Separation of poly(ADP-ribosylated) nuclear proteins by polyacrylamide gel electrophoresis at acidic pH and low temperature

A method for the extraction and electrophoresis of poly(ADP-ribosylated) nuclear proteins is described. An extraction method using lithium dodecyl sulfate as detergent at pH 2.4 and room temperature is shown to fully extract nuclear proteins under conditions where full stability of protein-linked polymer is ensured. The polyacrylamide gel electrophoresis is performed again under conditions where full stability is ensured. This work provides a technique whereby misinterpretation of relative ADP ribosylation of nuclear proteins can be avoided.     

Cholera-toxin-dependent ADP-ribosylation of the adenylate cyclase regulatory protein in turkey erythrocyte membranes

Incubation of turkey erythrocyte membranes with cholera toxin and [³²P]NAD caused toxin-dependent incorporation of ³²P into a 42,000 M_r peptide which could be distinguished from toxin-independent ³²P incorporation into other membrane proteins. The radiolabeled 42,000 M_r peptide could be extracted from the membranes using Lubrol PX. When toxin-treated membranes were incubated with isoproterenol and GMP before detergent solubilization, the 42,000 M_r labeled peptide was adsorbed by GTP-γ-agarose which, with the same conditions, adsorbed the adenylate cyclase guanine nucleotide regulatory protein. The labeled peptide and guanine nucleotide regulatory protein activity were coeluted from the affinity matrix by guanylyl-β,γ-imidodiphosphate, GDP, and GMP. Guanosine 5′-O-(2-thiodiphosphate), an analog of GDP which blocks guanine nucleotide- and fluoride-stimulated adenylate cyclase activity, caused elution of labeled peptide which exhibited no regulatory protein activity. Our data support the view that the 42,000 M_r peptide is part of the adenylate cyclase guanine nucleotide regulatory protein. The labeled peptide allows identification of both active and inactive regulatory protein and should be useful in monitoring the purification of the regulatory protein from turkey erythrocytes.     

Interactions between dissociated rat sympathetic neurons and skeletal muscle cells developing in cell culture. I. Cholinergic transmission

Sympathetic neurons, dissociated from superior cervical ganglia of newborn rats, and skeletal muscle cells were grown together in mass cultures containing many neurons (ca. 1000–3000) and myotubes, and in microcultures containing only one to three neurons and one or a few myotubes. When these neurons grow under the influence of certain nonneuronal cells many of them acquire cholinergic functions; in the absence of this influence they remain adrenergic. In the present study, the influence of the skeletal muscle cells was so effective that under certain conditions more than 75% of the neurons expressed cholinergic function as judged by their ability to form excitatory cholinergic synapses with myotubes (from rat and chick) and with each other. Stimulation of single neurons often gave rise in the myotubes to simple (direct) postsynaptic potentials (ejp's) and/or complex responses comprising a burst of ejp's that evoked one or more spikes; it appeared that these complex responses involved the activation of interneuronal pathways. In microcultures, a single neuron often made cholinergic synapses with itself (“autapse”) and/or with another neuron as well as with one or more myotubes. The nicotinic blocking agents, tubocurare (dTC), α-bungarotoxin (α-BuTx), and hexamethonium (C₆), attenuated or abolished the ejp's at moderate concentrations; the muscarinic blocker, atropine, was effective only at high concentrations. At several neuron-myotube junctions, the acetylcholine (ACh) receptors had dTC sensitivity similar to adult extrajunctional receptors; however, when different junctions were pooled the average dTC sensitivity was intermediate between that of adult end plate and extrajunctional receptors. The junctional C₆ sensitivity was much higher than expected from the action of the drug at the adult mammalian end plate. As in other studies, chemical transmission from neuron to neuron was also nicotinic cholinergic, but the nicotinic receptors on the myotubes were pharmacologically distinct from those on the neurons.     

A sulfhydryl group of the canine cardiac beta-adrenergic receptor observed in the absence of hormone

Canine cardiac beta-adrenergic receptors contain a free sulfhydryl group in the adrenergic ligand binding site. [125 I]-Iodohydroxybenzylpindolol [( 125 I]-IHYP) binding to cardiac beta-receptors was inhibited 80% by treatment with 1 mM p-chloromercuribenzoic acid (pCMB). Occupation of the beta-receptors by an antagonist prior to treatment with pCMB prevented this effect suggesting that a sulfhydryl group is present in or near the ligand binding site of the cardiac beta-receptor. In the presence of agonists, the sensitivity of cardiac beta-receptors to pCMB was increased. Incubation of isoproterenol-occupied cardiac beta-receptors, resulted in a 57% inhibition of [125 I]-IHYP binding measured after extensive washing to remove bound agonist. The ability of isoproterenol to increase the reactivity of cardiac beta-adrenergic receptors supports the hypothesis that agonists produce a conformational change upon binding.     

