Multiple forms of the glucocorticoid receptor steroid binding protein identified by affinity labeling and high-resolution two-dimensional electrophoresis

Potential charge heterogeneity within the glucocorticoid binding protein (GBP) of the glucocorticoid receptor was examined by a combination of affinity labeling, immunopurification, and high-resolution two-dimensional (2D) gel electrophoresis. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of [3H]dexamethasone 21-mesylate ([3H]DM) labeled cytosol identified a major, competable, component of Mr approximately equal to 92 000 (92K). This component was recognized by anti-human glucocorticoid receptor antibodies but not by nonimmune serum, indicating that the 92K component was the reduced denatured GBP. Examination of [3H]DM-labeled GBP by conventional 2D electrophoresis utilizing equilibrium isoelectric focusing in the first dimension failed to resolve the 92K GBP into discrete isoelectric components. This behavior was not representative of other, nonspecifically [3H]DM-labeled proteins or proteins in general. Nonequilibrium pH gradient electrophoresis (NEPHGE) was therefore employed to achieve separation in the first dimension. Immunopurified, [3H]DM-labeled GBP subjected to NEPHGE reached isoelectric equilibrium after 6 h of electrophoresis at 400 V. A single, broad peak of radioactivity was identified at pH approximately equal to 6.3. Second-dimension analysis of the NEPHGE-separated GBP by SDS-PAGE resolved this peak into two discrete, 92K, isoforms of apparent pI = 5.7 and 6.0-6.5. The GBP charge heterogeneity was confirmed by NEPHGE 2D analysis of [3H]DM-labeled GBP prepared directly from crude cytosol. Two isoforms indistinguishable from those observed in immunopurified samples were identified. An additional, more acidic, isoform (apparent pI approximately equal to 5.2) was also identified. Thus, there are at least two, and perhaps three, isoforms of the GBP. These data therefore suggest that there is significant charge heterogeneity in the GBP of the glucocorticoid receptor.     

Paramagnetic 1H and 13C NMR studies on cobalt-substituted human carbonic anhydrase I carboxymethylated at active site histidine-200: molecular basis for the changes in catalytic properties induced by the modification

Using bromo[1-13C]acetate to modify N tau of His-200 of human carbonic anhydrase isozyme I leads to the introduction of a useful 13C NMR probe into the active site. To complement our previous diamagnetic NMR studies with this probe, we have now succeeded in directly observing the paramagnetically perturbed resonance of the carboxylate in the cobalt-substituted modified enzyme above pH 8. In the pH range 8-10, the resonance undergoes a pH-dependent slow-exchange process, with the more alkaline form having a much smaller pseudocontact shift and a narrower line width. Below pH 8, the resonance apparently undergoes a very large paramagnetic downfield shift that was estimated by extrapolation. An ionization of approximate pK of 6 appears to control this process. Paramagnetic spin-relaxation studies on the resonance under conditions where it was directly observed yielded distance measurements between the carboxylate carbon and the active site cobalt ion. In inhibitor complexes, this distance was in the range of 5-7 A. In the absence of inhibitors, the distance was approximately 3.0-3.2 A at pH 7.9, consistent with the coordination of the carboxylate to the metal. However, at pH 10, the distance was increased to 4.8 A. These distance determinations were aided by relaxation measurements of a paramagnetically shifted proton resonance at 60-65 ppm downfield assigned by others to a proton of a ligand histidine of metal and confirmed by us to be 5.2 +/- 0.1 A from the metal. Our findings provide a molecular basis for the observed changes in catalytic properties that accompany the carboxymethylation.     

The effects of starvation and re-feeding on glycogen metabolism in mouse tail skin.

Although the glycogen content of mouse tail skin was decreased during starvation and was restored on re feeding, the proportion of glycogen synthase in the I form remained constant throughout at about 10% of the total. During the phase of net glycogen synthesis 1.5h after access to food was restored, the concentration of UDP glucose was markedly increased and the proportion of phosphorylase in the a form was significantly decreased.     

