- Email: [email protected]
Evidence for stimulation of glucose-6-phosphate dehydrogenase synthesis during initiation of periodic fruit body growth inAgaricus bisporus
EXPERIMENTAL
MYCOLOGY
(1985)
9, 116-121
Evidence for Stimulation of Glucose-6-phosphate Dehydrogenase Synthesis during Initiation of Periodic Fruit Body Growth in Agaricus bisporus TAKAHISA Glasshouse
Crops
Research
MINAMIDE’
Institute,
Worthing
AND JOHN B. W. HAMMOND~ Road,
Littlehampton,
Sussex
BN16
3PU,
United
Kingdom
Accepted for publication January 25, 1985 T., AND HAMMOND, J. B. W. 1985. Evidence for stimulation of glucose-6-phosphate synthesis during initiation of periodic fruit body growth in Agaricus bisporus. Experimental Mycology, 9, 116-121. Agaricus bisporus fruit bodies were harvested over several cycles of periodic fruiting (flushes) and assayed for glucose 6-phosphate dehydrogenase (G6PD) activity and for G6PD protein by enzyme-linked immunosorbent assay. The peaks of G6PD activity observed during flush emergence were accompanied by increases in enzyme protein. There was also an increase in G6PD molecular specific activity at flush emergence. Polyacrylamide gel electrophoresis and immunoassay suggested that this was due to the synthesis of a new form of G6PD at flush emergence, which was not antigenic to antibody raised against the constitutive enzyme. G6PD activity in the mycelium was low at all stages of the flushing cycle, as was G6PD protein. However, there was some evidence for the presence of the “new” form of G6PD in the mycelium at the time of flush emergence. 0 1985 Academic Press, IK. INDEX DESCRIPTORS: Agaricus bisporus; glucose-6-phosphate dehydrogenase; fruit body; mycelium; enzyme synthesis; isozyme; morphogenesis. MINAMIDE,
dehydrogenase
The cultivated mushroom Agaricm bisporus produces mature fruit bodies in periodic flushes at approximately weekly intervals. The initiation of flushes appears to be due to the release of a proportion of already formed fruit body initials for further development (Flegg, 1979). The stimulus for further development of initials has been suggested to be connected with levels of intracellular substrate, possibly glycogen and trehalose, in the mycelium (Hammond and Nichols, 1979; Chanter, 1979). In a previous report it was shown that glucose-6-phosphate dehydrogenase (G6PD)3 activity in fruit body extracts was
at a maximum at the time of flush emergence (Hammond, 1981). It was suggested that a resultant increase in pentose phosphate pathway flux could be important in initiating the early growth of the fruit bodies in a flush. In the work reported here, we explored the cause of the variations in G6PD activity in fruit body extracts and measured G6PD levels in the mycelium. The results suggest that flush initiation is accompanied by the synthesis in the fruit body of G6PD, some of which is of molecular form different from that found constitutively in this organ.
i Present address: Department of Horticulture, College of Agriculture, University of Osaka Prefecture, Sakai, Osaka 591, Japan. * To whom correspondence should be addressed at Physiology Department, Glasshouse Crops Research Institute, Worthing Road, Littlehampton, Sussex BN17 6LP, UK 3 Abbreviations used: ELISA, enzyme-linked immunosorbent assay; G6PD, glucose-6-phosphate dehydrogenase; MSA, molecular specific activity; PAGE, polyacrylamide gel electrophoresis.
A. bisporus (strain “Darlingtons 649”) was grown on compost under normal commercial conditions at the GCRI Mushroom Unit. Samples of 50-100 fruit bodies were taken as they reached stage 1 (Hammond and Nichols, 1976; “pinhead,” 5-mm diam, velum not differentiated) through the cropping period. They were cleaned, weighed, and frozen in liquid nitrogen. Mycelium
116 0147-5975185 $3.00 Copyright 0 1985 by Academic Press. Inc. All rights of reproduction in any form reserved.
