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G Milligan

Publications and source records attributed to G Milligan.

At least 271 records · Page 15Linked to original sources
PubMed1987

Antibodies directed against synthetic peptides distinguish between GTP-binding proteins in neutrophil and brain.

Antisera AS/6 and 7, raised against a synthetic peptide KENLKDCGLF corresponding to the carboxyl-terminal decapeptide of transducin-alpha, react on immunoblots with purified transducin-alpha and with proteins of 40-41 kDa in all tissues tested. The latter represent one or more forms of Gi alpha but not Go alpha, since a synthetic peptide, KNNLKDCGLF, corresponding to the carboxyl-terminal decapeptide of two forms of Gi alpha blocks AS/6 and 7 reactivity with transducin-alpha and Gi alpha on immunoblots, whereas the corresponding Go-related peptide, ANNLRGCGLY, does not. Antisera LE/2 and 3, raised against the synthetic peptide LERIAQSDYI, corresponding to an internal sequence predicted by one form of Gi alpha cDNA (Gi alpha-2) and differing by 3 residues from the sequence of another form, Gi alpha-1, react strongly with a 40-kDa protein abundant in neutrophil membranes and with the major pertussis toxin substrate purified from bovine neutrophils. LE/2 and 3 reveal a relatively faint 40-kDa band on immunoblots of crude brain membranes or of purified brain Gi/Go. LE/2 and 3 do not react with transducin-alpha or Go alpha nor with the 41-kDa form of pertussis toxin substrate in brain, Gi alpha-1. These antisera distinguish between the major pertussis toxin substrates of brain and neutrophil and tentatively identify the latter as Gi alpha-2.

Amino Acid Sequence↗
PubMed1987

Differential distribution of signal-transducing G-proteins in retina.

We used specific antibodies in immunoblot studies of membrane fractions derived from bovine retina, and in immunohistochemical studies of sections of rat retina to determine the distribution of two guanine nucleotide binding proteins Go (a G-protein of unknown function discovered in the brain) and transducin, in retina. Both Go and transducin were readily detected in membranes derived from whole retina, and in crude rod outer segment membranes. Purification of rod outer segment membranes by sucrose density gradient centrifugation resulted in enrichment in transducin and depletion of Go immunoreactivity. Transducin-alpha immunoreactivity was localized to photoreceptor inner and outer segments and the outer nuclear layer. In contrast, Go-alpha immunoreactivity was localized in the inner and outer plexiform layers and ganglion cell layer. The results indicate that Go unlike transducin, is not associated with rod outer segment membranes and is therefore unlikely to function in phototransduction. Go is, however, relatively abundant in neural layers of retina where it may be involved in signal transduction.

Animals↗
PubMed1987

Chemically induced hypothyroidism produces elevated amounts of the alpha subunit of the inhibitory guanine nucleotide binding protein (Gi) and the beta subunit common to all G-proteins.

Adipocytes of hypothyroid rats display an increased responsiveness to agents which function by inhibiting the production of cyclic AMP. Anti-peptide antisera which selectively recognise the alpha subunit of the inhibitory guanine nucleotide binding protein (Gi) detected a 40 kDa polypeptide in adipocyte plasma membranes of both euthyroid and hypothyroid rats. Amounts of the alpha subunit of Gi were elevated some 2-fold in the hypothyroid preparations in comparison with the euthyroid controls, when equal amounts of membrane protein of the two treatments were examined. As cells from the hypothyroid animals contained 2.7 times as much membrane protein as those from the control animals, the amounts of alpha subunit of Gi are elevated some 5.6-fold per cell in adipocytes of the hypothyroid animals compared with the euthyroid controls. Amounts of the 36 kDa beta subunit of G-proteins were also elevated in plasma membranes of adipocytes of hypothyroid animals, in this case by some 50% when compared on a protein basis. These results provide direct evidence for alterations in the amounts of the subunits of Gi caused by the hypothyroid state.

Adipose Tissue↗
PubMed1987

Tissue-specific regulation of GTP-binding protein and muscarinic acetylcholine receptor levels during cardiac development.

