J. W. Hastings

[14] Bacterial luciferase: Assay, purification, and properties

Publisher Summary This chapter focuses on bacterial luciferase. The activity of bacterial luciferase is measured as the initial maximum light intensity upon mixing the enzyme, aldehyde, and oxygen with reduced flavin mononucleotide (FMNH2). Luciferase activity may be measured by means of the total light produced. One of the principles involved in the assay of bacterial luciferase differs completely from that for most enzymes, including many other luciferases: no catalytic turnover is involved. The activity measured is the result of intermediates formed in the luciferase-reduced flavin reaction during the first fraction of a second. A second important feature of the reaction is that luciferases exhibit both oxidase and monooxygenase activities. A suitable soluble flavin reductase may be isolated and purified from extracts of luminous bacteria

A postulated mechanism for the bioluminescent oxidation of reduced flavin mononucleotide

Abstract A mechanism is presented for the luciferase catalyzed oxidation of reduced flavin mononucleotide with oxygen in the presence of long-chain aldehyde. The mechanism involves the formation of a flavin peroxy anion which attacks aldehyde. A Baeyer-Villiger type shift leads to oxidation of aldehyde to acid, and to formation of hydroxide and excited protonated flavin which emits a photon. The mechanism is consistent with known details of the bioluminescent reaction and with known reactions of flavins and allows several verifiable predictions to be made.

Circadian Changes in Enzyme Concentration Account for Rhythm of Enzyme Activity in Gonyaulax

A circadian rhythm in the activity of luciferase is partly responsible for rhythmic bioluminescence in the dinoflagellate alga Gonyaulax polyedra. The cyclic activity of this enzyme can be attributed to a corresponding rhythm in the concentration of immunologically reactive luciferase protein. Hence protein turnover (synthesis or degradation or both) is used by the endogenous clock to control the daily rhythm of bioluminescence.

Conditionality of circadian rhythmicity: Synergistic action of light and temperature

SummaryWith cells which have been grown at 20°C, the circadian rhythm of bioluminescence inGonyaulax polyedra disappears at a critical temperature, which is about 12°C. The transition from the rhythmic to the arrhythmic state is very sharp with temperature: the two states are separated by only 1–2°C. Following a return to a higher temperature (20°C) under otherwise constant conditions, the rhythm resumes with its new phase defined by the time of the cool to warm transition. Loss of rhythmicity also occurs in constant bright light, with a similar resumption and phase determination upon transfer to darkness. The experiments described here show that the effects of light and low temperature are additive: rhythmicity is lost under combined low temperature and light intensity treatments which are ineffective individually.

Critical pulses of anisomycin drive the circadian oscillator inGonyaulax towards its singularity

SummaryDose and phase response curves for phase shifting the circadian oscillator in the dinoflagellateGonyaulax polyedra were measured with pulses of the antibiotic anisomycin (an inhibitor of protein synthesis on 80 S ribosomes), using the bioluminescent glow rhythm as the assay. The three dimensional surface of final phase, initial phase, and concentration was found to be a right handed helix, with the axis at a critical initial phase near circadian time 12 h, and critical concentration near 0.2 micromolar anisomycin (for 1 h pulses). The normally rhythmic glow of populations ofGonyaulax was significantly disrupted by pulses with these critical parameters, and in many instances appeared nearly arrhythmic.With increasing drug concentration, phase response curves appear to move bodily to earlier phases, and no saturation is evident in the phase shifting effect. These results are interpreted as indicating that anisomycin at sufficiently high doses causes an immediate strong (type 0) phase shift, then holds the clock stationary for a time interval that increases with concentration.the possibility that the 80 S ribosomal complex may be centrally involved in the fundamental circadian oscillation is put forward.