MATERIALS
AND METHODS
G6PD
SYNTHESIS
AND
Agaricus
was sampled from the upper “casing” layer of the substrate using a 15mm-diameter cork borer, washed with iced water to remove traces of peat, and frozen in liquid nitrogen. Fruit body and mycelium samples were stored at - 20°C. Enzyme extraction. The frozen material was homogenized in Tris/HCl buffer (50 mM, pH 7.7) containing 50 mM 2-mercaptoethanol, 1 mM EDTA, 12 mM p-aminobenzamidine, 2 m44 phenylmethylsulfonyl fluoride, and 8 mM e-amino caproic acid. The homogenate was centrifuged at 20,OOOg for 20 min and the supernatant used for G6PD assay. All operations were carried out at 4°C. G4PD assay. G6PD was assayed as described previously (Hammond, 1981). Units of enzyme activity were defined as micromoles NADP+ reduced per minute under the conditions of the assay. A molar extinction coefficient of 6.2 x lo3 M-l cm-’ was assumed for NADPH. Protein content of extracts was determined by the Coomassie blue binding technique (Bradford, 1976), using bovine serum albumin standards ~ Measurement of G6PD protein by enzyme-linked immunosorbent assay (ELISA). G6PD was purified from stage 2 fruit bodies harvested during a flush, and antibody raised against the purified enzyme as described previously (Hammond, 198.5). The globulin fraction was purified and conjugated with alkaline phosphatase and the G6PD protein determined using the ELlSA method as described by Clark and Adams (1977). Extracts were diluted as necessary before assay to allow the response to fall on the calibration curve. Purified A. bisporus G6PD was used for calibration. Polyacrylamide gel electrophoresis (PAGE), Nondenaturing PAGE was carried out in 5% gels and the enzyme activity stained as described previously (Hammond, 1985). Laurel1 rocket immunoelectrophoresis of gel slices. After staining for G6PD activity,
FLUSH
INITIATION
117
the stained bands were cut singly from the rod gel and used for rocket immunoelectrophoresis (Laurel1 and McKay, 1981). The slices were laid on 1% agarose in Tris/barbiturate buffer (pH 8.6, 100 mM) containing 30 ~1 of antiserum/l2 ml agarose gel. Electrophoresis was carried out at 2 V/cm for 18 h. Plates were stained for protein wit Coomassie brilliant blue R-250. RESULTS
G6PD Activity in Fruit Body and Mycelium The results shown here are typical of data taken over four flushes in two separate crops. Each datum point is the mean of three determinations from each sample. G6PD activity in extracts of stage 1 fruit bodies sampled daily during the cropping period showed the same pattern of variation as that reported previously (Hammond, 1981), with a peak in activity at flush emergence (Fig. 1). The activity in mycelial extracts was considerably lower than that in sporophore extracts, on a soluble protein basis, over most of the experimental period (Fig. I). The activities of G6PD in fruit body and mycelium were only similar during flush growth when the activity in fruit bodies was very low. Smalt variations were seen in mycelial G6PD activity, wit some evidence of higher activity during the periods of high sporophore G6PD activity. Immunological Determination of G6PD Protein G6PD protein was determined by ELISA in fruit body and mycelial extracts. The increases in enzyme activity seen in fruit body extracts at flush emergence were accompanied by increases in the amount of G6PD protein on a total soluble protein basis (Fig. 2). In mycelial extracts the quantity of G6PD protein was lower than that seen in the fruit body. As with mycelial enzyme activity there were small variations with apparently higher levels around the period of flush emergence.
MINAMIDE Flush emergence
AND HAMMOND
tracts from material sampled at flush emergence showed an MSA for G6PD two to three times greater than extracts from material sampled during the periods of low G6PD activity. The pattern was seen most clearly in the fruit bodies. In mycelium, similar cyclic variations were observed, although due to the small amount of material that it was possible to isolate and to the low specific activity, the probable level of experimental error was greater.
Flush emergence
Polyacrylamide
IO
0 Time
20
(days)
FIG. 1. G6PD activity in stage 1 fruit bodies (0) and mycelium ((I) during flushing. Bars show range of mean.
