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.     

Reconstitution of the Turkey erythrocyte adenylate cyclase sensitivity to l-epinephrine upon re-insertion of the Lubrol solubilized components into phospholipid vesicles

Turkey erythrocyte adenylate cyclase was activated by GppNHp and l-epinephrine to its stable, highly active form. In this form the enzyme could be solubilized by Lubrol-PX and subsequently re-inserted into phospholipid vesicles concomitantly with the removal of up to 99.3% of the Lubrol. The ability of GTP and l-epinephrine to reverse the GppNHp/epinephrine activated state was taken as a measure for the reappearance of hormone sensitivity in the reconstituted vesicles. An incomplete but significant reappearance of hormone sensitivity in the reconstituted adenylate cyclase was achieved. This hormone sensitivity was found to be stereospecific for (?)epinephrine. The ¹²⁵I-cyanopindolol binding properties of the reconstituted β-receptor depend on the nature of the detergent and the phospholipids used in the reconstitution.     

Interaction of N-ethyl-N'-nitro-n-nitrosoguanidine with nucleic acids and proteins in comparison with N-methyl-N'-nitro-N-nitrosoguanidine

Reactivity of N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) was studied in comparison with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The radioactivity of [guanidino-14C]-ENNG was incorporated only into the protein fraction and that of [ethyl-14C]ENNG was incorporated into DNA, RNA and protein fractions in ascites hepatoma AH7974 cells, as were those of [guanidino-14C]- and [methyl-14C]MNNG, respectively. The amounts of the binding of ENNG were less than those of MNNG, especially in the corporation of the ethyl moiety of ENNG into nucleic acid fractions. In a non-cellular system, the radioactivity of [guanidino-14C]ENNG was incorporated into proteins, preferentially into basic proteins such as cytochrome c, but was not incorporated into nucleic acids. This behavior is similar to that of [guanidino-14C]MNNG, while the amount of binding of the former was about half of that of the latter. The radioactivity of [ethyl-14C]ENNG was also incorporated into basic proteins to almost the same extent as that of [methyl-14C]MNNG. However, the binding of the ethyl moiety of ENNG to nucleic acids was much lower than that of the methyl moiety of MNNG. Horse heart cytochrome c, bovine pancreatic RNase A and regenerating rat liver chromatin had altered their biological activities to various degrees after modification by ENNG or MNNG.     

Intracellular binding of lead in the kidney: The partial isolation and characterization of postmitochondrial lead binding components

Gel chromatography of kidney postmitochondrial fractions from control rats 2 hr after injection of ²⁰³Pb or after in vitro incubation with ²⁰³Pb disclosed the presence of two fractionated Pb-binding components plus binding in the void volume and total volume regions. The binding of Pb to the two components, with molecular weights of 11,500 and 63,000 daltons, was markedly decreased in Pb-pretreated rats. Sodium dodecyl sulfate-gel electrophoresis and autoradiography showed the presence of one major ²⁰³Pb band with an estimated molecular weight of 60,000 daltons. The 11,500-dalton peak did not incorporate ¹⁴C-leucine nor did concomitant administration of cycloheximide with the ²⁰³Pb inhibit incorporation of ²⁰³Pb activity, suggesting that the component is a preformed constituent of the kidney. In vitro incubation of brain, liver and lung postmitochondrial supernatants with ²⁰³Pb disclosed that these two binding components were also present in brain but not in liver or lung, suggesting a target tissue-specific localization for these Pb-binding macromolecules.     

Diphtheria toxin-receptor interaction: A polyphosphate-insensitive diphtheria toxin-binding domain

Inositol hexaphosphate, and other polyphosphates, inhibit diphtheria toxin-mediated cytotoxicity by binding to the toxin at a highly cationic site called the P site and preventing toxin binding to cell surface receptors. The binding of diphtheria toxin to a solubilized cell surface glycoprotein (150,000 daltons) is also inhibited by these polyphosphates. Treatment of this 150,000 dalton diphtheria toxin-binding cell surface glycoprotein with papain yielded an 88,000 dalton and a 74,000 dalton diphtheria toxin-binding glycoprotein whose binding to toxin was no longer inhibited by inositol hexaphosphate. This result suggests a model of diphtheria toxin-receptor interaction in which the toxin receptor possesses one binding site which interacts with the P site of the toxin in a $\text{polyphosphate-sensitive}$ fashion, and another binding site (located within the papain-derived 74,000–88,000 dalton glycoproteins) which can interact with the toxin at a site distinct from the P site (the X site) in a $\text{polyphosphate-insensitive}$ fashion. This X site-receptor interaction may be involved in the binding of CRM proteins that bind to the toxin receptor but that do not bind polyphosphates, or it may be involved in the entry process of the toxin.     

