Molecular Weight of Human Gamma Interferon Is Similar to that of Other Human Interferons

Yip, Y. K.; Barrowclough, Barbara S.; Urban, Carl; Vilček, Ján

doi:10.1126/science.6173921

Cited by 99 publications

(23 citation statements)

References 33 publications

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“…Another factor that contributed to the successful purification of IFN-y described in this paper was the recent demonstration that the biological activity of human IFN-y was not completely destroyed by treatment with NaDodSO4 (8). Residual biological activity after NaDodSO4 treatment could thus be used as a marker for the identification of IFN-y proteins under conditions precluding noncovalent interactions among protein molecules.…”

mentioning

confidence: 88%

Purification of two subspecies of human gamma (immune) interferon.

Yip¹,

Barrowclough²,

Urban³

et al. 1982

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

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Interferon (IFN)-y was produced in cultures of human leukocytes by combined stimulation with 12-0-tetradecanoylphorbol 13-acetate (TPA) and phytohemagglutinin (PHA).IFN-ywas purified by sequential adsorption and elution from controlled-pore glass and concanavalin A-Sepharose and by subsequent adsorptive removal of contaminating proteins on DEAESephacel at pH 8.0. Treatment of such partially purified IFN-y preparations with the anionic detergent NaDodSO4 (0.1% at 20-250C) decreased biological activity to approximately 5-20%.When analyzed by NaDodSO4polyacrylamide gel electrophoresis the bulk of IFN activity not destroyed by NaDodSO4 treatment was recovered from two peaks with apparent molecular weights of 20,000 and 25,000. The two activity peaks showed close correspondence with Coomassie blue-stained bands regularly demonstrable in purified supernatants from induced cultures but absent from culture supernatants from uninduced cells. Available evidence suggests that the two bands, isolated in pure form, represent subspecies of IFN-y. Native IFN-y was found to have a lower affinity for alkyl agarose columns than human IFN-a or IFN-fi did, suggesting that IFN-y is a relatively hydrophilic protein. Sulfhydryl-specific binding of native IFN-y to an Affi-Gel 501 column suggested that this IFN contains free sulfhydryl.Until recently virtually no information was available about physicochemical properties ofthe interferon (IFN) species produced in human lymphocytes upon stimulation with specific antigens (1) or with various other mitogens (2, 3). The identification of mitogen-induced IFN as IFN-y and its differentiation from the other two major species, termed IFN-a and IFN-/3 (4), is based mainly on antigenic differences among the three IFN species and on the known relative instability of IFN-y at pH 2 (3).The scarcity of information about IFN-y was mainly due to the lack of suitable methods for its production and purification. Recently, we reported a method of human IFN-y production based on combined stimulation of human lymphocytes with a phorbol ester (12-O-tetradecanoylphorbol 13-acetate, TPA) and the T cell mitogen phytohemagglutinin (PHA) (5, 6). This method enables the production of IFN-y preparations with relatively high initial specific activity. The use of lymphocyte-rich plateletpheresis residues as the cell source for production of IFN-y makes it possible to prepare batches yielding several liters of starting material for purification. We showed earlier that a relatively simple three-step purification procedure led to approximately a 1,000-fold purification of IFN from this starting material (7).Another factor that contributed to the successful purification of IFN-y described in this paper was the recent demonstration that the biological activity of human IFN-y was not completely destroyed by treatment with NaDodSO4 (8). Residual biological activity after NaDodSO4 treatment could thus be used as a marker for the identification of IFN-y proteins under conditions precluding noncovalent interactions am...

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mentioning

confidence: 88%

Purification of two subspecies of human gamma (immune) interferon.

Yip¹,

Barrowclough²,

Urban³

et al. 1982

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

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“…Purified recombinant interferon alpha A was the immunogen for preparation of the antibody to interferon alpha. After a three-step purification process (21), the interferon gamma used to prepare the latter antibody was virtually free of interleukin 1 Phenotyping of PBMC. No patient's PBMC spontaneously produced interferon gamma at either bleed.…”

Section: Methodsmentioning

confidence: 99%

“…were kindly provided by Sydney Pestka (Nutley, NJ) and Jan Vilcek (New York), respectively (20,21). Purified recombinant interferon alpha A was the immunogen for preparation of the antibody to interferon alpha.…”

Section: Methodsmentioning

confidence: 99%

Evolution of recurrent herpes simplex lesions. An immunohistologic study.

