Free RadicalBiology& Medicine, Vol. 13, pp. 305-318, 1992 Printed in the USA. All rights reserved.
0891-5849/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd.
Original Contribution SELECTION
AND
ANALYSIS OF SUPEROXIDE OF NEUROSPORA
DISMUTASE
MUTANTS
KENNETH D. MUNKRES Laboratory of Molecular Biology and Department of Genetics, The University of Wisconsin, Madison, WI 53706, U.S.A.
(Received 30 August 1991; Revised 25 February 1992; Accepted 27 April 1992) A b s t r a c t - - A survey of 12 genetically distinct, heat-sensitive mutants of Neurospora revealed three (un- 1, un-3, and un- 17) that are specifically deficient in the superoxide dismutase (SOD) isozymes SOD-2 (mitochondrial), SOD-3 (mitochondrial), SOD-4 (exocellular), respectively. Genetic analysis of the three mutants indicates that the enzyme deficiencies are probably the cause of the heat-sensitive phenotype. The phenotypes of the mutants are (1) no growth at the normally optimal temperature 35°C and comparatively inferior growth at 15-30°C; (2) inferior resistance to the oxidants paraquat or oxygen; (3) female sterility; and (4) inferior conidial viability and longevity. Paraquat or 02 inhibition is alleviated respectively by desferrioxamine-Mn (a SOD mimic) and tocopherol. Diverse antioxidants, including tocopherol, are therapeutic for the heat-sensitive and female-sterile phenotypes, and for inferior growth of wild type at stressfully high temperatures. The results support previous theories that heat stress is a form of oxyradical/oxidant stress and that antioxidant enzymes such as SOD are essential for normal growth, development, and longevity. Since the three genes may encode the three enzymes and are not closely linked to either one another or the family of antioxidant-enzyme regulatory genes Age-1, the latter apparently trans-regulate their expression. Keywords--Antioxidants, Tocopherol, Free radicals, Heat stress, Oxidative stress, Cell differentiation, Cell longevity, Oxyregulon
stress is a form of superoxide-mediated stress and (2) the enzymes are essential for normal growth and development. The results, together with previous genetic analysis, also further define the mechanisms involved in the regulation of the enzymes' synthesis. The selection method employed in the isolation of the described mutants is based on the theory that heat stress is a form of oxidative stress and the corollary that at a permissive temperature, some heat-sensitive mutants may be specifically deficient in one of the five known SOD isozymes. A class of heat-sensitive mutants described in this article is characterized by incapacity to grow at normally optimal temperatures 34-36°C and capacity to grow at 20-25°C without special nutritional supplement. Those mutants which are not able to grow at the higher temperatures on a "complete" medium consisting of minimal medium supplemented with malt, yeast extract, and amino acids are termed unknown (un) to indicate their unknown genetic defect, is'x6 Twenty-two un genes have been located in the nuclear genome by recombinational analysis, x5 Here, 12 un mutants are randomly selected from the collection of 22, grown at 24°C, and assayed for the five SOD isozymes. Three mutants
INTRODUCTION