When these data were recalculated on the basis of units of G6PD activity per milligram G6PD protein (G6PD MSA), the MSA varied through the flushing cycle in both fruit bodies and mycelium (Fig. 3). ExFlush emergence
Flush emergence
3.0
1
Gel Electrophoresis
The results of polyacrylamide gel electrophoresis of native protein in extracts, followed by staining for G6PD activity, are shown in Fig. 4. Fruit bodies sampled at the maxima and minima af G6PD activity were analyzed. G6PD from fruit bodies sampled at the minimum G6PD activity, i.e, during flush growth, resolved into three bands of activity (Bands 3-5, Fig. 4) as observed for the previously purified enzyme (Hammond, 1985), Extracts from fruit bodies sampled at the maximum of G6PD activity, i.e., at flush emergence, resolved into five bands of activity, with the two additional bands migrating more slowly than the other three (Bands 1 and 2, Fig. 4). The intensity of Flush emergence
Flush emergence
E
al 2.0 ij h 7 E” 2 $ 1.0 9
t-'4---~),_4-)--~-I--~.~~-. 0
I
I 0
10 Time
I 20
I 10
(days)
FZG. 2., Quantity of G6PD protein (determined by ELISA) in stage 1 fruit bodies (0) and mycelium (0) during flushing.
Time
(days)
FIG. 3. G6PD molecular specific activity in stage 1 fruit bodies (0) and mycelium (0) during flushing.
G6PD SYNTHESIS
a
b
AND
c
Agaricus
FLUSH INITIATION
most strongly staining and 4 (Figs. 4 and 5).
antigenic
ban
DISCUSSION
FIG. 4. Polyacrylamide gels stained for G6PD activity of extracts from stage 1 fruit bodies sampled (b) during flush growth and (c) at flush emergence. Blank of extract incubated without G6P is also shown (a). Bands l-5 G6PD activity: unnumbered band is an artifact.
staining suggests that the additional bands constitute one-third to one-half of the total G6PD activity in the sample. The individual bands were sliced from the gel and run in a Laurel1 rocket immunoelectrophoresis system to test the antigenicity of each to G6PD antiserum. As expected, the three bands found in both gels, and seen in the purified enzyme, showed rockets of similar height (Fig. 5), signifying similar degrees of antigenicity. The remaining two bands, observed only in samples taken at the time of flush emergence, gave rockets which were very small or not visible despite the fact that the intensity of activity staining was as great as that of the
The increase of G6PD activity in fruit bodies at flush emergence reported previously (Hammond, 1981) could have bee brought about through one or more of several mechanisms. Enzyme may have been translocated into the fruit body from the mycelium, synthesized in the fruit activated from an inactive precurso the periods of high activity. The data given here show that, while large variations in enzyme activity are seen in the fruit body during the flushing cycle, the activity of the enzyme in the myce~~um supporting the fruit body remains low. Thus it appears that the stimulation of G6PD activity at flush emergence is largely confine to the fruit body. A similar picture was seen when the level of enzyme protein was termined immunologically. Thus increase in enzyme activity was correlated with increase in enzyme protein Since negligible increases in enzyme protein were seen in the mycelium, the increases in GQPD are probably due to de n~vu synthesis of enzyme in the fruit body. However, the increase in G6PD MSA during flush emergence suggests that some other process also occurs. The resuhs PAGE and immunoelectrophoresis o resulting bands suggest that the reason for
FIG. 5. Laurel1 rocket immunoelectrophoresis of excised bands showing G6PD activity from polyacrylamide gels. Numbers refer to bands shown in Fig. 4.