The effects of protein synthesis inhibitors on theGonyaulax clock

SummaryCycloheximide has been shown to be potent in phase-shifting the circadian glow rhythm of the dinoflagellateGonyaulax polyedra. In experiments in which the cells were exposed to drug pulses of varying concentration (0.35 μM to 10 μM) and duration (0.5 h to 8 h), the phase shift produced was linear with the log of the product of pulse strength and duration. The sensitivity to drug-induced phase shifting varies as a function of time of day; both advances and delays occurred and, depending on the strength of the perturbation, resulted in strong or weak-type phase response curves. Pulses given at different times after the light/dark to constant dim transition resulted in a crossover from delays to advances at about 15.5 h; this crossover point was the same at 19 °C and 24°C. The occurrence of extended transients following cycloheximide-induced phase advances (but not delays) appears to be the first example of such transients in a microbial circadian system.SummaryRhythmic cultures ofGonyaulax polyedra Stein (Strain 70) were placed in constant dim light and analyzed for ultrastructural changes over a period of 32 h. Circadian changes were observed in chloroplast shape and distribution within the cell, as measured quantitatively by the percentage of chloroplasts covering the surface area of the cell, the length of the chloroplasts and their penetration into a central area which is occupied by chloroplasts at only one time of day. Chloroplast portions penetrated this area during the entire subjective day but not during the subjective night. Chloroplasts within this region showed expanded interlamellar distances, whereas only slight changes in the interlamellar distances were observed in the other portions (nearer to the periphery of the cell) of the chloroplast. No changes were observed in the intralamellar distances over the course of a day.In addition, circadian rhythms were detected in the percentage of two and three-stacked thylakoid lamellae and in the number of starch granules present in a defined inner and outer region of the cell. Few if any changes could be observed in the measured diameters of the tubular cristae or in the distances between outer and inner membranes of the mitochondria.Exposure to 1 μM cycloheximide for one hour did not immediately affect the ultrastructural parameters measured or other structural features of the cell. However, such pulses phase-shifted the measured rhythms by 8 to 12 h, the amount being dependent on the circadian phase at which the pulses occurred.

An Inhibitor of Protein Phosphorylation Stops the Circadian Oscillator and Blocks Light-Induced Phase Shifting in Gonyaulax polyedra

The expression of circadian rhythmicity in Gonyaulax polyedra is strikingly altered by an inhibitor of protein phosphorylation. The effects of 6-dimethylaminopurine (6-DMAP), known to reversibly block cell division in many systems through inhibition of protein kinase activity, are described here for Gonyaulax. Its action appears to be exclusively tonic in nature; in cells continuously exposed, the period is lengthened in a concentration-dependent fashion. Shorter treatments at a higher concentra tion of 6-DMAP (5 mM) apparently stop the circadian oscillator, but reversibly so, since the rhythm resumes after drug removal with a phase delay approximately equal to the duration of the treatment. Pulses of the inhibitor are effective in causing phase delays at all times of the circadian cycle. In addition, 6-DMAP completely blocks light-induced phase advances and is effective in inhibiting many Gonyaulax protein kinases in vitro.

Purification of the yellow fluorescent protein from vibrio fischeri and identity of the flavin chromophore

A low molecular weight protein (approximately 25,000 D) exhibiting a yellow fluorescence emission peaking at approximately 540 nm was isolated from Vibrio fischeri (strain Y-1) and purified to apparent homogeneity. FMN is the chromophore, but it exhibits marked red shifts in both the absorption (lambda max = 380, 460 nm) and the fluorescence emission. When added to purified luciferase from the same strain, which itself catalyzes an emission of blue-green light (lambda max approximately 495 nm), this protein induces a bright yellow luminescence (lambda max approximately 540 nm); this corresponds to the emission of the Y-1 strain in vivo. This yellow bioluminescence emission is thus ascribed to the interaction of these two proteins, and to the excitation of the singlet FMN bound to this fluorescent protein.

Inhibitors of Serine/Threonine Phosphoprotein Phosphatases Alter Circadian Properties in Gonyaulax polyedra

Protein serine/threonine phosphatases were implicated in the regulation of circadian rhythmicity in the marine dinoflagellate Gonyaulax polyedra based on the effects of three inhibitors specific for protein phosphatases 1 and 2A (okadaic acid, calyculin A, and cantharidin). Chronic exposure to okadaic acid resulted in a significant period lenghtening, as measured by the bioluminescent glow rhythm, whereas cantharidin and calyculin A caused large phase delays but no persistent effect on period. Short pulses of the phosphatase inhibitors resulted in phase delays that were greatest near subjective dawn. Unlike 6-dimethylaminopurine, a protein kinase inhibitor, okadaic acid, calyculin A, and cantharidin did not block light-induced phase shifts. The inhibitors tested also increased radiolabeled phosphate incorporation into Gonyaulax proteins in vivo and blocked protein phosphatase 1 and 2A activities in Gonyaulax extracts. This study indicates that protein dephosphorylation catalyzed by protein serine/threonine phosphatases is necessary for proper functioning of the circadian system.

Precision of the Gonyaulax circadian clock.

Under constant conditions, the circadian bioluminescent glow rhythm in populations (105 cells) ofGonyaulax polyedra is accurate to within 2 min/day. On successive days following the transfer to constant conditions, however, the glow exhibits a progressively broader waveform, implying that individual clocks in the population are drifting out of synchrony. Analysis of the glow waveform suggests that the standard deviation in circadian period among individual clocks is about 18 min and that the period of a given clock varies by less than this from one day to the next.