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D R Manning

Publications and source records attributed to D R Manning.

50 records · Page 3Linked to original sources
PubMed1987

Structural characteristics of the 35- and 36-kDa forms of the beta subunit common to GTP-binding regulatory proteins.

The beta subunit common to GTP-binding regulatory proteins can be resolved into immunologically distinct proteins typically referred to as 35- and 36-kDa forms of the subunit. The extent to which these two forms are structurally related was examined by isoelectric focusing, nonequilibrium pH gradient electrophoresis (NEPHGE), and two-dimensional peptide mapping. Each of the two forms of the beta subunit isolated from rabbit liver was found to comprise species of protein having isoelectric points of 5.7, 5.8, and 5.95; those having an isoelectric point of 5.8 were the most prominent. The beta subunits isolated from bovine brain and rod outer segments exhibited species having identical isoelectric points. Similar patterns of species were also noted for forms of the beta subunit detected immunologically within diverse types of cells. Analysis of the 35- and 36-kDa forms of the beta subunit by NEPHGE confirmed the absence, with only minor exceptions, of other constituent proteins. Subjection of chymotryptic peptides to high voltage electrophoresis and chromatography on cellulose revealed similarities, as well as differences, between the two forms of the beta subunit. These data provide evidence that the 35- and 36-kDa forms of the beta subunit are indeed structurally related and are conserved among diverse types of cells.

Animals↗
PubMed1986

Alterations in components of adenylate cyclase associated with transformation of chicken embryo fibroblasts by Rous sarcoma virus.

Regulation of adenylate cyclase coincident with transformation of chicken embryo fibroblasts by Rous sarcoma virus is manifest as a 10-50% decrease in basal, Mg2+-, and forskolin-stimulated activities; activities elicited by fluoride and guanosine 5'-O-(3-thiotriphosphate) are unaltered. The level of the catalytic component of adenylate cyclase, assessed with activated stimulatory guanine nucleotide-binding protein (Gs), increases approximately 1.5-fold. The level of the beta subunit common to Gs and the inhibitory regulatory protein assessed by enzyme-linked immunotransfer blotting, increases 2.7-fold. The isoelectric behavior of the beta subunit is unaltered. The amount of radiolabel incorporated into the alpha subunit of Gs (Mr = 45,000) upon incubation of membranes with 32P-labeled NAD and cholera toxin increases 3-fold upon transformation. Detergent extracts prepared from membranes of untransformed and transformed fibroblasts nevertheless exhibit equivalent abilities to reconstitute fluoride-stimulated activities to membranes of the cyc-variant of mouse S49 lymphoma cells. Islet-activating protein catalyzes incorporation of radiolabel from 32P-labeled NAD into 39,000- and 41,000-dalton proteins; the extent of radiolabel incorporation does not change upon transformation. Modest alterations in the isoelectric behaviors of substrates for cholera toxin and islet-activating protein occur.

Adenylate Cyclase Toxin↗
PubMed1986

Antisera of designed specificity for subunits of guanine nucleotide-binding regulatory proteins.

Antisera were raised against purified subunits of regulatory GTP-binding proteins (G proteins) and against synthetic peptides that correspond to defined regions of G proteins. Peptide antisera were generated that recognized all alpha or all beta subunits from Gs, Gi, Go, and transducin; others recognized only Gs alpha or Go alpha. Such cross-reaction or complete specificity for a given alpha subunit was not obtained when purified subunits were injected. Peptide antisera were used to identify G protein subunits in selected tissue membrane preparations by immunoblots.

Animals↗
PubMed1986

Fractionation of the beta subunit common to guanine nucleotide-binding regulatory proteins with the cytoskeleton.

Gs and Gi are guanine nucleotide-binding proteins that mediate the stimulation and inhibition, respectively, of adenylate cyclase. The extent to which the beta subunit common to these proteins may be associated with the cytoskeleton of S49 mouse lymphoma cells was assessed by procedures of differential detergent extraction and immunotransfer blotting. Treatment of cells with 1% Triton X-100 results in nearly quantitative extraction of cellular protein, phospholipid, tubulin, and the catalytic component of adenylate cyclase. Approximately 65% of the beta subunit is refractory to extraction. This population of the beta subunit, along with a population of actin presumed to originate from actin filaments, is subsequently solubilized with hypotonic medium containing 0.5% sodium deoxycholate and 1% Tween-40. The pattern of distribution of the beta subunit among sequential detergent extracts is corroborated by that of generated immunoreactive tryptic fragments. These results are consistent with the interaction of guanine nucleotide-binding proteins with the cytoskeleton.

