Transblot identification of biotin-containing proteins in rat liver

Peroxidase-conjugated avidin was used to detect biotin-containing carboxylases in rat liver. By a transblot method, avidin-peroxidase interacted with liver proteins with estimated molecular masses of 120 and 74 kDa. The proteins were identified as pyruvate carboxylase (120 kDa, 6.4 pI) and methylcrotonyl-CoA carboxylase (74 kDa, 7.2 pI) by two-dimensional gel electrophoresis and transblot method. An additional band with estimated molecular mass of 220 kDa was detected in the cytosol fraction of rat liver, compatible with acetyl-CoA carboxylase. Rat liver proteins were prepared and treated with avidin and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transblot with avidin-peroxidase. A 190-kDa band was found with a parallel decrease in the 120-kDa band determined by Coomassie blue staining; however, these proteins did not stain by the transblot avidin-peroxidase method. When the transblot of parallel proteins was incubated with biotin and subsequently with avidin-peroxidase, two additional bands, namely 190 and 145 kDa, were detected while the 74-kDa band disappeared correlated with decreased staining of the 120-kDa band. The present procedure is a simple, rapid, and inexpensive method for detecting biotin-containing proteins in various tissues and organs and in determining the occurrence of nonspecific staining with the avidin-biotin complex method of immunoblot.     

Identification of pyruvate carboxylase in 3T3-L1 cells by transblot and its correlation with differentiation into adipocytes.

The 3T3-L1 preadipocytes treated with insulin, dexamethasone and 3-methyl-1-isobutylxanthine (IBMX) two days before reaching monolayer undergo differentiation into adipocytes. Cell lysates were prepared from these cells under various conditions and analyzed by SDS-PAGE and transblot. Peroxidase-conjugated avidin used to detect endogenous proteins interacted strongly with a protein with an estimated molecular weight of 120 kDa, corresponding to pyruvate carboxylase, in the differentiated 3T3-L1 cells. On the other hand, this protein was not detected in undifferentiated 3T3-L1 cells.     

Multiple biotin-containing proteins in 3T3-L1 cells.

Extracts of 3T3-L1 cells prepared after labelling the monolayer cultures with [3H]biotin contained numerous protein bands that were detected by fluorography of dried SDS/polyacrylamide electrophoresis gels. All labelled proteins in the extracts could be removed by avidin affinity chromatography. The biotin-containing subunits of acetyl-CoA carboxylase, pyruvate carboxylase, methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase, with molecular masses of approx. 220, 120, 75 and 72 kDa respectively, were detected together with minor bands at 100, 85 and 37 kDa that did not appear to be partial degradation products. Additional labelled bands increased in amount during incubation of cell extracts or did not occur in extracts prepared with trichloroacetic acid, 9.5 M-urea or proteolytic inhibitors, and were tentatively classified as partial degradation products. The unknown bands were not removed by incubation of cell monolayers for 24 h, a treatment that gave degradation rate constants of 0.47 day-1 for acetyl-CoA carboxylase and 0.28 day-1 for pyruvate carboxylase. Upon two-dimensional electrophoresis, pyruvate carboxylase, methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase had isoelectric points of 6.4, 7.2 and 6.4 respectively. Several additional discrete spots with isoelectric points below 6.2 were also present. All the unknown biotin-containing proteins banded with intact mitochondria during density-gradient centrifugation. We conclude that several unknown biotin-containing proteins are present in the mitochondria of 3T3-L1 cells, whereas others are partial breakdown products of mitochondrial proteolysis.     

Characterization of a murine monoclonal antibody that detects a C-terminal fragment of the raf oncogene product

A murine mAb, STEGI 1, was generated against a 30-kDa raf protein purified from an Escherichia coli expression vector. Immunoblot analysis confirmed that this antibody recognized the original immunizing protein as well as a 44- to 48-kDa protein from several raf-transformed cell lines. Immunoprecipitation experiments isolated a 48-kDa protein from a cell line transfected with a c-raf construct as well as from normal NIH 3T3 fibroblasts. Parallel experiments with polyvalent antiserum prepared against E. coli-derived v-raf (C terminus)-precipitated proteins with apparent Mr of 48 and 74 kDa, as had been described previously. Immunofluorescence flow cytometry of raf-transformed cell lines revealed intense intracytoplasmic staining. This staining was specifically inhibited by preincubation of STEGI 1 with purified raf 30-kDa protein. It should now be possible to more easily assess the role of the raf oncogene product in malignant transformation.     

