Soluble NAD- and NADP-Linked Oxidoreductases in Filamentous Marine Fungi: Adaptation of Fluorescent Screening Technique and Development of Supporting Methodology

The metabolism of filamentous marine fungi has received little attention
either from the specific or comparative standpoints. As part of
an overall investigation of the carbon metabolism of this heterogeneous
group of Ascomycetes and Deuteromycetes, a rapid, semi-quantitative
method has been adapted to the screening of their NAD- and NADP-linked
dehydrogenases. The Ascomycete, Halosphaeria mediosetigera (700), and
Deteromycetes, Culcitalna achraspora (230) and Humicola alopallonella
(710), were grown in a semi-synthetic, artificial sea water broth in submerged
culture, from quantitatively standardized inocula, and harvested
in the linear growth phase when respirometric data indicated maximum
endogenous oxygen uptakes. Based upon dry weight, culture 700 gave
QO2 maxima of 9.3-14.6 at 72 hr growth, while culture 710 values were
12.4-16.2 for the same time; culture 230 gave values of 13.8-18.4 at
96 hr growth. Harvested mycelia were washed, quantitated and subjected
to sonic disintegration at 21 kHz for 20 minutes at 5-10 C in pH 7.0
phosphate buffer containing cysteine. Debris was removed by centrifuging
at 12,000 X g at 4 C for 30 minutes and the resulting turbid, yellow
supernatant fraction clarified by two successive 2 hr centrifugings at
4 C and 41,000 X g. Whatman #1 filter paper sheets, divided into 1 cm. squares, were pretreated with pH 8.0 Tris buffer containing MgCl2, and
air-dried. The papers were spotted at the grid intersections with appropriate substrates. Cell extracts were mixed with NAD or NADP in Tris and
the mixture superimposed upon the substrates. Within 2 hr the papers
were viewed under ultraviolet light at 3600 A for spot fluorescence from
the reduced coenzymes. In all extracts (5-10 mg protein per ml),
dehydrogenases were indicated for glucose, glucose-6-phosphate, gluconate,
6-phosphogluconate, isocitrate and malate. Less definite were indications
of oxidoreductases for galactose, lactate and glycerol. It is therefore indicated that these filamentous marine fungi utilize glucose carbon via
the hexose monophosphate pathway and the tricarboxylic acid cycle.