Characterization of a new, highly specific, beta 3-adrenergic receptor radioligand, [3H]SB 206606.

The RR-enantiomer of the beta 3-adrenergic receptor agonist BRL 37344 was tritiated to yield a high specific activity compound, [3H]SB 206606. This new, potentially specific, beta 3-adrenergic receptor ligand was characterized by binding studies using membranes from both Chinese hamster ovary K1 cells transfected with the rat beta 3-adrenergic receptor and rat interscapular brown adipose tissue, where beta 1-, beta 2-, and beta 3-adrenergic receptor subtypes are known to coexist. [3H]SB 206606 was found to bind to a single population of binding sites in both preparations. The Kd values for [3H]SB 206606 binding to membranes from Chinese hamster ovary K1 cells and brown adipose tissue were quite comparable (58 and 38 nM, respectively). At 37 degrees, the time courses of association and dissociation of [3H]SB 206606 with membranes of brown adipose tissue were quite short. At 4 degrees, the T1/2 were found to be 13 and 40 min, respectively. The Ki values for various beta-adrenergic agonists and antagonists in brown adipose tissue membranes were similar to those obtained in Chinese hamster ovary K1 cell membranes with both [3H]SB 206606 and [125I]iodocyanopindolol. The order of binding affinity was BRL 37344 >> (-)-isoproterenol = (-)-norepinephrine > (-)-epinephrine = (+)-isoproterenol. The similarity of the Kd values and of the Ki values for various beta-adrenergic agonists and antagonists in both systems tested indicates that, in a complex membrane system, [3H]SB 206606 binds selectively to the beta 3-adrenergic receptor. The affinity of [3H]SB 206606 is 76 times higher for the beta 3-adrenergic receptor than for the beta 1/beta 2-adrenergic receptors, thus allowing, under controlled conditions, measurement of interactions only with the beta 3-adrenergic receptor in complex membrane systems.

A polyclonal antibody preparation with Michaelian catalytic properties.

1. 4-Nitrophenyl 4'-(3-aza-2-oxoheptyl)phenyl carbonate (I), an amide conjugate (XI) involving the carboxy group of 4-nitrophenyl 4'-carboxymethylphenyl phosphate and an amino group of keyhole-limpet haemocyanin, and a fluorescein derivative (XVII) were synthesized. 2. The conjugate (XI) was used as an immunogen with which to raise polyclonal antibodies in multigeneration cross-bred sheep; the fluorescent derivative (XVII) was used for the initial assessment of the antisera via binding assays monitored by fluorescence polarization; the carbonate ester (I) was used as a chromogenic substrate for the investigation of catalytic activity. 3. The IgG from the antiserum of sheep no. 270 was isolated by Na2SO4 precipitation and chromatography on Protein G-Sepharose. 4. This preparation of IgG catalysed the hydrolysis of the carbonate ester (I); the catalysis at pH 8.0 and 25 degrees C obeyed Michaelis-Menten kinetics with at least 25 turnovers, Km = 3.34 microM, and lower limits for kcat. of 0.029 s-1 and for kcat./Km of 8.77 x 10(3) M-1.S-1, on the unlikely assumption that the concentration of catalytic antibody is provided by twice the total IgG concentration (two sites per molecule); probable estimates of the fraction of the total IgG that is anti-haptenic IgG and of the fraction of this that is catalytically active suggest that the values of kcat./Km are actually very much larger than these lower limits. 5. The failure of the antibody preparation to catalyse the hydrolysis of the isomeric 2-nitrophenyl carbonate (II), which differs from compound (I) only in the position of the nitro substituent in the leaving group, compels the view that catalytic activity is due to antibody rather than contaminant enzyme; this conclusion is supported by (a) the failure of the following to discriminate effectively between the isomeric substrates (I) and (II): pig liver carboxylesterase, rabbit liver carboxylesterase (collectively EC 3.1.1.1), whole serum from a non-immunized sheep and whole serum from a sheep immunized with a derivative of 3-O-methylnoradrenaline and (b) the lack of catalytic activity in IgG preparations from sheep immunized with sulphoxide or sulphone analogues of immunogen (XI). 6. The various parameters used for the comparison of the kinetic characteristics of hydrolytic catalytic antibodies are discussed. 7. The characteristics of hydrolysis of compound (I) catalysed by the present polyclonal antibody preparation are shown to be substantially better in most respects than those of analogous reactions of two other carbonate esters catalysed by monoclonal antibodies.