Cleavage of nucleolin and argyrophilic nucleolar organizer region associated proteins in apoptosis-induced cells

To investigate the behavior of nuclear proteins in apoptotic cells, we examined the changes in nucleolin and proteins of the nucleolar organizing region during apoptosis in human osteoblastic cell lines, Saos-2 and MG63. Apoptosis was induced by treatment of these cells with okadaic acid. Proteins prepared from apoptotic cells were subjected to Western blot analysis and a modified Western blot method using silver nitrate. The anti-nucleolin antibody recognized the 110-kDa band and the staining intensity of this band decreased in the proteins prepared from the okadaic acid-treated apoptotic cells. The additional band of an 80-kDa was also detected in the proteins prepared from the apoptotic cells. Two major silver nitrate-stained bands, 110-kDa and 37-kDa, were detected among the proteins obtained from control cells. Like the Western blot analysis, the intensity of the 110-kDa silver nitrate-staining band decreased; an 80-kDa band appeared and its staining intensity increased in the lysate from the okadaic acid-treated cells. The signal intensity of the 37-kDa protein did not change in the sample from the apoptotic cells. In a cell-free apoptotic system, the 80-kDa protein was also detected and the amount of the 110-kDa protein decreased in the extract of Saos-2 cell nuclei incubated with apoptotic cytosol. The change in nucleolin in Saos-2 cells induced to undergo apoptosis was examined by an immunocytochemical procedure using the anti-nucleolin antibody and Hoechst 33342. Nucleolin was visible as dots in nucleoli in the control cells; however, it was not detected in the cells undergoing apoptosis. The dual-exposure view of Hoechst 33342 and anti-nucleolin staining cells confirmed that nucleolin had disappeared from the apoptotic nuclei of Saos-2.     

Identification of antigen in rat spermatogenic cells interacting with an anti-human sperm monoclonal antibody

A monoclonal antibody (MAb) raised against human sperm protein, designated YWK-II, was used to determine the distribution of antigens in rat spermatozoa and rat testicular germ cells. By an indirect immunofluorescent method, the antibody localized over the rat spermatozoal head, except for the postacrosomal region. In paraffin sections of adult and immature rat testis, germ cells, at every developmental stage, and Sertoli cells stained, while interstitial cells and peritubular myoid cells remained unstained. When cocultures of Sertoli and germ cells were tested, only the germ cells stained intensely. Sertoli cells and peritubular myoid cells in cultures did not stain. In the epididymal sections, strong staining occurred with spermatozoa in the lumen and epididymal epithelial cells, with moderate staining in the myoid layers of epididymis. To determine the sperm antigen interacting with the YWK-II antibody, rat spermatozoa proteins were prepared and analyzed by an immunoblot technique. The monoclonal antibody interacted with a single protein, with an estimated molecular weight of 115,000, present in the cauda epididymal spermatozoa. Among the proteins of the caput epididymal spermatozoa, however, the antibody interacted with a major and a minor band with molecular weights of 115,000 and 88,000, respectively. On the other hand, with proteins prepared from the membrane fraction of adult and immature rat testis, the antibody reacted with two bands with estimated molecular weights of 88,000 and 115,000. In the lysate prepared from germ cells dissociated from Sertoli-germ cell cocultures, the antibody recognized only the 88,000 protein. The present results show that the YWK-II MAb interacts with two proteins with different molecular weights. The amount of the interacting proteins in spermatozoa varied with their location within the epididymis.     

Asb6, an adipocyte-specific ankyrin and SOCS box protein, interacts with APS to enable recruitment of elongins B and C to the insulin receptor signaling complex

The APS adapter protein plays a pivotal role in coupling the insulin receptor to CAP and c-Cbl in the phosphatidylinositol 3-kinase-independent pathway of insulin-stimulated glucose transport. Yeast two-hybrid screening of a 3T3-L1 adipocyte library using APS as a bait identified a 418-amino acid ankyrin and SOCS (suppressor of cytokine signaling) box protein Asb6 as an interactor. Asb6 is an orphan member of a larger family of Asb proteins that are ubiquitously expressed. However, Asb6 expression appears to be restricted to adipose tissue. Asb6 was specifically expressed in 3T3-L1 adipocytes as a 50-kDa protein but not in fibroblasts. In Chinese hamster ovary-insulin receptor (CHO-IR) cells Myc epitope-tagged APS interacted constitutively with FLAG-tagged Asb6 in the presence or absence of insulin stimulation and insulin stimulation did not alter the interaction. In 3T3-L1 adipocytes, insulin receptor activation was accompanied by the APS-dependent recruitment of Asb6. Asb6 did not appear to undergo tyrosine phosphorylation. Immunofluorescence and confocal microscopy studies revealed that Asb6 colocalized with APS in CHO cells and in 3T3-L1 adipocytes. In immunoprecipitation studies in CHO cells or 3T3-L1 adipocytes, the Elongin BC complex was found to be bound to Asb6, and activation of the insulin receptor was required to facilitate Asb6 recruitment along with Elongins B/C. Prolonged insulin stimulation resulted in the degradation of APS when Asb6 was co-expressed but not in the absence of Asb6. We conclude that Asb6 functions to regulate components of the insulin signaling pathway in adipocytes by facilitating degradation by the APS-dependent recruitment of Asb6 and Elongins BC.     