A quantitative immunoblot assay was developed by using affinity-purified monospecific antibodies to quantitate levels of guanine nucleotide binding regulatory protein (G-protein) subunits in atria and ventricles during embryonic chicken cardiac development. The muscarinic acetylcholine receptor (mAChR) number was measured with [3H]quinuclidinyl benzilate. On day 10 of embryonic development (day 10E) there was no difference between the atrial and ventricular membrane concentrations of beta-subunit, G0 alpha subunit, or mAChR. The level of Gi alpha was found to be 44% greater in atria than in ventricles on day 10E. The atrial membrane concentration of beta-subunit increased 80% between day 13E and 15E, G0 alpha increased 46% between day 10E and 15E, mAChR increased 61% between day 10E and 12E, and Gi alpha decreased 34% between day 10E and 13E. The atrial levels of beta-subunit, G0 alpha, Gi alpha, and mAChR did not change further through day 20E. The ventricular membrane concentration of these proteins did not change between day 10E and 20E, except for that of G0 alpha, which increased 47% between day 15E and 20E. The atrial specific increase in beta-subunit correlated with a loss of GTP inhibition of basal adenylate cyclase activity. The difference in Gi alpha levels between atria and ventricles on day 10E correlated with a difference in carbachol sensitivity of atrial and ventricular basal adenylate cyclase activity. Thus, the levels of several components of the cholinergic neuroeffector pathway are regulated in a tissue-specific manner at a time that coincides with the onset of functional parasympathetic innervation of the embryonic chicken heart.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclase Inhibitors↗
PubMed1987

Foetal-calf serum stimulates a pertussis-toxin-sensitive high-affinity GTPase activity in rat glioma C6 BU1 cells.

Cellular proliferation of rat glioma C6 BU1 cells in tissue culture is dependent on the presence of either calf or foetal-calf serum in the medium. Foetal-calf serum stimulated a high-affinity GTPase in membranes derived from C6 BU1 cells. Pretreatment of the cells with pertussis toxin decreased the high-affinity GTPase activity substantially, and attenuated the foetal-calf-serum-stimulated increase in this GTPase activity. Cholera toxin, in contrast, did not modulate the response to foetal-calf serum. Foetal-calf serum did not inhibit adenylate cyclase activity in membranes of these cells, indicating that the G-protein that was stimulated by foetal-calf serum was not Gi (the inhibitory one). Although the nature of the specific component of foetal-calf serum responsible for this pertussis-toxin-sensitive receptor-mediated stimulation of high-affinity GTPase activity has not been identified, it was mimicked neither by bombesin, which can stimulate inositol phospholipid turnover via a guanine nucleotide binding protein, nor by platelet-derived growth factor, which is present in substantial concentrations in foetal-calf serum. This report represents the first demonstration of a pertussis-toxin-substrate-mediated response in this cell line and provides further evidence that G-proteins other than Gi can be functionally inactivated by pertussis toxin.

Adenosine Diphosphate↗
PubMed1987

Guanine nucleotide regulation of the pertussis and cholera toxin substrates of rat glioma C6 BU1 cells.

Rat glioma C6 BU1 cells contain a pertussis toxin substrate of 40 kDa which does not appear to be identical with Gi,Go or transducin. The GTP analogue, GTP[gamma S], inhibited the rate of pertussis toxin-catalysed ADPribosylation of this protein, while the GDP analogue GDP[beta S] stimulated this reaction. A protein of the same kDa value was ADPribosylated by cholera toxin in the absence of added guanine nucleotides. It is suggested that this 40 kDa protein can be a substrate for both cholera and pertussis toxins under appropriate conditions.

Adenosine Diphosphate Ribose↗
PubMed1987

Development of opiate receptors and GTP-binding regulatory proteins in neonatal rat brain.

