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In the present study, a new polyclonal antibody (TxAb) was raised against native thromboxane A2 (TXA2)/prostaglandin H2 (PGH2) receptor protein. Previously developed anti-peptide antibodies (P1Ab, P2Ab) and TxAb were then used to prepare immunoaffinity columns to purify TXA2/PGH2 receptors from platelets, brain, and aorta. In platelets, SDS-polyacrylamide gel electrophoresis revealed the purification of a 55-kDa protein by each affinity column. Identification of this protein as the TXA2/PGH2 receptor was based on: 1) an identical electrophoretic mobility to authentic receptor; 2) immunoblotting of TxAb against P1Ab and P2Ab-purified protein; 3) immunoblotting of P1Ab/P2Ab against TxAb-purified protein; and 4) specific [3H]SQ29,548 binding to TxAb-purified protein. P1Ab/TxAb purification of receptors from brain revealed a major protein band at 55 kDa. Furthermore, the eluates from ligand affinity chromatography confirmed the presence of this 55-kDa protein in brain (which was immunoblotted with TxAb), and contained specific [3H]SQ29,548 binding. In addition to the 55-kDa protein, P1Ab/TxAb also purified a minor protein in brain at 52 kDa, which when concentrated, cross-blotted with TxAb and P1Ab. This finding indicates sequence homology between the 55- and 52-kDa proteins. Independent identification of brain TXA2/PGH2 receptors was provided by P2Ab/TxAb immunohistochemistry, which demonstrated specific labeling of discrete myelin-containing fiber tracts. P2Ab/TxAb purification of TXA2/PGH2 receptors from aorta also revealed a major protein band at 55 kDa and a minor band at 52 kDa. These results represent the first purification of TXA2/PGH2 receptors from either brain or aorta.

The hydrolysis of sodium 3-dansylamidomethyl-7-beta (thienyl-2')-acetamido-ceph-3-em-4-oate, catalyzed by the beta-lactamase of Staphylococcus aureus PC1, has previously been shown (Anderson, E. G., and Pratt, R. F. (1981) J. Biol. Chem. 256, 11401-11404) to follow the reaction scheme Formula; see text. where ES' is an enzyme-substrate complex in which the substrate has undergone nucleophilic attack at the beta-lactam carbonyl group and P is product. Acid quenching of the reaction mixture has now been shown to yield, in amounts predicted by the rate constants, a covalent enzyme-substrate complex. The liability of this complex in alkaline solution is suggestive of that of an ester. Together, all of these results prove that the turnover of this apparently normal substrate by a class A beta-lactamase involves an acyl-enzyme intermediate. In the case of another fluorescent substrate, dansylcephalexin, no intermediate analogous to ES' accumulated during catalysis; presumably here, acylation of the enzyme is rate-determining. The pH profiles (pH 4-9) of the pre-steady state rate constants for hydrolysis of the former substrate have also been determined. Binding (1/K8) is pH invariant except at low pH where it weakens, probably because of substrate protonation and/or a protein conformational change. The rate constants, k2, k-2, and k3, are pH invariant at low pH but decrease at higher pH in a way which can be described by ionization of an essential acid of pKa around 7.7. This may be the same acid for each constant, being either an active participant at the active site, or a more distant acid which controls an essential conformational change.

The pre-steady state kinetics of the hydrolysis of sodium 3-dansylamidomethyl-7-beta (thienyl-2')-acetamidoceph-3-em-4-oate, catalyzed by the beta-lactamase of Staphylococcus aureus PC1, has been studied by the stopped flow method. Fluorescence measurements indicated the presence of two intermediate enzyme-substrate complexes, i.e. at least a three-step mechanism. The disappearance of the characteristic cephem chromophore, which reflects nucleophilic attack at the beta-lactam carbonyl group, correlated with the second step. These results show that an intermediate species with a covalently altered substrate occurs on the reaction path prior to the last step which includes release of product. This intermediate is most likely an acyl-enzyme. The data have been analyzed numerically and the derived rate constants are in accord with the steady state parameters.