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E J Neer

Publications and source records attributed to E J Neer.

104 records · Page 6Linked to original sources
PubMed1978

Size and detergent binding of adenylate cyclase from bovine cerebral cortex.

Adenylate cyclase from the bovine cerebral cortex has been solubilized with the nonionic detergent Triton X-100. The physical properties of the enzyme have been determined. These are: sedimentation coefficient, 8.1 S; Stokes radius, 70 A; partial specific volume, 0.79 ml/g; mass, 305,000 daltons; f/f0 1.5. The high partial specific volume measured in the presence of detergent indicates that the enzyme binds a large quantity of detergent (0.28mg of Triton X-100/mg of protein). The mass of the enzyme protein alone is 220,000 daltons. The size and detergent-binding properties of adenylate cyclase from bovine cerebral cortex are different from those previously reported for adenylate cyclase from the rat renal medulla (Neer, E. J. (1974) J. Biol. Chem. 249, 6527-6531). The renal enzyme is smaller by 60,000 daltons than the cerebral enzyme and does not bind measurable amounts of detergent. The amount of detergent bound by cerebral adenylate cyclase is characteristic of intrinsic membrane proteins. It suggests that there is a hydrophobic region on the surface of the molecule by which it is held in the plasma membrane.

Adenylyl Cyclases↗
PubMed1976

Two soluble forms of guanosine 5'-(beta,gamma-imino)triphosphate and fluoride-activated adenylate cyclase.

Soluble and membrane-bound adenylate cyclase from the canine renal medulla are very slowly activated by guanosine 5'-(beta,gamma-imino)triphosphate (Gpp(NH)p) or fluoride. At 25 degrees, 8 to 10 h of incubation of the soluble enzyme, and 6 to 8 h of incubation of the membrane-bound enzyme are required to reach the maximal activity. The dependence of activation on concentration of Gpp(NH)p changes with time. Half-maximal activation of soluble adenylate cyclase occurs at 3 +/- 1 X 10(-6) M Gpp(NH)p if the activity is measured without preincubation with Gpp(NH)pp and at 4 +/- 2 X 10(-8) M if it is measured after 6 to 22 h of incubation with Gpp(NH)p at 25 degrees. The activation occurs over a rather broad range of nucleotide concentration and cannot, therefore, be simply interpreted to reflect a function of nucleotide binding. Two forms of soluble adenylate cyclase are resolved by Sepharose gel filtration. One form, with a Stokes radius of 71 A, is rapidly activated by Gpp(NH)p; the other, with a Stokes radius of 56 A, requires long incubation with Gpp(NH)p to be activated. The sedimentation coefficient of the two forms is 7.3 S. The apparent molecular weight of the rapidly activated form is 200,000 while that of the slowly activated form is 160,000. The interrelationship of these two species of adenylate cyclase is not clear and is under investigation.

Adenylyl Cyclases↗
PubMed1976

The size of adenylate cyclase and guanylate cyclase from the rat renal medulla.

The size distribution of adenylate cyclase from the rat renal medulla solubilized with the nonionic detergents Triton X-100 and Lubrol PX was determined by gel filtration and by centrifugation in sucrose density gradients made up in H2O or D2O. The physical parameters of the predominant form in Triton X-100 are s20,w, 5.9S; Strokes radius, 62 A; partial specific volume (v), 0.74 ml/g; mass, 159,000 daltons; f/f0, 1.6; axial ratio (prolate ellipsoid), 11. For the minor form the values are: s20w, 3.0; Stokes radius, 28 A; mass, 38,000 daltons; f/f0, 1.2. The corresponding values determined in Lubrol PX are similar. The value for V for the enzyme indicates that it binds less than 0.2 mg detergent/mg protein. Since interactions with detergents probably substitute for interactions with lipids and hydrophobic amino acid side chains, these findings suggest that no more than 5% of the surface of adenylate cyclase is involved in hydrophobic interactions with other membrane components. Thus, most of the mass of the enzyme is not deeply embedded in the lipid bilayer of the plasma membrane. Similar studies have been performed on the soluble guanylate cyclase of the rat renal medulla. In the absence of detergent, the molecular properties of this enzyme are: s20w, 6.3S; Stokes radius, 54 A, V, 0.75 ml/g; mass, 154,000 daltons f/f0, 1.4; Axial ratio, 7. The addition of 0.1% Lubrol PX to this soluble enzyme increases it activity two- to fourfold and changes the physical properties to: s20,w, 5.5S; Stokes radius, 62 A; V, 0.74 ml/g; mass, 148,000 daltons, f/f0, 1.6; axial ratio, 11. These results show that Lubrol PX activates the enzyme by causing a conformational change with unfolding on the polypeptide chain. Guanylate cyclase from the particulate cell fraction can be solubilized with Lubrol PX but has properties quite different from those of the enzyme in the soluble cell fraction. It is a heterogeneous aggregate with s20,w, 10S; Stokes radius, 65 A; mass about 300,000 daltons. The conditions which solubilize guanylate cyclase also solubilize adenylate cyclase and the two activities can be separated on the same sucrose gradient.

Adenylyl Cyclases↗
PubMed1975

Guanylate cyclase from the rat renal medulla. Physical properties and comparison with adenylate cyclase.

Guanylate cyclase from the rat renal medulla is found in both the soluble and particulate fractions of the cell. Sucrose density gradient centrifugation and gel filtration in H2O and D2O indicate that the enzyme from the soluble cell fraction has the following properties: S20w, 6.3 S; Stokes radius, 54 A; partial specific volume, 0.75 ml/g; mass, 154,000 daltons; f/fo, 1.4; axial ratio (prolate ellipsoid), 7. The addition of 0.1% Lubrol PX to this fraction activates the enzyme and changes thartial specific volume, 0.74 ml/g; mass, 148,000 daltons; f/fo, 1.6; axial ratio (prolate ellipsoid), 11. These findings show that detergent activates the enzyme by changing its conformation and not simply by dispersing nonsedimentable membrane fragments. The dimensions of this guanylate cyclase in detergent are very similar to those of detergent-solubilized adenylate cyclase from the same tissue (Neer, E.J. (1974) J. Biol. Chem. 249, 6527-6531). Guanylate cyclase can be solubilized from the particulate cell fraction with 1% Lubrol PX but has properties quite different from those of the guanylate cyclase in the soluble cell fraction. It is a large aggregate with a value of S20,w of about 10 S, Stokes radius of 65 A, and a mass of approximately 300,000 daltons. However, the peaks of guanylate cyclase activity in column effluents and sucrose density gradients are very broad indicating a mixture of different size proteins. The conditions used to solubilize guanylate cyclase from the particulate fraction also solubilize adenylate cyclase, and the two activities can be separated on the same sucrose gradient. Studies of this sort require a rapid, accurate guanylate cyclase assay. We have developed an assay for guanylate cyclase activity which meets these criteria by adapting the competitive protein binding assay for guanosine cyclic 3':5' monophosphate originally described by Murad et al. (Murad, F., Manganiello, V., and Vaughn, M. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 736-739).

Adenylyl Cyclases↗
PubMedDate not supplied

The beta gamma subunits of GTP-binding proteins activate the muscarinic K+ channel in heart.

Subunits of guanine nucleotide regulatory proteins purified from bovine cerebral cortex were used to perfuse the intracellular surface of excised patches of chick embryonic atrial cells. Single-channel current measurements unexpectedly indicate that the beta gamma, and not the alpha subunits, are responsible for activating the muscarinic-gated potassium channel.

Animals↗