Regulation of glucose-6-phosphate dehydrogenase under salt-stress condition in Aspergillus sydowii

Physiological responses of organisms to particular stresses are well understood in only a few cases (Bachofen, R. 1986 Experientia 42:1179-1182) This regular paper is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/vol36/iss1/12 Table 2. Kinetics of condia production upon nitrogen starvation. Hyphae grown 16 h in reduction standard medium with 50 mM NH4Cl were transferred to standard medium without nitrogen. Estimation of the percent hyphae septated into conidia was done on two pieces obtained from the edge of the agglomerate (average of two experiments each with two replicates). hours after addition % of hyphae pH mg dry weight transfer to septated per 75 ml nitrogeninto culture** free medium conidia* 0 0 4 . 5 1 0 3 5:6 2 0 5.8 136 4 20 156 8 80 5.6 196 6 5 0 5 . 6 1 8 3 1 2 9 0 5 . 6 2 1 0 12 50 mM NaCl 90 12 50 mM NH4Cl 0 3.9 12 50 mM NaNO3 0 6.9 * standard error of the mean was about 20% ** standard error of the mean was about 5% 5.5 213 312 298 Our experiments indicate that starvation for nitrogen or limitation for glucose induces formation of conidia in the ascomycete Neurospora crassa. Using similar stepdown experiments it was already shown that nitrogen regulates another morphogenetic process, the formation of protoperithecia (Sommer et al. 1987. Planta 170:205-208). So, nitrogen starvation plays a key role in triggering both the asexual and sexual life cycles of Neurospora. Acknowledgements: We thank Uta Marchfelder for typing and Niketan Pandit for critically reading the manuscript. B.T.M. is an fellow of the Studienstiftung des deutschen Volkes. This work was partially supported by the Deutsche Forschungsgemeinschaft Max-Planck Institut für molekulare Genetik, D-1000 Berlin 33, Federal Republic of Germany. Parekh. T. and H.S. Chhatpar Physiological responses of organisms to particular stresses are well understood in only a Regulation of glucose-6-phosphate few cases (Bachofen, R. 1986 Experientia 42:11791182). Most of the salt-stress in nature is due dehydrogenase under salt-stress to sodium salts, particularly NaCl (Strongonov, B.P. 1973. condition in Aspergillus sydowii Structure and function of plant cells i n saline habitats. Halsted Press, New York). Little information is available regarding the effect of salt on the regulation of enzymes in marine fungal systems. In our studies efforts have been made to understand the salt-mediated regulation of glucose-6-phosphate dehydrogenase (G6PDH) in halotolerant Aspergillus sydowii. Aspergillus sydowii was isolated from salt pans. The growth conditions employed were the same as described earlier (Karlekar et al. 1985 J. Biosci 9:197-201) except that casemino acid and yeast extract were replaced by asparagine (1.0%). Zn2+was added to the synthetic medium as ZnSO4 at the desired concentration. Zinc deficient, zinc suboptimal, zinc optimal and zinc supraoptimal conditions indicate no addition, addition of 0.1 mg, 1 mg and 10 mg per 100 ml of ZnS04 to the above growth medium, respectively. Methods for the preparation of cell-free extract, assay of G6PDH and protein were the same as described earlier (Savant et al. 1982 Experientia 38:310-311). Mycelial ash was prepared by heat drying the mycelia at 8OO°C for 5 hours. Earlier studies with A. sydowii grown in the presence of 2M NaCl showed significantly higher levels of G6PDH compared with control cultures. Km was found to decrease while Vmax increased when NaCl was added to the growth medium (Parekh and Chhatpar 1986 In: Contemporary themes in biochemistry (Kon et al. eds.) ICSU Press Cambridge pg. 334-335). Further studies on the in vitro effect of NaCl on kinetic constants of G6PDH in cell-free extracts of a 2M NaCl grown culture showed a decrease in Vmax without a change in Km values, suggesting a non-competitive type of inhibition of G6PDH by NaCl (Table 1). Various possibilities for regulation of G6PDH are; (a) Higher Na+ accumulation in the culture grown in the presence of 2M NaCl condition might be responsible for increasing the activity of G6PDH; (b) A different pattern of accumulation of other electrolytes may also be responsible for altering enzyme activity; and/or (c) The control culture may be synthesizing or accumulating inhibitor(s) of G6PDH. Table 1: Effect of NaCl on kinetic constants of G6PDH from A. sydowii grown in the presence of 2M NaCl. Cell-free extract Km Vmax 2M NaCl-grown culture 3.7 x 10^-5 M 6333 2M NaCl-grown culture + 0.2M NaCl 3.7 x 10^-5 M 400 A. sydowii showed greater accumulation of Na+ when grown in the presence of 2M NaCl (Parekh and Chhatpar 1986). Addition of NaCl to the growth medium caused an increase in intracellular Na+ as well as G6PDH activity. On the contrary, in vitro addition of NaCl resulted in a significant inhibition of G6PDH activity (Fig. 1). Many enzymes from halophytes and glycophytes have been shown to be inhibited by salt concentration greater than 100 mM (Flowers et al. 1977 Ann. Rev. Plant Physiol. 