Sizes and mass distributions of clathrin-coated vesicles from bovine brain

Clathrin-coated vesicles obtained from bovine brain have been studied by ultracentrifugation and dynamic light scattering techniques to provide information on their sedimentation and mass distributions and their average diffusion coefficients. "Uncoated" vesicles, obtained by removing the protein coat from coated vesicles, have been similarly characterized. For typical preparations, maximal values of approximately 210 and 95 S are observed for the sedimentation coefficients of coated and uncoated vesicles, respectively. Corresponding values for the average molecular weights, determined from values of average sedimentation and diffusion coefficients, are 49 X 10(6) and 13 X 10(6); values obtained by equilibrium sedimentation are 37.2 X 10(6) and 10.6 X 10(6). In order to obtain these results, some minor modifications of sedimentation and light-scattering techniques have been devised which may have application to other studies of size distributions of large particles.     

Interleukin 2-driven T lymphocyte proliferation is dependent upon a surface antigen distinct from the interleukin 2 receptor: requirements for inhibition of T-cell proliferation by monoclonal antibody 5C3

A monoclonal antibody (5C3) to an antigen expressed on activated guinea pig T lymphocytes that did not react with the interleukin 2 (IL-2) receptor, but inhibited IL-2-driven proliferative responses has been previously characterized. The present study provides further analysis of the inhibitory capacity of 5C3 for T-cell proliferation and of the relationship between the expression of the antigen defined by 5C3 and the capacity of cells to respond to IL-2. 5C3 inhibited proliferation of T-cell blasts to IL-2-containing fluids when added as late as 8 hr prior to termination of a 26-hr culture. 5C3 pretreatment of the IL-2-responsive blast cells was also sufficient to detect significant inhibition of proliferation. FACS analysis of these blasts indicated that maximal 5C3 binding was required for pretreatment to result in inhibition of IL-2-driven proliferation. Delayed addition of 5C3 to culture or pretreatment with 5C3 of responding cells also resulted in inhibition of proliferation of immune T lymphocytes to antigen-pulsed-presenting cells. Lastly, although modulated 5C3- blasts failed to proliferate to IL-2, induction of the 5C3-bearing molecule on these 5C3- blasts correlated with restoration of the ability of these cells to proliferate to IL-2. Collectively, these results further support the hypothesis that monoclonal antibody 5C3 interferes with a critical signal in the IL-2 growth pathway.     

Activation of human B lymphocytes. XVII. Synergy between nonspecific and specific signals in the antigen-specific responses of human B cells

The incubation of human peripheral blood lymphocytes (PBL) with the natural killer (NK)-sensitive MOLT-4 cell line results in PBL-target cell conjugate formation by certain lymphocyte subpopulations. Following velocity sedimentation, the PBL depleted of these conjugate-forming subpopulations are markedly diminished in the ability to mediate either antibody-dependent cellular cytotoxicity (ADCC) or NK activity. The immediate testing of highly pure PBL subpopulations isolated from the NK target conjugates does not reveal the expected recovery of augmented ADCC or NK levels. Following in vitro incubation, however, the PBL NK target-binding subpopulations do manifest augmented levels of both NK and ADCC, whereas the depleted PBL continue to display diminished NK and ADCC levels. In addition, the degree of augmented NK and ADCC levels recovered by the NK target-binding PBL subpopulations appears dependent on both the time and the temperature of in vitro incubation. Moreover, the ADCC recovery patterns are identical to those observed for NK activity regardless of the time and temperature of in vitro incubation. These results directly demonstrate that the PBL subpopulations isolated from certain NK target cells are functionally enriched in the ability to mediate from ADCC and NK activity.     

Mitogen-induced suppressor factor(s) from human lymphocytes: effects on lymphoid and nonlymphoid cells and biophysical properties

Concanavalin A (Con A) or phytohemagglutinin activate a population of human circulating lymphocytes to exert suppressive functions. We found that supernates from the activated human lymphocytes suppress lymphocyte responses to Con A, the mixed lymphocyte reaction and pokeweed mitogen-induced IgM production. Mitogen stimulated suppressor lymphocytes, or their supernates, inhibit also the spontaneous proliferation of human retinoblastoma cells (Y-79 line) and primary cultures of human keratocytes. A correlation was always noted between the levels of inhibitory activities of the lymphocytes and their supernates. Furthermore, a good correlation was found between the levels of inhibition by the supernates of lymphocyte functions (proliferation and IgM production) and of the nonlymphoid cells' proliferation. Some of the properties of this suppressor factor(s) are: (i) produced only by the T-cell population; (ii) appears after 8 hr of Con A stimulation, peaks at 24 to 48 hr and declines later on; (iii) stable at 56 °C and labile to 70 °C; (iv) nondialyzable and present in the 40K–100K dalton fraction of a G-200 Sephadex column; (v) labile to pH 2 treatment.     