In eucaryotes, five superoxide dismutase (SOD) isozymes have been identified and are defined by their subcellular location and cyanide sensitivity (Table 1). In addition to the well-characterized "cytosolic," cyanide-sensitive Cu/Zn-containing isozyme SOD- 1 and the mitochondrial-matrix-space, cyanide-resistant Mn-containing isozyme SOD-2 (Ref. 1), cyanide-sensitive SOD-3 has been found to reside between the outer and inner mitochondrial membranes? -s Exocellular (EC) SOD-4 is bound to the cell wall in Neurospora 1°-~ and Saccharomyces 9"~3and mammals.~4 EC SOD-5 is found in the culture media of Neurospora (this article) and Saccharomyces ~3and in extracellular fluid of mammals. 9'~4 This article describes the selection and analysis of three heat-sensitive Neurospora mutants, each respectively deficient in one of three SOD isozymes. The objectives are to test previous theories that (l) heat Address correspondence to: Kenneth D. Munkres, Laboratory of Molecular Biology, University of Wisconsin, 1525 Linden Drive, Madison, WI 53706. 305
306
K.D. MUNKRES
Table 1. Subcellular Location, Cyanide Sensitivity, and Mutants of N e u r o s p o r a Superoxide Dismutase Isozymesa
Culture media and methods
Subcellular Location
Vogel's minimal medium 21 with 2% reagent-grade sucrose was used for liquid and solid media. Solid medium contained 2% agar (Difco-Bacto, Detroit, MI). Conidia were produced at 22-25°C on 20 mL of solid medium in 125 mL Erlenmeyer flasks, harvested, and counted as described. 8'~8 Mycelia for enzyme analysis (Table 3) were obtained by inoculating 100 mL of liquid medium in 250-mL Erlenmeyer flasks with 108 fresh conidia, and incubating at 24°C on a rotary shaker at 250 rpm for 24 h, in the case of wild type, or 36 h for the mutants. (Since the mutants generally grow slower than wild type, they were incubated longer to obtain about equivalent yields.) Standing, nonaerated liquid cultures (Tables 4, 5, 7) were 18 × 150 mm test tubes with 10 mL of medium. Growth in aerated liquid cultures (Table 4) was measured with 125-mL Erlenmeyer flasks containing I0 mL of medium on a rotary shaker at 250 rpm. Extensional growth in pure oxygen was measured on solid medium in petri plates (Table 4). The plates were centrally inoculated with fresh conidia, incubated overnight at 24°C to initiate growth, and placed in a steel chamber (Torbal, Model AJ.2, Torsion Balance Co., Clifton, N J). The chamber was flushed with oxygen for a period of 15-30 min, adjusted to 1.7 atm of oxygen, and the cultures were incubated at 24°C. Control plates were incubated in air. Periodically, the position of the growing mycelial frontier was marked on the bottom of the plate with a felt pen to measure extensional growth rate (mm h-l). Steady-state extensional growth rates as a function of temperature were measured with 30 cm race tubes containing 25 mL of Vogel's minimal, 2% sucrose, and 2% purified agar (Difco-Bacto, Detroit, M1). 22 The cultures were started at a low temperature (1215 °C) and periodically moved to higher temperatures after steady-state growth rates were established: That procedure is essential to obtain maximal growth rates of wild type at temperatures above 35°C since the cells adapt to graded heat stress. In addition, at incubation temperatures of 40°C and above, wild-type mycelia were grown in a chamber with 85-100% relative humidity to avoid desiccation. Mating procedures and the collection and enumeration of ascospores were performed as described, is.19 Supplements were added to media from aseptic solutions in sterile-distilled water after the medium was sterilized by autoclaving and cooled. Highly concentrated stock solutions oftocopherol, tocopherol succinate, and ascorbyl palmitate were freshly prepared
Cytosol Mitochondria Exocellular
Isozyme Abbreviation
Cyanide Sensitivity
SOD- 1 SOD-2 SOD-3 SOD-4 SOD-5
+ + +
Deficient Mutants b
un- 1 un-3 u n - 17
+
a See text for additional description. b See Table 3.
