Transient kinetics of nicotinamide-adenine dinucleotide phosphate-linked isocitrate dehydrogenase from bovine heart mitochondria.

Pre-steady-state studies of the isocitrate dehydrogenase reaction show that the rate constant for the hydride-transfer step is above 990s-1, and that both subunits of the enzyme are simulataneously active. After the fast formation of NADPH in amounts equivalent to the enzyme subunit concentration, the rate of NADPH formation is equal to the steady-state rate if the enzyme has been preincubated with isocitrate and Mg2+. If the enzyme has been preincubated with NADP+ and Mg2+, in 0.05 M-triethanolamine chloride buffer, pH 7.0, with the addition of 0.1 M-NaCl, the amount of NADPH formed in the fast phase is only 60% of the enzyme subunit concentration, and the turnover rate is at first lower than the steady-state rate. In 0.05 M-triethanolamine chloride buffer, pH 7.0, if the enzyme is preincubated with NADP+ or NADPH, the turnover rate increases 3-fold to reach the steady-state rate after about 5 s. Preincubation of the enzyme with isocitrate and Mg2+ abolishes this lag phase, the steady-state rate being reached at once. It is suggested that the enzyme exists in at least two conformational forms with different activities, and that the lag phase represents the transition (k = 0.4s-1) from a form with low activity to the fully active enzyme, induced by the binding of isocitrate and Mg2+.     

A deactivating conformational change induced by reduced nicotinamide-adenine dinucleotide phosphate in a Neurospora glutamate dehydrogenase.

Stopped-flow fluorescence techniques have been used to observe the formation of the binary comples of E-NADPH. At pH 7.5 there is a protein conformational change after the formation of the binary complex. This conformational change can be detected by a decrease in the fluorescence intensity of the complex at 350 nm and by an increase in its fluorescence intensity at 450 nm.     

A nicotinamide-adenine dinucleotide assay utilizing liver alcohol dehydrogenase

An assay for the determination of NAD has been developed utilizing the coupled oxidoreductase activity of liver alcohol dehydrogenase. The coupled reaction between ethanol and lactaldehyde is driven by the removal of one of the products, acetaldehyde, into a semicarbazide solution. Under the stated conditions, a linear relationship exists between the absorbance of acetaldehyde semicarbazone and NAD concentration in the reaction mixture. The principal advantages of this method are speed and simplicity. NAD⁺ and NADH are assayed by the same procedure, which is also used to measure NADP⁺ and NADPH after these nucleotides have been converted to NAD⁺ and NADH, respectively.     

The kinetics and reaction mechanism of the nicotinamide-adenine dinucleotide phosphate-specific glycerol dehydrogenase of rat skeletal muscle

The NADP-specific glycerol dehydrogenase of rat skeletal muscle has been partially purified by ammonium sulphate fractionation. The enzyme has been studied kinetically by initial-velocity analysis, product inhibition and inhibition by fluoride. The experimental results indicate that the reaction mechanism for the enzyme is ordered such that the first product leaves the enzyme before the addition of the second substrate.     

Induced transcription-dependent synthesis of mitochondrial reduced nicotinamide-adenine dinucleotide dehydrogenase in Drosophila.

The 23S rRNA of Agrobacterium tumefaciens contains at least two nicks which result in the formation of RNA components with mol.wts. of 0.52 X 10(6) and 0.48 X 10(6). Thus under the usual conditions of extraction and analysis, no 23S rRNA was recovered from the bacterium. The experiments show that 23S rRNA is synthesized as a continuous chain, in which one or two nicks are formed almost immediately near the ends of the molecule and an additional nick in the middle at a later time.     

Nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase of Neurospora.

Neurospora NADP-specific glutamate dehydrogenase that was treated with iodoacetate, iodoacetamide, or N-ethylmaleimide to block the thiol groups was cleaved with cyanogen bromide. Of the expected 10 peptides, based on a methionine content of 9 residues, 8 were obtained in pure form and 2 were handled as a mixture. The fragments ranged in size from 9 to 109 residues. In addition, there were isolated 6 peptides, produced by anomalous cleavage at the carboxyl groups of tryptophan residues, and two by hydrolysis of an aspartyl-proline bond. Preliminary separation of these peptides was accomplished by gel filtration followed by either ion-exchange chromatography of the larger peptides or by paper chromatography and paper electrophoresis of the smaller fragments. Ordering of the CNBr fragments in sequence was based upon sequences of tryptic and chymotryptic peptides obtained in another laboratory. The complete sequence of the protein is presented. The amino acid sequences of the bovine and chicken liver glutamate dehydrogenases previously determined show considerable homology with the NADP-specific enzyme of Neurospora in the NH2-terminal half of the molecule; this includes the region of the specifically reactive lysine residue and the portion of the sequence that has been implicated in coenzyme binding. Particularly striking is the fact that most of the residues conserved among the three homologous proteins would be expected to be important for conformational, rather than catalytic, effects. This implies that the conformation of the Neurospora enzyme must be similar in parts of its structure to the vertebrate enzymes but undoubtedly differs in some regards.