C I Ragan

THE PHARMACOLOGY OF THE BENZODIAZEPINE SITE OF THE GABA-A RECEPTOR IS DEPENDENT ON THE TYPE OF GAMMA -SUBUNIT PRESENT

The pharmacology of native and recombinant GABA-A receptors containing either gamma1, gamma2 or gamma3 subunits has been investigated. The pharmacology of native receptors has been investigated by immunoprecipitating receptors from solubilised preparations of rat brain with antisera specific for individual gamma-subunits and analysing their radioligand binding characteristics. Receptors containing a gamma1-subunit do not bind benzodiazepine radioligands with high affinity. Those containing either a gamma2 or gamma3 subunit bind [3H]flumazenil with high affinity. Some compounds compete for these binding sites with multiple affinities, reflecting the presence of populations of receptors containing several different types of alpha-subunit. Photoaffinity-labelling of GABA-A receptors from a cell line stably expressing GABA-A receptors of composition alpha1beta3gamma2 followed by immunoprecipitation of individual subunits revealed that the alpha and gamma but not the beta-subunit could be irreversibly labelled by [3H]flunitrazepam. The properties of recombinant receptors have been investigated in oocytes expressing gamma1, gamma2, or gamma3 subunits in combination with an alpha and a beta-subunit. Some compounds such as zolpidem, DMCM and flunitrazepam show selectivity for receptors containing different gamma-subunits. Others such as CL 218,872 show no selectivity between receptors containing different gamma-subunits but exhibit selectivity for receptors containing different alpha-subunits. These data taken together suggest that the benzodiazepine site of the GABA-A receptor is formed with contributions from both the alpha and gamma-subunits.

Bovine inositol monophosphatase: development of a continuous fluorescence assay of enzyme activity.

This paper describes a continuous assay for the enzyme inositol monophosphatase which has been developed using a new substrate, the fluorescent compound 4-methylumbelliferyl phosphate. The hydrolysis of the phosphate group from this compound can be readily detected by a resultant large red shift in the emission spectrum from 390-450 nm. The kinetic constants for the enzyme using this new substrate are described.

Bovine inositol monophosphatase: proteolysis and structural studies

Bovine brain inositol monophosphatase is inactivated when trypsin catalyses the cleavage of a single peptide bond between Lys‐36 and Ser‐37. This proteolysis is closely followed by cleavage at two other sites in the protein between Lys‐78 and Ser‐79 and between Lys‐156 and Ser‐157 suggesting that all of these sites are exposed in the native conformation of the protein. All of these residues are predicted to lie at the ends of α helices. The most susceptible bond (Lys‐36‐Ser‐37) is predicted to lie in a highly flexible region of the protein. Circular dichroism studies suggest that approximately 40% of the secondary structure of this protein is helical which is similar to that predicted by the algorithm of Gamier et al. [(1978) J. Mol. Biol. 120, 97‐120].

Bovine inositol monophosphatase: The identification of a histidine residue reactive to diethylpyrocarbonate

The inositol monophosphatase from bovine brain is inactivated by the histidine‐specific reagent diethylpyrocarbonate. Using 4 mM reagent at pH 6.5, the reaction results in the modification of 3 equivalents of histidine per polypeptide chain. The loss of activity occurs at the same rate as the slowest reacting of these residues. Site directed mutagenesis studies have been used to generate a mutated enzyme species bearing a His‐217→Gln replacement and have shown that it is the modification of histidine 217 which results in the inactivation of the enzyme.