Biomedical subjects

G H Perdew

Publications and source records attributed to G H Perdew.

At least 55 records · Page 3Linked to original sources

PubMed1993

Localization and characterization of the 86- and 84-kDa heat shock proteins in Hepa 1c1c7 cells.

The 90-kDa heat shock protein (hsp90) is present in cells at high levels in the cytoplasm and is composed of two separate gene products, hsp86 and hsp84. Rabbit polyclonal antibodies to the murine N-terminal sequences of the 86- and 84-kDa heat shock proteins were isolated from serum by peptide affinity chromatography. Antibodies against each form of hsp90 are capable of immunoprecipitating hsp90. Each antibody preparation is specific against either hsp86 or hsp84 when tested on a protein blot of Hepa 1c1c7 cytosol. The over-all ratio of hsp84/hsp86 in Hepa 1 cytosol was estimated to be 2 to 1. Each antibody preparation was used to immunoprecipitate hsp84 or hsp86 from Hepa 1 cytosol to test whether hsp86/84 exists as a homo- and/or heterodimer. After electrophoresis, silver staining revealed that anti-hsp86 antibodies immunoprecipitated both hsp86 and hsp84. This result would suggest that hsp86 forms heterodimers with hsp84. In contrast, the anti-hsp84 antibodies immunoprecipitated almost entirely hsp84, suggesting that hsp84 exists largely as homodimers. Both anti-hsp86 and hsp84 antibodies were able to immunoprecipitate the 2-azido-3-[125I]iodo-7,8-dibromodibenzo-p-dixoin-labeled Ah receptor from Hepa 1 cytosol, indicating that these antibodies are able to bind to hsp90 when it is complexed with other proteins. Both antibody preparations recognize hsp90 in mouse, rat, and human cell lines. Immunofluorescence and confocal microscopy were performed using both antibody preparations, and the results indicated that both hsp86 and hsp84 were located in the cytoplasm and nucleus of Hepa 1 cells. Hsp86 was found to localize unevenly in the cytoplasm, while hsp84 was found evenly dispersed throughout the cytoplasm. Hsp86 also appeared to be localized to a greater degree than hsp84 in the vicinity of the nuclear envelope.

Amino Acid Sequence↗

PubMed1992

Reconstitution of the multiprotein complex of pp60src, hsp90, and p50 in a cell-free system.

A rabbit reticulocyte lysate system that has been used to reconstitute functional complexes between steroid receptors and the 90-kDa heat shock protein (hsp90) has been used here to form complexes between the pp60src tyrosine kinase and hsp90. Reticulocyte lysate forms complexes between hsp90 and a temperature-sensitive mutant of Rous sarcoma virus pp60v-src, which is normally present in cytosol virtually entirely in the multiprotein complex form. In addition, hsp90 in the lysate complexes with wild-type pp60v-src, of which only a small portion is normally recovered in cytosol in the native multiprotein complex, and with the cellular homolog, pp60c-src, which has never been recovered in cytosol in the form of a native multiprotein complex with hsp90. Moreover, the reticulocyte lysate-reconstituted complex also contains the 50-kDa phosphoprotein component of the native pp60v-src multiprotein complex. The native and reconstituted pp60src-hsp90 complexes have similar thermal stability and, like steroid receptor heterocomplexes, they are stabilized by molybdate. As previously shown with reticulocyte lysate-reconstituted steroid receptor heteroprotein complexes, the reconstituted pp60src multiprotein complex contains hsp70, which is a major candidate for providing the protein unfoldase activity required for hsp90 association.

Animals↗

PubMed1992

Chemical cross-linking of the cytosolic and nuclear forms of the Ah receptor in hepatoma cell line 1c1c7.

