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E J Neer

Publications and source records attributed to E J Neer.

At least 91 records · Page 5Linked to original sources
PubMed1985

Physical and immunological characterization of a guanine nucleotide-binding protein purified from bovine cerebral cortex.

ADP-ribosylation by pertussis toxin has been used to identify the alpha subunit of Ni, the guanine nucleotide-binding protein which mediates hormone and GTP inhibition of adenylate cyclase. Two proteins have been purified from bovine cerebral cortex which are substrates for ADP-ribosylation by pertussis toxin, a 41-kDa protein (alpha 41) and a 39-kDa protein (alpha 39). The 41-kDa protein is very similar to the subunit of Ni purified from other tissues while the function of the 39-kDa protein is unknown (Neer, E. J., Lok, J. M., and Wolf, L. G. (1984) J. Biol. Chem. 259, 14222-14229; Sternweis, P. C., and Robishaw, J. D. (1984) J. Biol. Chem. 259, 13806-13813). We now show that the purified alpha 39 protein from bovine brain is a relatively hydrophilic protein which associates with a hydrophobic beta gamma component. The complex can be dissociated by guanosine 5'-(3-O-thio)triphosphate. The alpha 39 component binds guanosine 5'-(3-O-thio)triphosphate with a KD of 27 nM. We have developed polyclonal antibodies to alpha 39 and beta. The antibodies to alpha 39 cross-react weakly with alpha 41 in an immunoblot assay indicating some homology between the two proteins but making it unlikely that alpha 39 is derived from alpha 41. Using the antibodies for quantitation we found that alpha 39 is 0.5% and beta is 0.7% of membrane proteins. While the antibodies cross-react with alpha 39 and beta proteins in many different species, central nervous system tissues always have more immunoreactivity than membranes from peripheral organs. Anti-beta antibody recognizes the beta subunit when it is associated with alpha 39 or alpha 41 and can immunoprecipitate both alpha . beta gamma trimers. The guanine nucleotide-dependent dissociation of the alpha 39 . beta gamma trimer suggests that the complex could inhibit adenylate cyclase by liberating free beta gamma units. The function of alpha 39 may not, however, be exclusively to regulate adenylate cyclase but may include coupling hormone receptors to other effectors. Antibodies specific for alpha 39 and beta will be useful tools in determining the functions of alpha 39 and beta in hormone-responsive cells.

Animals↗
PubMed1985

Partial purification and characterization of a pp60v-src-related tyrosine kinase from bovine brain.

We have identified and substantially purified a tyrosine protein kinase from normal bovine brain that is immunologically related to the product of the Rous sarcoma virus oncogene (pp60v-src). The enzyme, a 61-kDa protein (p61), is solubilized with detergent from bovine cerebral cortical membranes and purified by column chromatography. In the purest preparations, this protein is phosphorylated only on tyrosine, but it can also be a substrate for serine- and threonine-specific protein kinases. The p61 protein phosphorylates the heavy chain of immunoglobulins from rabbits bearing Rous sarcoma virus-induced tumors (TBR IgG) but not normal IgG. TBR IgG precipitates the 61-kDa phosphoprotein and protein kinase activity from purified preparations. The activity of the purified brain tyrosine kinase is 10 times higher in the presence of 7-10 mM Mn2+ and 6 mM Mg2+ than it is with 6 mM Mg2+ alone. With Mn2+, the p61 enzyme has a Km for ATP of 2 microM. All preparations of p61 also contain a 64-kDa protein (p64) that is phosphorylated on tyrosine. Measurement of the Stokes radius of p61 and p64 by gel filtration shows that they are not physically associated in buffer containing the nonionic detergent Lubrol 12A9. The p64 protein is not precipitated by TBR IgG. We do not know whether p64 is only a substrate for the p61 tyrosine kinase or is itself a kinase.

Adenosine Triphosphate↗
PubMed1984

Purification and properties of the inhibitory guanine nucleotide regulatory unit of brain adenylate cyclase.