Antigenic determinant and interspecies cross-reactivity of a monoclonal antibody to poly(ADP-ribose) synthetase

A monoclonal antibody (1F4) was prepared against calf thymus poly(ADP-ribose) synthetase. It was classified as IgG1/kappa and its antigenic determinant was localized on the 46 kDa portion of the enzyme molecule which contains the site for the binding of DNA. When calf thymus DNA-binding proteins were subjected to immunostaining after electrophoresis and transblotting to a nitrocellulose filter, the native enzyme (120 kDa) and its endogenous degradation products (80, 64 and 32 kDa) were detected. When the interspecies cross-reactivity was examined using DNA-binding proteins from 6 different sources, 1F4 reacted with the 120- and 32-kDa protein bands in HeLa cells, mouse testis and chicken liver as in the case of calf thymus. These results indicate that the antigenic structures of poly(ADP-ribose) synthetase and its degradation products are highly conserved in various animal cells.     

Crossreaction with an anti-Bax antibody reveals novel multi-endocrine cellular antigen.

We found a novel protein that has crossreactivity with a polyclonal anti-Bax antibody (SCBAX antibody). The protein was localized exclusively in the endocrine cells of hypothalamus, pituitary gland, and pancreatic islets. Immunohistochemical (IHC) double labeling revealed that the cells showing crossreactivity with this antibody corresponded precisely to oxytocin neurons and ACTH, alpha-MSH, and glucagon cells in rat and gerbil. By immunoelectron microscopy, the protein was localized predominantly in and just around the secretory granules in the cytoplasm but not in the mitochondria. Double-labeling IHC with the anti-Bax SCBAX antibody and two anti-Bax monoclonal antibodies (MAbs) showed that cells stained with the anti-Bax SCBAX antibody were not stained with anti-Bax MAbs except for very few cells (probably apoptotic cells). Western blotting analysis revealed that the molecular mass of the protein was approximately 55 kD, which differs from that of Bax protein (21 kD). These findings indicate that the anti-Bax SCBAX antibody recognizes not only pro-apoptotic Bax protein (a 21-kD mitochondrial protein) but also an unknown substance present in one endocrine cell group in each endocrine organ. Therefore, the protein is designated as multi-endocrine cellular antigen (MECA). MECA is probably a 55-kD protein secreted from the particular differentiated cell groups of endocrine tissues.     

Biotin carboxylases in mitochondria and the cytosol from skeletal and cardiac muscle as detected by avidin binding

Biotin carboxylases in mammalian cells are regulatory enzymes in lipogenesis and gluconeogenesis. In this study, endogenous biotin in skeletal and cardiac muscle was detected using avidin conjugated with alkaline phosphatase and applied in high concentrations to muscle sections. The avidin binding was subsequently visualized by histochemical demonstration of the alkaline phosphatase activity. All cardiac muscle cells showed high affinity for avidin with only the nuclei and the intercalated discs remaining unstained. In skeletal muscle a diffuse reaction could be detected in the sarcoplasm of the muscle fibres. A granular reaction was noted in the same fibres that showed activity for succinic dehydrogenase. The specificity of the coloured reaction product in the muscle sections was investigated and is suggested to be caused by avidin binding to biotin moieties in mitochondria and the cytosol. Mitochondrial and cytosolic preparations of skeletal muscle were electrophoresed in sodium dodecyl sulphate gels. After blotting and incubation with conjugated avidin, two bands with molecular weights of 75 kDa and 130 kDa respectively were evident in the mitochondrial preparation. It is suggested that the 75-kDa band represents comigration of the biotin-containing subunits of propionyl-CoA carboxylase and methylcrotonyl-CoA carboxylase. The 130-kDa band may represent the biotin-containing pyruvate carboxylase. In the cytosolic preparation a 270-kDa band was stained in blots that had been incubated with conjugated avidin; this band is suggested to represent acetyl-CoA carboxylase. A 190-kDa cytosolic band might be a cleavage product of acetyl-CoA carboxylase. We propose that using alkaline phosphatase-conjugated avidin it is possible to detect the mitochondrial and cytosolic biotin-dependent carboxylases in striated muscle.     

