Proteolytic activation of membrane-bound guanylate cyclase

2001 ◽  
Vol 61 (7) ◽  
pp. 915-920 ◽  
Author(s):  
Zi-Jiang Chen ◽  
Dong-Li Song ◽  
Zhenhua Miao ◽  
Chung-Ho Chang
Keyword(s):  
Guanylate Cyclase ◽  
Proteolytic Activation ◽  
Membrane Bound

scholarly journals Proteolytic activation and solubilization of endoplasmic-reticulum cyclic AMP phosphodiesterase activity

1983 ◽  
Vol 213 (1) ◽  
pp. 99-105 ◽  
Author(s):  
S R Wilson ◽  
M D Houslay
Keyword(s):  
Endoplasmic Reticulum ◽  
Proteinase Inhibitors ◽  
Smooth Endoplasmic Reticulum ◽  
Limited Proteolysis ◽  
Proteolytic Activation ◽  
Freeze Thaw ◽  
Membrane Bound ◽  
Time Courses ◽  
Triton X ◽  
Thiol Proteinase

Dithiothreitol led to the activation and solubilization of the cyclic nucleotide phosphodiesterase activities associated with the smooth and various rough subfractions of rat liver endoplasmic reticulum. The activity in each of the subfractions exhibited somewhat different time courses, and sensitivities to dithiothreitol concentration, in respect of their solubilization and activation. Both activation and solubilization by dithiothreitol could be blocked by either thiol proteinase inhibitors or excess bovine serum albumin. Freeze-thaw solubilization was not blocked by the thiol proteinase inhibitor antipain and did not lead to the activation of the enzyme. After dithiothreitol-induced solubilization, all of the enzymes exhibited non-linear Lineweaver-Burk plots indicative of apparent negative co-operativity. In contrast, after freeze-thaw solubilization the enzyme in the smooth-endoplasmic-reticulum-plus-Golgi fraction still obeys Michaelis kinetics, as does the membrane-bound enzyme. It is possible to mimic the action of dithiothreitol in solubilizing and activating the enzyme by limited proteolysis with trypsin. Triton X-100 is highly efficient at solubilizing these enzymes, yet has little effect on their activities. Charged detergents exhibit highly selective effects on the enzymes as regards their solubilization and activity expressed.

scholarly journals Mutations in Tyr808 reveal a potential auto-inhibitory mechanism of guanylate cyclase-B regulation

2013 ◽  
Vol 33 (3) ◽  
Author(s):  
Takeshi Katafuchi
Keyword(s):  
Guanylate Cyclase ◽  
Sequence Similarity ◽  
Activation Mechanism ◽  
Phosphorylation Sites ◽  
Wild Type ◽  
Tyrosine Residues ◽  
Sphingosine 1 Phosphate ◽  
Membrane Bound ◽  
Partial Fragment ◽  
Insight Into

In this study, Tyr808 in GC-B (guanylate cyclase-B), a receptor of the CNP (C-type natriuretic peptide), has been shown to be a critical regulator of GC-B activity. In searching for phosphorylation sites that could account for suppression of GC-B activity by S1P (sphingosine-1-phosphate), mutations were introduced into several candidate serine/threonine and tyrosine residues. Although no novel phosphorylation sites that influenced the suppression of GC-B were identified, experiments revealed that mutations in Tyr808 markedly enhanced GC-B activity. CNP-stimulated activities of the Y808F and Y808A mutants were greater than 30-fold and 70-fold higher, respectively, than that of WT (wild-type) GC-B. The Y808E and Y808S mutants were constitutively active, expressing 270-fold higher activity without CNP stimulation than WT GC-B. Those mutations also influenced the sensitivity of GC-B to a variety of inhibitors, including S1P, Na3VO4 and PMA. Y808A, Y808E and Y808S mutations markedly weakened S1P- and Na3VO4-dependent suppression of GC-B activity, whereas Y808E and Y808S mutations rather elevated cGMP production. Tyr808 is conserved in all membrane-bound GCs and located in the niche domain showing sequence similarity to a partial fragment of the HNOBA (haem nitric oxide binding associated) domain, which is found in soluble GC and in bacterial haem-binding kinases. This finding provides new insight into the activation mechanism of GCs.