Both cytosolic and high salt nuclear extracts were isolated from Hepa 1c1c7 cells incubated with 2-azido-3[125I]iodo-7,8-dibromo-dibenzo-p-dioxin ([125I]N3Br2DpD). The [125I]N3Br2DpD-labeled cytosolic fraction was subjected to chemical cross-linking with dimethyl pimelimidate and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Chemical cross-linking of the cytosolic form of the AhR revealed monomeric (97 kDa), dimeric (185 kDa), trimeric (281 kDa), and tetrameric (327 kDa) complexes. In a time course of exposure to the cross-linking reagent, the largest form given above became the predominant AhR form observed in the cytosolic extracts. The 327 kDa cytosolic species apparently consists of a 97 kDa AhR, an approximately 88 kDa protein, an approximately 96 kDa protein, and an approximately 46 kDa protein. Nuclear extracts from [125I]N3Br2DpD-labeled Hepa 1c1c7 cells were applied to sucrose density gradients. The 6 S nuclear receptor peak fractions were pooled and subjected to chemical cross-linking. Analysis by SDS-PAGE revealed a monomeric (97 kDa) ligand binding protein and a dimeric (182 kDa) complex. This would suggest that the nuclear 6 S AhR consists of a 97 kDa AhR and an approximately 85 kDa protein. These findings would indicate that the AhR exists in cytosol as a tetrameric species, while in the nucleus the AhR exists as a heterodimer.

Carcinoma, Hepatocellular↗

PubMed1991

A 50-kDa cytosolic protein complexed with the 90-kDa heat shock protein (hsp90) is the same protein complexed with pp60v-src hsp90 in cells transformed by the Rous sarcoma virus.

We have previously shown that a 50-kDa protein is one component of a heteromeric complex immunoprecipitated by the 90-kDa heat shock protein (hsp90) monoclonal antibodies 8D3 and 3G3 (Perdew, G. H., and Whitelaw, M. L. (1991) J. Biol. Chem. 266, 6708-6713). In this report, we compare the 50-kDa protein with that found in pp60v-src-hsp90-p50 complexes immunoprecipitated from Rous sarcoma virus-transformed cells with antibodies to pp60v-src. 35S- and 32P-labeled p50 proteins from each system were identical in their mobilities by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis. The profile of N-chlorosuccinimide cleavage products derived from each 32P-labeled p50 protein were also identical when resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We have developed a mouse monoclonal antibody, 3M/1B5p50, capable of detecting p50 on Western blots. This antibody detected the 50-kDa protein which co-purified with the pa104 pp60v-src mutant of the avian sarcoma virus oncoprotein in 44A rat fibroblasts. We did not detect p50 in association with native glucocorticoid receptor in L cells or with the overexpressed glucocorticoid receptor in Chinese hamster ovary cells. Two experiments utilizing immunochemical staining implied that essentially all cytosolic p50 is associated with hsp90. Firstly, immunoprecipitating hsp90 from Hepa 1 cytosol with monoclonal antibody 3G3 left the cytosol depleted of p50. Secondly, cytosol fractionated by sucrose gradient revealed that p50 cosedimented with hsp90, confirming the existence of p50 only in association with hsp90.

Animals↗

PubMed1991

Retinoic acid receptor belongs to a subclass of nuclear receptors that do not form "docking" complexes with hsp90.

We have recently reported that, in contrast to the glucocorticoid receptor, the thyroid hormone receptor does not bind to hsp90 when the receptor is translated in rabbit reticulocyte lysate [Dalman, F. C., Koenig, R. J., Perdew, G. H., Massa, E., & Pratt, W. B. (1990) J. Biol. Chem. 265, 3615-3618]. All of the steroid receptors that are known to bind hsp90 are recovered in the cytosolic fraction when hormone-free cells are ruptured in hypotonic buffer. In contrast, unliganded thyroid hormone receptors and retinoic acid receptors are tightly associated with nuclear components. In this paper, we translated the human estrogen receptor and the human retinoic acid receptor in reticulocyte lysate and then immunoadsorbed the [35S]methionine-labeled translation products with the 8D3 monoclonal antibody against hsp90. The estrogen receptor is bound to hsp90, as indicated by coimmunoadsorption, but the retinoic acid receptor is not. Translation and immunoadsorption of chimeric proteins containing the DNA binding domain of one receptor and the N-terminal and COOH-terminal segments of the other show that the DNA binding finger region of the estrogen receptor is neither necessary nor sufficient for hsp90 binding. These observations suggest that there are two classes within the steroid receptor family. In one class (e.g., glucocorticoid, mineralocorticoid, sex hormone, and dioxin receptors), the receptors bind to hsp90 and remain in some kind of inactive "docking" mode until hormone-triggered release of hsp90 occurs. In the retinoic acid/thyroid hormone class, the unligated receptors do not bind to hsp90, and the receptors appear to proceed directly to their high-affinity nuclear acceptor sites without entering the "docking" state.