deficient in SOD-2 (un-1), SOD-3 (un-3), and SOD-4 (un-17), respectively, were characterized with respect to temperature sensitivity, oxidant resistance, fertility, and growth responses in the presence of antioxidants. The results support the previous theory that antioxidant enzymes of Neurospora, including SOD, play a defensive role which is essential for its normal growth, development, and longevity,s'1°-~2'~7The specific roles of each of the three SOD isozymes in growth and sexual differentiation are indicated here. Previous analysis indicates that the conidia of the un mutants are inferior in viability and longevity, t8 Evidence in support of the theory that heat stress is a form of oxy-radical (oxidant) stress is presented. The results, in conjunction with linkage data about the three un genes, IS which probably encode each of the three SOD isozymes, and the antioxidant-enzyme, regulatory-gene family Age-1 (Ref. 19) provide additional definition of the oxy-regulon, a global genetic unit that regulates the synthesis of many antioxidant enzymes. ~°-12 An abstract of this research was published. 2° MATERIALS AND METHODS
Strains
The wild type A-1-9A was from inbred Oak-Ridge wild type. t8 The wild type 2-2A was newly isolated after a second generation of inbreeding. The un mutants were obtained from the Fungal Genetics Stock Center, University of Kansas Medical Center, Kansas City, KS, and from D. D. Perkins, Stanford University (Table 2). Perkins's strains, inbred to Oak Ridge, were inbred once again (Table 2). The origins and characteristics of the mutants were reviewed. 15 Master stocks were stored on culture slants at -20°C. (All temperatures are measured in centigrade.) Fresh subcultures were grown for each experiment.
SOD-deficient Neurospora mutants
307
Table 2. Neurospora Strains Used in This Research
Strain or Locus No. Wild type (Oak Ridge) un locus no.: 1 2 3 5 6 7 10 14 16 17 21 22 un-lA un-la un-3A un-3a un-lA un-la un-3A un-3a
Allele or Isolate No.
FGSC Stock No., Perkins No., or Munkres No.
Linkage Group
A- I-9A 2.2A
---
---
308 1956 636 4000 1328 2175 2341 2348 4306 2356 3307 4323 P 1722 P 1723 P 1432 P 1774 MI-13 F5 M 1-1 F5 M 3-1 F4 M 3-4 F5
1R IC IL IL IIIR IR VIIR IIIR IL IIIR IIIR VII IR IR IL IL IR IR IL IL
44409 46006 55701 b39 83106 T53M50 T42M45 T54M55 T42M38 T51M171 T53M26 61C 44409 44409 55701 55701 44409 44409 55701 55701
(t) (t) (t) (t) (t) (t) (t) (t) (t) (t) (t) (t) (t) (t) (t) (t) (t) (t) (t) (t)
Genetic Background* SL SL AXL SL3 AXC SL6 AXL SL SL SL SL SL SL2 SL SL4 SL4 SL3 SL4 SL5 SL5 SL4 SL5
a All of the strains, except FGSC 308 and 636, are in St. Lawrence wild-type background. (St. Lawrence and Oak Ridge wild types are customarily assumed to be equivalent.) The F4 and F5 lines of un- 1 and un-3 are obtained by additional backcrosses of the Perkins isolates to Oak Ridge wild type. The original un- 1 and un-3 mutants are in mixed genetic background (i.e., Abbot x Lindegren and Abbot x Chilton).
with 95% ethanol and serially diluted in sterile distilled water.
