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

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Date Issued:
1970
Summary:
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.
Title: Soluble NAD- and NADP-Linked Oxidoreductases in Filamentous Marine Fungi: Adaptation of Fluorescent Screening Technique and Development of Supporting Methodology.
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Name(s): Rodrigues, Joanna
Sguros, Peter L., Thesis advisor
Florida Atlantic University, Degree grantor
Type of Resource: text
Genre: Electronic Thesis Or Dissertation
Date Created: 1970
Date Issued: 1970
Publisher: Florida Atlantic University
Place of Publication: Boca Raton, Fla.
Physical Form: application/pdf
Extent: 71 p.
Language(s): English
Summary: 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.
Identifier: FA00000821 (IID)
Degree granted: Thesis (M.S.)--Florida Atlantic University, 1970.
Note(s): Charles E. Schmidt College of Science
Subject(s): Marine fungi
Dehydrogenases
Held by: Florida Atlantic University Libraries
Sublocation: Digital Library
Persistent Link to This Record: http://purl.flvc.org/fau/fd/FA00000821
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Host Institution: FAU
Is Part of Series: Florida Atlantic University Digital Library Collections.