28:89-121). 0 0.2 0.4 U.6 0.8 1.0 NaCl concentration (M) Fig. 1: In vitro effect of NaCl on the activity of G6PDH from A. sydowii These observations suggested that there might be some factor(s) which significantly contribute to decreased activity of G6PDH in control cultures or increased activity in 2M the NaCl-grown condition. To determine if an inhibitor was present in control cultures, cell-free extract from a control culture was mixed with cell-free extract of a 2M NaClgrown culture and the activity of G6PDH was measured. The observed significant decrease in the activity of G6PDH suggested the possibility of the presence of inhibitor(s) in cell-free extracts of the control culture (Table 2). This inhibitory activity was lost by dialyzing the control cell-free extract but not by boiling it for 10 minutes. The non-lipid and non-proteinic nature of the putative inhibitor was suggested from the observations that the inhibitory activity was not affected after lipase treatment or removal of proteins with (NH4)2 SO4 precipitation. Treatment with lysozyme of control cellfree extract also did not affect the inhibitory activity. Moreover, inhibitory activity was regained when dialysate of control cell-free extract concentrated appropriately and added in the assay system. Addition of ash prepared from control grown culture to the 2M NaCl-grown culture cell-free extract also showed inhibition, when added into the assay system (Table 2). Table 2: Characterization of inhibitor of G6PDH from A. sydowii NaCl. G6PDH activity Cell-free extract (U/ml) A) 2M NaCl-grown culture^a 2450 B) Control-grown culture 490 80.0 C) A + 0.2 ml of B 2107 14.0 E) A + 100% (NH4)2 SO4 precipitates of B 2455 F) A + 0.2 ml of B (lipase treated)^b 1863 24.0 G) A + 0.2 ml of B (lysozyme treated)^b 1812 26.0 H) A + 0.2 ml of B (dialyzed) 2450 I) A + dialysate of B 1960 20.0 J) i) A + 2 mg mycelial ash^c of A 2452 ii) A + 2 mg mycelial ash of B 1617 34.0 K) A + 100 ug of ZnSO4 1568 36.0 On the basis of these experiments it was inferred that the grown in the absence of % Inhibition/ decrease of G6PDH D) A +0.2ml of B (boiled for 10 min) 2009 18.0 inhibitor accumulated in the control-grown culture is inorganic in nature and that it is heat stable. As reported earlier, control grown A. sydowii showed a 7-8 fold higher accumulation of Zn2+ (Parekh and Chhatpar 1985). Out of inorganic ingredients of the growth medium tested viz. ZnSO4, FeSO4, MgSO4, MnCl2, CaCl2, ammonium molybdate and borax (data not shown), Zn2+ was found to be responsible for regulation of G6PDH activity (Table 3). Table 3: Effect of Zn2+ on G6PDH activity from A. sydowii grown in the presence of 2M NaCl Growth condition G6PDH activity (U/mg protein) Zinc deficient 320 Zinc suboptimal 979 Zinc optimal 927 Zinc supraoptimal 520 These results indicated that higher accumulation of zinc in the control condition might be responsible for the reduced level of G6PDH. However, the possibility of the presence of other inhibitor(s) cannot be ruled out, since addition of 2 mg ash (approx. 0.15 ug of Zn2+) to the assay systems gave 34% inhibition of G6PDH, which was the same as observed by the addition of 100 ug of ZnSO4 (approx. 23 ug of Zn2+). Earlier, Rouxel et al. (1987 Physiol. Plantarum 69:330-336) demonstrated that RNase from the halophyte Suaeda was not affected by the salinity of the growth medium but was totally inhibited by 10mMZn2+. The results presented in Table 3 illustrate the influence of zinc (in the growth medium) on the level of G6PDH in A. sydowii. A lower level of G6PDH was observed when growth medium was supplemented at a supraoptimal concentration of Zn2+. However, a low concentration (suboptimal) of Zn2+ was required for the optimum activity of G6PDH. 2M NaCl-grown cells allow less accumulation of Zn2+ in the cytoplasm which may increase the activity of GGPDH, while a higher accumulation of Zn2+ in the control-grown cells could be one of the factors responsible for lowered G6PDH activity. This work was supported by a Senior Research Fellowship to Trilok Parekh from the Council of Scientific and Industrial Research, New Delhi, India. Department of Microbiology, Faculty of Science, M.S. University of Baroda, Baroda 390 002, India.


Parekh. T. and H.S. Chhatpar
Physiological responses of organisms to particular stresses are well understood in only a Regulation of glucose-6-phosphate few cases (Bachofen, R. 1986Experientia 42:1179-1182.
Most of the salt-stress in nature is due dehydrogenase under salt-stress to sodium salts, particularly NaCl (Strongonov, B.P. 1973. condition in Aspergillus sydowii Structure and function of plant cells i n saline habitats. Halsted Press, New York). Little information is available regarding the effect of salt on the regulation of enzymes in marine fungal systems. In our studies efforts have been made to understand the salt-mediated regulation of glucose-6-phosphate dehydrogenase (G6PDH) in halotolerant Aspergillus sydowii.