Enzyme extraction and assay Mycelia were collected on a Biachner funnel, pressed dry with paper towels, and stored at -20°C. SOD-5 was isolated from fresh culture filtrate.13 SOD4 was extracted with KBr from a measured weight of mycelia.9-12 Intracellular enzymes were isolated as follows. Mycelia (1 g) were minced with scissors, dispersed with a mechanical homogenizer in 20 mL of an ice-cold solution of 10 mM N-morpholinopropane sulfonate buffer, pH 7.6, containing 1 mM ethylenediaminetetraacetic acid (EDTA) and 0.44 M sucrose, and broken in a carborundum grind mill. 8 After centrifugation of the extract at 4°C for 10 rain at 1000 x g to remove cell walls and unbroken nuclei, the supernatant was centrifuged 20 rain at 20,000 X g. The 20,000 x g supernatant ("cytosol") containing SOD- 1 was stored not more than 1 or 2 d at 5°C before assay or at -20°C for not more than 1-2 weeks. The crude mitochondrial pellet was suspended in 1 mL of the extraction medium, purified by density equilibrium centrifugation in a sucrose step-gradient containing the ex-
traction buffer, diluted fourfold with the extraction buffer without sucrose, broken by sonication, 8 and centrifuged 20 min at 20,000 x g. The supernatant, containing SOD-2, SOD-3, and submitochondrial particles, was stored at 5 or - 2 0 ° C for periods of 2 days or 1-2 weeks, respectively. Superoxide dismutase (EC 1.15.1.1) was assayed at 36°C by the method of Misra and Fridovich.23 Differential cyanide sensitivity of SOD isozymes in mixtures is assayed with and without 100 mM cyanide. 13 Misra 24 noted that the activities of cyanide-resistant and cyanide-sensitive superoxide dismutases are respectively diminished and enhanced by the higher pH (10.2) of the assay, relative to the activities in the xanthine oxidase-cytochrome c assay at pH 7.8. Since the latter is physiologically more relevant, the activities are normalized to pH 7.8, using Misra's correction factors.24 Since the yields of protein in the exoceUular, cytosolic, and mitochondrial fractions from a given amount of cells differ greatly, the SOD activities were expressed in terms of unit cell weight to estimate the relative proportions of the isozymes in the subceUular fractions. Those estimates are useful for comparison of strains, but they are not absolute because the additional manipulations of the mitochondria during purl-
308
K. D. MUNKRES Table 3. Enzyme Activity Profiles of Neurospora Wild-Type and Heat-Sensitive Mutants Enzyme-Specific Activityc Subcellular Localization ~ Exocellular
Exp. No?
Strain or Locus
Isolate No. b
wild un-1 un-3 un-17 wild un-I un-3 wild un-I un-3
A-1-9A 308 636 2356 2-2A 1-13F5 3-4 F~ 2-2A 1-13 F~ 3-4 F~
1
2a
2b
Cytosol
Mitochondria
SOD-5
SOD-4
CAT
POX
SOD-2
SOD- 1
CRD
710
7,500 9,000 7,300 0
775 1,325 900 1,100 660 860 1,700
25 32 II 32
0891-5849/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd.
Original Contribution SELECTION
AND
ANALYSIS OF SUPEROXIDE OF NEUROSPORA
DISMUTASE
MUTANTS
KENNETH D. MUNKRES Laboratory of Molecular Biology and Department of Genetics, The University of Wisconsin, Madison, WI 53706, U.S.A.
(Received 30 August 1991; Revised 25 February 1992; Accepted 27 April 1992) A b s t r a c t - - A survey of 12 genetically distinct, heat-sensitive mutants of Neurospora revealed three (un- 1, un-3, and un- 17) that are specifically deficient in the superoxide dismutase (SOD) isozymes SOD-2 (mitochondrial), SOD-3 (mitochondrial), SOD-4 (exocellular), respectively. Genetic analysis of the three mutants indicates that the enzyme deficiencies are probably the cause of the heat-sensitive phenotype. The phenotypes of the mutants are (1) no growth at the normally optimal temperature 35°C and comparatively inferior growth at 15-30°C; (2) inferior resistance to the oxidants paraquat or oxygen; (3) female sterility; and (4) inferior conidial viability and longevity. Paraquat or 02 inhibition is alleviated respectively by desferrioxamine-Mn (a SOD mimic) and tocopherol. Diverse