The mu and delta opiate receptors present in rat brain were measured independently during postnatal development. The numbers of delta receptors were almost undetectable at birth and increased substantially during the first few weeks, whereas the numbers of mu receptors remained relatively constant. Activation of either of these receptors caused inhibition of adenylate cyclase, but inhibition coupled to mu receptors was much smaller than that associated with delta receptors at all ages. Attempts to use pertussis toxin-catalyzed ADP-ribosylation as an assay for the GTP-binding proteins Ni and No were hampered by the development of an NADase with age. However, specific antibodies directed against the alpha subunits of Ni or No allowed separate quantitation of these transducer proteins. Both increased with age. No is present at levels at least 5-fold higher than Ni in the adult rat brain. The N proteins are in vast excess over receptors and as such are unlikely to be limiting factors in receptor function. The data further suggest that the number of opiate receptors present throughout neonatal development is in excess over that required for optimal function.

Aging↗
PubMed1987

The D2-dopamine receptor of anterior pituitary is functionally associated with a pertussis toxin-sensitive guanine nucleotide binding protein.

Dopaminergic inhibition of prolactin release from the anterior pituitary may be mediated through both the adenylate cyclase and Ca2+ mobilization/phosphoinositide pathways. The D2-dopamine receptor of the bovine anterior pituitary has been partially purified by affinity chromatography on CMOS-Sepharose (immobilized carboxymethyleneoximinospiperone). Reinsertion of these partially purified receptor preparations into phospholipid vesicles reconstituted guanine nucleotide-sensitive high affinity agonist binding, agonist-promoted GTPase and 35S-labeled guanosine 5'-O-(thiotriphosphate) [( 35S]GTP gamma S) binding activity in these preparations. Pertussis toxin treatment of the purified receptor preparation abolished agonist-stimulated GTPase and guanine nucleotide-sensitive high affinity agonist binding. These observations suggest that the receptor copurifies with an endogenous, pertussis toxin-sensitive guanine nucleotide binding protein (N). [32P]ADP-ribosylation of affinity-purified D2 receptor preparations by pertussis toxin revealed the presence of a substrate of Mr 39,000-40,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Peptide maps generated using elastase of the [32P]ADP-ribosylated endogenous N protein, transducin, and Ni and No from brain revealed similarities but not identity between the endogenous pituitary N protein and brain Ni and No. Immunoblotting of the partially purified D2 receptor preparations showed an Mr 39,000-40,000 band with an Ni-specific antiserum raised against a synthetic peptide, and with RV3, an No-specific anti-serum, but not with CW6, an antiserum strongly reactive with brain Ni. Several lines of evidence indicate that endogenous pituitary N protein is functionally coupled to the D2 receptor. As measured by [35S]GTP gamma S binding, ratios of 0.2-0.6 mol N protein/mol receptor were observed. Association of N protein with the D2 receptor was increased by agonist pretreatment and decreased by guanine nucleotides. These results suggest that No and/or a form of Ni distinct from the Mr 41,000 pertussis toxin substrate (Ni) is the predominant N protein functionally coupled with the D2-dopamine receptor of anterior pituitary.

Adenylate Cyclase Toxin↗
PubMed1987

The insulin receptor tyrosyl kinase phosphorylates holomeric forms of the guanine nucleotide regulatory proteins Gi and Go.

An affinity purified human insulin receptor preparation was shown to phosphorylate the alpha- and beta-subunits of the guanine nucleotide-regulatory proteins Gi and Go, derived from bovine brain. The presence of insulin stimulated the rate of their phosphorylation some 2-fold. The presence of Gi and Go did not affect the degree of autophosphorylation of the beta-subunit of the insulin receptor. Under conditions known to cause the dissociation of Gi and Go into their constituent subunits then phosphorylation of Gi and Go by the insulin receptor was abolished. The alpha-subunits of Gi and Go could be selectively phosphorylated by the insulin receptor tyrosyl kinase using appropriate concentrations of Mg2+ and GTP-gamma-S.

Cell Membrane↗
PubMed1986

Detection of the major pertussis toxin substrate of human leukocytes with antisera raised against synthetic peptides.

Antisera raised against the carboxy-terminal decapeptide (KENLKDCGLF) of transducin-alpha detected the 40 kDa, major pertussis toxin substrate of human neutrophils. The antisera also detected this protein in undifferentiated HL-60 and U937 cells, and revealed an approx. 2-fold increase in protein/mg membrane protein with differentiation into mature phagocytic cells. The results provide direct immunochemical evidence for the presence of a novel, pertussis toxin-sensitive guanine nucleotide-binding protein in human leukocytes.