Multiple expressions of the activity of guanine nucleotide regulatory protein in human thyroid adenylate cyclase

Adenylate cyclase (ATP pyrophosphate-lyase, EC 4.6.1.1) in plasma membranes from human thyroid was highly responsive to thyrotropin. Pretreatment of thyroid plasma membranes with 5′-guanylylimidodiphosphate (Gpp(NH)p) in the presence of Mg²⁺ led to a temperature-dependent activation, which was seen neither in the absence of Mg²⁺ nor at 4 °C. By contrast, thyrotropin bound to its receptors regardless of the temperature and produced its maximal effect after 2 min of preincubation in the absence or presence of Mg²⁺. Furthermore, activation was seen after treatment with thyrotropin and Gpp(NH)p even carried out in the absence of Mg²⁺ or at 4 °C. However, the full activation by Gpp(NH)p required Mg²⁺, hormone, and elevated temperature. These observations suggest that there appears to be two types of nucleotide interaction responsible for the Gpp(NH)p activation in human thyroid membrane; one type seen in the absence of hormone may represent the system uncoupled from hormone receptor, while the fully coupled hormone-sensitive adenylate cyclase accounts for the second type of interaction which requires the presence of hormone.     

Radioimmunoassay for pig angiotensin I converting enzyme: a comparison of immunologic with enzymatic activity.

Angiotensin I converting enzyme in body fluids and extracts of various pig tissues was measured by radioimmunoassay and by enzymic assay. Based on the ratios of enzymic to immunologic activity, the extracts could be separated into two groups. One group, with ratios around 4 U/mg, included urine and extracts from the adrenal, choroid plexus, epididymis, gall bladder, heart, liver, retina, spleen, and testis. The other group, with ratios around 12 U/mg, contained serum and extracts from lung and kidney. Explanations are offered for why one group had a lower enzymic to immunologic ratio than the other.     

Purification and characterization of protein synthesis initiation factors IF1, IF2, and IF3 from Escherichia coli.

A simple procedure is described for the purification in high yields of protein synthesis initiation factors IF1, IF2, and IF3 from Escherichia coli strain MRE 600. IF2 was separated from IF1 and IF3 by ammonium sulfate fractionation and was purified by column chromatography on phosphocellulose and diethylaminoethyl (DEAE) Sephadex. IF1 and IF3 were separated by phosphocellulose column chromatography. IF1 was purified by molecular sieve chromatography, and IF3 by phosphocellulose column chromatography in urea buffer. Each factor was analyzed by sodium dodecyl sulfate or urea polyacrylamide gel electrophoresis and was greater than 98% pure. Only one form of IF1 and IF3 was found, with molecular weights of 8,500 and 22,500, respectively. Two forms of IF2 were isolated: IF2a with a molecular weight of 118,000 and IF2b with a molecular weight of 90,000. The amino acid composition of each factor was determined, and their stimulation in a variety of assays for initiation of protein synthesis is reported.     

Purification of human liver cytochrome P-450 and comparison to the enzyme isolated from rat liver

Human liver cytochrome P-450 was isolated from autopsy samples using cholate extraction and chromatography on n-octylamino-Sepharose 4B, hydroxylapatite, and DEAE-cellulose gels. Purified preparations contained as much as 14 nmol cytochrome P-450 mg^?1 protein, were free of other hemoproteins, and were active in the mixed-function oxidation of d-benzphetamine and 7-ethoxycoumarin when coupled with either rat or human liver NADPH-cytochrome P-450 reductase. Some of the preparations were apparently homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; apparent subunit M_rs estimated for several preparations were 53,000 or 55,500. The amino acid composition of one preparation was determined and found to resemble those of rat liver cytochromes P-450, although some variations were noted. Rabbit antibodies raised to phenobarbital-treated rat liver cytochrome P-450 were more effective in inhibiting d-benzphetamine N-demethylase activity in human liver microsomes than were antibodies raised to 3-methylcholanthrene-treated rat liver cytochrome P-450. These antibodies also inhibited benzo(a)pyrene hydroxylation in human liver microsomes, although the inhibition patterns did not follow a general pattern as in the case of benzphetamine demethylase activity. Microsomes prepared from three different human liver samples were more effective in eliciting complement fixation with antibodies raised to phenobarbitalthan to 3-methylcholanthrene-treated rat liver cytochrome P-450. Complement fixation in such systems appears to result from similarity of certain rat and human liver cytochrome P-450 antigenic determinants, as fixation could be inhibited by removal of cytochrome P-450-directed antibodies from the total immunoglobulin population and purified human cytochrome P-450 was more effective (on a protein basis) than liver microsomes in producing fixation. Human liver microsomes prepared from five different individuals all produced ≥ 90% complement fixation, but variations were observed in the fixation curves plotted either versus microsomal protein or versus spectrally detectable microsomal cytochrome P-450.These results indicate that human liver microsomal cytochromes P-450 can be isolated using modifications of techniques developed for laboratory animals and that human and rat liver cytochromes P-450 share certain features of structural, functional, and immunological similarity. The available data suggest the existence of multiple forms of human liver microsomal cytochrome P-450, but possible artifacts associated with the use of autopsy samples suggest caution in advancing such a conclusion.     