Animals↗

PubMed1991

Evidence that the 90-kDa heat shock protein (HSP90) exists in cytosol in heteromeric complexes containing HSP70 and three other proteins with Mr of 63,000, 56,000, and 50,000.

Monoclonal antibody (mAb) 8D3 and 3G3 are unique antibodies capable of precipitating both free 90-kDa heat shock protein (HSP90) and HSP90-protein complexes. Immunoprecipitation of [35S]methionine-labeled Hepa 1c1c7 cytosolic extracts were performed using mAb 8D3 or 3G3. The resulting immunoprecipitates can be dissociated from the mAb with a 500 mM NaCl wash. These washes were subjected to both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. Five major protein spots were detected in addition to HSP90 with the following relative molecular weights: 68,000, 63,000, 56,000, 50,000, and 188,000. On Western blots mAb 3G3 was capable of specifically binding to HSP90. Each of these proteins was localized on two-dimensional gels. Using one-dimensional gel electrophoresis and immunochemical localization on Western blots, the p68 spot was identified as HSP70, and the p56 spot was found to cross-react with polyclonal antibody JP-1 raised against a 59-kDa protein. This 59-kDa protein has been found previously to be associated with several steroid hormone receptors in rabbit uterine cytosol. Immunoprecipitation of [32P]orthophosphate-labeled Hepa 1c1c7 cytosol with mAb 8D3 or 3G3 revealed two major phosphorylated proteins with relative molecular weights of 90,000 and 50,000. The identities of p63 and p188 are currently unknown. This is the first report examining the major proteins that are complexed with HSP90 in mammalian cells.

Antibodies, Monoclonal↗

PubMed1991

Comparison of the nuclear and cytosolic forms of the Ah receptor from Hepa 1c1c7 cells: charge heterogeneity and ATP binding properties.

2-[125I]iodo-7,8-dibromo-p-dioxin ([125I]Br2DpD) and 2-[125I]iodo-3-azido-7,8-dibromo-p-dioxin ([125I]N3Br2-DpD) are both capable of binding to the Ah receptor (AhR) with a high degree of specificity in cultured Hepa 1c1c7 cells. After incubation with either [125I]N3Br2DpD or [125I]Br2DpD Hepa 1c1c7 cytosolic and high salt nuclear extracts were analyzed by sucrose density gradient analysis with the following results: (i) With both radioligands an approximately 9 S form of the AhR was observed in cytosolic extracts. (ii) Nuclear extracts labeled with [125I]N3Br2DpD revealed both approximately 6 S and approximately 9 S forms of the AhR. (iii) In contrast, analysis of nuclear extracts labeled with [125I]Br2DpD revealed only an approximately 6 S form of the AhR. The approximately 9 S [125I]N3Br2DpD-labeled AhR was preferentially extracted with 100 mM KCl from a nuclear fraction and mixed with monoclonal antibody 8D3, an anti-90-kDa heat shock protein antibody. Monoclonal antibody 8D3 was able to bind to the approximately 9 S nuclear form of the AhR and caused the receptor to sediment as a heavier complex on sucrose density gradients. This would indicate that the AhR can reside in the nucleus bound to 90-kDa heat shock protein. The [125I]N3Br2DpD-labeled approximately 6 S peak fractions were collected and subjected to denaturing two-dimensional gel electrophoresis. A comparison of [125I]N3Br2DpD-labeled cytosolic (9 S) AhR preparations with the nuclear (6 S) AhR by 2-D gel electrophoresis was performed. The cytosolic form of the AhR was present in the apparent pI range of 5.2-5.7; the nuclear form focused between 5.5 and 6.2. The [125I]N3Br2DpD-labeled nuclear extracts were incubated with ATP-agarose and 43% of the photoaffinity-labeled AhR bound to the affinity gel. In contrast, approximately threefold lower binding of [125I]N3Br2DpD-labeled receptor was obtained when GTP-, AMP-, or ADP-agarose was used. Only 2% of the [125I]N3Br2DpD-labeled cytosolic AhR was able to bind to ATP-agarose. These results suggest that after the AhR translocates into the nucleus the following biochemical changes occur: (i) The sedimentation value for the AhR changes from an approximately 9 S to an approximately 6 S species. (ii) The AhR attains the ability to bind with specificity to ATP. (iii) The AhR undergoes a shift to a more basic pI.

