Temperature-dependent inactivation of nucleic acid binding and aggregation of the 1,25-dihydroxyvitamin D3 receptor

The interaction of the 1α,25-dihydroxyvitamin D₃ receptor with immobilized calf thymus DNA has been compared with its sedimentation properties on hypotonic sucrose gradients. Forty to sixty percent of total hormone:receptor complexes formed at 4 °C were retained by DNA-cellulose and could be eluted by 0.18 to 0.2 m KCl. In contrast, heating preparations to 25 °C rapidly and irreversibly converted receptor to a form which bound hormone and DEAE-cellulose normally, but was unable to associate with DNA. Similarly, the ability of receptor to aggregate to a 6 S species was labile at 25 °C. Stabilization of receptor in the DNA binding aggregating form was accomplished using Ca²⁺, Mg²⁺, Mn²⁺, or Na₂MoO₄ while several protease and phosphatase inhibitors were ineffective. An examination of DNA binding properties of aggregating and nonaggregating receptor forms revealed that only receptor competent to enter into aggregates could bind DNA suggesting that a functional nucleic acid binding site, and, hence, a nucleic acid interaction is necessary for aggregate formation. Consistent with this view, an RNA:receptor interaction appears to be involved in formation of the 6 S complex since removal of RNA by ribonuclease treatment or purification of receptor reduced aggregation, an effect that could be reversed by addition of purified RNA.     

Isocitrate lyase: artifacts and multiple enzyme forms

Multiple enzyme forms of isocitrate lyase from various sources have been frequently reported. Protease action after cell rupture was sporadically claimed to explain the observed multiple enzyme forms. In this communication studies which are consistent with a protease action in vitro on isocitrate lyase of Pinus pinea germinating seeds are reported. Moreover, changes in DEAE-Sephacel patterns, mainly related to the age of germination, were observed. Differences regarding the heat stability of the detected enzyme forms were also found. The results indicate that isocitrate lyase from P. pinea may be detected in at least three different forms, one of which is heat stable and may be obtained only at the early stages of germination.     

Synthesis of phage-specific transfer RNA molecules by vibriophage phi 149

32P-Labelled tRNA was isolated from uninfected and phage phi 149-infected Vibrio cholerae cells. These tRNA preparations were then hybridised with DNA isolated from phage phi 149. Significant hybridisation was observed only with tRNA from phage phi 149-infected cells. This strongly suggests that infection of classical vibrio with phage phi 149 results in the synthesis of phage-specific tRNA molecules.     

Structural investigation of the capsular polysaccharide produced by a novel Klebsiella serotype (SK1). Location of O-acetyl substituents using NMR and MS techniques

The capsular polysaccharide of Klebsiella SK1 was investigated by methylation analysis, Smith degradation, and ¹H NMR spectroscopy. The oligosaccharides (P1 and P2) obtained by bacteriophage ΦSK1 degradation of the polymer were studied by methylation analysis, and 1D- and 2D-NMR spectroscopy. The resulting data showed that the patent repeating unit is a branched pentasaccharide having a structure identical to the revised structure recently proposed for Klebsiella serotype K8 capsular polysaccharide.

The 2D-NMR data showed that one third of the glucuronic acid residues in the SK1 polymer are acetylated at O-2, O-3, or O-4. FABMS studies confirmed the presence of monoacetylated glucuronic acid residues. Thus, the relationship between the Klebsiella K8 and SK1 polymers is akin to that found for Klebsiella polysaccharides K30 and K33, which have been typed as serologically distinct yet their structures differ only in the degree of acetylation.     

Regulation of insulin receptor kinase activity by insulin mimickers and an insulin antagonist

Three agents which mimic insulin action in intact cells (concanavalin A, wheat germ agglutinin, and polyclonal insulin receptor antibody), mimicked insulin's ability to stimulate the kinase activity of purified insulin receptors. In contrast, monoclonal insulin receptor antibody, an antagonist of insulin action, did not stimulate the phosphorylation of the insulin receptor either in intact IM-9 cells or in purified receptor preparations. This antibody, however, antagonized the ability of insulin to stimulate the phosphorylation of the receptor both in intact cells and in the purified receptor. These studies with insulin mimickers and an insulin antagonist are consistent with a role for the kinase activity of the receptor mediating the actions of insulin.     

Monoclonal antibody-directed immunopurification and identification of cytochromes P-450

A 28 amino acid peptide with diuretic and natriuretic activity has been purified from rat atrial muscle. The primary structure of this atrial peptide is H-Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly- (sequence in text) Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-(Arg)-Tyr-OH. The biological activity of this peptide is identical to that of atrial natriuretic factor and cardionatrin I isolated from rat atria.     

