Rat and human embryo and post-natal sera contain a potent endogenous competitor of estrogen-rat alpha-fetoprotein interactions

A highly active inhibitor of the binding of estrone and estradiol-17β to rat alpha-fetoprotein is demonstrated for the first time in embryo, immature and adult rat sera as well as in fetal and adult human sera. The competitive character and the narrow specificity of this inhibition effect is shown. The major compound responsible for this activity is isolated by successive column Sephadex LH₂₀ and thin layer chromatography : it is characterized as a nonpolar, nonphenolic, dialysable and thermostable substance, unreactive towards anti-estrone and anti-estradiol-17β anti-bodies. The possible biological role of an endogenous non-estrogen ligand of rodent fetoproteins is discussed.     

Action of alkyl cations and the natural ATPase inhibitor from mitochondria on soluble mitochondrial ATPase

The effect of the natural ATPase inhibitor and octylguanidine on the ATPase activity of soluble oligomycin-insensitive mitochondrial F₁ were compared. Both compounds induced a maximal inhibition of 60–80% in various preparations of F₁ studied. The inhibition was of the uncompetitive type with respect to MgATP, and the action of the compounds was partially additive. The data suggest that octylguanidine reproduces the action of the natural ATPase inhibitor. Alkylammonium salts also affect the ATPase activity in a similar form. F₁ bound to Sepharose-hexylammonium is largely inactive, whilst free hexylammonium at higher concentrations induces only a partial inhibition of the activity. This suggests that the degree of immobilization of F₁ is related to the magnitude of inhibition of ATPase activity induced by alkyl cations. The binding of F₁ to Sepharose-hexylammonium is prevented by high concentrations of Na⁺ or K⁺.     

Changes in liver nuclear protein metabolism after a single dose of urethane to suckling mice

Treatment of 8-9-day-old C57BL/A mice with a single carcinogenic dose of urethane, at 1.2 mg/g body wt., resulted in an immediate decrease in liver DNA synthesis reaching a maximum at about 16-18 h after injection, the rate of synthesis returning to normal after 48 h. When the nuclear proteins were radiolabelled, the non-histone protein (NHP) fraction showed a significant decrease in specific activity 8-18 h after injection of urethane and slight increase in specific activity after 24 h. Histone and residual proteins did not show any significant change. The liver NHP were analysed by isoelectric focusing (IEF) and sodium dodecyl sulphate (SDS) electrophoresis in polyacrylamide gels. The latter technique failed to show any distinctive differences but IEF results indicated some quantitative and qualitative changes in protein content and synthesis were induced by the urethane treatment. The most noticeable change in the stained gels was an increase in a protein component having a pI of 7.35 and the appearance of new bands at pI's of 7.85 and 5.55 in the 18 h treated livers. However, the [3H]tryptophan labelling pattern indicated that this was not due to an increased synthesis of these components. 24 h after urethane there appeared to be an increased rate of synthesis of some of the major components of the mixture, particularly at the pI 5.65 region. Histone and residual protein fractions were also analysed by electrophoresis and showed no difference between treated and control livers.     

Kinetics of protein-mediated transfer of rat pancreatic microsomal phosphatidylinositol to liposomes

Pancreatic microsomes were isolated from fasted and pilocarpine-injected rats and the microsomal phosphatidylinositol radiolabelled with myo-[2-³H]inositol by isotopic exchange. A standard reaction mixture was established in which partially purified rat liver phosphatidylinositol exchange proteins sustain a maximal rate of phosphatidylinositol transfer from rat pancreatic microsomes to liposomes. Determination of the transfer kinetics shows (1) that pancreatic microsomal phosphatidylinositol is partitioned approximately equally between a non-exchangeable and a single exchangeable pool and (2) that cholinergic stimulation does not significantly change the relative sizes of the two pools nor the exchange half-life of the latter pool.     

Glycosaminoglycan synthesis by mammary tumor spheroids

Multicellular aggregates of tumorigenic mouse mammary epithelial cells contain a hyaluronate-rich matrix, both at the aggregate periphery and within the growing spheroid. It is proposed that the establishment of a hyaluronaterich matrix is essential to spheroid growth in vitro, and that the spheroid is a good model system for analysis of this aspect of early tumor development.     

Binding, internalization and intracellular processing of 125I-epidermal growth factor purified by isoelectric focusing

When epidermal growth factor (EGF) which had been extensively purified by HPLC was subjected to iodination with sodium ¹²⁵iodide, 5 major species of differing isoelectric points were produced. Some of these species bound to rat fibroblasts with different affinities but were internalized with equal efficiency. Examination of the internalized ¹²⁵I-labelled molecules revealed processing of all the ¹²⁵I-EGF species to macromolecules with more acidic isoelectric points. The ¹²⁵I-EGF species with a pI of 4.5 corresponded in electrofocusing behavior with intact non-iodinated EGF. Other EGF species probably represented molecules which were covalently modified as a result of the iodination procedure.     

Androgen receptor complex binding in murine skeletal muscle nuclei

Examination of binding of androgen-receptor complexes from murine skeletal muscle cytosol was performed by modified nuclear retention assay and modified nuclear acceptor assay. The experiments showed the binding of androgen-receptor complexes to the nuclear acceptor sites to be a cooperative process. Hill analysis of the data obtained resulted in a Hill coefficient of 3,6. The apparent dissociation constant for binding of cytosolic [³H]-testosterone-receptor complexes to nuclei was found to be in the range of K_D = 6 ? 8 × 10^?11 M. The nuclear matrix was able to bind androgen-receptor complexes in a saturable way, too.     

Co-purification of soluble human galactosyltransferase and immunoglobulins

Preparations of human malignant effusion galactosyltransferase activity purified according to previously published techniques using enzyme-specific affinity chromatography consistently produced antibodies directed toward immunoglobulins with no detectable antigalactosyltransferase. Double immunodiffusion analysis of the antigen showed the presence of both IgG and IgA. Affinity chromatography with anti-human IgG-Sepharose and anti-human serum-Sepharose resulted in a 48,000-fold purification of galactosyltransferase activity with no detectable IgG by radioimmunoassay. Immunization of rabbits with this preparation produced antibodies directed against galactosyltransferase activity and minimal anti-Ig. The persistence of immunoglobulins during the purification of soluble galactosyltransferase activity through two enzyme-specific affinity chromatographic steps suggests an association of immunoglobulins with galactosyltransferase activity.     

Synthesis of charged amphipathic nitroxide lipid spin labels and an example of their application in membrane studies

The synthesis of a series of amphipathic nitroxide lipid spin labels is reported. Thus, 12-proxylhexadecanol has been converted into the versatile fatty acid spin label 14-proxylstearic acid. This substance was used to prepare 14-proxylstearyltrimethylammonium methanesulfonate, a positively charged label, and 14-proxylstearylmethyl phosphate sodium salt, a negatively charged label. Also prepared in the doxyl series were quaternary ammonium salts derived from 16-doxyl- and 7-doxylstearic acid. The positively charged and negatively charged proxyl labels were used in a preliminary experiment to investigate the role of charge in their interaction with reconstituted cytochrome oxidase. The average binding affinity of the negatively charged label is approximately 2-fold higher than that of the positively charged label at pH 7.4. At pH 5.5 the average relative affinity for negatively charged label is about 3.5-fold higher than that of positively charged label, suggesting that the ionizable group(s) on the protein can interact with the lipid headgroup.