Structural analysis of covalently labeled estrogen receptors by limited proteolysis and monoclonal antibody reactivity

We have used limited proteolysis of affinity-labeled estrogen receptors (ER), coupled with antireceptor antibody immunoreactivity, to assess structural features of ER and the relatedness of ER from MCF-7 human breast cancer and rat uterine cells. MCF-7 ER preparations covalently labeled with [3H]tamoxifen aziridine [( 3H]TAZ) were treated with trypsin (T), alpha-chymotrypsin (C), or Staphylococcus aureus V8 protease prior to electrophoresis on sodium dodecyl sulfate gels. Fluorography revealed a distinctive ladder of ER fragments containing TAZ for each protease generated from the Mr 66,000 ER: for T, fragments of 50K, 38K, 36K, 31K, 29K, and 28K that with longer exposure generated a 6K fragment; for C, fragments of 50K, 38K, 35K, 33K, 31K, 19K, and 18K that with longer exposure generated 14K and 6K fragments; and for V8, ca. 10 fragments between 62K and 28K. Two-dimensional gels revealed charge heterogeneity (two to three spots between pI 5.5 and 6.2) of the 66K ER and the T-generated 28K meroreceptor form. Immunoblot detection with the primate-specific antibody D75P3 gamma revealed that all immunoreactive fragments corresponded to TAZ-labeled fragments but that some small TAZ-labeled fragments (V8-generated forms less than 47K and T-generated forms less than 31K) were no longer immunoreactive. In contrast, use of the antibody H222Sp gamma revealed a correspondence between TAZ-labeled and immunoreactive fragments down to the smallest fragments generated, ca. 6K for T and C and 28K for V8. MCF-7 nuclear and cytosol ER showed very similar digest patterns, and there was a remarkable similarity in the TAZ-labeled and H222-immunoreactive fragments generated by proteolysis of both MCF-7 and rat uterine ER. These findings reveal great structural similarities between the human (breast cancer) and rat (uterine) ER and between nuclear and cytosol ER, indicate charge heterogeneity of ER, and allow a comparison of the immunoreactive and hormone attachment site domains of the ER. The observation that T and C generate a ca. 6K TAZ-labeled fragment that is also detectable with the H222 antibody should be of interest in studies determining the hormone binding domain of the ER and in amino acid sequencing of this region.     

Characteristics of different cytoplasmic and nuclear estrogen receptors appearing with continuous hormonal exposure

Biochemical properties of cytosol estrogen receptor (ERC) and nuclear estrogen receptor (ERN) from rat uteri continuously exposed in vivo to 17 beta-[2,4,6,7-3H] estradiol ( [3H]E2) for 6 h have been studied on the basis of immunological recognition and chromatographic elution patterns. Overall concentrations of ERC and ERN did not change during this time period when receptor-saturating concentrations of [3H]E2 were maintained (Jakesz, R., Kasid, A., and Lippman, M. E. (1983) J. Biol. Chem. 258, 11798-11806); however, biochemical characteristics were different in ERC and ERN after short or long term hormonal exposure. When ERC from rats treated with estradiol for 30 min was applied to HAP or DEAE columns, two different ER binding components were seen. DNA binding in a cell-free system revealed that these binding components represented an activated and a nonactivated ERC population. After long term hormonal exposure (6 h), only one component of ERC with low DNA binding could be shown despite the preservation of an equivalent quantity of cytoplasmic binding activity. This binder does not react with a monoclonal antibody directed against extranuclear estrogen receptor species. These data suggest disappearance of the activated ERC population, with appearance of a new, immunologically nonrecognizable ERC species with 6 h of continuous hormonal exposure. Elution profiles of ERN on HAP chromatography reveal 2 different binding components at 30 min and at 6 h of continuous [3H]E2 exposure. There is an increase of the population eluted at higher molarity after 6 h of in vivo treatment. This later eluting binding component is the major DNA binder in vitro. ERN from both time points are recognized immunologically by monoclonal antibody. After reaction with the antibody, the sedimentation coefficient shifted to 8-9 S on sucrose gradients, but the previously described faster sedimentation of ERN extracted 6 h after injection persisted. We conclude that ER in both cellular compartments undergoes time-dependent alterations, which may be involved in the initiation of hormone action.     