120
MINAMIDE
AND
the increase in MSA is synthesis of electro.phoretically and immunologically distinct forms of G6PD (G6PD-2). The possibility of artifactual enzyme staining bands in PAGE of fungal developmental systems has been raised by Evers and Ross (1983). The G6PD-2 bands do not fall into this category since gels of extracts incubated without G6P showed only the single artifactual band of Fig. 4 in all extracts. Additionally, the inclusion of phenazine methosulfate in the incubation medium rules out the possibility of the diffusion of NADPH leading to artifacts as suggested by Evers and Ross (1983). G6PD-2 resolves separately on PAGE from the normal enzyme and has low antigenicity for G6PD antiserum. Thus although an increase in activity due to G6PD2 is observed, the increase in enzyme protein would not be seen in the ELISA, leading to an apparent increase in MSA. The antibody used in this assay was prepared from fruit bodies sampled at the period of maximum yield, i.e., during flush growth, when G6PD activity is low and G6PD-2 does not appear to be present. Thus the antibody used here was prepared from material which did not contain G6PD2. Although no evidence for the presence of G6PD-2 in mycelium could be obtained from PAGE, because of the low levels of materials available, the apparent increase in MSA of mycelial G6PD at flush emergence suggests that G6PD-2 is present in the mycelium at this time, and in a similar ratio to normal G6PD as observed in the fruit body. In a previous study three bands of G6PD activity were observed on nondenaturing polyacrylamide gels. These corresponded with protein bands when pure A. bisporus enzyme was electrophoresed (Hammond, 1985). All bands gave identical peptide maps, and only one band was seen on sodium dodecyl sulfate (SDS)-PAGE. Thus it was suggested that the three bands were due to different aggregation states of the same enzyme molecule. The two bands of
HAMMOND
G6PD-2 on native PAGE could similarly be due to different aggregation states of the G6PD-2 molecule. Forms of enzymes, including G6PD, specific to particular stages in development have been observed in other fungi, e.g., Coprinus cinereus (Moore and Jirjis, 1,981) and Sclerotinia sclerotiorum (Wong and Willetts, 1974). However, it appears that G6PD-2 in A. bisporus is produced at a specific stage of the flushing cycle rather than at a specific stage of fruit body development. Because of this, it may be more comparable with the synthesis of a new G6PD isozyme in Aspergillus oryzae in response to the presence of ribose (Muino-Blanc0 et al., 1983). The lack of antigenicity of G6PD-2 to constitutive G6PD antibody suggests that it has a different antigenic structure and therefore may be a different gene product. Further work is necessary to confirm this. If this proves to be the case, our report of G6PD-2 is the first recorded instance of a protein which is synthesized only during the period of flush emergence. This property should allow the protein to be used as a tool to study the mechanism by which flushing is stimulated. REFERENCES
BRADFORD,M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal. Biochem.
71: 248-254.
CHANTER, D. 0. 1979. Harvesting the mushroom crop: A mathematical model. J. Gen. Microbial. 115: 79-87.
CLARK, M. F., AND ADAMS, A. N. 1977. Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant vimses. J. Gen. Virol. 34: 475-483. EVERS, D. C., AND Ross, I. K. 1983. Isozyme patterns and morphogenesis in higher basidiomycetes. Exp. Mycol. 7: 9-16. FLEGG, P. B. 1979. Effects of competition on the development of the mycelium, mycelial aggregates and sporophores of Agaricus bisporus. Sci. Hortic. (Amsterdam) 11: 141-149. HAMMOND, J. B. W. 1981. Variations in enzyme ac-
G6PD SYNTHESIS
AND Agaricus FLUSH INITIATION
tivity during periodic fruiting of Agaricus bisporus. New Phytol. 89: 419-428. HAMMOND, J. B. W. 1985. Glucose 6-phophate dehydrogenase from Agaricus bisporus; purification and properties. J. Gen. Microbial. 131: 321-328. HAMMOND, J. B. W., AND NICHOLS, R. 1976. Carbohydrate metabolism in Agaricus bisporus (Lange) Sing.: Changes in soluble carbohydrates during growth of mycelium and sporophore. J. Gen. Microbiol. 93: 309-32.0. HAMMOND, J. B. W., AND NICHOLS, R. 1979. Carbohydrate metabolism in Agaricus bisporus: Changes in non-structural carbohydrates during periodic fruiting (flushing). New Phytol. 83: 723-730. LAURELL, C. B., AND MCKAY, E. J. 1981. Electroimmunoassay. In iMethods in Enzymology (J. J. Lan-
gone and H. Van Vunakis, Eds.), Vol. 73, pp. 339369. Academic Press, New York. MOORE, D., AND JIRJIS, R. I. 1981. Electrophoretic studies of carpophore development in the basidiomycete Coprinus cinereus. New Phytol. 87: 101113. MUINO-BLANCO, I., CEBRIAN-PEREZ, J. A., AND PEREZ-MARTOS, A. 1983. Regulation of the oxidative phase of the pentose phosphate cycle in Aspergillus oryzae (Ahlberg). I. Induction of glucose-6-phosphate dehydrogenase. Arch. Microbial. 136: 39-41. WONG, A. L., AND WILLETTS: H. J. 1974. Polyacrylamide-gel etectrophoresis of enzymes during morphogenesis of sclerotia of Sclerotinia scierotiorwm. J. Gen. Microbioi. 81: 101-109.
