CELL SURFACE NADH OXIDASE ACTIVITY OF BRINE SHRIMP OSCILLATES WITH A PERIOD OF 25 MIN AND IS ENTRAIN

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CELL SURFACE NADH OXIDASE ACTIVITY OF BRINE
SHRIMP OSCILLATESWITH A PERIOD OF 25 MIN AND IS
ENTRAINED BY LIGHT

• Chalko, C. J., D. M. Morre and D. J. Morre
• Jan 22, 2000
• abstracted by
• David Fu
• Feb 20, 2002

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Key terms

• NADH
• NADH is the abbreviation used for Nicotinamide
  Adenine Dinucleotide, one of the most important
  coenzymes in the human brain and body.
• All living cells contain nicotinamide adenine
  dinucleotide, or NADH, a substance synthesized
  from niacin. In humans, NADH stimulates the
  production of ATP (adenosine triphosphate)
  molecules, which store energy. The more NADH a
  cell has, the more energy it produces. Increased
  concentrations of NADH in the brain may boost the
  production of important neurotransmitters.
• A coenzyme is the active, or working form of a
  vitamin. NADH is the reduced (electron- energy
  rich) coenzyme form of vitamin B3, while NAD is
  the oxidized (burned) coenzyme form of B3.

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Key terms

• NAD and NADH are converted into each other in
  numerous different metabolic activities. In some
  metabolic reactions it is NAD which is the needed
  catalyst, with NADH a useful by-product, in other
  reactions the situation is reversed.
• NAD and NADH also serve to activate various
  enzymes, NAD for example, activates alcohol
  dehydrogenase and acetaldehyde dehydrogenase that
  are the two enzymes needed to detoxify the
  alcohol we drink into carbon dioxide and water.
• NADH is the first of five enzyme complexes of the
  electron transport chain, where much of the ATP
  bioenergy that runs every biological process of
  our lives is formed.

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Reduced Form, High energy
NADH
ATP A-Pi-Pi-Pi
NAD
ADP A-Pi-Pi
Pi gt H3 PO3-
H
Oxidized Form
Pi
Reduced Form, High energy
NADH
CoEnzymeForm
NAD
CoEnzymeForm
Oxidized Form
H
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Introduction

• Plants
• Surface NADH oxidase
• Measure time / oscillating period gt24-min
• Synchronized by light
• After 12-min a maximum is observed
• Animal ?
• Brine shrimp
• The findings shows it is similar to plant

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Introduction

• A cell surface hydroquinone (NADH) oxidase with
  protein disulfide isomerase activity was
  described in recently research.
• It has the potential to serve as an ultradian
  (period less than 24 h) time keeping molecule.
• The protein is located at the external cell
  surface and can be used to assayed directly using
  impermeant substrates such as NADH .
• It is the first such enzymatic activity
  associated uniquely with the external plasma
  membrane surface of plant and animal cells.

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• Synchrony (entrainment) of the cell surface NOX
  activity in plants is achieved by light .
• This study was to determine if animals exhibited
  a similar periodicity in NADH oxidation and to
  answer the question, does the cell surface NADH
  oxidase of animals respond to light?

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• Study organism Brine shrimp (Artemia saliva).
• Easy to grow and manipulate
• Dont require mechanical mixing
• Survived intact for several h under the
  conditions of assay.
• Isolated plasma membranes (plant or animal)
• Not respond to light
• photoreceptor associations are disrupted during
  cell fractionation.

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• Measurements of NADH oxidation
• Brine shrimp exhibited a surface NADH oxidase
  activity
• Oscillated with a period of approximately 25 min.
  
• White light gt a new period corresponding to the
  time of light exposure.

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Materials and Methods

• Growth of shrimp.
• Eggs of brine shrimp, Artemia salina (Leach), in
  brine solution (ca. 3.8 g sea salt per liter)
  (San Francisco Bay Brand, Newark, CA)
• Treated under continuous incandescent light (25
  pmoles rn2s-1) at 25 C for hatching
• After 2 to 3 days, the larvae (nautili) were
  concentrated with a net and assayed at a density
  of about 100 larvae/ml.

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Materials and Methods

• Spectrophotometric assay.
• NADH oxidase activity was determined from the
  disappearance of NADH at 340 nm as reference
  using a Milton Roy Spec 20 spectrophotometer with
  readings taken at 30 set intervals at 25 C over
  about 50 min.
• The reaction mixture contained the sample and 150
  pM NADH in a total volume of 3 ml.
• The millimolar extinction coefficient of 6.22
  used to calculate NADH disappearance was
  unaffected by the dilute brine solution compared
  to Tris-MES buffer pH 7.

