Immunological Heterogeneity of Rabbit Antibody Fragments against Taka-amylase A

Okada, Yoshimi; Ikenaka, Tokuji; Yagura, T; Yamamura, Yuichi

doi:10.1093/oxfordjournals.jbchem.a127737

Cited by 13 publications

(2 citation statements)

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“…It was found that in the presence of antibody, the rate of catalysis of L-glutamine was significantly increased over normal serum controls. This is similar to the results of Pollock (24) with penicillinase, Okada et al (22) with amylase, Rotman and Celada (25) with ,8-galactosidase, and Suzuki et al (28) with ribonuclease. A probable mechanism for this effect is that constraint placed on the enzyme by bound antibody prevents conformative changes usually imposed by normal substrate (30,31).…”

Section: Discussionsupporting

confidence: 89%

Effects on specific antibodies on the catalytic activity of L-asparaginase from Serratia marcescens and Escherichia coli

Ferguson¹,

Phillips²,

Marucci³

1975

J Bacteriol

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Rabbit antisera against highly purified L-asparaginase from Serratia marcescens and from Escherichia coli showed up to 60% inhibition of the catalytic amidohydrolysis of L-asparagine when combined with the homologous enzyme. This inhibition was diminished somewhat against the heterologous enzyme. Kinetic studies in the presence of these antisera showed an increased Km,&P for both homologous and heterologous enzymes using L-asparagine as substrate. In contrast, kinetic studies employing the poor substrate, L-glutamine, showed activation attributable to specific antibodies. This was seen in lower KmaPP values and up to twofold increases in the Vi.. over the normal rabbit serum controls. The high degree of cross-inhibition ('80%) and the low degree of cross-reactivity in the quantitative precipitin test (-34%) suggest that these two enzymes possess structural similarities located mainly in the regions of the catalytic sites.trifuged. The clear supernatant was mixed with 0.4 ml of 1.5 N KOH and assayed. To standardize protein 424

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Section: Discussionsupporting

confidence: 89%

Effects on specific antibodies on the catalytic activity of L-asparaginase from Serratia marcescens and Escherichia coli

Ferguson¹,

Phillips²,

Marucci³

1975

J Bacteriol

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“…Since this stimulatory activity is also present in the gamma globulin fractions of the antisera, it is likely due to the interaction of a specific antibody with the tubulin molecule, although verification of this point will rest on the purification of the specific anti-tubulin immunoglobulin from the total globulin fractions used here. While the nature of this interaction is not understood at present, it is possible that just as antibodies against various enzymes have been observed to increase the activity of the enzymes by causing conformational changes when the antibody binds to the enzyme (21)(22)(23)(24)(25), so antibody in our antisera may alter the colchicine-binding site in a similar way.…”

Section: Resultsmentioning

confidence: 99%

Antisera against electrophoretically purified tubulin stimulate colchicine-binding activity.

Aubin¹,

Subrahmanyan²,

Kalnins³

et al. 1976

Proc. Natl. Acad. Sci. U.S.A.

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Several rabbit antisera have been prepared against reduced and alkylated, electrophoretically purified tubulin isolated from chick brain. These antisera give a single precipitin line in Ouchterlony double diffusion plates when tested against partially purified tubulin, and label specifically microtubule-and tubulin-containing structures, such as mitotic spindles, cilia, and vinblastine-induced crystals, in a variety of cells. The same antisera also display the unique ability to stimulate the colchicine-binding activity of tubulin preparations from chick brain and Chinese hamster ovary tissue culture cells. This specific stimulation of colchicinebinding activity is also obtained with the gamma globulin fractions purified by ammonium sulfate precipitation of these antisera.Intensive effort has been directed at characterizing the distribution, ultrastructure, and mode of action of microtubules; their assembly from tubulin subunits; and their response to various agents, for example, temperature, pressure, and the plant alkaloids colchicine and vinblastine (1-5). Recent efforts have centered upon the raising of specific antisera (8-12) so that microtubule structure can be studied immunologically. The antisera have been prepared in different ways against intact microtubules or tubulin, using for immunization preparations having various degrees of purity obtained from a number of sources, including vinblastine-induced paracrystals, flagellar outer doublets, and tubulin isolated from brain. By the fluorescein-labeled antibody technique, the antisera have been observed to stain specifically microtubule-and tubulin-containing structures such as vinblastine paracrystals (6), mitotic spindles (6, 7, 10), and fibrous networks in the cytoplasm (11). Whether or not these antisera affect the rather specific binding of colchicine to tubulin (3, 4) has not been reported.We report here the preparation and initial characterization of antisera produced against electrophoretically purified, reduced, and alkylated tubulin from chick brain. bulin bands were located by scanning the gels at 280 nm, they were cut out and the tubulin was eluted and used for immunization. About 30% of the original amount placed on the preparative gels was recovered. When the recovered tubulin was tested by analytical gel electrophoresis, only the two tubulin bands were detected.Antisera. Each of several rabbits was injected subcutaneously with a total of 1 mg of the reduced-alkylated, electrophoretically purified tubulin in complete Freund's adjuvant, over a period of 6 weeks. A detailed method for preparing the antisera will be published elsewhere (manuscript submitted for publication). The antisera obtained were tested by Ouchterlony double diffusion in 1% agarose gels at 40 for 24 hr. Control sera were obtained from the same rabbits prior to immunization.Cells. Mouse embryo fibroblasts, HeLa cells, and CHO cells were grown on coverslips in a-minimal Eagle's medium and 10% fetal calf serum (Flow Laboratories). To induce crystal formation, 10 ,ug/m...

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Conformational Adaptability in Enzymes

Citri¹

1973

Advances in Enzymology - And Related Areas of Molecular Biology

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Immunological Heterogeneity of Rabbit Antibody Fragments against Taka-amylase A

Cited by 13 publications

References 0 publications

Effects on specific antibodies on the catalytic activity of L-asparaginase from Serratia marcescens and Escherichia coli

Effects on specific antibodies on the catalytic activity of L-asparaginase from Serratia marcescens and Escherichia coli

Antisera against electrophoretically purified tubulin stimulate colchicine-binding activity.

Conformational Adaptability in Enzymes

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