MYCOLOGY
(1985)
9, 116-121
Evidence for Stimulation of Glucose-6-phosphate Dehydrogenase Synthesis during Initiation of Periodic Fruit Body Growth in Agaricus bisporus TAKAHISA Glasshouse
Crops
Research
MINAMIDE’
Institute,
Worthing
AND JOHN B. W. HAMMOND~ Road,
Littlehampton,
Sussex
BN16
3PU,
United
Kingdom
Accepted for publication January 25, 1985 T., AND HAMMOND, J. B. W. 1985. Evidence for stimulation of glucose-6-phosphate synthesis during initiation of periodic fruit body growth in Agaricus bisporus. Experimental Mycology, 9, 116-121. Agaricus bisporus fruit bodies were harvested over several cycles of periodic fruiting (flushes) and assayed for glucose 6-phosphate dehydrogenase (G6PD) activity and for G6PD protein by enzyme-linked immunosorbent assay. The peaks of G6PD activity observed during flush emergence were accompanied by increases in enzyme protein. There was also an increase in G6PD molecular specific activity at flush emergence. Polyacrylamide gel electrophoresis and immunoassay suggested that this was due to the synthesis of a new form of G6PD at flush emergence, which was not antigenic to antibody raised against the constitutive enzyme. G6PD activity in the mycelium was low at all stages of the flushing cycle, as was G6PD protein. However, there was some evidence for the presence of the “new” form of G6PD in the mycelium at the time of flush emergence. 0 1985 Academic Press, IK. INDEX DESCRIPTORS: Agaricus bisporus; glucose-6-phosphate dehydrogenase; fruit body; mycelium; enzyme synthesis; isozyme; morphogenesis. MINAMIDE,
dehydrogenase
The cultivated mushroom Agaricm bisporus produces mature fruit bodies in periodic flushes at approximately weekly intervals. The initiation of flushes appears to be due to the release of a proportion of already formed fruit body initials for further development (Flegg, 1979). The stimulus for further development of initials has been suggested to be connected with levels of intracellular substrate, possibly glycogen and trehalose, in the mycelium (Hammond and Nichols, 1979; Chanter, 1979). In a previous report it was shown that glucose-6-phosphate dehydrogenase (G6PD)3 activity in fruit body extracts was
at a maximum at the time of flush emergence (Hammond, 1981). It was suggested that a resultant increase in pentose phosphate pathway flux could be important in initiating the early growth of the fruit bodies in a flush. In the work reported here, we explored the cause of the variations in G6PD activity in fruit body extracts and measured G6PD levels in the mycelium. The results suggest that flush initiation is accompanied by the synthesis in the fruit body of G6PD, some of which is of molecular form different from that found constitutively in this organ.
i Present address: Department of Horticulture, College of Agriculture, University of Osaka Prefecture, Sakai, Osaka 591, Japan. * To whom correspondence should be addressed at Physiology Department, Glasshouse Crops Research Institute, Worthing Road, Littlehampton, Sussex BN17 6LP, UK 3 Abbreviations used: ELISA, enzyme-linked immunosorbent assay; G6PD, glucose-6-phosphate dehydrogenase; MSA, molecular specific activity; PAGE, polyacrylamide gel electrophoresis.
A. bisporus (strain “Darlingtons 649”) was grown on compost under normal commercial conditions at the GCRI Mushroom Unit. Samples of 50-100 fruit bodies were taken as they reached stage 1 (Hammond and Nichols, 1976; “pinhead,” 5-mm diam, velum not differentiated) through the cropping period. They were cleaned, weighed, and frozen in liquid nitrogen. Mycelium
116 0147-5975185 $3.00 Copyright 0 1985 by Academic Press. Inc. All rights of reproduction in any form reserved.
