Rapid degradation of FAD following lysis of Neurospora crassa cells: Consequences for evaluation of flavin composition in vivo

Rapid degradation of FAD following lysis of Neurospora crassa cells: Consequences for evaluation of flavin composition in vivo. This regular paper is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/vol31/iss1/10 Ram, S., B.G. Nair and H.S. Chhatpar From our earlier studies on the effect of inorganic phosphate on alterations in phospholipids (Nair and Chhatpar, Phosphate mediated changes in fatty 1983, Neurospora Newsletter 30: 11) and changes in sugar uptake in Neurospora crassa (Savant, Parikh and Chhatpar, 1982, acid composition in Neurospora crassa. Experientia 38: 31O-311), we suggested that phosphate might play an important role at the membrane level with respect to uptake and permeability functions. Further, we were interested in seeing the effect of inorganic phosphate on membrane fatty acid composition since alterations in fatty acid composition have been shown to result in changes in ion permeability and enzyme activity (Davis and Silbert, 1974, Biochim. Biophys. Acta 373: 224; Dekruyff, et al., 1973, Biochim. Biophys. Acta 298: 479). The synthetic liquid medium employed for the growth of N. crassa (wild type, carotenogenic) contained per litre: glucose, 50 g; trisodium citrate, 2.5 g ; (NH4)2SO 2.5 g; MgS04.7H2O, 0.5 g; ZnS04.7H2O, 2.5 mg; FeCl3, 5.0 mg; CaCl2, 10 mg; and biotin 100 pg. The pH was adjusted to 5.6. 'High phosphate' condition indicates the addition of KH2PO KH2PO4 to the above medium. 1.0 g% whereas 'Low phosphate' condition indicates the addition of 0.01 g% These phosphate conditions did not change the pH of the medium. Growth temperature was 30° C. Culture density at harvest was determined by drying mycelial mats at 50° C to constant weight: for the low phosphate culture dry mat weight was 0.24 g/50 ml flask, and for the high phosphate culture it was 0.28 g/50 ml flask.


Scheideler, L., B. Hahne and H. Ninnemann
In an attempt to judge the possible participation of free flavins in blue light photoreceotion processes in Neurospora, we determined size and composition of intracellular flavin pools. To minimize artificial liberation of in vivo proteinbound (or associated ) flavins during handling we worked with the cell wall-less mutant "slime" (FGSC #1118) which can be easily lysed by osmotic shock.

Rapid degradation of FAD following lysis of Neurospora crassa cells: consequences for evaluation of flavin composition in vivo.
Cells were cultivated in a gyratory shaker (100 rpm) in darkness at 30° C in 250 ml Erlenmeyer flasks containing 50 ml Vogel's minimal medium (Vogel 1956 Microbial. Genet. Bull. 13: 42-43) with 10% sorbitol and 2% sucrose.
Under these conditions maximal cell number is reached after 60 h; thus from the third day on, cultures are in the stationary phase.
Cultures 2 to 9 d old were harvested (centrifugation 10 min/l90 g) and lysed with 4 to 8 ml cold double-distilled water. After centrifugation (Beckman Spinco L 50, 120 min/100,000 g), 2-4 ml of the resulting supernatant were run overnight at 5° C on a Sephadex G-50 medium column (1.55 cm, 90 cm length) in 50 mM KH 2 PO 4 buffer at pH 7.0: fraction size was 1.8 ml.
as A 280 nm and also tested by the Lowry method.
The protein content was monitored The elution profile of flavins was traced by measuring fluorescence emission at 525 nm upon excitation at 466 nm.
Fractions containing flavoprotein or free flavin were pooled separately. Analysis of individual flavins in both pools was performed by phenol-extraction (modified after Yagi, 1962. In Meth. Biochem. Anal. Vol. X, Glick ed.: 319-356). After saturation of samples with ammonium sulfate, flavins were extracted twice with phenol,thenre-extracted into a small volume of water and separated by TLC on Merck silica gel H type 60 with 135 mM Na2HPO4 as solvent. Flavin concentrations were determined after alkaline photolysis (Yagi 1962) as lumiflavin fluorescence.

From cell lysates of a cell culture of slime, bound-and free flavins were separated by Sephadex G-50 filtration.
A representative fractionation pattern is shown where bound flavins appeared in fractions no. 21-29 and free flavins in fractions no. 58-70 (Fig. 1). The fluorescence of fractions 21-29 coincided with the protein peak and with the major absorbance maximum at 280 nm. A minor protein peak (peptides?) was observed in fractions 53-56 which appeared before the peak of free flavins; the latter was free of protein.
The abosrobance at 280 nm in fractions 64-72 is likely due to nonproteinaceous components since there could be no more than 5 µg/ml protein in these fractions as determined by the Lowry method.
--X-U In the bound flavin fraction, ll-28% FAD was detected and around 55% and 25% of FMN and RF respectively.

F R A C T I O N N U M B E R
1.0 to 3.3 nmoles of total flavin were put onto the gel.
The unexpectedly low concentration of free FAD in both fractions could have been due to degradation during processing.
Adding exogenous FAD to a cell lysate of a 5 d culture and exposing this mixture to our experimental conditions showed fast degradation of the added FAD. Conditions were: a) leaving the mixture for maximally 5 set after addition of FAD at 20° C and heating to 80° C. This resulted in a decrease in the FAD content from 89% of total flavin to 63%; b) incubation of mixtures for 10 min at 5° C before heating: this resulted in a decrease in the FAD concentration to 28%; c) after gel filtration at 5° C overnight on Sephadex G-50 (i.e. under the same conditions as in the experiments mentioned earlier) only 25% of total flavin in the eluate was detected as FAD; the amount of FMN increased correspondingly (Table I).  The heat-treated sample was cooled to 23° C, 500 µl of unpurified FAD, added to each sample and to a third vial containing 25 ml water.
After 15 min at 23° C samples and control were heated for 15 min at 75° C. After cooling and centrifugation of the crude lysates for 20 min/50,000 g, flavins were extracted from the supernatants and the contra1 by the phenol method and separated.
The difference of total flavins in the FAD control compared with the heat treated and untreated samples is probably due to loss of flavins during pelleting of membranes and organelles after incubation.
The rapid degradation of FAD in the cell lysate has not been shown to be heat-labile and is likely to be enzyme-catalysed (Table II}. These results can be explained by phosphatase(s) splitting off the adenosine group of FAD.
The enzyme(s) is reacting fast even at low temperature and within the shortest possible handling time of a few seconds or minutes.
Enzyme activities of this kind must be taken into account before making statements on the composition of intracellular flavin pools in Neurospora or other organisms.
Data obtained after standard extraction procedures can be perturbed by enzymatic degradation of FAD and/or FMN after decompartmentation following lysis of the cells and may not represent in vivo conditions.
We are investigating the possibilities of specfic inhibition of the FAD degrading reaction(s). ---Insitut fur Chemische Pflanzenphysiologie der Universitat Tubingen, FRG.