antioxidants, including tocopherol, are therapeutic for the heat-sensitive and female-sterile phenotypes, and for inferior growth of wild type at stressfully high temperatures. The results support previous theories that heat stress is a form of oxyradical/oxidant stress and that antioxidant enzymes such as SOD are essential for normal growth, development, and longevity. Since the three genes may encode the three enzymes and are not closely linked to either one another or the family of antioxidant-enzyme regulatory genes Age-1, the latter apparently trans-regulate their expression. Keywords--Antioxidants, Tocopherol, Free radicals, Heat stress, Oxidative stress, Cell differentiation, Cell longevity, Oxyregulon
stress is a form of superoxide-mediated stress and (2) the enzymes are essential for normal growth and development. The results, together with previous genetic analysis, also further define the mechanisms involved in the regulation of the enzymes' synthesis. The selection method employed in the isolation of the described mutants is based on the theory that heat stress is a form of oxidative stress and the corollary that at a permissive temperature, some heat-sensitive mutants may be specifically deficient in one of the five known SOD isozymes. A class of heat-sensitive mutants described in this article is characterized by incapacity to grow at normally optimal temperatures 34-36°C and capacity to grow at 20-25°C without special nutritional supplement. Those mutants which are not able to grow at the higher temperatures on a "complete" medium consisting of minimal medium supplemented with malt, yeast extract, and amino acids are termed unknown (un) to indicate their unknown genetic defect, is'x6 Twenty-two un genes have been located in the nuclear genome by recombinational analysis, x5 Here, 12 un mutants are randomly selected from the collection of 22, grown at 24°C, and assayed for the five SOD isozymes. Three mutants
INTRODUCTION
In eucaryotes, five superoxide dismutase (SOD) isozymes have been identified and are defined by their subcellular location and cyanide sensitivity (Table 1). In addition to the well-characterized "cytosolic," cyanide-sensitive Cu/Zn-containing isozyme SOD- 1 and the mitochondrial-matrix-space, cyanide-resistant Mn-containing isozyme SOD-2 (Ref. 1), cyanide-sensitive SOD-3 has been found to reside between the outer and inner mitochondrial membranes? -s Exocellular (EC) SOD-4 is bound to the cell wall in Neurospora 1°-~ and Saccharomyces 9"~3and mammals.~4 EC SOD-5 is found in the culture media of Neurospora (this article) and Saccharomyces ~3and in extracellular fluid of mammals. 9'~4 This article describes the selection and analysis of three heat-sensitive Neurospora mutants, each respectively deficient in one of three SOD isozymes. The objectives are to test previous theories that (l) heat Address correspondence to: Kenneth D. Munkres, Laboratory of Molecular Biology, University of Wisconsin, 1525 Linden Drive, Madison, WI 53706. 305
306
K.D. MUNKRES
Table 1. Subcellular Location, Cyanide Sensitivity, and Mutants of N e u r o s p o r a Superoxide Dismutase Isozymesa
Culture media and methods
Subcellular Location
Vogel's minimal medium 21 with 2% reagent-grade sucrose was used for liquid and solid media. Solid medium contained 2% agar (Difco-Bacto, Detroit, MI). Conidia were produced at 22-25°C on 20 mL of solid medium in 125 mL Erlenmeyer flasks, harvested, and counted as described. 