ADP-ribosylation of brain synaptosomal proteins correlates with adenylate cyclase activation

ADP-ribosylation of the adenylate cyclase $\text{G}\text{F}$ regulatory subunit by cholera toxin is a major tool for the study of this enzyme. Investigation of the brain enzyme has been hampered up to now by the failure to demonstrate cholera toxin-dependent ADP-ribosylation of membrane-bound proteins. Synaptosomes prepared by flotation from fresh brains homogenized in the presence of protease inhibitors yielded membranes of which several proteins could be ADP-ribosylated by the toxin. The same membranes subjected to mild proteolysis could not be ADP-ribosylated. Adenylate cyclase activation and ADP-ribosylation were simultaneous processes. The major labeled species was of 47,000 M_r. It was solubilized by Lubrol-PX, together with other labeled polypeptides. As analyzed on sucrose gradients, the 47,000 M_r protein was found both in the 3S region, and in the adenylate cyclase containing fraction (9.1S).     

Methionine adenosyltransferase activity in cultured cells and in human tissues

We have investigated methionine adenosyltransferase activity (MAT) in extracts of a variety of normal and malignant human tissues and cultured cell lines. MAT activity assayed from 17 different cultured cell lines varied to a great extent. Ramos (human, Burkitt's lymphoma) and EL4 (mouse, T cell lymphoma) cell showed MAT activity near 300 pmol/mg per min. Daudi (human, Burkitt's lymphoma) and almost all monolayer cells had MAT activity below 100 pmol/mg per min. Human peripheral blood lymphocytes had MAT activity of 36 pmol/mg permin. The MAT activity of the cell lines can be related to doubling time: cell lines with short doubling times have much higher MAT activity than other cell lines. A large variation in MAT activity in different human tissues was observed. In autopsy samples MAT activity was highest in the brain and in the colon. Malignant tissue samples gave much higher MAT activity than normal tissues. Lung cancer (carcinoma squamocellulare pulmonis) had MAT activity of 30.7 pmol/mg per min, while in normal lung it was 2.4 pmol/mg per min.     

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The photoaffinity ligand 8-azidoadenosine 3′,5-monophosphate was employed to label cAMP binding proteins in both fractionated and freeze-thawed rabbit gastric glands. Fractionated glands incorporated the azido-cAMP label primarily into two cytosolic proteins with apparent molecular weights of 58 000 and 48 000. No enrichment of label was found in fractions containing basolateral or apical membranes. DEAE-cellulose chromatography of the cytosolic proteins resulted in the separation of two cAMP-dependent protein kinase peaks. Azido cAMP labelling of each peak suggested the initial peak contained type I cAMP-dependent protein kinase while the second peak contained the type II kinase. Labelling of ‘resting’ gastric glands resulted in radioactive proteins of apparent molecular weights of 58 000.and 48 000. When gastric glands were stimulated to produce acid by the addition of 10^?4 M histamine or 1 mM dibutyryl cAMP there was 32–44% dimunition of ligand incorporation compared to control glands. The results strongly suggest that histamine- mediated stimulus-secretion coupling in gastric glands involves activation of parietal cell cAMP-dependent protein kinases.     

Solubilization and partial purification of protein kinase systems from brain membranes that phosphorylate calspectin: A spectrin-like calmodulin-binding protein (fodrin)

In brain tissue a spectrin-like calmodulin-binding protein calspectin, or fodrin, is concentrated in a synaptosome fraction, where most of the calspectin is associated with the synaptic membranes. This endogenous calspectin was phosphorylated by protein kinase system(s) associated with the membranes. Here, we report the solubilization and partial purification of the membrane-associated calspectin kinase activity. The activity was resolved on a gel filtration column into two fractions, peaks I and II having estimated M_r of 800 000 and 88 000. The activity of peak I was dependent on the presence of both Ca²⁺ and calmodulin. Peak II revealed a basal activity in the absence of Ca²⁺ and calmodulin, which was stimulated 2-fold by addition of Ca²⁺. Calmodulin had no effect on the peak II activity.