Hormonal inhibition of adenylate cyclase is mediated by a guanine nucleotide regulatory protein (Ni) which is different from the one which mediates hormonal stimulation. There is substantial evidence that the active component of Ni (termed alpha i can be ADP-ribosylated by a toxin from Bordetella pertussis. We have found that in bovine cerebral cortex there are three proteins of similar molecular weight (39,000-41,000) which are modified by pertussis toxin. We have purified these proteins and have resolved the 41,000-dalton protein from the 40,000/39,000-dalton doublet. All three forms of pertussis toxin substrate can be isolated in free form or together with a 36,000 beta component. We have also purified this beta component. ADP-ribosylation of the three pertussis toxin substrates is greatly enhanced by the addition of the purified beta component. This makes possible an assay of beta subunit activity based on its interaction with alpha i. The three forms of pertussis toxin substrate which we have purified differ in two functions: susceptibility to ADP-ribosylation and GTPase activity. The 41,000-dalton protein is more readily ADP-ribosylated by pertussis toxin than the smaller forms. The 39,000-dalton protein has GTPase activity with a low Km (0.3 microM) for GTP. The GTPase activity can be doubled by phospholipids. The GTPase activity of the 41,000-dalton protein is almost undetectable. It is not yet known what the relationship of the forms is to each other. The smaller forms may be derived from the larger by proteolysis or it may be intrinsically different. It remains to be shown whether one of the forms represents a different type of regulatory protein which transmits a hormonal signal to effectors other than adenylate cyclase.

Adenosine Diphosphate Ribose↗
PubMed1984

Reconstitution of a hormone-sensitive adenylate cyclase system. The pure beta-adrenergic receptor and guanine nucleotide regulatory protein confer hormone responsiveness on the resolved catalytic unit.

A hormone responsive adenylate cyclase has been reconstituted in phosphatidylcholine vesicles from its isolated protein components. The proteins used were the affinity chromatography purified (500-2000-fold) or pure Mr = 64,000 beta-adrenergic receptors (beta AR) isolated from hamster and guinea pig lung membranes, the pure heterotrimeric (Mr: alpha = 42,000; beta = 35,000; gamma approximately equal to 5,000) guanine nucleotide regulatory protein (Ns) isolated from human erythrocyte membranes; and the catalytic unit of the adenylate cyclase (C) solubilized from bovine brain caudate nucleus and resolved from beta AR and Ns by gel filtration. Adenylate cyclase activity in vesicles containing C alone was stimulated by forskolin but not by guanine nucleotides or by the beta-adrenergic agonist isoproterenol. Reconstitution of Ns and C interactions in the lipid vesicles resulted in guanine nucleotide but not beta-adrenergic agonist sensitivity. When beta AR was inserted together with Ns and C into lipid vesicles, the catalytic unit became responsive to beta-adrenergic agonists as well and this stimulation was blocked in a stereoselective manner by the beta-adrenergic antagonist alprenolol. Regulation of adenylate cyclase activity in the reconstituted system by beta-adrenergic agonists, guanine nucleotides, and Mg2+ showed properties similar to those observed in native membranes. The interactions of the various protein components in the reconstituted system were also monitored by GTPase activity. Such activity was observed to occur primarily as a result of receptor-Ns interactions. The results described in this report document the feasibility of studying hormone-responsive adenylate cyclase in a totally reconstituted system which retains the major regulatory properties of the enzyme in its native membrane-bound environment.

Adenylyl Cyclases↗
PubMed1982

Non-co-ordinate development of beta-adrenergic receptors and adenylate cyclase in chick heart.

We have studied the properties of beta-adrenergic receptors and of their interaction with adenylate cyclase in the chick myocardium during embryogenesis. Between 4.5 and 7.5 days in ovo the number of receptors determined by (-)-[3H]dihydroalprenolol ([3H]DHA) binding is constant at approx. 0.36 pmol of receptor/mg of protein. By day 9 the density decreases significantly to 0.22 pmol of receptor/mg of protein. At day 12.5--13.5 the number was 0.14--0.18 pmol of receptor/mg of protein. This number did not change further up to day 16. The same results were obtained with guanosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppG) added to the assay mixtures. There was no significant change in receptor affinity for the antagonist [3H]DHA between days 5.5 and 13. Despite the decrease in numbers of beta-adrenergic receptors, there was no change in basal, p[NH]ppG-, isoprenaline- or isoprenaline-plus-p[NH]ppG-stimulated adenylate cyclase activity between days 3 and 12 of development. We conclude that beta-adrenergic receptors and adenylate cyclase are not co-ordinately regulated during early embryonic development of the chick heart. Some of the beta-adrenergic receptors present very early in the ontogeny of cardiac tissue appear not to be coupled to adenylate cyclase since their loss is not reflected in decreased activation of the enzyme.