Adenosine Triphosphate↗

PubMed1991

Detection of the Ah receptor in rainbow trout: use of 2-azido-3-[125I]iodo-7,8-dibromodibenzo-p-dioxin in cell culture.

The Ah receptor was detected in RTG-2 cells (rainbow trout embryonic gonad cells) following the addition of the photoaffinity ligand, [125I]2-azido-3-iodo-7,8-dibromodibenzo-p-dioxin, to cells in culture. Cytosolic and nuclear extracts were prepared and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and revealed one radiolabeled band. Very little non-specific binding was observed under the conditions employed when compared to photoaffinity labeling RTG-2 cytosolic extracts in vitro. The photoaffinity-labeled Ah receptor in RTG-2 cytosol was analyzed by sucrose density centrifugation. The cytosolic form was observed to sediment at approximately 9.8S and the high salt nuclear extract form at approximately 7.5S. The relative molecular weight of the Ah receptor was determined to be 145 kDa under denaturing conditions and is considerably larger than the Ah receptor from mammalian sources. Inhibition of photoaffinity ligand binding to the RTG-2 cytosolic Ah receptor by competing ligands revealed the same rank order of ligand affinity as that previously demonstrated for the mouse Ah receptor.

Affinity Labels↗

PubMed1991

Production of Ah receptor ligands in rat fecal suspensions containing tryptophan or indole-3-carbinol.

An assay system was developed to test whether bacteria in the gastrointestinal tract are capable of metabolizing tryptophan to compounds that are able to bind to the aryl hydrocarbon (Ah) receptor. Tryptophan (1 mM) was added to feces diluted 1:1,000 in phosphate-buffered saline and incubated at 37 degrees C overnight. The suspensions were extracted with chloroform to obtain the hydrophobic compounds. To test for the presence of Ah receptor ligands, a competition binding assay using [2-125I]iodo-7, 8-dibromodibenzo-p-dioxin and Hepa 1c1c7 cytosol was employed; it was capable of detecting picogram levels of a competing ligand with similar affinity. Fecal suspensions in the presence of 1 mM tryptophan and oxygen are capable of producing greater than 60% inhibition of radioligand binding per 10 micrograms of feces. In contrast, oxygen-equilibrated fecal suspensions without tryptophan and argon-equilibrated fecal suspensions with tryptophan exhibited 10% inhibition of radioligand binding per 10 micrograms of feces in the competition binding assay. Other indolylic compounds and amino acids were similarly tested. Histidine, tyrosine, phenylalanine, glycine, indole-3-acetic acid, and tryptamine were all negative in this assay. Indole-3-carbinol was capable of forming compounds that bind to the Ah receptor under a variety of conditions: in fecal suspensions with or without oxygen, in 50 mM HCl for 80 minutes, and in neutral pH buffer overnight at 37 degrees C. Addition of oxygenated tryptophan-fecal incubation extracts to Hepa 1 and Hepa c4 mutant (defective Ah receptor) cell cultures resulted in the induction of ethoxyresorufin O-deethylase activity in Hepa 1 cells, but no induction was observed in Hepa c4 cells. These results suggest that bacteria in the gastrointestinal tract under the proper conditions are able to metabolize tryptophan to compounds that are Ah receptor ligands.

