Purification and characterization of cGMP binding protein-phosphodiesterase from rat lung
The cGMP binding protein-phosphodiesterase (cG-BPP) with a phosphodiesterase specific activity of 7 ..mu..M/min/mg has been purified from rat lung by sequential chromatography on DEAE-cellulose, Blue-Sepharose, zinc chelate affinity adsorbent and HPLC-DEAE. Migration of the major band on SDS-PAGE corresponds to a MW of approx.93,000. Both cGMP phosphodiesterase activity and cGMP binding from the HPLC-DEAE profile correlate with this band. Since the authors previous work has determined the native MW to be approx.177,000, this suggests a dimeric structure comprised of two 93,000 MW subunits for the rat lung cG-BPP. At low cGMP concentrations, cGMP binding is stimulated approx.20-fold by histone and approx.5-fold by 3-isobutyl-1-methylxanthine(IBMX). The purified protein has one component of cGMP dissociation with a rate constant of 0.045/min. Photolysis of the purified protein in the presence of /sup 32/P-cGMP labels the 93,000 MW band and this labeling is increased by IBMX, indicating that the 93,000 MW band is a subunit of the cGMP-BPP. This implies that the enzyme preparation is nearly homogeneous, a conclusion also supported by a minimum (/sup 3/H)-cGMP binding stoichiometry of 0.5 mol per 93,000 subunit. An additional protein band with a MW of approx.90,000 also occurs in these preparations which exhibits behavior similar to the 93,000 MW protein. N/sup 2/-Hexyl-cGMP inhibits phosphodiesterase activity by competing with cGMP for hydrolysis at the catalytic site but not at the binding site. N/sup 2/-Hexyl cGMP actually increases cGMP binding. This provides the first evidence that cGMP binding is increased by compounds hydrolyzed at the catalytic site. This interaction between the binding and phosphodiesterase sites could be important in the regulation of the functions of these sites in vivo.
- Research Organization:
- Vanderbilt Univ. School of Medicine, Nashville, TN
- OSTI ID:
- 6992298
- Report Number(s):
- CONF-8606151-
- Journal Information:
- Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Vol. 45:6; Conference: 76. annual meeting of the Federation of American Society for Experimental Biology, Washington, DC, USA, 8 Jun 1986
- Country of Publication:
- United States
- Language:
- English
Similar Records
Activated G-protein releases cGMP from high affinity binding sites on PDE from toad rod outer segments (ROS)
cGMP is tightly bound to bovine retinal rod phosphodiesterase
Related Subjects
PHOSPHODIESTERASES
PURIFICATION
BIOCHEMICAL REACTION KINETICS
ELECTROPHORESIS
LIQUID COLUMN CHROMATOGRAPHY
LUNGS
PHOSPHORUS 32
RATS
STOICHIOMETRY
TRACER TECHNIQUES
ANIMALS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BODY
CHROMATOGRAPHY
DAYS LIVING RADIOISOTOPES
ENZYMES
ESTERASES
HYDROLASES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LIGHT NUCLEI
MAMMALS
NUCLEI
ODD-ODD NUCLEI
ORGANS
PHOSPHORUS ISOTOPES
RADIOISOTOPES
REACTION KINETICS
RESPIRATORY SYSTEM
RODENTS
SEPARATION PROCESSES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques