antiserum to human gamma GTP reacted only very weakly with rat gamma GTP. It is concluded that gamma GTP of human liver, kidney, and bile are immunologically identical and that rat gamma GTP and human gamma GTP have certain antigenic determinants in common. 相似文献
18.
T. Sayers A. Leoutsakos P. Berg H. Baum 《Journal of bioenergetics and biomembranes》1981,13(5-6):255-267
Antimitochondrial antibodies are found in a variety of autoimmune liver diseases, particularly primary biliary cirrhosis. The antigen against which these antibodies are directed is localized on the inner mitochondrial membrane. Earlier work suggested that this antigen was associated with the mitochondrial ATPase. However, we have succeeded in separating the enzyme activity from the antigenic activity using gel filtration and ion-exchange chromatography. Furthermore, the antigenic activity is not affected by modulators of ATPase enzymatic activity like aurovertin or oligomycin. The antigenic activity is, however, very susceptible to reagents which block thiol groups. The mitochondrial antigen, in contrast to the ATPase enzyme, is found in high amounts in brown fat mitochondria. Identification of this antigen may help to explain why specific antimitochondrial antibodies arise in the sera of patients with primary biliary cirrhosis.Abbreviations ATPase
adenosine triphosphatase
- PBC
primary biliary cirrhosis
- AMA
antimitochondrial antibodies
- SMPs
submitochondrial particles
- CFT
complement fixation test
- SDS
sodium dodecyl sulfate
- BSA
bovine serum albumin
- BAT
brown adipose tissue 相似文献
19.
A R Shikhman L V Nikolaeva O A Kondrakova E P Savel'ev 《Zhurnal mikrobiologii, epidemiologii, i immunobiologii》1986,(2):73-76
The use of the competitive enzyme immunoassay (EIA) has made it possible to demonstrate that antiserum to the ribosomes of group A streptococcus, type 29 M, contains antibodies to homologous protein M and does not contain antibodies to group A polysaccharide, lipoteichoic acid and peptidoglycan. Ribosomal antiserum has been found to bind with the surface of heterologous type M streptococci in EIA, while forming no precipitation lines with heterologous proteins M in the double immunodiffusion test, which indicates that the binding of ribosomal antibodies with the surface of group A streptococcal cells is not type-specific. Antibodies to the ribosomes of group A streptococcus recognize some of the antigenic determinants of E. coli ribosomes. 相似文献
20.
Previous work based on double immunodiffusion assays had shown that there are common antigenic determinants for nitrate reductase from Escherichia coli and component I of nitrogenase from Azotobacter vinelandii. Further work reported herein using a variety of immunoelectrophoretic techniques indicates that the cross-reaction between nitrate reductase and antiserum to component I of nitrogenase results from a contaminant antigen co-purified with nitrate reductase. 相似文献