Adenylyl Cyclases↗
PubMed1981

Calmodulin activates the isolated catalytic unit of brain adenylate cyclase.

The catalytic and guanine nucleotide regulatory (G/F) units of solubilized bovine brain adenylate cyclase were separated by gel filtration as described by Strittmatter, S., and Neer, E. J. ((1980) Proc. Natl. Acad. Sci. U. S. A. 77, 6344-6348). The isolated catalytic unit is activated 4 +/- 1-fold (n = 11) by pure bovine brain calmodulin and is stabilized by calmodulin against thermal inactivation. The separated G/F unit can be freed of endogenous calmodulin by gel filtration in buffer containing 1 mM EDTA and no divalent cations. The calmodulin-free G/F unit still activates the catalytic unit. Re-addition of calmodulin does not affect the rate or extent of activation of the G/F unit by guanosine 5'-(beta, gamma-imino)triphosphate. The activation by calmodulin and the G/F unit together is additive, not synergistic. These studies show that calmodulin interacts with the adenylate cyclase catalytic unit but does not seem to affect the function of the G/F unit.

Adenylyl Cyclases↗
PubMed1980

Increase in the size of soluble brain adenylate cyclase with activation by guanosine 5'-(beta, gamma-imino)triphosphate.

Adenylate cyclase solubilized from bovine brain with Lubrol 12A9 or Triton X-100 can be resolved into two forms by gel filtration or sucrose density gradient centrifugation. The activity of one of these forms is not stimulatable by guanosine 5'-(beta, gamma-imino)triphosphate (Gpp(NH)p) and represents the "basal" adenylate cyclase activity. In Lubrol 12A9, this form has Mr = 330,000 (total, protein and detergent) and Mr (protein only) = 265,000. The other form of adenylate cyclase can be activated by Gpp(NH)p and has a smaller molecular weight: Mr (protein and detergent) = 293,000, Mr (protein only) = 199,000. Upon activation by Gpp(NH)p, the size of the Gpp(NH)p-responsive form of adenylate cyclase increases: Mr (protein and detergent) = 330,000, Mr (protein only) = 251,000. Similar values were obtained in Triton X-100. The kinetics of heat inactivation are different in the two forms of the enzyme. Both forms are activated about 2-fold by 5 mM MnCl2. Neither of the forms is associated with measurable low Km GTPase activity. On the basis of these studies, we propose that the catalytic unit (C) of adenylate cyclase and the guanine nucleotide regulatory unit (G/F) may exist in solution in the following rapid equilibrium: (formula: see text) We propose that activation by Gpp(NH)p stabilizes the C . G/F complex and this accounts for the greater mass of the enzyme measured after activation with Gpp(NH)p. The size of the enzyme which represents the basal activity is very similar to that of the C . G/F complex. We suggest, therefore, that the basal activity is the result of a stable association of the catalytic unit with the guanine nucleotide regulatory site.

Adenylyl Cyclases↗
PubMed1980

The site of alpha-chymotryptic activation of pigeon erythrocyte adenylate cyclase.

Treatment of intact pigeon erythrocytes with trypsin or alpha-chymotrypsin does not alter the isoproterenol-dependent adenylate cyclase activity in plasma membranes prepared after proteolysis. However, both proteases affect adenylate cyclase activity when isolated membranes are digested. Thus, the proteases probably act at the cytoplasmic side of the membranes. This conclusion is supported by the finding that proteases are able to inhibit NADH cytochrome c oxidoreductase, an enzyme located on the inner face of the plasma membrane. In isolated membranes, trypsin inhibits adenylate cyclase. Chymotrypsin (2.5 microgram/ml, 10 min, 37 degrees C) activates adenylate cyclase about 3-fold when the enzyme activity is measured with NaF, guanosine 5'-(beta, gamma-imino)-triphosphate, or guanosine 5'-(beta, gamma-imino)-triphosphate and isoproterenol. Chymotrypsin also activates adenylate cyclase in membranes pretreated with cholera toxin. Activation by chymotrypsin is not expressed when adenylate cyclase is assayed with 5 mM Mn2+ without guanine nucleotides or fluoride. However, the chymotryptic activation is expressed when guanosine 5'-(beta, gamma-imino)-triphosphate is present together with Mn2+. We conclude that interaction of the guanine nucleotide regulatory subunit with the catalytic subunit of adenylate cyclase is required for expression of chymotryptic activation.