Action of Lipases of Staphylococcus aureus on Milk Fat

The activity of the lipase(s) of two strains of coagulase-positive Staphylococcus aureus was determined in milk fat incubated at 15, 22, and 30 C for 8 days. Total fat hydrolysis was measured by acid degree values (ADV). Neutral lipids were separated into component groups on a Florisil column. Free fatty acids were determined by temperature-programmed gas-liquid chromatography. The ADV were 25 to 50% greater at 22 than at 15 C and 4 to 7 times greater at 30 than at 22 C. The lipases liberated as much as 0.48 g of fatty acids per gram of fat during 8 days at 30 C. The enzyme showed a predilection for the palmitic acid-glycerol bond. Addition of fatty acids C₁₄ to C₁₈ inclusive to inoculated sterile skim milk caused inhibition of S. aureus as follows: (i) complete at 0.05 and 0.10% concentration of C₁₀ and (ii) partial at 0.05 and complete at 0.10% concentration of C₈. The samples showing inhibition were negative for peptonization, coagulase, and change in pH. Addition of oleic and stearic acid to sterile skim milk inoculated with S. aureus resulted in an increase in nonprotein nitrogen, and the C₄ to C₁₂ acids caused a decrease in protease activity.     

Patterns of Root Colonization in Epacridaceous Plants Collected from Different Sites

Root colonization was studied in ten species of the Epacridaceaeat three sites in Victoria by morphological and cross-inoculationexperiments. The sites and genera chosen were Cranbourne [Epacrisimpressa Labill. andLeucopogon ericoides(Smith) R. Br.] andRye [L. parviflorus(Andrews) Lindley] on the Mornington Peninsula,and the Grampians[Astroloma conostephioides(Sond.) Benth.,A.humifusum(Cav.) R. Br.,A pinifolium(R. Br.) Benth,Brachylomadaphnoides(Smith) Benth.,E. impressa, E. impressavar.grandifloraBenth.andStyphelia adscendensR. Br.] in western Victoria. For morphologicalstudies, samples of roots from each species at each site werecleared and stained and examined microscopically. For cross-inoculationstudies, cuttings from each site were struck in potting mediuminoculated with soil from the same and other sites. The ericoidmycorrhizae in the roots of plants found at or grown in Cranbourneand Rye soils were similar. Both were significantly differentfrom the internal hyphae found in the roots of plants foundat or grown in Grampians soils, which were three times largerin diameter and formed dense coils which filled the host celland invaded adjacent epidermal cells. This suggests that morethan one fungus is involved in the relationships, that the MorningtonPeninsula sites had a different fungus from the Grampians siteand that host specificity is low. Vesicular structures werealso found commonly on plants at the Grampians site, in contrastwith other sites. Epacridaceae; root; fungus; mycorrhiza; morphology; inoculation     

Binding of sulfosuccinimidyl fatty acids to adipocyte membrane proteins: Isolation and ammo-terminal sequence of an 88-kD protein implicated in transport of long-chain fatty acids

Summary We recently reported (Harmon et al., J. Membrane Biol. 124:261–268, 1991) that sulfo-N-succinimidyl derivatives of long-chain fatty acids (SS-FA) specifically inhibited transport of oleate by rat adipocytes. These compounds bound to an 85–90 kD membrane protein which was also labeled by another inhibitor of FA transport [³H]DIDS (4,4-diisothiocyanostilbene-2-2-sulfonate). These results indicated that the protein was a strong candidate as the transporter for long-chain fatty acids. In this report we determined that the apparent size of the protein is 88 kD and its isoelectric point is 6.9. We used [³H]SS-oleate (SSO), which specifically labels the 88-kD protein, to isolate it from rat adipocyte plasma membranes. Identification of 15 amino acids at the N-terminus region revealed strong sequence homology with two previously described membrane glycoproteins: CD36, a ubiquitous protein originally identified in platelets and PAS IV, a protein that is enriched in the apical membranes of lipidsecreting mammary cells during lactation. Antibody against PAS IV cross-reacted with the adipocyte protein. This, together with the N-terminal sequence homology, suggested that the adipocyte protein belongs to a family of related intrinsic membrane proteins which include CD36 and PAS IV.     