Localization of Endogenous Biotin-Containing Proteins in Mouse Bergmann Glial Cells

A peroxidase-conjugated avidin–biotin complex was used to detect endogenous biotin-containing proteins in mouse cerebellum. By this method, Bergmann glial cells were found to be strongly labelled in the adult mouse cerebellum. Developmentally, cells in the granular layer, probably astrocytes, appeared to be labelled around postnatal 10-day (P10). Their labelling decreased after P20, although the positive-labelling remained in the Bergmann glial cells up to the adult stage. The findings were confirmed by using a Alexa Fluor 488-conjugated streptavidin technique. The labelling was not affected by routine hydrogen peroxide treatment, but it was eliminated by avidin–biotin blocking. By another transblot method, the reactive proteins in the mouse cerebellum were found to be 120?kDa (the strongest one) and 75?kDa. For electron microscopy, a gold-conjugated anti-biotin antibody was immunoreacted to the mitochondria of Bergmann glial cells. These results suggest that endogenous biotin-containing proteins are abundant in the Bergmann glial cells. Therefore, the avidin–biotin complex method is useful for detecting Bergmann glial cells, probably because of the difference of biotin metabolism in the cerebellar glial cells.     

Disruption of Ha_BtR alters binding of Bacillus thuringiensis delta-endotoxin Cry1Ac to midgut BBMVs of Helicoverpa armigera

Disruption of the Ha_BtR (a cadherin gene) is genetically linked to resistance to Cry1Ac delta-endotoxin of Bacillus thuringiensis in the GYBT strain of Helicoverpa armigera. Brush border membrane vesicles (BBMVs) prepared from midguts of both the Cry1Ac-resistant GYBT strain (homozygous for a deletion knockout of Ha_BtR) and the susceptible GY strain (homozygous for the wild type of Ha_BtR) possessed saturable and specific binding ability to (125)I-Cry1Ac. The binding constant (K(d)) of the GY strain was significantly lower than that of the resistant GYBT strain, whereas their binding site concentrations (B(max)) were similar. When midgut BBMVs were reacted directly with streptavidin conjugated to horseradish peroxidase, the GY strain had very clear 120- and 85-kDa protein bands, which indicated that the 120- and 85-kDa bands are endogenous biotin-containing proteins. However, the GYBT strain almost completely lost these two biotin-containing proteins. Ligand blotting with biotinylated Cry1Ac toxin showed midgut BBMVs of the GY strain contain five protein bands of 210-, 190-, 150-, 120-, and 85-kDa, respectively, while BBMVs of the GYBT strain contain only two protein bands of 150- and 120-kDa. 120-kDa bands may consist of two proteins with coincidentally the same molecular weight (putatively, an APN and a biotin-containing protein). Our results showed that the binding pattern of Cry1Ac to midgut BBMVs of H. armigera was altered quantitatively and qualitatively by knockout of Ha_BtR. There are multiple Cry1Ac-binding proteins in the midgut of susceptible H. armigera, but only the Ha_BtR can be considered as a putative functional receptor of Cry1Ac. Possible involvement of other receptor proteins in the intoxication process in vivo could not be excluded.     

Cellular and subcellular distribution of PBP72/74, a peptide-binding protein that plays a role in antigen processing