The binding of pentaammineruthenium (III) to RNase B and RNase A+d(pA)4 in the crystalline state

The binding of pentaammineruthenium (III) to ribonuclease A and B both free and complexed with d(pA)₄ has been examined in the crystalline state through the application of X-ray diffraction and difference Fourier techniques. In crystals of native RNase B, the reagent was observed to have many binding sites, some entirely electrostatic in nature and others consistent with coordination to histidine residues. The primary histidine in the latter case was 105 with 119 also partially substituted. In crystals of RNase A+d(pA)₄ complex only a single, extremely strong site of substitution was observed, and this was 2.4 Å from the native position of the imidazole ring of histidine 105. Thus, the results of these X-ray diffraction studies appear to be quite consistent with the findings of earlier NMR studies and with the results obtained in crystals of the gene 5 DNA binding protein.     

A Novel Algorithm for Identification of Activated Cryptic 5′ Splice Sites

Abstract

The resonances of the protonated carbons of [d(TAGCGCTA)]₂ have been assigned by the two-dimensional proton-detected double-quantum heteronuclear correlation experiment ([¹H-^l3C]-DQCOSY). ¹³C-coupled and ^l3C-decoupled versions of the experiment were used. The assignment method is discussed in detail. The deoxyribose cross peaks segregate into five well-resolved regions, and the base cross peaks have distinct features that are helpful for assignments. The cross peaks from the ¹H-¹³C pairs at the Cyd5, Ado2 and ThdCH₃ base positions fall in separate regions of the spectrum from each other; they also are resolved from the closely spaced Ado8, Guo8, Cyd6 and Thd6. Additional parameters for distinction of the base signals are their differing J-coupling values and long-range coupling patterns.     

A developmental study of rat sperm and testis selenoproteins

Essentially all of the selenium in the rat spermatozoon is bound to a polypeptide of Mr 15,000-17,000 confined to the capsule that surrounds the sperm mitochondria. Isoelectric focussing of isolated 75Se-labelled, carboxymethylated mitochondrial capsule protein (MCP) reveals the presence of at least four radioactive components, with a predominant charge isomer at pI4.6. The sperm selenoprotein appears to be identical with MCP, as judged by the exact coincidence of radioactivity and protein stain during two-dimensional electrophoresis. The temporal pattern of 75Se-labelling of rat caput epididymal spermatozoa after intratesticular 75Se injection suggests that maximum incorporation of 75Se into MCP occurs in step 7-step 12 spermatids and that 75Se uptake ceases during step 15 of spermiogenesis. The developmental appearance of sperm selenoprotein in rat testis therefore appears to lag several days behind that reported for MCP in mouse testis, suggesting the presence of selenium-free MCP in immature germ cells. SDS gel electrophoretic analysis of testis subcellular fractions 24 h after 75Se injection into rat testis at 21, 28 and 90 days of age indicates that sperm selenoprotein first appears in very low concentration during late meiosis and that its concentration increases sharply during early spermiogenesis. Additional 75Se-labelled polypeptides were detected on the gels, most of them of higher molecular weight than MCP. At least two of these (Mr 47,000 and 54,000) displayed a marked decrease in labelling between 5 and 24 h after injection into adult testis, coincident with a comparable increase in 75Se-labelled MCP, indicating that they may be precursors of MCP.     

Cytotoxic effects of ethylene glycol monomethyl ether in the forelimb bud of the mouse embryo

The role of cytotoxicity in digital maldevelopment in CD-1 mouse embryos was examined following dosage with ethylene glycol monomethyl ether (EGME) on gestation day (gd) 11. Patterns of cell necrosis in the forelimb buds of embryos collected from dams given EGME orally at doses of 100, 250 or 350 mg/kg were characterized by staining with Nile blue A. Cell death was induced in the mesenchymal tissue and to some extent in the limb bud ectoderm, including the apical ectodermal ridge in a dose-related manner. The area of preaxial physiological cell necrosis was enlarged by EGME, and the shape of the limb buds was altered 24 hr after treatment. Preaxial tissue and the predigital chondrocyte condensations were reduced or missing following 250 and 350 mg EGME per 1 kg. Light and electron microscope evaluations of forelimb buds revealed the presence of phagocytic vacuoles and condensed, fragmented cytoplasm, which indicate cytotoxicity, as early as 2 hr following EGME, a maximum effect being observed 6 hr after the dose was administered. Although the severity of the cytotoxic response appeared to be dose-related, comparison with the incidence of digital malformations in near-term fetuses indicates that the loss of mesenchymal tissue is partially compensated for as formation of the limb progresses.