Antigen-Antibody Complex↗
PubMed1986

Immunochemical evidence for a novel pertussis toxin substrate in human neutrophils.

Pertussis toxin catalyzes incorporation of 20.2 pmol of ADP-ribose/mg of protein into approximately 40-kDa protein(s) in human neutrophil membranes compared with 14.1 pmol/mg in bovine brain membranes. Based on these measurements we estimate that pertussis toxin substrate(s) should represent at least 0.085% of total membrane protein in neutrophils. Both brain and neutrophil membranes show high concentrations (0.34 versus 0.16% of total membrane protein, respectively) of the common beta subunit of guanine nucleotide binding proteins. Affinity purified antibodies specific for Go-alpha fail to detect any protein in immunoblots of neutrophil membranes (150 micrograms) under conditions where as little as 10 ng of purified Go-alpha is detectable, and Go-alpha is readily detected in brain membranes (100 micrograms). An antiserum against transducin that cross-reacts strongly with Gi-alpha, detects as little as 5 ng of purified Gi-alpha and readily detects Gi-alpha in brain membranes, but in neutrophil membranes, the antiserum detects an approximately 40-kDa band that corresponds to less than 10% of the expected amount of pertussis toxin substrate(s). The results show that human neutrophil membranes contain relatively large amounts of pertussis toxin substrate(s), but that the predominant pertussis toxin substrate is immunochemically distinct from previously identified substrates, transducin, Gi, and Go.

Adenosine Diphosphate Ribose↗
PubMed1986

Changes in the guanine nucleotide-binding proteins, Gi and Go, during differentiation of 3T3-L1 cells.

Differentiation of 3T3-L1 cells from fibroblasts to adipocytes is accompanied by increased adenylate cyclase response to lipolytic agents. We used pertussis toxin and specific antibodies to measure the inhibitory guanine nucleotide-binding protein, Gi, and the novel G-protein, Go, in membranes from 3T3-L1 cells. Pertussis toxin-dependent labeling of a 39-40 kDa protein showed an initial 30% rise, followed by an 80% fall during differentiation. Immunoblots showed that 3T3-L1 cells contain Go, as well as Gi, and that changes in the former parallel the changes in pertussis toxin labeling. Changes in Gi and GO may contribute to altered adenylate cyclase response during 3T3-L1 cell differentiation.

1-Methyl-3-isobutylxanthine↗
PubMed1986

The GTP-binding regulatory proteins of neuroblastoma x glioma, NG108-15, and glioma, C6, cells. Immunochemical evidence of a pertussis toxin substrate that is neither Ni nor No.

Amounts of the guanine nucleotide binding regulatory proteins which are also pertussis toxin substrates (such as Ni and No) were measured in rat glioma, C6BU-1, cells and in neuroblastoma X glioma, NG108-15, hybrid cells. Measurements were performed both by quantitating pertussis toxin catalyzed ADP-ribosylation and by quantitative immunoblotting with affinity purified antibodies specific for Ni or No. The amounts of pertussis toxin substrate in C6 and NG108-15 cells are 7.5 and 0.6 pmol/mg membrane protein, respectively. These levels are minimum values and higher estimates of the total amounts of N proteins in the two cells are obtained by quantitative immunoblot analysis of the beta-subunit common to all N proteins. Immunoblots with specific antibodies show that NG108-15 cells contain 3.8 pmol/mg of No and detectable but small (less than 0.1 pmol/mg) amounts of Ni. In contrast, C6 cell membranes contain no detectable No and only 0.14 pmol/mg Ni. Thus, C6 cells contain large amounts of a pertussis toxin substrate which is neither Ni nor No.

Animals↗
PubMed1986

The beta subunit of the guanine nucleotide regulatory proteins: identification of its location on two-dimensional gels of brain tissue and its regional and subcellular distribution in brain.