Animals↗

PubMed1990

Analysis of photoaffinity-labeled aryl hydrocarbon receptor heterogeneity by two-dimensional gel electrophoresis.

The level of charge heterogeneity in the aryl hydrocarbon receptor (AhR) was examined by high-resolution denaturing two-dimensional (2D) gel electrophoresis. Hepa 1c1c7 cell cytosolic fraction was photoaffinity-labeled with 2-azido-3-[125I]iodo-7,8-dibromodibenzo-p-dioxin and applied to isoelectric focusing (IEF) tube gels. After optimization of focusing conditions a broad peak of radioactivity was detected in the apparent pI range of 5.2-5.7. IEF tube gels were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by visualization of the radiolabeled AhR by autoradiography; three distinct isoforms were detected. The same 2D electrophoretic isoform pattern was obtained when the AhR from Hepa 1c1c7 was photoaffinity-labeled in cell culture. BPrCl cells, a mutant line derived from Hepa 1c1c7 cells, contain an AhR that is unable to bind to DNA. Photoaffinity-labeled BPrCl cytosolic fractions were subjected to 2D gel electrophoretic analysis resulting in essentially the same molecular weight and isoform pattern as seen in Hepa 1c1c7 cytosol. This result would suggest that if a mutation is present in the BPrCl AhR it has not caused a significant change in its IEF pattern, although a small shift in the pI values was observed. Two-dimensional gel electrophoresis of photoaffinity-labeled cytosolic fractions from HeLa cells, the rat liver tumor cell line McA-RH7777, and buffalo rat thymus revealed three isoforms, essentially the same isoform pattern as in Hepa 1c1c7 cells. This would indicate that despite the considerable molecular weight polymorphism between species the level of charge heterogeneity is highly conserved.

Affinity Labels↗

PubMed1990

In contrast to the glucocorticoid receptor, the thyroid hormone receptor is translated in the DNA binding state and is not associated with hsp90.

We have recently reported that the glucocorticoid receptor (GR) becomes bound to the 90-kDa heat shock protein (hsp90) at or near the end of receptor translation in vitro (Dalman, F. C., Bresnick, E. H., Patel, P. D., Perdew, G. H., Watson, S. J., Jr., and Pratt, W. B. (1989) J. Biol. Chem. 264, 19815-19821). In this paper we compare the hsp90 binding and DNA binding activities of the thyroid hormone receptor (TR) to those of the GR after cell-free translation of the two receptors in rabbit reticulocyte lysate. In contrast to the newly translated GR, which is bound to hsp90 and must be transformed to the DNA binding state, the TR is not bound to hsp90 and is translated in its DNA binding form without any requirement for transformation. When the GR is translated in wheat germ extract, which does not contain hsp90, it is translated in its DNA binding form in the same manner as the TR synthesized in reticulocyte lysate. These observations provide direct evidence that binding of GR to hsp90 is associated with repression of its DNA binding function. The fact that the TR does not bind to hsp90 and is translated in its DNA binding form is consistent with the different behavior of this receptor with respect to classic steroid receptors in the intact cell. We propose that binding to hsp90 may account for the fact that most of the steroid receptors are recovered in the cytosolic fraction after lysis of hormone-free cells in low salt buffer whereas the hormone-free TR is recovered in tight association with the nucleus.

Animals↗

PubMed1989

Direct evidence that the glucocorticoid receptor binds to hsp90 at or near the termination of receptor translation in vitro.

