Ox liver glutamate dehydrogenase. The role of lysine-126 reappraised in the light of studies of inhibition and inactivation by pyridoxal 5''-phosphate.

The time-course of inactivation of bovine liver glutamate dehydrogenase by pyridoxal 5'-phosphate was studied in the presence of varied amounts of 2-oxoglutarate or NADH. Pseudo-first-order analysis reveals that the protection by both these compounds is competitive with respect to the chemical modifier. The competition is only partial, however: saturation with either NADH or 2-oxoglutarate decreases the rate constant for inactivation to a finite minimum and not to zero. Similarly, the plot of activity at equilibrium as a function of the concentration of the protecting substrate or coenzyme reveals that neither NADH nor 2-oxoglutarate protects completely against inactivation. In initial-rate experiments, pyridoxal 5'-phosphate, used as an instantaneous inhibitor rather than a long-term inactivator, displayed non-competitive inhibition with respect to both 2-oxoglutarate and NADH. These results clearly indicate that, although there is mutual hindrance between the binding to the enzyme of pyridoxal 5'-phosphate, on the one hand, and 2-oxoglutarate or NADH on the other, binding is not mutually exclusive. These findings are discussed in terms of the two-step mechanism for inactivation by pyridoxal 5'-phosphate. It is concluded that lysine-126 cannot be solely responsible for binding either the substrate or the coenzyme, but could be essential for the catalytic step.     

Dogfish M4 lactate dehydrogenase: reversible inactivation by pyridoxal 5''-phosphate and complete protection in complexes that mimic the active ternary complex.

Dogfish M4 lactate dehydrogenase, like the corresponding pig enzyme, is inactivated by pyridoxal 5'-phosphate through modification of a single essential lysine residue. The activity is completely protected in the complexes E-NAD+-oxalate, E-NADH-oxamate and E-(NAD+-pyruvate adduct), but only partially protected in E-NAD+, E-NADH, E-NAD+-oxamate and E-NADH-oxalate.     

Anotia, facial paralysis, heart abnormalities: specific triad or variant of Goldenhar syndrome?

A girl was born with anotia, facial palsy and cardiac malformations. Her case is compared with three other similar ones described in the literature, and the question is raised as to whether this is a new clinical entity or represents a variation of Goldenhar syndrome.     

Estimation of binding parameters of enzyme-ligand complex from fluorometric data by a curve fitting procedure: seryl-tRNA synthetase-tRNA Ser complex

A curve fitting procedure for the determination of the association constant, number of binding sites, and theoretical titration end point of an enzyme-ligand interaction monitored by fluorescence quenching titration is described and compared with classical graphical evaluation processes.     

A novel bufadienolide synthesis

A simple two-step synthesis of bufadienolides is reported. It consists in the addition of the dimethyl acetal of chloroketene to a steroidal 20-methylene 21-aldehyde and in the treatment of the resulting 2,2-dimethoxy 3-chloro 2,3-dihydropyran with sodium methoxide in dimethylsulfoxide. This method is exemplified by the synthesis of 3β-hydroxy-5α,14α-bufadienolide from 3β-acetoxy-20-methylene-5α-pregnan-21-al, prepared from 3β-acetoxy-5α-androstan-17-one. The new procedure represents the most efficient bufadienolide synthesis yet known.