The four half-transamination reactions [the pyridoxal form of Escherichia coli aspartate aminotransferase (AspAT) with aspartate or glutamate and the pyridoxamine form of the enzyme with oxalacetate or 2-oxoglutarate] were followed in a stopped-flow spectrometer by monitoring the absorbance change at either 333 or 358 nm. The reaction progress curves in all cases gave fits to a monophasic exponential process. Kinetic analyses of these reactions showed that each half-reaction is composed of the following three processes: (1) the rapid binding of an amino acid substrate to the pyridoxal form of the enzyme; (2) the rapid binding of the corresponding keto acid to the pyridoxamine form of the enzyme; (3) the rate-determining interconversion between the two complexes. This mechanism was supported by the findings that the equilibrium constants for half- and overall-transamination reactions and the steady-state kinetic constants (Km and kcat) agreed well with the predicted values on the basis of the above mechanism using pre-steady-state kinetic parameters. The significant primary kinetic isotope effect observed in the reaction with deuterated amino acid suggests that the withdrawal of the alpha-proton of the substrates is rate determining. The pyridoxal form of E. coli AspAT reacted with a variety of amino acids as substrates. The Gibbs free energy difference between the transition state and the unbound state (unbound enzyme plus free substrate), as calculated from the pre-steady-state kinetic parameters, showed a linear relationship with the accessible surface area of amino acid substrate bearing an uncharged side chain.(ABSTRACT TRUNCATED AT 250 WORDS)

To elucidate the mechanism of resistance of hypoalbuminemic patients to furosemide, the effect of this diuretic on urine volume of normal and analbuminemic rats (NAR) and of hypoalbuminemic patients was studied. Intravenous administration of furosemide rapidly enhanced sodium diuresis in normal rats but not in NAR. Total plasma clearance and distribution volume of furosemide were much larger in NAR than in normal rats, while no significant difference in these pharmacokinetic parameters was observed for the unbound fraction of the diuretic between the two animal groups. In contrast, urinary secretion of furosemide was significantly lower in NAR than in normal rats. Injected furosemide bound to albumin markedly promoted diuresis in NAR, while the same dose of albumin alone had no effect, indicating that binding to albumin is essential for the delivery of furosemide to the kidney, the site for its action. Injection of the complex rapidly increased the urine volume of hypoalbuminemic patients who showed a marked resistance to this diuretic. Thus, the resistance to furosemide in both NAR and hypoalbuminemic patients may be explained on the same basis.

Both cytosolic (c-AAT) and mitochondrial (m-AAT) isozymes of aspartate aminotransferase (EC 2.6.1.1) appear in serum in some diseases including hepatobiliary dysfunction. The present study aimed at elucidation of the mechanism by which AAT isozymes are cleared from blood. Intravenous injection into rats of m-AAT and c-AAT purified from rat liver exhibited a biphasic clearance curve with an overall half-life of 42 min and 4.7 hr, respectively. The tissue distribution of the radioactivity following intravenous administration of 125I-labeled isozymes revealed that the liver is a major organ involved in plasma clearance of these isozymes. This conclusion was also supported by the significant retardation in plasma clearance of m-AAT in hepatectomized as well as CCl4-intoxicated rats. Furthermore, clearance rate of each AAT isozyme in an isolated perfused liver exhibited a single exponential process with the uptake rate for m-AAT being much faster than that for c-AAT. Separation of hepatocytes and sinusoidal liver cells from the rat intravenously injected with 125I-labeled AAT isozymes revealed that sinusoidal cells were responsible for the plasma clearances. In vitro uptake study showed that both isozymes were exclusively taken up by sinusoidal liver cells. The uptake rate for m-AAT was considerably greater than that for c-AAT. Endocytotic index for uptake by sinusoidal cells was 16 times with c-AAT and 34 times with m-AAT as compared with that for inulin or dextran which are taken up by fluid-phase endocytosis, suggesting involvement of adsorptive endocytosis in the uptake of the isozymes.