MATERIALS
AND METHODS
G6PD
SYNTHESIS
AND
Agaricus
was sampled from the upper “casing” layer of the substrate using a 15mm-diameter cork borer, washed with iced water to remove traces of peat, and frozen in liquid nitrogen. Fruit body and mycelium samples were stored at - 20°C. Enzyme extraction. The frozen material was homogenized in Tris/HCl buffer (50 mM, pH 7.7) containing 50 mM 2-mercaptoethanol, 1 mM EDTA, 12 mM p-aminobenzamidine, 2 m44 phenylmethylsulfonyl fluoride, and 8 mM e-amino caproic acid. The homogenate was centrifuged at 20,OOOg for 20 min and the supernatant used for G6PD assay. All operations were carried out at 4°C. G4PD assay. G6PD was assayed as described previously (Hammond, 1981). Units of enzyme activity were defined as micromoles NADP+ reduced per minute under the conditions of the assay. A molar extinction coefficient of 6.2 x lo3 M-l cm-’ was assumed for NADPH. Protein content of extracts was determined by the Coomassie blue binding technique (Bradford, 1976), using bovine serum albumin standards ~ Measurement of G6PD protein by enzyme-linked immunosorbent assay (ELISA). G6PD was purified from stage 2 fruit bodies harvested during a flush, and antibody raised against the purified enzyme as described previously (Hammond, 198.5). The globulin fraction was purified and conjugated with alkaline phosphatase and the G6PD protein determined using the ELlSA method as described by Clark and Adams (1977). Extracts were diluted as necessary before assay to allow the response to fall on the calibration curve. Purified A. bisporus G6PD was used for calibration. Polyacrylamide gel electrophoresis (PAGE), Nondenaturing PAGE was carried out in 5% gels and the enzyme activity stained as described previously (Hammond, 1985). Laurel1 rocket immunoelectrophoresis of gel slices. After staining for G6PD activity,
FLUSH
INITIATION
117
the stained bands were cut singly from the rod gel and used for rocket immunoelectrophoresis (Laurel1 and McKay, 1981). The slices were laid on 1% agarose in Tris/barbiturate buffer (pH 8.6, 100 mM) containing 30 ~1 of antiserum/l2 ml agarose gel. Electrophoresis was carried out at 2 V/cm for 18 h. Plates were stained for protein wit Coomassie brilliant blue R-250. RESULTS
G6PD Activity in Fruit Body and Mycelium The results shown here are typical of data taken over four flushes in two separate crops. Each datum point is the mean of three determinations from each sample. G6PD activity in extracts of stage 1 fruit bodies sampled daily during the cropping period showed the same pattern of variation as that reported previously (Hammond, 1981), with a peak in activity at flush emergence (Fig. 1). The activity in mycelial extracts was considerably lower than that in sporophore extracts, on a soluble protein basis, over most of the experimental period (Fig. I). The activities of G6PD in fruit body and mycelium were only similar during flush growth when the activity in fruit bodies was very low. Smalt variations were seen in mycelial G6PD activity, wit some evidence of higher activity during the periods of high sporophore G6PD activity. Immunological Determination of G6PD Protein G6PD protein was determined by ELISA in fruit body and mycelial extracts. The increases in enzyme activity seen in fruit body extracts at flush emergence were accompanied by increases in the amount of G6PD protein on a total soluble protein basis (Fig. 2). In mycelial extracts the quantity of G6PD protein was lower than that seen in the fruit body. As with mycelial enzyme activity there were small variations with apparently higher levels around the period of flush emergence.
MINAMIDE Flush emergence
AND HAMMOND
tracts from material sampled at flush emergence showed an MSA for G6PD two to three times greater than extracts from material sampled during the periods of low G6PD activity. The pattern was seen most clearly in the fruit bodies. In mycelium, similar cyclic variations were observed, although due to the small amount of material that it was possible to isolate and to the low specific activity, the probable level of experimental error was greater.
Flush emergence
Polyacrylamide
IO
0 Time
20
(days)
FIG. 1. G6PD activity in stage 1 fruit bodies (0) and mycelium ((I) during flushing. Bars show range of mean.