A 72/74-kDa peptide binding protein (PBP72/74) was previously described which plays a role in the processing and/or presentation of Ag, possibly by facilitating the association of processed Ag with the MHC class II molecules. PBP72/74 was recently shown to be related to the 70-kDa family of heat shock proteins (hsp70), whose members show the general characteristic of binding to denatured or inappropriately folded proteins. Here we describe the cellular and subcellular distribution of PBP72/74. By flow cytometry with PBP72/74-specific rabbit antisera, PBP72/74 is detected on the surfaces of mouse Ig+ B cells and MAC-1+ macrophages. PBP72/74 74 was not detected on the surfaces of Thy-1+ T cells or NK1.1+ NK cells. The cell surface expression of PBP72/74 does not require MHC class II expression. Indeed, the Ia- variant B cell lymphoma cell line, M12.C3, expresses PBP72/74 at levels equivalent to that of the Ia+ parent cell line, M12.4.1, from which it was derived. Furthermore, the fibroblast L cell line, DAP.3, shows no cell surface expression of PBP72/74, nor do DAP.3 lines transfected with and expressing genes encoding the alpha- and beta-chain of the I-Ad and I-Ed molecules. Moreover, treatment of B cells with either IL-4 or LPS, which increases Ia expression severalfold, does not affect PBP72/74 expression. Thus, PBP72/74 cell surface expression appears to be a property of B cells and macrophages, independent of Ia expression. In addition, the B cell surface expression of PBP72/74 is not altered by stress in the form of heat shock. Thus, PBP72/74 appears to be a constitutive noninducible member of the hsp70 family. By immunoelectron microscopy, PBP72/74 is detected in approximately 36% of early endocytic vesicles into which surface Ig is internalized after binding to anti-Ig antibodies. This compartment was previously shown to contain class II en route to the cell surface associated with invariant chain and the proteases cathepsin B and D and is suggested to be a subcellular site of antigen processing. PBP72/74 is also found associated with the plasma membrane, endoplasmic reticulum, and membranes proximal to the Golgi stacks. The cellular and subcellular distribution of PBP72/74 is consistent with its playing a role in the processing of presentation of Ag with the MHC class II molecules.     

Cross-linking of a polyomavirus attachment protein to its mouse kidney cell receptor.

We used photoaffinity cross-linking with the heterobifunctional cross-linker N-hydroxysuccinimidyl 4-azidobenzoate (HSAB) to covalently link polyomavirus to a mouse kidney cell surface component. The virus-HSAB combination was adsorbed to the cells and then cross-linked and isolated in monopinocytotic vesicles from the cells after endocytosis. The cross-linked product was identified on sodium dodecyl sulfate-polyacrylamide gels by the presence of a new band carrying 125I-labeled virion protein with a higher molecular mass than the normal virion protein bands. A single new band, with an apparent molecular mass of 120 kilodaltons (120 kDa), was identified by this procedure. This band was formed only in the presence of the HSAB cross-linker when virions were bound to the cells. The band also copurified with cross-linked virions when virion-containing vesicles were treated with detergent to remove the cell membrane. Antibody treatments that blocked up to 100% of virus binding and internalization also blocked cross-linking, as measured by the formation of the 120-kDa band. The 120-kDa band was characterized by preparation of antibody against the excised band from the gel. This antibody was shown to have the expected dual specificity for polyomavirus VP1 sequences and plasma membrane proteins, as analyzed on Western blots. The anti-120-kDa antibody was also shown by immunofluorescence to bind to the surface of mouse kidney cells. These data have demonstrated that molecules of possible biological significance in the binding of polyomavirus to mouse kidney cells have been cross-linked and that cell surface molecules have been identified that may be characterized further for possible receptor function in polyomavirus attachment.     

Reversible cAMP-induced translocation of cytoskeleton-associated 300- to 350-kDa proteins from nucleus to cytoplasm

We previously reported that treatment of SV-3Y1 cells in an exponential growth state with 1 mM db-cAMP plus 1 mM theophylline induced reversible disappearance of nuclear dots stained by monoclonal anti-microtubule-associated protein (MAP)-1 antibody [T. Nakayama, K. Nishizawa, G. Kimura, and C. Sato (1986) Exp. Cell Res. 163, 246]. In the present study, we examined the relation between the intracellular localization and phosphorylation of 300- to 350-kDa proteins that are intracellular antigens for our anti-MAP-1 and -2 antibodies. Treatment with 1 mM db-cAMP plus 1 mM theophylline was found to result in a reversible decrease in immunofluorescent staining of the nucleus with polyclonal MAP-1 or -2 antibody, and a reversible increase in that of the cytoplasm. Simultaneous treatment with 2.5 microM colchicine, 2.5 microM colcemid, 20 microM putrescine, or 3 mM alpha-naphthyl phosphate in the presence of db-cAMP plus theophylline almost prevented this effect of db-cAMP plus theophylline. We examined the cytoplasmic and nuclear fractions by immunoperoxidase staining, immunoprecipitation, and 125I-protein A with anti-MAP-1 and -2 antibodies. Treatment with db-cAMP plus theophylline resulted in the increase of 300- to 350-kDa proteins in the cytoplasm and a decrease in the nucleus. This treatment also caused the dephosphorylation of 300- to 350-kDa proteins. The present research indicated that treatment with db-cAMP plus theophylline resulted in the reversible translocation of 300- to 350-kDa proteins from the nucleus to the cytoplasm accompanied by the dephosphorylation of these proteins.     

