Extraction and purification of 1H, 2H, and isotope hybrid algal cytochrome, ferredoxin and flavoprotein

Crespi, Henry L.; Smith, Ursula; Gajda, L.; Tisue, T.; Ammeraal, R.M.

doi:10.1016/0005-2728(72)90196-x

Cited by 38 publications

(22 citation statements)

References 22 publications

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“…The yield of flavodoxin from iron-limited cultures of P. tricornutum wa.s approximately 1 mgofflavodoxing"'(wet weight) of algae. This is in the same range as yields reported for the thermophilic cyanobacterium Synechococcus lividus (Crespi et al 1972). Both preimmune and flavodoxin antisera were tested against proteins from nitrate-, phosphate-and iron-limited P. tricornutum.…”

Section: Resultsmentioning

confidence: 88%

FLAVODOXIN EXPRESSION AS AN INDICATOR OF IRON LIMITATION IN MARINE DIATOMS¹

Roche

Murray

Orellana

et al. 1995

Journal of Phycology

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We have prevously shown that a marine chlorophyte expressed flavodoxin under iron limitation but not under other nutrient stress conditions. Here we use polyclonal antiserum raised against the diatom Phaeodactylum tricornutum Bohlin to show that a similar response is observed in this species. Using our antibody, western blotting techniques, and standard colorimetric detection (4‐chloro‐1‐naphthol), we can detect at least a 25–50‐fold increase in flavodoxin in iron‐depleted compared to iron‐replete cells. In iron‐limited batch cultures of P. tricornulum, flavodoxin accumulation was inversely proportional to growth rate and was not detectable in cultures containing initially more than 750 nm of iron. We demonstrated that the accumulation of flavodoxin under iron stress is widespread among marine diatoms and that it may be possible to use the presence or absence of flavodoxin in natural marine diatom assemblages to detect iron limitation. However, our polyclonal antisera appears to be specific for diatoms and did not cross‐react with Synechococcus sp., Micromonas pusilla (Butcher) Manton et Parke, Dunaliella tertiolecta Butcher, Chlorella sp., Emiliania huxleii (Lohm.) Hay et Parke, or Isochrysis galbana Parke. A reverse bioassay experiment was conducted with natural phytoplankton assemblages containing mainly diatoms from Long Island Sound and in shelf waters near Cape Hatteras, two areas not suspected to be iron‐limited. Although flavodoxin was not detected in situ in these areas, natural populations of diatoms driven into iron limitation expres.sed flavodoxin. Flavodoxin was detected in mats of the diatom Rhizosolenia castracanei Cleve collected from the Equatorial Pacific during a JGOFS cruise in 1992, consistent with the hypothesis that iron may be limiting in this high‐nutrient, low‐chlorophyll region.

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Section: Resultsmentioning

confidence: 88%

FLAVODOXIN EXPRESSION AS AN INDICATOR OF IRON LIMITATION IN MARINE DIATOMS¹

Roche

Murray

Orellana

et al. 1995

Journal of Phycology

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“…Growth conditions are not always well described in early experiments and it is difficult to assess the degree to which iron affects flavodoxin levels. This is clearly acknowledged by Crespi et al (1972). Although these authors detected flavodoxin in S. lividus under their 'normal' growth conditions, they reported variability in the level of flavodoxin between algae grown in different batches of media.…”

Section: Physiological and Molecular Diagnosticsmentioning

confidence: 93%

The role of iron in phytoplankton photosynthesis, and the potential for iron-limitation of primary productivity in the sea