beta-Adrenergic receptor induction in HeLa cells: synergistic effect of 5-azacytidine and butyrate

³H-Labeled leukotriene C₃ was efficiently taken up by the isolated, perfused rat liver and excreted into the bile. The isolated, perfused kidney eliminated leukotriene C₃ from the perfusate slower and excreted only a fraction of the radioactivity into the urine. Isolated hepatic, intestinal and renal cells also took up leukotriene C₃, the renal cells being the most effective in accumulating the label. Anthglutin, an inhibitor of γ-glutamyl transferase, decreased the uptake by kidney cells but had no effect on the uptake by the other cell types. In liver cells, the uptake rate was sensitive to temperature and to cellular ATP content. Chromatographic analyses indicated that renal cells metabolized leukotriene C₃ more rapidly than hepatic and intestinal cells. Leukotriene D₃ and E₃ were formed during the incubations with kidney cells, whereas intestinal cells produced mainly more polar metabolites.     

Inhibition of aminoacyl-tRNA synthetases by p-chloroamphetamine and its role in protein synthesis inhibition

p-Chloroamphetamine inhibited to some degree all amino acid-dependent pyrophosphate-exchange activities which could be detected in a rabbit reticulocyte extract. A detailed kinetic analysis of the reaction catalyzed by reticulocyte leucyl-tRNA synthetase demonstrated that the inhibitor affected only amino acid binding. Less rigorous studies of other synthetases from both rabbit reticulocyte and Escherichia coli could be similarly interpreted, suggesting that this compound interacts in a common manner with these several enzymes. The contribution of such effects to the inhibition of protein synthesis by the drug was investigated using cell-free translation systems in which rates of amino acid incorporation were limited to varying degrees by the synthesis and availability of aminoacyl-tRNA. In a wheat germ system programmed with globin mRNA, in which levels of amino acids and aminoacyl-tRNAs were shown to limit the rate of protein synthesis, the inhibition produced by p-chloroamphetamine could be partially reversed by increasing the concentration of the limiting amino acid. In a reticulocyte lysate, in which amino acid concentrations were not limiting, inhibition failed to show an amino acid-reversible component. Thus, while the inhibition of aminoacyl-tRNA synthetases by amphetamines can be shown in some cases to play a role in the effects of these compounds on in vitro protein synthesis, other sites of interference with initiation and/ or elongation reactions may predominate, depending on the construction of the system under study.     

Two pools of beta-endorphin-like immunoreactivity in blood: plasma and erythrocytes

While attempting to delineate the reason for the reported extreme variability of beta-endorphin-like immunoreactivity (beta-ir) in human plasma (eg., nondetectable to 1 ng/ml) by standard radioimmunoassay, we noted that a substantial portion of circulating beta-ir was associated with erythrocytes. That erythrocyte associated beta-ir is authentic beta-endorphin (beta-EP) was confirmed by high performance liquid chromatography (HPLC). Analysis of blood samples from rabbits, rats and mice revealed the presence of beta-ir in erythrocytes from these species as well. These results suggest that there are two pools of beta-endorphin-like immunoreactivity in blood: plasma and erythrocytes.     

