cGMP-binding Protein Research Articles

Identification of cyclic guanosine monophosphate-binding proteins in Drosophila by direct photoaffinity labeling

A procedure for direct photoaffinity labeling with [ 32P]cGMP has been used to identify cGMP-binding proteins in Drosophila. This method provides better sensitivity and resolution than previously described direct methods, because the proteins can be visualized by autoradiography following sodium dodecyl sulfate-gel electrophoresis. Labeling is observed with cGMP concentrations as low as 4 × 10 −8 m and is specific for cGMP. The sensitivity of the technique is sufficient to permit detection of cGMP-binding proteins in crude extracts. With this technique a single cytoplasmic cGMP-binding protein of subunit M r 108,000 has been identified in Drosophila embryos and cultured cells.

Identification of cGMP-stimulated cyclic nucleotide phosphodiesterase in lung tissue with monoclonal antibodies.

Monoclonal antibodies (CGS-1, 2, 3, and 4) and rabbit antisera have been produced against bovine heart cyclic GMP-stimulated cyclic nucleotide phosphodiesterase. The monoclonal antibodies were all of the IgG1 subclass and had relatively high affinities (kd values, 1-5 X 10(-10) M), making them suitable for many sensitive immunological and analytical procedures. Most of the monoclonal antibodies did not inhibit the enzyme, which allowed a rapid and specific assay for this form of phosphodiesterase to be developed. All of the activity could be adsorbed from purified preparations of cGMP-stimulated phosphodiesterase by each of the antibodies indicating antigenic homogeneity of the cGMP-stimulated phosphodiesterase. Neither the monoclonal antibodies nor the antisera inhibited cGMP-binding to the phosphodiesterase. Substrate specificity, Electrophoresis, and cyclic GMP binding studies carried out on immunoadsorbed material indicated that a major part of the cyclic nucleotide phosphodiesterase activity present in bovine lung was due to the cyclic GMP-stimulated form. The presence of this enzyme was difficult to identify by other more common procedures and has not been previously demonstrated in this tissue. These studies also indicated that the cGMP-stimulated phosphodiesterase was immunologically distinct from the other major cGMP-binding proteins present in lung extract and that it could be separated from these binding proteins by chromatography on DEAE-cellulose. Finally, the data suggest that none of the other peaks of cyclic nucleotide phosphodiesterase activity, seen upon DEAE-cellulose fractionation of lung extracts, contain the immunological determinants present on the cGMP-stimulated form which are recognized by the antibodies.

Nonequilibrium kinetics of a cyclic GMP-binding protein in Dictyostelium discoideum.

Chemoattractants added to cells of the cellular slime mold dictyostelium discoideum induce a transient elevation of cyclic GMP levels, with a maximum at 10 s and a recovery of basal levels at approximately 25 s after stimulation. We analyzed the kinetics of an intracellular cGMP binding protein in vitro and in vivo. The cyclic GMP binding protein in vitro at 0 degrees C can be described by its kinetic constants K(1)=2.5 x 10(6) M(- 1)s(-1), k(-1)=3.5 x 10(-3)s(-1), K(d)=1.4 x 10(-9) M, and 3,000 binding sites/cell. In computer simulation experiments the occupancy of the cGMP binding protein was calculated under nonequilibrium conditions by making use of the kinetic constants of the binding protein and of the shape of the cGMP accumulations. These experiments show that under nonequilibrium conditions by making use of the kinetic constants of the binding protein and the shape of the cGMP accumulations. These experiments show that under nonequilibrium conditions the affinity of the binding protein for cGMP is determined by the rate constant of association (k(1)) and not by the dissociation constant (k(d)). Experiments in vivo were performed by stimulation of aggregative cells with the chemoattractant cAMP, which results in a transient cGMP accumulation. At different times after stimulation with various cAMP concentrations, the cells were homogenized and immediately thereafter the number of binding proteins which were not occupied with native cGMP were determined. The results of these experiments in vivo are in good agreement with the results of the computer experiments. This may indicate that: (a) The cGMP binding protein in vivo at 22 degrees C can be described by its kinetic constants: K(1)=4x10(6)M(-1)s(-1) and K(-1)=6x10(-3)s(-1). (b) Binding the cGMP to its binding protein is transient with a maximum at about 20-30 s after chemotactic stimulation, followed by a decay to basal levels, with a half-life of approximately 2 min. (c) The cGMP to its binding proteins get half maximally occupied at a cGMP accumulation of delta[cGMP](10)=2x10(-8) M, which corresponds to an extracellular stimulation of aggregative cells by 10(-10) M cAMP. (d) Since the mean basal cGMP concentration is approximately 2x10(-7) M, the small increase of cGMP cannot be detected accurately. Therefore the absence of a measurable cGMP accumulation does not argue against a cGMP function. (e) There may exist two compartments of cGMP: one contains almost all the cGMP of unstimulated cells, and the other contains cGMP binding proteins and the cGMP which accumulates after chemotactic stimulation. (f) From the kinetics of binding, the cellular responses to the chemoattractant can be divided into two classes: responses which can be mediated by this binding protein (such as light scattering, proton extrusion, PDE induction, and chemotaxis) and responses which cannot be (solely) mediated by this binding protein such as rlay, refractoriness, phospholipids methylation, and protein methylation.

Characterization of a novel cGMP binding protein from rat lung.

A specific cGMP binding protein from rat lung has been partially purified and characterized. The binding protein is clearly distinct from the cGMP-dependent protein kinase as determined by several of its characteristics such as chromatographic behavior and spatial requirements for cGMP binding. The protein has a sedimentation coefficient of 7.8 S, a Stokes radius of 5.5 nm, and an estimated M, = 177,000. A cGMP phosphodiesterase activity co-purifies with the binding protein through several steps (ion exchange chromatography, gel filtration, and sucrose density gradient centrifugation) and the possibility exists that these two activities may be functions of the same protein. Several lines of evidence indicate that the cGMP binding site is distinct from the phosphodiesterase catalytic site. First, 1-methyl-3-isobutylxanthine is a competitive inhibitor of cGMP for phosphodiesterase activity, while under similar conditions this compound increases cGMP binding. Other phosphodiesterase inhibitors also increase cGMP binding and these effects are not due to substrate protection. Second, the cGMP analogue 2’-0monobutyryl cGMP is an effective inhibitor of cGMP phosphodiesterase activity, but at the same concentration this compound has no effect on cGMP binding. Third, heat or trypsin treatment of the protein causes loss of cGMP phosphodiesterase activity, while both treatments increase cGMP binding. The characteristics of the cGMP binding protein-phosphodiesterase and its presence in several cell types, ie. sea urchin sperm and rat platelets, which appear to contain very low levels of cGMP-dependent protein kinase, suggest that this protein may have a physiological role in mediating cGMP action.

A simple and sensitive competitive protein-binding assay for cyclic GMP

A simple and sensitive competitive protein-binding assay for cyclic GMP (cGMP) has been developed, using a specific cGMP-binding protein isolated from rat lungs. The contamination of cAMP-binding protein was partially removed by fractionated precipitation with ammonium sulfate. The most active binding was observed when cGMP-binding protein was extracted with phosphate buffer (50 mM) containing 2-mercaptoethanol. Magnesium (40 mM) and zinc (4 and 40 mM) ions enhanced the binding of cGMP to the protein. The amount of protein which bound to cGMP decreased when the protein was extracted with buffer but without 2-mercaptoethanol. The binding constant of this protein was 5.5–16.3 × 10 −9 M. The sensitivity of the assay was 0.2 picomole of cGMP. Other nucleotides exerted little or no interference.