Specific interactions of chromatin with the nuclear envelope: positional determination within the nucleus in Drosophila melanogaster.

Specific interactions of chromatin with the nuclear envelope (NE) in early embryos of Drosophila melanogaster have been mapped and analyzed. Using fluorescence in situ hybridization, the three-dimensional positions of 42 DNA probes, primarily to chromosome 2L, have been mapped in nuclei of intact Drosophila embryos, revealing five euchromatic and two heterochromatic regions associated with the NE. These results predict that there are approximately 15 NE contacts per chromosome arm, which delimit large chromatin loops of approximately 1-2 Mb. These NE association sites do not strictly correlate with scaffold-attachment regions, heterochromatin, or binding sites of known chromatin proteins. Pairs of neighboring probes surrounding one NE association site were used to delimit the NE association site more precisely, suggesting that peripheral localization of a large stretch of chromatin is likely to result from NE association at a single discrete site. These NE interactions are not established until after telophase, by which time the nuclear envelope has reassembled around the chromosomes, and they are thus unlikely to be involved in binding of NE vesicles to chromosomes following mitosis. Analysis of positions of these probes also reveals that the interphase nucleus is strongly polarized in a Rabl configuration which, together with specific targeting to the NE or to the nuclear interior, results in each locus occupying a highly determined position within the nucleus.     

Identification of Ser-543 as the major regulatory phosphorylation site in spinach leaf nitrate reductase.

Spinach leaf NADH:nitrate reductase (NR) responds to light/dark signals and photosynthetic activity in part as a result of rapid regulation by reversible protein phosphorylation. We have identified the major regulatory phosphorylation site as Ser-543, which is located in the hinge 1 region connecting the cytochrome b domain with the molybdenum-pterin cofactor binding domain of NR, using recombinant NR fragments containing or lacking the phosphorylation site sequence. Studies with NR partial reactions indicated that the block in electron flow caused by phosphorylation also could be localized to the hinge 1 region. A synthetic peptide (NR6) based on the phosphorylation site sequence was phosphorylated readily by NR kinase (NRk) in vitro. NR6 kinase activity tracked the ATP-dependent inactivation of NR during several chromatographic steps and completely inhibited inactivation/phosphorylation of native NR in vitro. Two forms of NRk were resolved by using anion exchange chromatography. Studies with synthetic peptide analogs indicated that both forms of NRk had similar specificity determinants, requiring a basic residue at P-3 (i.e., three amino acids N-terminal to the phosphorylated serine) and a hydrophobic residue at P-5. Both forms are strictly calcium dependent but belong to distinct families of protein kinases because they are distinct immunochemically.     

Interactions of U1 RNP with heterogeneous nuclear RNA in rat Novikoff hepatoma nuclei

Summary The nature of the association of U1 RNA with rapidly sedimenting RNP structures in rat hepatoma nuclei was investigated. The effects of salt and proteinase K treatment on the stability of this bound form of U1 RNA were studied using sucrose density gradient analyses. Quantitation of the amount of U1 RNA remaining associated with large structures after treatment was used to assess the relative contribution of protein-protein(and protein-RNA) versus RNA-RNA interactions. Forty-eight percent of the total nuclear U1 RNA released by sonication was found in a bound form when the sonicate was centrifuged through gradients containing 50 mM NaCl. Fifty percent of this bound U1 RNA remained associated with rapidly sedimenting RNPs when the NaCl concentration was raised to 500 mM. To assess the contribution of protein independent interactions, large RNPs were completely deproteinized and their RNA moieties were then recentrifuged on gradients. By this analysis, 27% of the U1 RNA originally bound to hnRNPs was associated with rapidly sedimenting (>30 S) RNA (at 50 mM NaCl) suggesting their association by RNA-RNA hydrogen bonds. When the concentration of NaCl was 500 mM, 31% of the U1 RNA was associated with large RNA. Hence, approximately 30% of the U1 RNA molecules originally bound (or about 15% of the total nuclear U1 RNA) were found to be associated by RNA-RNA hydrogen bonds while the remainder of the binding of U1 RNP to hnRNP was by protein-protein and/or protein-RNA interactions.