1994

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Iron supply has been suggested to influence phytoplankton biomass, growth rate and species composition, as well as primary productivity in both high and low NO3 (-) surface waters. Recent investigations in the equatorial Pacific suggest that no single factor regulates primary productivity. Rather, an interplay of bottom-up (i.e., ecophysiological) and top-down (i.e., ecological) factors appear to control species composition and growth rates. One goal of biological oceanography is to isolate the effects of single factors from this multiplicity of interactions, and to identify the factors with a disproportionate impact. Unfortunately, our tools, with several notable exceptions, have been largely inadequate to the task. In particular, the standard technique of nutrient addition bioassays cannot be undertaken without introducing artifacts. These so-called 'bottle effects' include reducing turbulence, isolating the enclosed sample from nutrient resupply and grazing, trapping the isolated sample at a fixed position within the water column and thus removing it from vertical movement through a light gradient, and exposing the sample to potentially stimulatory or inhibitory substances on the enclosure walls. The problem faced by all users of enrichment experiments is to separate the effects of controlled nutrient additions from uncontrolled changes in other environmental and ecological factors. To overcome these limitations, oceanographers have sought physiological or molecular indices to diagnose nutrient limitation in natural samples. These indices are often based on reductions in the abundance of photosynthetic and other catalysts, or on changes in the efficiency of these catalysts. Reductions in photosynthetic efficiency often accompany nutrient limitation either because of accumulation of damage, or impairment of the ability to synthesize fully functional macromolecular assemblages. Many catalysts involved in electron transfer and reductive biosyntheses contain iron, and the abundances of most of these catalysts decline under iron-limited conditions. Reductions of ferredoxin or cytochrome f content, nitrate assimilation rates, and dinitrogen fixation rates are amongst the diagnostics that have been used to infer iron limitation in some marine systems. An alternative approach to diagnosing iron-limitation uses molecules whose abundance increases in response to iron-limitation. These include cell surface iron-transport proteins, and the electron transfer protein flavodoxin which replaces the Fe-S protein ferredoxin in many Fe-deficient algae and cyanobacteria.

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“…The algal cytochrome was isolated according to the procedure described by Crespi et al [6] using water throughout the preparation so that only the nonexchangeable hydrogens remain deuterated in the protein isolated from 2H20-grown alga. Horse heart cytochrome c (type VI) was obtained from Sigma and used without further purification.…”

Section: Methodsmentioning

confidence: 99%

Resonance raman studies of a c type algal cytochrome deuterium shifts and a comparison with mammalian cytochrome c

Yamamoto

Palmer

Crespi

1976

Biochimica et Biophysica Acta (BBA) - Protein Structure

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A c type cytochrome isolated from Synechococcus lividus grown on water and 2H2O media, has been studied by resonance Raman spectroscopy. The spectra were taken on the oxidized and reduced protein with excitation within the Soret band at 441.6 nm to determine whether individual resonance Raman bands of the heme shift upon deuterium substitution and also to provide a comparison with the spectra of horse heart cytochrome c. Some of the shifts observed with the deuterated heme c are larger than the corresponding shifts in meso-deuterated metalloporphyrins suggesting mixing of peripheral substituent vibrations with the skeletal modes of the porphyrin macrocycle. The algal cytochrome exhibits resonance Raman spectra roughly similar to those of horse heart cytochrome c, consistent with its optical absorption spectra which is typical of c type cytochromes, although a detailed comparison reveals note-worthy differences between the spectra of the two proteins; this may be a reflection of the effect of non-methionine ligands and protein environment on the vibrations of the c type heme in the algal cytochrome.

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Extraction and purification of 1H, 2H, and isotope hybrid algal cytochrome, ferredoxin and flavoprotein

Cited by 38 publications

References 22 publications

FLAVODOXIN EXPRESSION AS AN INDICATOR OF IRON LIMITATION IN MARINE DIATOMS¹

FLAVODOXIN EXPRESSION AS AN INDICATOR OF IRON LIMITATION IN MARINE DIATOMS¹

The role of iron in phytoplankton photosynthesis, and the potential for iron-limitation of primary productivity in the sea

Resonance raman studies of a c type algal cytochrome deuterium shifts and a comparison with mammalian cytochrome c

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Extraction and purification of 1H, 2H, and isotope hybrid algal cytochrome, ferredoxin and flavoprotein

Cited by 38 publications

References 22 publications

FLAVODOXIN EXPRESSION AS AN INDICATOR OF IRON LIMITATION IN MARINE DIATOMS1

FLAVODOXIN EXPRESSION AS AN INDICATOR OF IRON LIMITATION IN MARINE DIATOMS1

The role of iron in phytoplankton photosynthesis, and the potential for iron-limitation of primary productivity in the sea

Resonance raman studies of a c type algal cytochrome deuterium shifts and a comparison with mammalian cytochrome c

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FLAVODOXIN EXPRESSION AS AN INDICATOR OF IRON LIMITATION IN MARINE DIATOMS¹

FLAVODOXIN EXPRESSION AS AN INDICATOR OF IRON LIMITATION IN MARINE DIATOMS¹