Exogenous Aldehyde Research Articles

Effects of 3' end deletions from the Vibrio harveyi luxB gene on luciferase subunit folding and enzyme assembly: generation of temperature-sensitive polypeptide folding mutants.

Ten recombinant plasmids have been constructed by deletion of specific regions from the plasmid pTB7 that carries the luxA and luxB genes, encoding the alpha and beta subunits of luciferase from Vibrio harveyi, such that luciferases with normal alpha subunits and variant beta subunits were produced in Escherichia coli cells carrying the recombinant plasmids. The original plasmid, which conferred bioluminescence (upon addition of exogenous aldehyde substrate) on E. coli carrying it, was constructed by insertion of a 4.0-kb HindIII fragment of V. harveyi DNA into the HindIII site of plasmid pBR322 [Baldwin, T.O., Berends, T., Bunch, T. A., Holzman, T. F., Rausch, S. K., Shamansky, L., Treat, M. L., & Ziegler, M. M. (1984) Biochemistry 23, 3663-3667]. Deletion mutants in the 3' region of luxB were divided into three groups: (A) those with deletions in the 3' untranslated region that left the coding sequences intact, (B) those that left the 3' untranslated sequences intact but deleted short stretches of the 3' coding region of the beta subunit, and (C) those for which the 3' deletions extended from the untranslated region into the coding sequences. Analysis of the expression of luciferase from these variant plasmids has demonstrated two points concerning the synthesis of luciferase subunits and the assembly of those subunits into active luciferase in E. coli. First, deletion of DNA sequences 3' to the translational open reading frame of the beta subunit that contain a potential stem and loop structure resulted in dramatic reduction in the level of accumulation of active luciferase in cells carrying the variant plasmids, even though the luxAB coding regions remained intact.

30] Aldose reductase from human tissues

This chapter describes an assay method for the synthesis of aldose reductase from human tissues. Aldose reductase and polyol dehydrogenase constitute the sorbitol pathway converting glucose to fructose in extrahepatic tissues. Aldose reductase catalyzes the reduction of other sugar aldehydes and of several aliphatic and aromatic aldehydes. Aldose reductase is assayed spectrophotometrically by recording the decrease in nicotinamide adenine dinucleotide phosphate dehydrogenase (NADPH) absorbance at 340 nm. With crude enzyme solutions, blank reactions occur with NADPH in the absence of any aldehyde substrate; consequently the rate of the reaction is recorded before the addition of exogenous aldehyde. Depending on the substrate and on the tissue analyzed, other dehydrogenases, notably aldehyde reductase, may interfere. Interference with aldehyde reductase is diminished by the addition of diphenylhydantoin or phenobarbitone to the assay medium. The ratio of activities measured with D-xylose, DL-glyceraldehyde, and D-glucuronate is used to distinguish between the two enzymes. The chapter discusses the procedure used to isolate and purify carbonyl reducing enzymes from human tissues, along with the preparation of brain aldose reductase. Brains, either whole or without cortex and cerebellum, are obtained from legal medical autopsies.

Luciferase inactivation in the luminous marine bacterium Vibrio harveyi.

Luciferase was rapidly inactivated in stationary-phase cultures of the wild type of the luminous marine bacterium Vibrio harveyi, but was stable in stationary-phase cultures of mutants of V. harveyi that are nonluminous without exogenous aldehyde, termed the aldehyde-deficient mutants. The inactivation in the wild type was halted by cell lysis and was slowed or stopped by O2 deprivation or by addition of KCN and NaF or of chloramphenicol. If KCN and NaF or chloramphenicol were added to a culture before the onset of luciferase inactivation, then luciferase inactivation did not occur. However, if these inhibitors were added after the onset of luciferase inactivation, then luciferase inactivation continued for about 2 to 3 h before the inactivation process stopped. The onset of luciferase inactivation in early stationary-phase cultures of wild-type cell coincided with a slight drop in the intracellular adenosine 5'-triphosphate (ATP) level from a relatively constant log-phase value of 20 pmol of ATP per microgram of soluble cell protein. Addition of KCN and NaF to a culture shortly after this drop in ATP caused a rapid decrease in the ATP level to about 4 pmol of ATP per microgram whereas chloramphenicol added at this same time caused a transient increase in ATP level to about 25 pmol/microgram. The aldehyde-deficient mutant (M17) showed a relatively constant log-phase ATP level identical with that of the wild-type cells, but rather than decreasing in early stationary phase, the ATP level increased to a value twice that in log-phase cells. We suggest that the inactivation of luciferase is dependent on the synthesis of some factor which is produced during stationary phase and is itself unstable, and whose synthesis is blocked by chloramphenicol or cyanide plus fluoride.