We have translated the rat glucocorticoid receptor in both reticulocyte lysate and in wheat germ extract. Receptor synthesized in the reticulocyte lysate is immunoadsorbed by the 8D3 monoclonal antibody directed against the 90-kDa heat shock protein (hsp90) and it has a normal ability to bind glucocorticoid in a high affinity manner. Although the wheat germ extract synthesizes the full length receptor, the receptor is not immunoadsorbed by 8D3 and we cannot demonstrate high affinity steroid binding. Receptor synthesized by the reticulocyte lysate can be immunoadsorbed by antibody directed against hsp90 as soon as the translation product is full length, suggesting that the receptor becomes associated with hsp90 late during translation or immediately at the termination of translation. When newly synthesized receptor is bound with steroid and incubated at 25 degrees C, it is converted to a form that binds to DNA. This study provides direct evidence that association of hsp90 with the glucocorticoid receptor is a very early event and that the newly formed heteromeric receptor-hsp90 complex is fully competent to undergo transformation.

Adsorption↗

PubMed1988

Association of the Ah receptor with the 90-kDa heat shock protein.

Partially purified Ah receptor preparations were used to produce a monoclonal antibody, designated as 8D3, that is capable of immunoprecipitating the Ah receptor. Hepa 1c1c7 cytosol was photoaffinity-labeled with [125I]-2-azido-3-iodo-7,8-dibromodibenzo-p-dioxin followed by immunoprecipitation, and the resulting precipitate was applied to a sodium dodecyl sulfate-polyacrylamide electrophoretic gel. These gels were stained with Coomassie Blue and revealed the presence of a major immunoprecipitated 90-kDa protein, and after autoradiography a radiolabeled 95-kDa protein (Ah receptor) was detected. The 90-kDa protein was determined to be the 90-kDa heat shock protein (HSP90) by western blot analysis using an antibody (AC88) previously shown to be specific for HSP90. An increase in the sedimentation of the Ah receptor on sucrose density gradients was seen upon addition of monoclonal antibody 8D3 to Hepa 1c1c7 cytosol. Monoclonal antibody 8D3 immunoprecipitates the Ah receptor from Hepa 1 cells (murine), HeLa cells (human), and rat liver cytosolic extracts, indicating that the Ah receptor is complexed with HSP90 in several mammalian species tested. These results illustrate another physicochemical property that the supergene family of soluble steroid receptors and the Ah receptor have in common.

Affinity Labels↗

PubMed1988

Purification of the Ah receptor from C57BL/6J mouse liver.

The photoaffinity ligand for the Ah receptor, [125I]-2-azido-3-iodo-7,8-dibromodibenzo-p-dioxin, previously has been shown to selectively label two peptides in the cytosol fraction of C57BL/6J mouse liver: a 95-kDa peptide, the ligand binding moiety of the Ah receptor, and a 70-kDa proteolytic fragment formed from the larger peptide (Poland, A., Glover, E., Ebetino, F. H., and Kende, A.S. (1986) J. Biol. Chem. 261, 6352-6365). These two peptides were partially purified to an approximately 20,000-fold enrichment with a 15-20% yield by the following scheme: 1) photoaffinity labeling of the 35-55% ammonium sulfate fraction of liver cytosol; 2) chromatography on polyethyleneimine-Sepharose coupled at low charge density and heparin/Mn2+ precipitation of the dilute column eluate; 3) DEAE-Sepharose chromatography to remove heparin; 4) chromatography on heparin-Sepharose; 5) preparative sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis followed by electroelution of the protein and ion pair extraction to remove sodium dodecyl sulfate; and 6) high performance liquid chromatography on a reverse-phase C-4 column. Following initial chromatography on polyethyleneimine Sepharose, it was found that substantial subsequent purification could only be achieved under denaturing conditions.

Affinity Labels↗

PubMed1988

Alterations in hepatic microsomal protein levels in rainbow trout fed cyclopropenoid fatty acids analyzed by two-dimensional gel electrophoresis.

