beta-Alanine synthase: purification and allosteric properties.

beta-Alanine synthase has been purified greater than 1000-fold to homogeneity from rat liver. The enzyme has a subunit molecular weight of 42,000 and a native size of hexamer. The enzyme undergoes ligand-induced changes in polymerization: association in response to the substrate, N-carbamoyl-beta-alanine, and the inhibitor, propionate; and dissociation in response to the product, beta-alanine. The ability of the substrate to associate the pure native enzyme to a larger polymeric species was exploited in the final purification step. The purified enzyme had a pI of 6.7, a Km of 8 microM, and a kcat/Km of 7.9 x 10(4) M-1 s-1. Positive cooperativity was observed toward the substrate N-carbamoyl-beta-alanine, with nH = 1.9. Such cooperativity occurred at substrate concentrations below 12 nM, so that this activation most likely occurs at a regulatory site, with a significantly stronger affinity for N-carbamoyl-beta-alanine than that shown by the catalytic site. The enzyme was sensitive to denaturation, which could be minimized by avoiding heat steps during the purification and by the presence of reducing agents. Such denatured enzyme had little change in Vmax, but had much higher Km, and had also lost the ability to associate or dissociate in response to effectors. After purification, enzyme stability was achieved by the addition of glycerol and detergent.

Regulation of N-carbamoyl-beta-alanine amidohydrolase, the terminal enzyme in pyrimidine catabolism, by ligand-induced change in polymerization.

N-Carbamoyl-beta-alanine (NC beta A) amidohydrolase (EC 3.5.1.6) is regulated in opposing fashion by the substrate, NC beta A and the product, beta-alanine. The native enzyme from rat liver has a molecular weight of 235,000 in the absence of ligands. NC beta A and substrate analogs (N-amidino-beta-alanine, N-carbamoyl-glycine) produced association of the enzyme. beta-Alanine and its analog gamma-aminobutyrate caused dissociation of the enzyme and produced inhibition. Negative cooperativity was observed for the binding of all ligands as measured by the change in polymerization of the enzyme, with an average Hill coefficient (napp) of 0.5. Enzyme that had been dissociated by preincubation with beta-alanine had little or no initial activity; only after a lag of 9 s was a steady state progress curve evident. The existence of a regulatory site is proposed as a model to explain physical and kinetic data. The enzyme activity was highest in rat liver and detectable in kidney; activity was not detected in brain, lung, muscle, or spleen of rat, nor in mouse Ehrlich ascites tumor cells. The rat liver enzyme has a pH optimum of 6.8, with a Km of 6.5 microM for NC beta A and a Ki of 1.08 mM for beta-alanine at this pH.

Clinical and coronary arteriographic profile of 100 black Americans: focus on subgroup with undiagnosed suspicious chest discomfort.

To help answer questions regarding the percentage of black patients catheterized for suspected cardiac ischemia who are found to have negative coronary arteriograms, and how this percentage compares with that of similarly classified white patients, the coronary arteriographic and left ventriculographic findings of 100 consecutive blacks were reviewed and compared with those of 226 contemporaneously catheterized whites. After excluding patients with prior histories of a clinical cardiac ischemic event, invasive therapeutic intervention for coronary artery disease (CAD), and nonatherosclerotic cardiac disease, a subgroup of 50 black and 104 white patients remained with undiagnosed chest discomfort that was suspicious for cardiac ischemia. Of this subgroup, 68 percent of the black patients showed no or insignificant CAD by arteriography, as compared with 21 percent of the white patients (P < .001). An incidental but note-worthy finding pertaining to this same subgroup was that 18 percent of the black patients who underwent arteriography and were found to have no CAD exhibited a subnormal left ventricular ejection fraction; in contrast, none of the white patients in the same category did so (P < .05).