Bacterial bioluminescence in vivo: control and synthesis of aldehyde factor in temperature-conditional luminescence mutants.

Bioluminescent marine bacteria possess luciferase, which catalyzes the oxidation of reduced flavin mononucleotide and long-chain aldehyde to produce light. Temperature-sensitive mutants of these bacteria can be obtained which require exogenous aldehyde for light production at higher temperatures. In Beneckea harveyi. two classes of such mutants were found which differed with regard to their response to temperature shifts. In one class, a shift from permissive to nonpermissive temperature in liquid cultures resulted in a rapid (t((1/2)) approximately 3 min) loss of luminescence. In the other, there was no immediate decline in luminescence; it was the increase of luminescence that was blocked. Through studies of these and other effects of temperature shifts on the in vivo luminescence of these mutants, we conclude that at least two genes are specifically involved in the in vivo biosynthesis of aldehyde for the luminescence reaction and that both genes are coordinately controlled with that for luciferase.

Monoamine oxidase activity in peripheral organs and adrenergic tissues of the rat

Monoamine oxidase activity in vitro was studied in the heart, kidneys, submaxillary glands, and superior cervical ganglia of control rats by the method of Lovenberg and co-workers ( J. Pharmacol. 1962). Comparison of monoamine oxidase activity in the absence and presence of exogenous aldehyde dehydrogenase revealed nearly saturating amounts of aldehyde dehydrogenase in the liver, submaxillary glands, lungs, spleen, and stomach (fundus, body, pyloric antrum). In the absence of exogenous aldehyde dehydrogenase, the uterus, ventricles, right atrium, left atrium, and proximal and distal small intestine showed activities of 24, 46, 58, 49, 65, and 68 per cent activity respectively. The activity in the superior cervical ganglia, stellate ganglia, thoracic chains, and retinas was 8.5, 11.0, 27, and 38 per cent. In the presence of exogenous aldehyde dehydrogenase, the greatest monoamine oxidase activity, based on the protein content, was noted in the superior cervical ganglia. On the wet weight basis, ganglionic monoamine oxidase activity was greater than that of all other tissues investigated except the liver. In the absence of exogenous aldehyde dehydrogenase, the reaction velocity of monoamine oxidase in ventricles, atria and pooled adrenergic tissue was linear for 30 min at the optimum substrate concentration. In the presence of exogenous aldehyde dehydrogenase, the monoamine oxidase reaction rates of the ventricles, atria, and adrenergic tissue were linear for up to 40 min at optimum substrate concentrations. Substrate inhibition became apparent at the highest substrate concentration used (11.67 mole/ml), and unsaturation of the enzyme was noted at the lowest substrate concentration (0.233 mole/ml). In homogenates of the ventricles, atria, and pooled adrenergic tissue, monoamine oxidase activity was inhibited by relatively small increases in enzyme concentration (tissue homogenate). This inhibition was not overcome by a tenfold increase in substrate concentration nor by the doubling of NAD and exogenous aldehyde dehydrogenase. Centrifugation at 50,000 g for 25 min localized the inhibitory component(s) in the pellet. The supernatant was devoid of monoamine oxidase activity.

Enzymic determination of aldehyde permeability in luminous bacteria. I. Effect of chain length on light emission and penetration

1. 1. A mutant of Achromobacter fischeri which requires exogenous aldehyde for light emission has been used to study the rate of penetration of a series of aliphatic aldehydes (heptanal to tetradecanal) through the bacterial cell membrane. 2. 2. As the chain length is increased, penetration into the cell is slower, affinity for luciferase increases, and the maximum light intensity is greater. The properties of the isolated enzyme agree with those observed for the activity in the whole cell. 3. 3. No special transport mechanism for the movement of these aldehydes through the bacterial membrane was revealed by the use of a variety of metabolic inhibitors. 4. 4. It is concluded that for this series of aldehydes lipide solubility is of minor importance for penetration and that the major controlling factors are membrane pore size and molecular mass of the aldehyde molecules.