Two-dimensional gel electrophoresis was used to analyze the effect of dietary cyclopropenoid fatty acids on hepatic microsomal polypeptide distribution patterns. Antibodies against rainbow trout type-LM2 cytochrome P-450 were employed to localize the corresponding polypeptide(s) by immunochemical staining. The LM2 antigen was purified from trout that had been fed beta-naphthoflavone. Microsomes from trout fed beta-naphthoflavone showed a decrease in a cytochrome P-450 polypeptide, detected with antibody against LM2. In contrast, microsomes from control fish contained two distinct cytochrome P-450 polypeptides, differing in their isoelectric points. Cyclopropenoid fatty acid treatment caused a preferential decrease in the additional isozyme seen in control samples. The distribution of concanavalin-A-binding glycopolypeptides was also assessed. Surprisingly, the two P-450 isozymes localized from control microsomes were found to bind concanavalin A. In addition to this, the cyclopropenoid fatty acid treatment generated a shift in a closely related group of microsomal glycopolypeptides, labeled gp80, gp82, gp80(1), and gp82(1). A decrease in the levels of gp80 and gp82 and a corresponding increase in gp80(1) and gp82(1) was observed.

Animals↗

PubMed1986

Alterations in the synthesis of proteins in hepatocytes of rainbow trout fed cyclopropenoid fatty acids.

Hepatocytes from rainbow trout reared on a diet containing cyclopropenoid fatty acids were analyzed for alterations in protein composition and synthesis by double label experiments. Both cytosolic and microsomal hepatocyte fractions were investigated. In the cytosolic fraction, the synthesis of proteins in the range of 68,000 to 74,000 daltons were significantly decreased. The identity of these proteins remains uncertain. A pronounced depression in both the mass and apparent synthesis of a 200,000 to 240,000 dalton microsomal protein was also observed. Immunoblotting with antibodies raised against goose acetyl-CoA carboxylase and avidin-peroxidase staining suggest that this protein is acetyl-CoA carboxylase. Moreover, synthesis of this protein as well as mass of the protein in cyclopropenoid fatty acid-fed fish were less than 20% of that found in control fish.

Acetyl-CoA Carboxylase↗

PubMed1983

Characterization of lipid-linked octa-, nona-, and decasaccharides formed during in vitro synthesis of mammary glycoproteins.

The lipid-linked octa-, nona-, and decasaccharides, isolated from incubations of a membrane preparation from the lactating bovine mammary tissue with GDP-[14C]mannose and UDP-N-acetylglucosamine were subjected to mild acid hydrolysis and purified extensively by repeated gel filtration and paper chromatography. Structural characterization of the oligosaccharides containing six to eight mannose residues linked to an N,N'-diacetylchitobiose unit utilizing digestions with alpha-mannosidase, beta-mannosidase, endo-beta-N-acetylglucosaminidase, D, H, and L, acetolysis, and methylation analysis revealed the presence of several isomers within each size species. Supplementation of the incubations with 0.1 mM dolichol phosphate reduces the number of isomers within these oligosaccharides; the predominant isomers of saccharides from these incubations appear to be similar to the saccharides isolated from in vivo preparations of Chinese hamster ovary cells.

Animals↗

PubMed1983

The use of a zwitterionic detergent in two-dimensional gel electrophoresis of trout liver microsomes.

CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-Propane sulfonate, a zwitterionic detergent, has been shown to exhibit superior membrane protein solubilizing characteristics as compared to nonionic detergents. Replacement of NP-40 with CHAPS in isoelectric focusing of rainbow trout liver microsomes has increased resolution markedly. The two-dimensional electrophoretic system described will allow effective resolution of up to 300 micrograms of crude microsomal protein. CHAPS exhibits no effect on the stability or type of pH gradient when compared to NP-40 during isoelectric focusing in the presence of urea.

Animals↗