8'~8 Mycelia for enzyme analysis (Table 3) were obtained by inoculating 100 mL of liquid medium in 250-mL Erlenmeyer flasks with 108 fresh conidia, and incubating at 24°C on a rotary shaker at 250 rpm for 24 h, in the case of wild type, or 36 h for the mutants. (Since the mutants generally grow slower than wild type, they were incubated longer to obtain about equivalent yields.) Standing, nonaerated liquid cultures (Tables 4, 5, 7) were 18 × 150 mm test tubes with 10 mL of medium. Growth in aerated liquid cultures (Table 4) was measured with 125-mL Erlenmeyer flasks containing I0 mL of medium on a rotary shaker at 250 rpm. Extensional growth in pure oxygen was measured on solid medium in petri plates (Table 4). The plates were centrally inoculated with fresh conidia, incubated overnight at 24°C to initiate growth, and placed in a steel chamber (Torbal, Model AJ.2, Torsion Balance Co., Clifton, N J). The chamber was flushed with oxygen for a period of 15-30 min, adjusted to 1.7 atm of oxygen, and the cultures were incubated at 24°C. Control plates were incubated in air. Periodically, the position of the growing mycelial frontier was marked on the bottom of the plate with a felt pen to measure extensional growth rate (mm h-l). Steady-state extensional growth rates as a function of temperature were measured with 30 cm race tubes containing 25 mL of Vogel's minimal, 2% sucrose, and 2% purified agar (Difco-Bacto, Detroit, M1). 22 The cultures were started at a low temperature (1215 °C) and periodically moved to higher temperatures after steady-state growth rates were established: That procedure is essential to obtain maximal growth rates of wild type at temperatures above 35°C since the cells adapt to graded heat stress. In addition, at incubation temperatures of 40°C and above, wild-type mycelia were grown in a chamber with 85-100% relative humidity to avoid desiccation. Mating procedures and the collection and enumeration of ascospores were performed as described, is.19 Supplements were added to media from aseptic solutions in sterile-distilled water after the medium was sterilized by autoclaving and cooled. Highly concentrated stock solutions oftocopherol, tocopherol succinate, and ascorbyl palmitate were freshly prepared
Cytosol Mitochondria Exocellular
Isozyme Abbreviation
Cyanide Sensitivity
SOD- 1 SOD-2 SOD-3 SOD-4 SOD-5
+ + +
Deficient Mutants b
un- 1 un-3 u n - 17
+
a See text for additional description. b See Table 3.
deficient in SOD-2 (un-1), SOD-3 (un-3), and SOD-4 (un-17), respectively, were characterized with respect to temperature sensitivity, oxidant resistance, fertility, and growth responses in the presence of antioxidants. The results support the previous theory that antioxidant enzymes of Neurospora, including SOD, play a defensive role which is essential for its normal growth, development, and longevity,s'1°-~2'~7The specific roles of each of the three SOD isozymes in growth and sexual differentiation are indicated here. Previous analysis indicates that the conidia of the un mutants are inferior in viability and longevity, t8 Evidence in support of the theory that heat stress is a form of oxy-radical (oxidant) stress is presented. The results, in conjunction with linkage data about the three un genes, IS which probably encode each of the three SOD isozymes, and the antioxidant-enzyme, regulatory-gene family Age-1 (Ref. 19) provide additional definition of the oxy-regulon, a global genetic unit that regulates the synthesis of many antioxidant enzymes. ~°-12 An abstract of this research was published. 2° MATERIALS AND METHODS
Strains
The wild type A-1-9A was from inbred Oak-Ridge wild type. t8 The wild type 2-2A was newly isolated after a second generation of inbreeding. The un mutants were obtained from the Fungal Genetics Stock Center, University of Kansas Medical Center, Kansas City, KS, and from D. D. Perkins, Stanford University (Table 2). Perkins's strains, inbred to Oak Ridge, were inbred once again (Table 2). The origins and characteristics of the mutants were reviewed. 15 Master stocks were stored on culture slants at -20°C. (All temperatures are measured in centigrade.) Fresh subcultures were grown for each experiment.
SOD-deficient Neurospora mutants
307
Table 2. Neurospora Strains Used in This Research
Strain or Locus No. Wild type (Oak Ridge) un locus no.: 1 2 3 5 6 7 10 14 16 17 21 22 un-lA un-la un-3A un-3a un-lA un-la un-3A un-3a
Allele or Isolate No.
FGSC Stock No., Perkins No., or Munkres No.