Adenylyl Cyclases↗
PubMed1985

Physical properties of chicken erythrocyte HMG-1, HMG-2 and HMG-E.

HMG-1, HMG-2 and HMG-E were purified from chicken erythrocyte chromatin without exposure to overt denaturing conditions and subjected to several types of physical measurement. The principal conclusions drawn from the measurements were: none of the proteins has a strong tendency to self-associate, although HMG-1 does weakly self-associate; the frictional properties of HMG-1 and HMG-E (and probably HMG-2) indicate that the proteins deviate significantly from compact, moderately hydrated spheres; and each of the proteins contains approximately 40% helix and little if any beta-pleated sheet.

Animals↗
PubMed1984

ADP-ribosylation of transducin by islet-activation protein. Identification of asparagine as the site of ADP-ribosylation.

Islet-activating protein catalyzes the ADP-ribosylation of transducin, a guanine nucleotide-binding regulatory protein that mediates activation of a retinal cyclic GMP-selective phosphodiesterase. Radiolabel from [adenylate-32P]NAD+ was incorporated specifically into the alpha subunit of purified transducin. Maximal levels of incorporation approximated 0.8 mol of ADP-ribose/mol of transducin. A peptide containing the ADP-ribosyl moiety was purified from a tryptic digest of radiolabeled transducin. This peptide was characterized by chemical and enzymatic procedures and by fast atom bombardment mass spectrometry. The primary structure of this peptide was Glu-Asn-Leu-Lys-Asn(ADP-ribose)-Gly-Leu-Phe. It is probable that the peptide originated from the carboxyl terminus of the alpha subunit and that the ADP-ribosyl moiety is attached by an N-glycosidic linkage to the asparagine residue. Transducin associated with retinal disc membranes is also ADP-ribosylated by cholera toxin. Cholera toxin and islet-activating protein sequentially catalyze the incorporation of 1.9 mol of ADP-ribose/mol of transducin, indicating two distinct sites of ADP-ribosylation within transducin.

Adenosine Diphosphate Ribose↗
PubMed1983

The regulatory components of adenylate cyclase and transducin. A family of structurally homologous guanine nucleotide-binding proteins.

G/F and transducin are guanine nucleotide-binding regulatory proteins that mediate activation of adenylate cyclase and of a rod outer segment cyclic GMP-specific phosphodiesterase, respectively. The substrate for islet-activating protein is a third guanine nucleotide-binding protein that is postulated to mediate inhibition of adenylate cyclase. The extent of structural homology among subunits of all three proteins was examined by analyses of amino acid compositions and electrophoretic patterns of proteolytic peptides. The lower molecular weight subunits (beta subunits; Mr = 35,000) of these proteins have identical amino acid compositions and yield similar peptides upon proteolysis with Staphylococcus aureus V8 protease and elastase. The higher molecular weight subunits (alpha subunits; Mr = 39,000, 41,000, and 45,000) are also similar to each other in these respects. Similarity between the subunits of transducin and the islet-activating protein (IAP) substrate is especially evident. Substantial differences do, however, exist between the lower and higher molecular weight subunits within each protein. In addition, evidence was obtained that the 41,000-Da subunit of the IAP substrate is not derived from the 45,000-Da subunit of G/F. It is concluded that transducin, the IAP substrate, and G/F represent a family of structurally homologous guanine nucleotide-binding regulatory proteins.

Adenylate Cyclase Toxin↗
PubMed1982

Myosin light chain phosphorylation-dephosphorylation in mammalian skeletal muscle.

Phosphorylation of the myosin light chain 2 (LC2) subunit was examined in rat fast-twitch and slow-twitch skeletal muscles in response to repetitive stimulation at 23 and 35 degrees C and on incubation of fast-twitch skeletal muscle with isoproterenol. After a 1-s tetany at 35 degrees C, LC2 phosphate content in extensor digitorum longus muscle increased rapidly and transiently from 0.21 to 0.51 mol phosphate/mol LC2. This pattern of phosphorylation was similar to that observed at 23 degrees C. Increases in LC2 phosphate content were dependent on the frequency and duration of stimulation. In soleus muscle LC2 phosphate content was minimal following a 1-s tetany but increased markedly following more prolonged tetanies. On incubation of extensor digitorum longus muscle with isoproterenol (20 microM), LC2 phosphate content did not change, whereas phosphorylase a levels increased. A positive correlation existed between LC2 phosphate content and potentiation of peak twitch tension in both types of muscles, suggesting a physiological function for LC2 phosphorylation.

Action Potentials↗
PubMed1980

Protein phosphorylation: quantitative analysis in vivo and in intact cell systems.