When these data were recalculated on the basis of units of G6PD activity per milligram G6PD protein (G6PD MSA), the MSA varied through the flushing cycle in both fruit bodies and mycelium (Fig. 3). ExFlush emergence
Flush emergence
3.0
1
Gel Electrophoresis
The results of polyacrylamide gel electrophoresis of native protein in extracts, followed by staining for G6PD activity, are shown in Fig. 4. Fruit bodies sampled at the maxima and minima af G6PD activity were analyzed. G6PD from fruit bodies sampled at the minimum G6PD activity, i.e, during flush growth, resolved into three bands of activity (Bands 3-5, Fig. 4) as observed for the previously purified enzyme (Hammond, 1985), Extracts from fruit bodies sampled at the maximum of G6PD activity, i.e., at flush emergence, resolved into five bands of activity, with the two additional bands migrating more slowly than the other three (Bands 1 and 2, Fig. 4). The intensity of Flush emergence
Flush emergence
E
al 2.0 ij h 7 E” 2 $ 1.0 9
t-'4---~),_4-)--~-I--~.~~-. 0
I
I 0
10 Time
I 20
I 10
(days)
FZG. 2., Quantity of G6PD protein (determined by ELISA) in stage 1 fruit bodies (0) and mycelium (0) during flushing.
Time
(days)
FIG. 3. G6PD molecular specific activity in stage 1 fruit bodies (0) and mycelium (0) during flushing.
G6PD SYNTHESIS
a
b
AND
c
Agaricus
FLUSH INITIATION
most strongly staining and 4 (Figs. 4 and 5).
antigenic
ban
DISCUSSION
FIG. 4. Polyacrylamide gels stained for G6PD activity of extracts from stage 1 fruit bodies sampled (b) during flush growth and (c) at flush emergence. Blank of extract incubated without G6P is also shown (a). Bands l-5 G6PD activity: unnumbered band is an artifact.
staining suggests that the additional bands constitute one-third to one-half of the total G6PD activity in the sample. The individual bands were sliced from the gel and run in a Laurel1 rocket immunoelectrophoresis system to test the antigenicity of each to G6PD antiserum. As expected, the three bands found in both gels, and seen in the purified enzyme, showed rockets of similar height (Fig. 5), signifying similar degrees of antigenicity. The remaining two bands, observed only in samples taken at the time of flush emergence, gave rockets which were very small or not visible despite the fact that the intensity of activity staining was as great as that of the
The increase of G6PD activity in fruit bodies at flush emergence reported previously (Hammond, 1981) could have bee brought about through one or more of several mechanisms. Enzyme may have been translocated into the fruit body from the mycelium, synthesized in the fruit activated from an inactive precurso the periods of high activity. The data given here show that, while large variations in enzyme activity are seen in the fruit body during the flushing cycle, the activity of the enzyme in the myce~~um supporting the fruit body remains low. Thus it appears that the stimulation of G6PD activity at flush emergence is largely confine to the fruit body. A similar picture was seen when the level of enzyme protein was termined immunologically. Thus increase in enzyme activity was correlated with increase in enzyme protein Since negligible increases in enzyme protein were seen in the mycelium, the increases in GQPD are probably due to de n~vu synthesis of enzyme in the fruit body. However, the increase in G6PD MSA during flush emergence suggests that some other process also occurs. The resuhs PAGE and immunoelectrophoresis o resulting bands suggest that the reason for
FIG. 5. Laurel1 rocket immunoelectrophoresis of excised bands showing G6PD activity from polyacrylamide gels. Numbers refer to bands shown in Fig. 4.