Characterization of bullous pemphigoid antigen: A unique basement membrane protein of stratified squamous epithelia

Bullous pemphigoid (BP) antigen is a normal basement membrane zone antigen of epidermis and other stratified squamous epithelia. It is defined immunologically by antibodies in the sera of patients with the subepidermal blistering disease BP. In this study we sought to demonstrate that epidermal cells synthesize this antigen, to determine the immunological specificity of BP antibodies and to characterize this antigen. Cultured human epidermal cells (HEC) and a spontaneously transformed mouse epidermal cell line (Pam) both demonstrated BP antigen by indirect immunofluorescence. To characterize the antigen, these cells were radiolabeled with ³⁵S-methionine or ¹⁴C-amino acids and extracts were immunoprecipitated using nine different BP sera. Immunoprecipitated proteins were identified using sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and fluorography. All nine BP sera precipitated a protein with disulfide-linked chains of apparent molecular weight approximately 220 kd. Eight normal human sera and six pemphigus vulgaris sera, as well as antibodies directed against fibronectin and laminin, did not precipitate this protein. Furthermore, it was not precipitated by BP sera from radiolabeled extracts of fibroblasts. The protein was soluble in Tris-HCI buffered saline but was not secreted into the culture medium. These studies demonstrate that BP antigen is synthesized by epidermal cells in culture, different patients with BP have antibodies against the same protein, and BP antigen can be identified on SDS-PAGE as a high molecular weight protein consisting of disulfide-linked chains of approximate molecular weight 220 kd.     

Induction of monocytic suppression after stimulation of peripheral human mononuclear cells with staphylococcal protein A and Staphylococcus aureus

Staphylococcal protein-A (SPA) and Staphylococcus aureus are known to be polyclonal human B-cell activators. It was noted that they induced plaque-forming-cell (PFC) responses lower than those induced by pokeweed mitogen (PWM) and the possibility of early triggering of a suppressor cell was investigated in the present series of experiments. Peripheral mononuclear cells (MNC) were passed through Sephadex G-10 columns to eliminate monocytes. The PFC responses to SPA and S. aureus were thereby increased. PWM-driven PFC responses are suppressed by the simultaneous presence of SPA in a dose-related way, if present in the early phases of the cultures. MNC precultured with SPA or S. aureus have the ability to suppress the PFC response of autologous MNC to PWM. Interestingly this suppressor cell activity was radiation resistant and could not be abrogated by treatment with anti-T-cell monoclonal antibody plus complement. The above experiments clearly demonstrate that the observed low PFC responses of MNC after stimulation with SPA and S. aureus are due to the induction of suppressor cells by these stimulants. The suppressor cells are apparently of monocytic origin.     

Inhibition of [3H]nitrendipine binding by phospholipase A2

Phospholipase A2 from several sources inhibited [3H]nitrendipine binding to membranes from brain, heart and ileal longitudinal muscle. The enzymes from bee venom and Russell's viper venom were most potent, having IC50 values of approximately 5 and 14 ng/ml, respectively, in all three membrane preparations. Inhibition of binding by bee venom phospholipase A2 was time- and dose-dependent. Mastoparan, a known facilitator of phospholipase A2 enzymatic activity, shifted the bee venom phospholipase A2 dose-response curve to the left. Pretreatment of brain membranes with bee venom phospholipase A2 (10 ng/ml) for 15 min caused a 2-fold increase in the Kd without changing the Bmax compared with untreated membranes. Extension of the preincubation period to 30 min caused no further increase in the Kd but significantly decreased the Bmax to 71% the value for untreated membranes. [3H]Nitrendipine, preincubated with bee venom phospholipase A2, was recovered and found to be fully active, indicating that the phospholipase A2 did not modify the ligand. It is concluded that phospholipase A2 acts on the membrane at or near the [3H]nitrendipine binding site and that phospholipids play a key role in the interactions of 1,4 dihydropyridine calcium channel antagonists with the dihydropyridine binding site.     

Interactions of erythrosin B (U.S. F,D & C Red 3) with rat cortical membranes

Erythrosin B inhibits Na, K-ATPase in rat brain tissue as demonstrated by studying glycoside binding, ATPase activity and ion fluxes. The potency of the noncompetitive inhibition of [³H]-ouabain binding by erythrosin B is influenced by glycoside concentration, monovalent cation concentration, and incubation time. [¹⁴C]- Erythrosin B binds to synaptic membranes prepared from rat cortex. Erythrosin B and some of its structural analogs inhibit both [³H]-ouabain and [¹⁴C]-erythrosin B binding, but ouabain and other glycosides do not inhibit the binding of [¹⁴C]-erythrosin B. Subcellular distributions of [³H]-ouabain and [¹⁴C]- erythrosin B binding in fractionated cortical tissue preparations are equivalent and parallel ATPase activity. The dissimilar response of [³H]-ouabain binding and [¹⁴C]-erythrosin B binding to changes in tissue preparation, incubation temperature, and partial solubilization of binding sites by deoxycholate (DOC) Suggests two separate binding sites for erythrosin and ouabain to rat cortical membranes.