Adenylyl Cyclases↗
PubMed1980

Properties of the separated catalytic and regulatory units of brain adenylate cyclase.

Adenylate cyclase from bovine brain cortex was solubilized with 14 mM cholate and 1 M (NH4)2SO4. Gel filtration over a column of Sepharose 6B separated the catalytic unit (CU) from a factor (G/F) that confers responsiveness to 5'-guanylyl imidophosphate (p[NH]ppG) or fluoride. The separated CU, which elutes with a Kav, of 0.48 +/- 0.01 (n=5), is not responsive to p[NH]ppG or fluoride and is relatively inactive when Mg . ATP is the substrate but activated 8-15-fold by Mn2+. The separated G/F elutes with a Kav of 0.70 +/- 0.02 (n=4). It restores the responsiveness of the CU to p[NH]ppG and fluoride. Activation of the enzyme by p[NH]ppG before solubilization does not decrease the amount of G/F eluting with a Kav of 0.7. Therefore, the G/F is probably present in brain cortex in excess over the CU. p[NH]ppG stabilizes the G/F but not the CU against thermal inactivation, suggesting that it interacts with G/F and not with CU. Incubation of the G/F with p[NH]ppG before addition of CU markedly increases the rate of activation of the reconstituted enzyme by p[NH]ppG. We propose, therefore, that the rate-limiting step in adenylate cyclase activation is a process in G/F alone and not a slow conformational change in CU or a slow association of G/F with CU. Binding of p[NH]ppG to the isolated G/F appears to be readily reversible; the ability of fully activated G/F to stimulate CU can be blocked if GDP is added before CU. In contrast, after the CU has been activated by interaction with G/F, GDP cannot reverse the activation. This suggests that association with the CU increases the affinity of G/F for p[NH]ppG.

Adenylyl Cyclases↗
PubMed1979

Separation of soluble adenylate and guanylate cyclases from the mature rat testis.

The mature rat testis contains both a soluble guanylate cyclase and a soluble adenylate cyclase. Both these soluble enzymes prefer manganous ion for activity. It is known that guanylate cyclase can, when activated by a variety of agents, catalyze the formation of cyclic AMP. The following experiments were performed to determine whether the testicular soluble adenylate and guanylate cyclase activities were carried on the same molecule. Analysis of supernatants from homogenized rat testis by gel filtration and sucrose density gradient centrifugation showed that the two activities were clearly separable. The molecular weight of guanylate cyclase is 143 000, while that of adenylate cyclase is 58 000. Treatment of the column fractions with 0.1 mM sodium nitroprusside allowed guanylate cyclase activity to be expressed with Mg(2+) as well as with Mn(2+). Sodium nitroprusside did not affect the metal ion or substrate specificity of adenylate cyclase. These experiments show that adenylate and guanylate cyclase activities are physically separable.

Adenylyl Cyclases↗
PubMed1978

Physical and functional properties of adenylate cyclase from mature rat testis.

The mature rat testis contains two forms of adenylate cyclase. One is membrane-bound and hormone-responsive; the other is water-soluble and not activated by hormones (Braun, T., and Dods, R. F. (1976) Pnoc. Natl. Acad. Sci. U.S.A. 72, 1097-1101). The water-soluble adenylate cyclase is a globular protein which is much smaller than the enzyme which is solubilized from membranes of mature rat testis by Triton X-100. Its physical properties are: sedimentation coefficient, 3.8 S; Stokes radius, 34 A; molecular weight, 56,000; frictional ratio, 1.2. Inclusion of protease inhibitors during enzyme extraction did not affect the fraction of total enzyme activity which was water-soluble, nor did autolysis for 1 h at 25 degrees C. The physical properties of the membrane-bound adenylate cyclase were determined after solubilization with Triton X-100. In detergent, the values are: sedimentation coefficient, 6.7 S; Stokes radius, 68 A; partial specific volume, 0.73 ml/g; molecular weight, 191,000; frictional ratio, 1.6. The fact that the partial specific volume of the enzyme in detergent is the same as that of a typical water-soluble protein shows that the Triton X-100-solubilized enzyme does not bind a large quantity of detergent. This indicates that it does not have extensive hydrophobic regions on its surface. The detergent-solubilized adenylate cyclase of the mature rat testis is similar in this respect to adenylate cyclase solubilized from the rat renal medulla (Neer, E. J. (1974) J. Biol. Chem. 249, 6527-6531).

Adenylyl Cyclases↗