Linkage Group
A- I-9A 2.2A
---
---
308 1956 636 4000 1328 2175 2341 2348 4306 2356 3307 4323 P 1722 P 1723 P 1432 P 1774 MI-13 F5 M 1-1 F5 M 3-1 F4 M 3-4 F5
1R IC IL IL IIIR IR VIIR IIIR IL IIIR IIIR VII IR IR IL IL IR IR IL IL
44409 46006 55701 b39 83106 T53M50 T42M45 T54M55 T42M38 T51M171 T53M26 61C 44409 44409 55701 55701 44409 44409 55701 55701
(t) (t) (t) (t) (t) (t) (t) (t) (t) (t) (t) (t) (t) (t) (t) (t) (t) (t) (t) (t)
Genetic Background* SL SL AXL SL3 AXC SL6 AXL SL SL SL SL SL SL2 SL SL4 SL4 SL3 SL4 SL5 SL5 SL4 SL5
a All of the strains, except FGSC 308 and 636, are in St. Lawrence wild-type background. (St. Lawrence and Oak Ridge wild types are customarily assumed to be equivalent.) The F4 and F5 lines of un- 1 and un-3 are obtained by additional backcrosses of the Perkins isolates to Oak Ridge wild type. The original un- 1 and un-3 mutants are in mixed genetic background (i.e., Abbot x Lindegren and Abbot x Chilton).
with 95% ethanol and serially diluted in sterile distilled water.
Enzyme extraction and assay Mycelia were collected on a Biachner funnel, pressed dry with paper towels, and stored at -20°C. SOD-5 was isolated from fresh culture filtrate.13 SOD4 was extracted with KBr from a measured weight of mycelia.9-12 Intracellular enzymes were isolated as follows. Mycelia (1 g) were minced with scissors, dispersed with a mechanical homogenizer in 20 mL of an ice-cold solution of 10 mM N-morpholinopropane sulfonate buffer, pH 7.6, containing 1 mM ethylenediaminetetraacetic acid (EDTA) and 0.44 M sucrose, and broken in a carborundum grind mill. 8 After centrifugation of the extract at 4°C for 10 rain at 1000 x g to remove cell walls and unbroken nuclei, the supernatant was centrifuged 20 rain at 20,000 X g. The 20,000 x g supernatant ("cytosol") containing SOD- 1 was stored not more than 1 or 2 d at 5°C before assay or at -20°C for not more than 1-2 weeks. The crude mitochondrial pellet was suspended in 1 mL of the extraction medium, purified by density equilibrium centrifugation in a sucrose step-gradient containing the ex-
traction buffer, diluted fourfold with the extraction buffer without sucrose, broken by sonication, 8 and centrifuged 20 min at 20,000 x g. The supernatant, containing SOD-2, SOD-3, and submitochondrial particles, was stored at 5 or - 2 0 ° C for periods of 2 days or 1-2 weeks, respectively. Superoxide dismutase (EC 1.15.1.1) was assayed at 36°C by the method of Misra and Fridovich.23 Differential cyanide sensitivity of SOD isozymes in mixtures is assayed with and without 100 mM cyanide. 13 Misra 24 noted that the activities of cyanide-resistant and cyanide-sensitive superoxide dismutases are respectively diminished and enhanced by the higher pH (10.2) of the assay, relative to the activities in the xanthine oxidase-cytochrome c assay at pH 7.8. Since the latter is physiologically more relevant, the activities are normalized to pH 7.8, using Misra's correction factors.24 Since the yields of protein in the exoceUular, cytosolic, and mitochondrial fractions from a given amount of cells differ greatly, the SOD activities were expressed in terms of unit cell weight to estimate the relative proportions of the isozymes in the subceUular fractions. Those estimates are useful for comparison of strains, but they are not absolute because the additional manipulations of the mitochondria during purl-
308
K. D. MUNKRES Table 3. Enzyme Activity Profiles of Neurospora Wild-Type and Heat-Sensitive Mutants Enzyme-Specific Activityc Subcellular Localization ~ Exocellular
Exp. No?
Strain or Locus
Isolate No. b
wild un-1 un-3 un-17 wild un-I un-3 wild un-I un-3
A-1-9A 308 636 2356 2-2A 1-13F5 3-4 F~ 2-2A 1-13 F~ 3-4 F~
1
2a
2b
Cytosol
Mitochondria
SOD-5
SOD-4
CAT
POX
SOD-2
SOD- 1
CRD
710
7,500 9,000 7,300 0
775 1,325 900 1,100 660 860 1,700
25 32 II 32