Protein phosphorylation-dephosphorylation appears to be an essential component in the regulation of many cellular processes by hormones and drugs. This concept has developed primarily from in vitro biochemical studies in which various purified proteins have been phosphorylated and dephosphorylated by distinct protein kinases and phosphoprotein phosphatases. However, the more difficult, but essential, task of demonstrating the physiological occurrence of these reactions in intact tissue or cell preparations in many cases has not been undertaken in a quantitative manner. There are 4 basic approaches for assessing the extent of protein phosphorylation in vivo and in intact cell systems, each having particular advantages and disadvantages. These are summarized in Table 2. The applicability of any one procedure will be highly dependent upon the protein under investigation. For instance, chemical measurements of total protein-bound phosphate may provide only limited information for proteins which are phosphorylated at multiple sites but could be highly useful for those proteins such as glycogen phosphorylase which are phosphorylated at single sites. The relative ease and the high sensitivity of measuring 32P incorporation into proteins will tempt many investigators to rely heavily on this approach. It is a very powerful procedure, particularly for the initial identification of phosphoproteins, but ultimately quantitative conclusions regarding 32P incorporation must be corroborated by one or more of the other procedures. There is no simple, single experimental approach that may be used under all circumstances, but by integrating these procedures firm conclusions may be drawn regarding the physiological importance of phorphorylation of specific proteins.

Adenosine Diphosphate↗
PubMed1980

Phosphorylation of contractile proteins in heart and skeletal muscle.

Contractile performance of cardiac and skeletal muscles may be regulated by cyclic AMP or Ca2+, two second messengers that stimulate the phosphorylation of specific myofibrillar proteins. Cyclic AMP-dependent protein kinase catalyzed the rapid phosphorylation of a single site in the inhibitory subunit of cardiac troponin in vitro and in perfused hearts. Skeletal muscle troponin was not phosphorylated by this enzyme in vivo. Although there was a correlation between cardiac troponin phosphorylation and the positive inotropic response to catecholamines, a biochemical mechanism that could account for a functional relationship between the two processes has not been discovered. Phosphorylation of skeletal muscle myosin was catalyzed by myosin light chain kinase in the presence of Ca2+ and the ubiguitous, multifunctional Ca2+-dependent regulator protein (CDR). The activation of kinase activity appeared to proceed via a trimolecular reaction process in which Ca2+ bound to CDR and the Ca2+.CDR complex then interacted with the enzyme. In rat extensor digitorum longus muscle, a 1 sec tetanic contraction resulted in phosphorylation of myosin light chain with the maximal phosphate incorporated 20 sec after the contraction. The light chain phosphate content declined slowly and correlated to post-tetanic potentiation of isometric twitch tension. Phosphorylation of skeletal muscle myosin may be important in modulating contraction.

Animals↗
PubMedDate not supplied

Identification of a G-protein in depolarizing rod bipolar cells.

Synaptic transmission from photoreceptors to depolarizing bipolar cells is mediated by the APB glutamate receptor. This receptor apparently is coupled to a G-protein which activates cGMP-phosphodiesterase to modulate cGMP levels and thus a cGMP-gated cation channel. We attempted to localize this system immunocytochemically using antibodies to various components of the rod phototransduction cascade, including Gt (transducin), phosphodiesterase, the cGMP-gated channel, and arrestin. All of these antibodies reacted strongly with rods, but none reacted with bipolar cells. Antibodies to a different G-protein, G(o), reacted strongly with rod bipolar cells of three mammalian species (which are depolarizing and APB-sensitive). Also stained were subpopulations of cone bipolar cells but not the major depolarizing type in cat (b1). G(o) antibody also stained certain salamander bipolar cells. Thus, across a wide range of species, G(o) is present in retinal bipolar cells, and at least some of these are depolarizing and APB-sensitive.

Amino Acid Sequence↗
PubMedDate not supplied

G proteins. Linkages with amine receptors.

Means of defining specific linkages among receptors and guanosine triphosphate (GTP)-binding regulatory proteins (G proteins) are discussed. The definition of such linkages relies upon the development of suitable G protein-directed antibodies and methods of resolving closely related subtypes of subunits. Conventional means of assessing interactions among receptors and G proteins are described, with an emphasis placed upon their application to receptors for serotonin. Emerging protocols are based upon agonist-effected decreases (or increases) in adenosine diphosphate (ADP) ribosylation, purification of receptor-G protein complexes, disruption of communication among receptors and G proteins, and activation-related alterations in protein conformation. Additional determinants of specificity are cotranslational and posttranslational modifications.

Animals↗