120
MINAMIDE
AND
the increase in MSA is synthesis of electro.phoretically and immunologically distinct forms of G6PD (G6PD-2). The possibility of artifactual enzyme staining bands in PAGE of fungal developmental systems has been raised by Evers and Ross (1983). The G6PD-2 bands do not fall into this category since gels of extracts incubated without G6P showed only the single artifactual band of Fig. 4 in all extracts. Additionally, the inclusion of phenazine methosulfate in the incubation medium rules out the possibility of the diffusion of NADPH leading to artifacts as suggested by Evers and Ross (1983). G6PD-2 resolves separately on PAGE from the normal enzyme and has low antigenicity for G6PD antiserum. Thus although an increase in activity due to G6PD2 is observed, the increase in enzyme protein would not be seen in the ELISA, leading to an apparent increase in MSA. The antibody used in this assay was prepared from fruit bodies sampled at the period of maximum yield, i.e., during flush growth, when G6PD activity is low and G6PD-2 does not appear to be present. Thus the antibody used here was prepared from material which did not contain G6PD2. Although no evidence for the presence of G6PD-2 in mycelium could be obtained from PAGE, because of the low levels of materials available, the apparent increase in MSA of mycelial G6PD at flush emergence suggests that G6PD-2 is present in the mycelium at this time, and in a similar ratio to normal G6PD as observed in the fruit body. In a previous study three bands of G6PD activity were observed on nondenaturing polyacrylamide gels. These corresponded with protein bands when pure A. bisporus enzyme was electrophoresed (Hammond, 1985). All bands gave identical peptide maps, and only one band was seen on sodium dodecyl sulfate (SDS)-PAGE. Thus it was suggested that the three bands were due to different aggregation states of the same enzyme molecule. The two bands of
HAMMOND
G6PD-2 on native PAGE could similarly be due to different aggregation states of the G6PD-2 molecule. Forms of enzymes, including G6PD, specific to particular stages in development have been observed in other fungi, e.g., Coprinus cinereus (Moore and Jirjis, 1,981) and Sclerotinia sclerotiorum (Wong and Willetts, 1974). However, it appears that G6PD-2 in A. bisporus is produced at a specific stage of the flushing cycle rather than at a specific stage of fruit body development. Because of this, it may be more comparable with the synthesis of a new G6PD isozyme in Aspergillus oryzae in response to the presence of ribose (Muino-Blanc0 et al., 1983). The lack of antigenicity of G6PD-2 to constitutive G6PD antibody suggests that it has a different antigenic structure and therefore may be a different gene product. Further work is necessary to confirm this. If this proves to be the case, our report of G6PD-2 is the first recorded instance of a protein which is synthesized only during the period of flush emergence. This property should allow the protein to be used as a tool to study the mechanism by which flushing is stimulated. REFERENCES
BRADFORD,M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal. Biochem.
71: 248-254.
CHANTER, D. 0. 1979. Harvesting the mushroom crop: A mathematical model. J. Gen. Microbial. 115: 79-87.
CLARK, M. F., AND ADAMS, A. N. 1977. Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant vimses. J. Gen. Virol. 34: 475-483. EVERS, D. C., AND Ross, I. K. 1983. Isozyme patterns and morphogenesis in higher basidiomycetes. Exp. Mycol. 7: 9-16. FLEGG, P. B. 1979. Effects of competition on the development of the mycelium, mycelial aggregates and sporophores of Agaricus bisporus. Sci. Hortic. (Amsterdam) 11: 141-149. HAMMOND, J. B. W. 1981. Variations in enzyme ac-
G6PD SYNTHESIS
AND Agaricus FLUSH INITIATION
tivity during periodic fruiting of Agaricus bisporus. New Phytol. 89: 419-428. HAMMOND, J. B. W. 1985. Glucose 6-phophate dehydrogenase from Agaricus bisporus; purification and properties. J. Gen. Microbial. 131: 321-328. HAMMOND, J. B. W., AND NICHOLS, R. 1976. Carbohydrate metabolism in Agaricus bisporus (Lange) Sing.: Changes in soluble carbohydrates during growth of mycelium and sporophore. J. Gen. Microbiol. 93: 309-32.0. HAMMOND, J. B. W., AND NICHOLS, R. 1979. Carbohydrate metabolism in Agaricus bisporus: Changes in non-structural carbohydrates during periodic fruiting (flushing). New Phytol. 83: 723-730. LAURELL, C. B., AND MCKAY, E. J. 1981. Electroimmunoassay. In iMethods in Enzymology (J. J. Lan-
gone and H. Van Vunakis, Eds.), Vol. 73, pp. 339369. Academic Press, New York. MOORE, D., AND JIRJIS, R. I. 1981. Electrophoretic studies of carpophore development in the basidiomycete Coprinus cinereus. New Phytol. 87: 101113. MUINO-BLANCO, I., CEBRIAN-PEREZ, J. A., AND PEREZ-MARTOS, A. 1983. Regulation of the oxidative phase of the pentose phosphate cycle in Aspergillus oryzae (Ahlberg). I. Induction of glucose-6-phosphate dehydrogenase. Arch. Microbial. 136: 39-41. WONG, A. L., AND WILLETTS: H. J. 1974. Polyacrylamide-gel etectrophoresis of enzymes during morphogenesis of sclerotia of Sclerotinia scierotiorwm. J. Gen. Microbioi. 81: 101-109.