Induction of 7-ethoxycoumarin o-deethylase activity in cultured human epithelial cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD): evidence for TCDD receptor

The responsiveness of 5 human squamous cell carcinoma (SCC) lines derived from tumors of the epidermis and tongue to 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) was assessed by measuring the induction of the cytochrome P1-450-mediated monooxygenase activity, 7-ethoxycoumarin O-deethylase (ECOD). In 4 of the SCC lines the EC50 for this response was approximately 10(-9)M, whereas in one line the EC50 was 10(-10)M. In each of the less sensitive lines a concentration of 10(-10)M TCDD elicited less than 5% of the maximal enzyme activity. Specific binding of radiolabeled TCDD was detected in the cytosol fraction from all the SCC lines. The relative amount of receptor measured in each line correlated with maximally-induced ECOD activity. The data indicate that human cell lines derived from a target tissue for TCDD toxicity contain the TCDD receptor and show differential sensitivity to TCDD analogous to the murine strain differences in sensitivity regulated by the Ah locus.     

alpha-N-acetyl-beta-endorphin1-26 from the neurointermediary lobe of the rat pituitary: isolation, purification, and characterization by high-performance liquid chromatography

The neurointermediary lobes from 190 rat pituitaries were homogenized in an acidic medium which inhibits peptidase activity and maximizes the solubilization of undamaged peptides. Octadecylsilyl-silica (ODS-silica) was used to extract the supernatant of the tissue homogenate. The ODS-silica eluate, now largely protein and salt free, was subjected to reversed-phase high-performance liquid chromatography (HPLC) employing 0.1% trifluoroacetic as counter ion. The column eluates were monitored for beta-endorphin immunoreactivity. Five immunoreactive components were observed. The most hydrophobic of these was repurified on the same HPLC column using 0.13% heptafluorobutyric acid as counter ion. Characterization of the purified peptide by gel permeation HPLC, amino acid analysis, and tryptic fragmentation indicated that it corresponded in structure to alpha-N-acetyl-beta-endorphin1-26. Amino acid analysis of the native peptide and its trypsin and carboxypeptidase fragments indicated that an alanyl residue occupies position 26. This finding is in contrast to the sequence predicted for the beta-lipotropin/corticotropin precursor by recombinant DNA techniques which suggests that the 26th residue of the beta-endorphin molecule should be valine.     

Chromophoric peptide substrates for activity determination of animal aspartic proteinases in the presence of their zymogens: A novel assay

Solid-phase synthesis was used for the preparation of pyroglutamyl-histidyl-p-nitrophenylalanyl-phenylalanyl-alanyl-leucine amide (I) and glycyl-glycyl-histidyl-p-nitrophenylalanyl-phenylalanyl-alanyl-leucine amide (II), two water-soluble and sensitive chromophoric substrates of chicken pepsin, hog pepsin A, and bovine spleen cathepsin D. The kinetic constants of hydrolysis of the p-nitrophenylalanyl-phenylalanyl bond of the substrates were measured by difference spectrophotometry at 308 nm (Δ? = 860 m^?1 cm^?1) and by ninhydrin colorimetry (substrate I, ?₅₇₀ = 2.31 × 10⁴m^?1 cm^?1). The pH optimum of cleavage is 5 for the pepsins and 3.7 for cathepsin D. Since all three proteinases still have a significant activity at pH 5.5–6 a new, simple assay was designed for submicrogram quantities of pepsins in the presence of pepsinogens without interference of the latter. The method is particularly suitable for the analyses of the zymogen activation mixtures.     

Rapid resolution of transferrin C subtypes through isoelectric focusing with 2-mercaptoethanol

The technique of choice currently used for the detection of serum transferrin molecular polymorphism is isoelectric focusing on polyacrylamide slab gels. However, this procedure is unsatisfactory for routine purposes, since a long pretreatment of the serum with iron-donor compounds or neuraminidase is necessary in order to obtain a complete resolution of the transferrin molecule. A very fast and highly economical standardized procedure for transferrin typing which enables a fair molecular resolution within only 3 1/2 h is reported. Protracted pretreatment of serum with neuraminidase or with iron-donor compounds can be totally avoided. An ultrathin layer of polyacrylamide gel is employed for the run, using pH ranges of 4-6.5 or 5-7. A short pretreatment of serum with a 13% solution of 2-mercaptoethanol is performed before the samples are placed on the gel. This technique has been used to perform transferrin typing in 396 cord serum samples from newborn infants of Arezzo (Tuscany), without occurrence of artifacts or the appearance of extra bands in transferrin patterns.