The beta subunit of the guanine nucleotide regulatory proteins (also termed G proteins) has been examined in both rat and human brain. Proteins contained within samples of fresh rat and human brain tissue were separated by two-dimensional gel electrophoresis and either stained with silver or reacted with various antisera raised against the G proteins. In both rat and human brain, a single protein of molecular weight 36,000 daltons and pI 5.8 reacted the antisera. This protein also comigrated with one of the proteins present in a purified preparation of bovine brain G proteins. Based upon molecular weight, pI, and reaction with specific antisera, it was concluded that this protein is the beta subunit of the G proteins in brain. Using this information, the regional and subcellular distribution of the G protein beta subunit was studied in rat brain. Of 25 distinct neuroanatomical areas examined, cortical regions were generally found to contain the largest amount of this protein. The subcellular distribution of the G protein beta subunit revealed that large amounts are present in the synaptic membrane, crude synaptic vesicles, and microsomes. These studies serve to identify another protein visible on silver-stained two-dimensional electrophoretograms of rat and human brain. The regional and subcellular distribution of the G protein beta subunit correlate well with the proposed physiological function of this protein.

Animals↗
PubMed1986

Use of specific antibodies to quantitate the guanine nucleotide-binding protein Go in brain.

We immunized rabbits with purified guanine nucleotide-binding proteins (G proteins) from bovine brain and obtained an antiserum, RV3, that reacts specifically with the alpha subunit (39 kDa) of a G protein of unknown function, termed Go, as well as with the beta subunit (35 kDa) common to all G proteins. RV3 showed no crossreactivity with the alpha subunits of the stimulatory (Gs) or inhibitory (Gi) G proteins associated with adenylate cyclase, nor with that of the rod outer segment G protein, transducin. Immunoblots with crude and affinity-purified antiserum showed that RV3 specifically recognizes the Go alpha subunit and the beta subunit in crude brain membranes. Using RV3, we found approximately equal amounts of Go in brain membranes from frog, chicken, rat, cow, and man. Quantitative immunoblotting gave Go alpha subunit/ beta subunit ratios approximately equal to 1 in cerebral cortex, raising the possibility that free Go alpha subunit (unassociated with beta subunit) may exist in brain. The concentration of Go alpha subunit in cortex is about 5 times that of Gi alpha subunit. The results show that Go is an immunochemically distinct, highly conserved protein distributed throughout the brain, with particularly high concentrations in forebrain.

Animals↗
PubMed1985

The inhibitory guanine nucleotide-binding protein (Ni) purified from bovine brain is a high affinity GTPase.

Using modifications of the methods of Bokoch et al. (Bokoch, G.M., Katada, T., Northup, J. K., Ui, M., and Gilman, A. G. (1984) J. Biol. Chem. 259, 3560-3567) and Codina et al. (Codina, J., Hildebrandt, J. D., Sekura, R. D., Birnbaumer, M., Bryan, J., Manclark, C. R., Iyengar, R., and Birnbaumer, L. (1984) J. Biol. Chem. 259, 5871-5886), we have purified a pertussis toxin substrate with the expected characteristics of the inhibitory guanine nucleotide-binding protein (Ni) essentially to homogeneity. The purified protein consists of 3 subunits of Mr 40,000, 35,000, and less than 10,000. The Mr 40,000 band is found, upon close examination, to consist of a poorly resolved doublet. Starting with the membranes from 1,320 g of bovine forebrain we purified the protein some 100-fold with approximately 20% yield to obtain 13 mg of a greater than 95% pure protein. Chromatography on octyl-Sepharose provided efficient separation of Ni from Ns (the stimulatory guanine nucleotide-binding protein). Analytical ultracentrifugation indicates an Mr of 82,000 and a sedimentation coefficient S20,w of 5.1. The protein is able to restore opiate-mediated inhibition of adenylate cyclase to membranes prepared from NG 108-15 cells which had been treated with pertussis toxin. Bovine brain Ni has the enzymatic properties of a low Km GTPase with a turnover number of 0.3 and affinities for nucleotides in the order GppNHp greater than or equal to GTP greater than or equal to GDP much greater than ATP, CTP, UTP, and GMP. Na+ specifically stimulates the GTPase and low concentrations of Mg2+ (less than 50 microM) are inhibitory. Some Mg2+ is apparently necessary because EDTA, but not EGTA, abolishes the GTPase activity.

Adenosine Diphosphate Ribose↗