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Materials and Methods

• Light treatment.
• Light grown shrimp were placed in darkness for
  about 45 min
• Illuminated with incandescent white light (50
  pmoles m2s-1) for 1 min prior to initiation of
  measurements.

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Results

• The oxidation of NADH showed periodic
  alternations of rapid and slow decline (Fig. 1).
• The periods of rapid decline were equivalent to
  an NADH oxidation rate of about 2.5
  nmoles/min/100 shrimp in a total volume of 3 ml.
• The NADH was largely consumed within the first 3
  h of incubation. Experiments were repeated at
  least three times with consistent results.

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Results

• The brine shrimp grown in dim incandescent light
  exhibited a regular period of about 25 min.
• This periodicity was determined to remain
  constant over the course of an afternoon and
  evening (Table 1).
• Maximum were estimated from the midpoint of the
  time of active NADH oxidation determined by
  linear regression of line segments as shown in
  Fig. 1.
• As illustrated by data of Table 1, the period
  length determined over 19 periods was 25.0 min.
• Period lengths of 24.9 or 25.1 min would have
  resulted in deviations of 1.9 min over 19
  periods.

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Discussion

• Circadian and other forms of periodicity have
  been studied widely.
• not known about biochemical or molecular
  reactions capable of a time-keeping function have
  been described either in animals or plants.
• Recently report had it that the enzymatic
  activity of a hormone-stimulated and
  growth-related NADH oxidase with protein
  disulfide isomerase activity oscillated with a
  temperature compensated period of about 24 min
  (60 times per 24 h day).
• The oscillations were temperature compensated
  such that the period remained constant at about
  24 min between 17 and 37 C, a temperature range
  over which enzymatic activity varied
  approximately four-fold (Q10 of 2).
• The oscillations were observed both with the
  oxidation of NADH and in the restoration of
  activity to scrambled ribonuclease which was used
  as a measure of the protein disulfide-thiol
  interchange activity also carried out by the
  protein .

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Discussion

• A similar activity has been observed in animal
  and with membranes of milk fat globules of the
  bovine, a derivative of the mammary epithelial
  cell membrane (IO).
• The period was about 24 min and was temperature
  compensated. The NOX protein from HeLa cells has
  been cloned and expressed in bacteria. The cloned
  and expressed protein exhibits the same
  periodicity and alternation of activities as
  given by the protein of cells and membranes .

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Discussion

• The plasma membrane-associated NADH oxidase (NOX)
  activity is highly synchronized from both plant
  and animal sources.
• In plants, the activity is synchronized by light
  .
• After the interruption of a dark period by white
  light, a new period is established with a maximum
  precisely 12 min (one-half period) after the dark
  interruption.

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Discussion

• In the present experiments with brine shrimp,
• The organisms also were observed to exhibit a
  cell surface NADH oxidase activity that exhibited
  periodic oscillations.
• In this instance, the period was about 25 min
  rather than 24 min.

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Discussion

• Shrimp gt moved to the dark for 45 min gt light
• surface NADH oxidase gt light-induced pattern of
  periodicity
• new maximum between 12 and 13 gt 36 and 38 min
  
• Period is 2425 minutes after the light
  exposure.
• The original period determined prior to the dark
  period also was retained to some degree but was
  usually of lesser magnitude than the period
  established in response to light.
• The lack of complete synchrony, in contrast to
  studies with plants, might indicate that coupling
  to the photoreceptor in brine shrimp is
  incomplete or that some of the receptor-NOX
  interactions were screened from the light and
  failed to respond.

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Discussion

• Biorhythms of both plants and animals are known
  to respond to light.
• However, this is the first demonstration of a
  light response not mediated either through the
  visual system or which might be attributed to
  sensing via some mechanism other than via the
  pineal gland.
• The nature of the photoreceptor or the potential
  photoreceptor for a light-responsive oscillating
  cell surface NADH oxidase activity in animal
  cells is unknown.
• Nor has the action spectrum of the response been
  determined. However, a light synchronized cell
  surface protein with an oscillatory behavior is
  of interest as a potentially entrainable
  biochemical oscillator of circadian time keeping.

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Discussion

• The light absorbing pigments of circadian
  photoreception in animals are thought to be
  cryptochromes.
• the mouse homologues of plant cryptochromes
  appear to be required for normal functioning of
  the mouse circadian clock (11).
• Mutant mice that lacked functional cryptochrome
  genes when maintained on a 24 h cycle of 12 h of
  light and 12 h of darkness, exhibited behavior on
  a running wheel that was identical to that of
  normal mice.
• However, when the mutant mice were placed in
  constant darkness, they ran randomly at all times
  of the day suggesting that the circadian clock
  was disrupted by the lack of cryptochromes.