Cunningham¹,

Turner²,

Miller³

et al. 1985

J. Clin. Invest.

224

162

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“…Indeed, it has been shown that IFN-y molecule produced by mitogen-stimulated T lymphocytes undergoes glycosylation (26), and a number of investigators have shown that mouse IFN-y derived from normal spleen cells stimulated with mitogens displays a broad charge heterogeneity and different degrees of glycosylation and hydrophobicity (26,30,31). Similar findings have also been reported for the human IFN-y by Yip et al (10,32). In particular, they found that IFN--y activity recovered from a partially purified preparation of human IFN-y run on NaDodSO4/PAGE corresponded to two bands with molecular weights of 21 and 25 kDa.…”

Section: Discussionmentioning

confidence: 56%

Monoclonal antibodies against murine gamma interferon.

Prat¹,

Gribaudo²,

Comoglio³

et al. 1984

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

136

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Monoclonal antibodies against murine immune interferon (IFN-y) were produced by fusing the murine nonsecreting myeloma cell line P3.X63.Ag8.653 with spleen cells from rats immunized with IFN-y-containing supernatants obtained by stimulating a T-cell lymphoma, L12-R4, with phorbol 12-myristate 13-acetate. Supernatants from a twicecloned hybridoma were found to neutralize and to adsorb in depletion experiments up to 27 units of mouse IFN-y but not equivalent amounts of mouse leukocyte or fibroblast IFNs. The AN-18.17.24 monoclonal antibody neutralized to the same extent mouse IFN-y from different sourcesnamely, (i) concanavalin A-stimulated spleen cells, (ii) alloantigen-stimulated spleen cells, and (iii) monkey fibroblasts transfected with the cloned gene of murine IFN-y. Moreover, the monoclonal antibody displayed species specificity, since it did not neutralize IFN-y of human origin. Binding inhibition experiments with murine IFN-y preparations exposed to enzymatic or physicochemical degradation demonstrated that the protein moiety and not the carbohydrate residues were responsible for the binding to the Activation of T lymphocytes by alloantigens (1, 2), mitogens (3), and conventional antigens (4) induces the release of an interferon (IFN) type classified as immune (IFN-y) that acts on a variety of somatic and immunorelated target cells in a genetically unrestricted manner (5). Some in vitro and in vivo studies indicate that IFN-y has a greater antiproliferative effect on neoplastic cells than do leukocyte and fibroblast IFNs (IFN-a and IFN-p, respectively) (6).Recently, genes of both human and murine IFN-y have been successfully cloned by recombinant DNA technology. The results of these experiments show that the molecular weights of human and mouse IFN-'y are 17,100 and 15,900, respectively (7-9). A limit of this kind of technology, however, is that it does not take into account the possible posttranslational modifications of the synthesized molecules. In fact, as far as IFN-y is concerned, discrepancies on molecular weights have been reported in studies using conventional physicochemical techniques for purification (10) as opposed to those using recombinant DNA technology (7-9).Monoclonal antibodies have proved to be valuable reagents for the unambiguous identification, quantitative analysis, and large-scale purification of a variety of proteins (11). Whereas different monoclonal antibodies have already been produced and successfully used for the purification and biological characterization of IFN-a and -,/ (12-15), few and conflicting reports exist in the case of monoclonal antibodies against human 16).In previous studies we established in vitro a murine T-cell lymphoma line producing, upon stimulation with phorbol 12-myristate 13-acetate (PMA), huge amounts of homogeneous IFN-y apparently not contaminated by other known lymphokines (17, 18). Taking advantage of this system, we now have produced a rat monoclonal antibody recognizing murine IFN-y but not murine IFN-a or -,3. MATERIALS AND METHODSIm...

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Molecular Weight of Human Gamma Interferon Is Similar to that of Other Human Interferons

Cited by 99 publications

References 33 publications

Purification of two subspecies of human gamma (immune) interferon.

Purification of two subspecies of human gamma (immune) interferon.

Evolution of recurrent herpes simplex lesions. An immunohistologic study.

Monoclonal antibodies against murine gamma interferon.

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