Guanyl‐Cyclase
Authors: Böhme Eycke 
Published: March 2005

An enzyme system catalyzing the formation of guanosine cyclic 3′:5′‐monophosphate (Guo‐3′:5′‐P) from GTP has been found in various tissues of the rat including kidney, spleen, liver, brain, heart, skeletal muscle and adipose tissue. The highest guanyl cyclase activity was measured in the lung.

Guanyl cyclase activity has been determined in crude tissue extracts by measuring [14C]Guo‐3′:5′‐P formed from [14C]GTP. These tissue extracts contain enzymes which degrade GTP and Guo‐3′:5′‐P. To compensate for these enzymatic interferences, the following procedures have been undertaken:

The degradation of GTP has been reversed by the addition of a GTP regenerating system. To exclude the possibility that during the incubation the substrate concentration decreases to levels which may significantly affect the rate of Guo‐3′:5′‐P formation, the remaining substrate has been estimated after separation on thin layer plates. Tritiated GTP has been included in this separation to control the recovery. Rapid enzymatic degradation of [14C]Guo‐3′:5′‐P formed has been reduced by the addition of Guo‐3′:5′‐P. The degradation of Guo‐3′:5′‐P has been determined in each sample by means of tritiated Guo‐3′:5′‐P. A calculation procedure is given to correct for this loss.

Guo‐3′:5′‐P is separated from GTP and other labelled products by thin layer chromatography on PEI cellulose. Compared to other degradation products, only small amounts of Guo‐3′:5′‐P are formed from GTP. Therefore, Guo‐3′:5′‐P eluted from thin layer plates had to be purified. For this purpose Guo‐3′:5′‐P has been adsorbed to an anion‐exchange resin. In the supernatant fluid disintegration rates of 3H and 14C have been determined. To eliminate interference by other labelled products, an identical control sample has been treated with phosphodiesterase and alkaline phosphatase to convert Guo‐3′:5′‐P to guanosine. This nucleoside is adsorbed to the resin to a smaller degree than Guo‐3′:5′‐P. [14C]Guo‐3′:5′‐P and [3H]Guo‐3′:5′‐P present at the end of the incubation period have been calculated from the difference of the disintegration rates between the sample parts. The recovery from this isolation procedure has been obtained from special samples containing [3H]Guo‐3′:5′‐P. These samples have been treated as indicated except for the omission of incubation with tissue extracts.

Some properties of guanyl cyclase have been investigated in the rat kidney. Formation of Guo‐3′:5′‐P measured in the presence of Mn++ is higher than the activities obtained with Mg++ or Ca++. In the absence of divalent cations no guanyl cyclase activity has been detected. More than 95% of the total guanyl cyclase activity has been found in the 100000 ×g supernatant fluid. The existence of a particulate form could not be excluded. The low guanyl cyclase activity measured in crude tissue extracts has been increased by the addition of Mn++ in the concentration of 10 mM instead of 1 mM or by dialysis of the tissue extracts. Guanyl cyclase activity appears also to be influenced by the Mn++ concentration present during the dialysis procedure. The rate of Guo‐3′:5′‐P formation measured in the presence of Mn++ has been stimulated by Ca++. Guanyl cyclase activity has been reduced by Zn++, anorganic pyrophosphate and nucleoside triphosphates. An apparent Michaelis constant of 0.4 mM has been measured for GTP in the presence of 1 mM Mn++. The non‐linearity in a log v versus 1/T plot may be due to the existence of two different forms of the active enzyme.