scholarly journals Adenylate cyclase, guanylate cyclase and cyclic nucleotide phosphodiesterases of guinea-pig cardiac sarcolemma

1976 ◽  
Vol 158 (3) ◽  
pp. 535-541 ◽  
Author(s):  
P J St Louis ◽  
P V Sulakhe
Keyword(s):  
Adenylate Cyclase ◽  
Guinea Pig ◽  
Guanylate Cyclase ◽  
Cyclic Gmp ◽  
Maximal Activity ◽  
Cardiac Sarcolemma ◽  
Guinea Pig Heart ◽  
Membrane Bound ◽  
Triton X ◽  
Assay Conditions

1. The activities of the enzymes involved in the metabolism of cyclic nucleotides were studied in sarcolemma prepared front guinea-pig heart ventricle; the enzyme activities reported here were linear under the assay conditions. 2. Adenylate cyclase was maximally activated by 3mM-NaF; NaF increased the Km for ATP (from 0.042 to 0.19 mM) but decreased the Ka for Mg2+ (from 2.33 to 0.9 mM). In the presence of saturating Mg2+ (15 mM), Mn2+ enhanced adenylate cyclase, whereas Co2+ was inhibitory. beta-Adrenergic amines (10-50 muM) stimulated adenylate cyclase (38+/-2%). When added to the assay mixture, guanyl nucleotides (GTP and its analogue, guanylyl imidophosphate) stimulated basal enzyme activity and enhanced the stimulation by isoproterenol. By contrast, preincubation of sarcolemma with guanylyl imidodiphosphate stimulated the formation of an ‘activated’ form of the enzyme, which did not reveal increased hormonal sensitivity. 3. The guanylate cyclase present in the membranes as well as in the Triton X-100-solubilized extract of membranes exhibited a Ka for Mn 2+ of 0.3 mM; Mn2+ in excess of GTP was required for maximal activity. Solubilized guanylate cyclase was activated by Mg2+ only in the presence of low Mn2+ concentrations; Ca2+ was inhibitory both in the absence and presence of low Mn2+. Acetylcholine as well as carbamolycholine stimulated membrane-bound guanylate cyclase. 4. Cylic nucleotide phosphodiesterase activities of sarcolemma exhibited both high-and low-Km forms with cyclic AMP and with cyclic GMP as substrate. Ca2+ ions increased the Vmax. of the cyclic GMP-dependent enzyme.

scholarly journals Phosphorylation of membrane-bound guanylate cyclase of sea urchin spermatozoa.

1986 ◽  
Vol 103 (1) ◽  
pp. 95-101 ◽  
Author(s):  
G E Ward ◽  
G W Moy ◽  
V D Vacquier
Keyword(s):  
Plasma Membrane ◽  
Enzymatic Activity ◽  
Guanylate Cyclase ◽  
Ammonium Hydroxide ◽  
Arbacia Punctulata ◽  
Direct Demonstration ◽  
Seawater Ph ◽  
Sperm Plasma Membrane ◽  
Membrane Bound

When Arbacia punctulata spermatozoa are incubated in seawater containing ammonium hydroxide (pH 8.8), the sperm plasma membrane-bound guanylate cyclase is dephosphorylated, its electrophoretic mobility increases (from an apparent molecular mass of 160 to 150 kD), and its enzymatic activity decreases 3.5-fold. Transfer of these cells into ammonium-free seawater (pH 7.4) results in the rephosphorylation of the cyclase, its reconversion to 160 kD, and recovery of the enzymatic activity lost upon dephosphorylation. This is the first direct demonstration that the activity of membrane-bound guanylate cyclase can be regulated by phosphorylation. A plasma membrane preparation is described that specifically supports the in vitro phosphorylation of the guanylate cyclase. This preparation will be useful in more detailed studies on the relationship between phosphorylation state and enzymatic activity of membrane-bound guanylate cyclase.

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