The effect of detergent treatment on methylene blue sensitized cytochrome b photoreduction in fractions from corn coleoptiles

Abstract

It was previously demonstrated that photoexcited methylene blue can act as electron donor in red light induced reduction of a particulate b-type cytochrome in fraction from etiolated corn coleoptiles (Zea mays L. WF 9 x Bear 38). It was postulated that the same cytochrome as the one active in blue light photoprocesses was involved. This study describes the effect of detergents upon such red light induced reductions in corn coleoptile preparations fractionated after differential centrifugation into 9 KP, 21 KP and 50 KP (500-9000g pellet, 9000-21,000g pellet and 21,000-50,000 g pellet, spun for 20, 20, and 45 min, respectively). Both Triton X-100 (more effective) and deoxycholate (less effective and somewhat destructive) could be used as solubilizers if concentrations above the critical micellar concentration were chosen. Tween 40 was ineffective and dodecyl sulphate affected the cytochrome so that it lost its accessibility to electrons from photoexcited methylene blue. The recovery, measured as the ratio between light induced absorbance change (LIAC) in the Soret region after and before solubilization, was highest in 9 KP (70%) and lower in 21 and 50 KP fractions (50% and 43% respectively). The band in the Soret region in light minus dark spectra had its peak at longer wavelengths compared to the dithionite reduced minus no addition absorption difference band, whether the sample was solubilized or not. Similar results were obtained when the material was separated on a discontinuous sucrose gradient (15/28/33/45% w/w sucrose). In such a separation, the distribution of LIAC between fractions (collected at the interfaces) was about the same after solubilization as before (solubilization brought about a slight shift towards heavier fractions). The ratios of LIAC to the dithionite reduced minus no addition absorbance difference decreased upon detergent treatment. The LIAC still had its peak at longer wavelengths compared to the peak obtained upon dithionite reduction.The usefulness of detergents in the purification of the particulate b-type cytochrome is discussed.

Distribution of cytochrome b photoreductions mediated by endogenous photosensitizer or methylene blue in fractions from corn and cauliflower

“…4; 9 KP L3, heavy fraction) may be explained by the fact that cyt c oxidase can be photoreduced, which results in a negative absorbance change in the region where LIAC is measured. This has been observed with both corn (Widell 1980) andcauliflower (Catibergs andde Greef 1982).…”

Comparison of two methods for the preparation of a membrane fraction of cauliflower inflorescences containing a blue light reducible b ‐type cytochrome

“…The 9KP was discarded and the 9KP supernatant centrifuged for one hour at 50' 000 g. The 50KP was resuspended in the homogenization buffer without mercaptoethanol (total volume of 0,133 ml g~^ original fresh weight of plant material). The material obtained in this way was similar to a combined 21KP and 50KP fraction as described by others (Britz et al, 1979, Widell 1980, Goldsmith et al, 1980, Leong and Briggs 1981, Caubergs and De Greef 1981, Aliquots of 2 ml of this membrane suspension were layered onto a 30 ml linear sucrose density gradient of 25 to 45% (w/w) sucrose dissolved in the MES-buffer, The sucrose gradient was centrifuged at 100 000 g in a 6x38 ml swing-out rotor (MSE) for 16 h. The gradients were fractionated in 1,5 ml portions. Those membrane fractions, containing LIAC activities (fractions 5 to 20) were pooled, diluted with 3 times the volume of sucrose-free MES buffer, pelleted and resuspended in 5 ml of the same buffer.…”

“…In the same way the upper phase was washed with fresh lower phase of the blatik. These transfers were repeated twice, A more detailed description of the procedure is found elsewhere (Widell and Larsson 1981), The washed upper and lower phases were diluted with 30 ml MES buffer without sucrose and pelleted. After resuspension in a volume of 0,017 ml g~' original fresh weight (which corresponds to 5 ml when 300 g fresh weight is used as starting material), a 4 ml aliquot of the resuspended material of the upper phase was divided onto 2 Urografin gradients and treated as described for the "post sucrose gradient" fractions.…”

Photocontrol of Fungal Development

“…olivicolor (F ARAJ SALMAN 1969). This is of special interest, as methylene blue can replace the flavin moeity of a b-type cytochrome in photoreducing cytochrome c (BRITZ et al 1979, WIDELL 1980.…”