The cellular proteins which can associate specifically with polyomavirus middle T antigen in human 293 cells include the major human 70-kilodalton heat shock proteins.

We compared the proteins which associate with middle T antigen (MT) of polyomavirus in human cells infected with Ad5(pymT), a recombinant adenovirus which directs the overexpression of MT, with the MT-associated proteins (MTAPs) previously identified in murine fibroblasts expressing MT. MTAPs of 27, 29, 36, and 63 kilodaltons (kDa) appeared to be fairly well conserved between the two species, as judged by comigration on two-dimensional gels. Several 61-kDa MTAP species detected in MT immunoprecipitates from both cell sources also comigrated on these gels. However, no protein comigrating precisely with the murine 85-kDa MTAP could be detected in the human cells. Furthermore, two proteins of 72 and 74 kDa associated with wild-type MT in the infected human cells but not in murine fibroblasts expressing MT. It had been previously reported for murine cells that the 70-kDa heat shock protein associates with a particular mutant MT but not with wild-type MT (G. Walter, A. Carbone, and W.J. Welch, J. Virol. 61:405-410, 1987). By the criteria of comigration on two-dimensional gels, tryptic peptide mapping, and immunoblotting, we showed that the 72- and 74-kDa proteins that associate with wild-type MT in human cells are the major human 70-kDa heat shock proteins.     

Biochemical characterization and biosynthesis of the Ki-1 antigen in Hodgkin-derived and virus-transformed human B and T lymphoid cell lines

The Hodgkin-associated Ki-1 antigen was analyzed in different cell lines. In Hodgkin analogous L428 cells, biosynthetically labeled with radioactive amino acids, the Ki-1 antibody precipitated three glycoproteins with 90, 105, and 120 kDa, respectively. Surface-labeling revealed that the two larger components were membrane-associated forms of the Ki-1 antigen, although the 90-kDa molecule was shown in pulse-chase experiments to be the precursor of the 105- and 120-kDa forms. All three forms of the Ki-1 antigen possess a tunicamycin-sensitive 6-kDa N-linked carbohydrate moiety. O-Linked oligosaccharides could not be detected. Thus, the differences in m.w. are probably not due to glycosylation. The ionophore monensin prevented the appearance of the membrane-associated molecules, which demonstrated that they are assembled between the transcompartment of the Golgi complex and their insertion into the cell membrane. The 90-kDa precursor molecule cannot be generated by disulfide reduction from the two larger forms. After internal labeling with P-32, only the 105- and 120-kDa bands became visible, indicating that the Ki-1 molecule is phosphorylated after its processing into the two larger membrane-associated forms. Analysis of the Ki-1 antigens from other cell lines demonstrated that after external labeling of two other Hodgkin-derived cell lines, six Epstein-Barr virus lymphoblastoid cell lines and one human T leukemia virus I-positive T cell line, both the 105- and the 120-kDa membrane molecules could be detected, regardless of the presence or type of virus integrated.     

Involvement of a non-hormone-binding 90-kilodalton protein in the nontransformed 8S form of the rabbit uterus progesterone receptor

Nontransformed 8S progesterone receptor (8S-PR) was purified by hormone-specific affinity chromatography from rabbit uterine low-salt cytosol containing 20 mM molybdate. In the eluate obtained with radioactive progestin, sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) showed the presence of several bands, including three that corresponded to approximately 90-, approximately 120-, and approximately 85-kDa proteins. None of these three proteins was found in the eluate of the affinity column when the molybdate-containing cytosol was chromatographed in the presence of nonradioactive progesterone ("mock purification"). Subsequent purification of the affinity eluate by DEAE-Sephacel chromatography gave a single radioactive receptor peak at 0.15 M KCl (approximately 20% yield, 19% purity on the basis of one binding site per approximately 100 kDa) with a sedimentation coefficient of 8.5 S. Silver staining after SDS-PAGE revealed that this purified 8S-PR fraction contained mainly the 120-, 90-, and 85-kDa proteins. [3H]R5020-labeled 8S-PR purified by DEAE-Sephacel column chromatography was UV irradiated, and after SDS-PAGE the 120- and 85-kDa proteins were revealed, but the 90-kDa protein was not.(ABSTRACT TRUNCATED AT 250 WORDS)