Aspergillus sydowii was isolated from salt pans.
The growth conditions employed were the same as described earlier (Karlekar et al. 1985 J. Biosci 9:197-201) except that casemino acid and yeast extract were replaced by asparagine (1.0%).
Zn²+was added to the synthetic medium as ZnSO4 at the desired concentration.
Zinc deficient, zinc suboptimal, zinc optimal and zinc supraoptimal conditions indicate no addition, addition of 0.1 mg, 1 mg and 10 mg per 100 ml of ZnS04 to the above growth medium, respectively. Methods for the preparation of cell-free extract, assay of G6PDH and protein were the same as described earlier (Savant et al. 1982 Experientia 38:310-311). Mycelial ash was prepared by heat drying the mycelia at 8OO°C for 5 hours.
Earlier studies with A. sydowii grown in the presence of 2M NaCl showed significantly higher levels of G6PDH compared with control cultures. Km was found to decrease while Vmax increased when NaCl was added to the growth medium (Parekh and Chhatpar 1986 In: Further studies on the in vitro effect of NaCl on kinetic constants of G6PDH in cell-free extracts of a 2M NaCl grown culture showed a decrease in Vmax without a change in Km values, suggesting a non-competitive type of inhibition of G6PDH by NaCl (Table 1). Various possibilities for regulation of G6PDH are; (a) Higher Na+ accumulation in the culture grown in the presence of 2M NaCl condition might be responsible for increasing the activity of G6PDH; (b) A different pattern of accumulation of other electrolytes may also be responsible for altering enzyme activity; and/or (c) The control culture may be synthesizing or accumulating inhibitor(s) of G6PDH. A. sydowii showed greater accumulation of Na+ when grown in the presence of 2M NaCl (Parekh and Chhatpar 1986). Addition of NaCl to the growth medium caused an increase in intracellular Na+ as well as G6PDH activity. On the contrary, in vitro addition of NaCl resulted in a significant inhibition of G6PDH activity (Fig. 1).
Many enzymes from halophytes and glycophytes have been shown to be inhibited by salt concentration greater than 100 mM (Flowers et al. 1977 Ann. Rev. Plant Physiol. 28:89-121). These observations suggested that there might be some factor(s) which significantly contribute to decreased activity of G6PDH in control cultures or increased activity in 2M the NaCl-grown condition. To determine if an inhibitor was present in control cultures, cell-free extract from a control culture was mixed with cell-free extract of a 2M NaClgrown culture and the activity of G6PDH was measured. The observed significant decrease in the activity of G6PDH suggested the possibility of the presence of inhibitor(s) in cell-free extracts of the control culture (Table 2). This inhibitory activity was lost by dialyzing the control cell-free extract but not by boiling it for 10 minutes. The non-lipid and non-proteinic nature of the putative inhibitor was suggested from the observations that the inhibitory activity was not affected after lipase treatment or removal of proteins with (NH4)2 SO4 precipitation. Treatment with lysozyme of control cellfree extract also did not affect the inhibitory activity. Moreover, inhibitory activity was regained when dialysate of control cell-free extract concentrated appropriately and added in the assay system. Addition of ash prepared from control grown culture to the 2M NaCl-grown culture cell-free extract also showed inhibition, when added into the assay system (Table 2). inhibitor accumulated in the control-grown culture is inorganic in nature and that it is heat stable. As reported earlier, control grown A. sydowii showed a 7-8 fold higher accumulation of Zn2+ (Parekh and Chhatpar 1985). Out of inorganic ingredients of the growth medium tested viz. ZnSO4, FeSO4, MgSO4, MnCl2, CaCl2, ammonium molybdate and borax (data not shown), Zn2+ was found to be responsible for regulation of G6PDH activity (Table 3). These results indicated that higher accumulation of zinc in the control condition might be responsible for the reduced level of G6PDH. However, the possibility of the presence of other inhibitor(s) cannot be ruled out, since addition of 2 mg ash (approx. 0.15 ug of Zn²+) to the assay systems gave 34% inhibition of G6PDH, which was the same as observed by the addition of 100 ug of ZnSO4 (approx. 23 ug of Zn2+). Earlier, Rouxel et al. (1987 Physiol. Plantarum 69:330-336) demonstrated that RNase from the halophyte Suaeda was not affected by the salinity of the growth medium but was totally inhibited by 10mMZn²+.
The results presented in Table 3 illustrate the influence of zinc (in the growth medium) on the level of G6PDH in A. sydowii. A lower level of G6PDH was observed when growth medium was supplemented at a supraoptimal concentration of Zn2+. However, a low concentration (suboptimal) of Zn2+ was required for the optimum activity of G6PDH. 2M NaCl-grown cells allow less accumulation of Zn2+ in the cytoplasm which may increase the activity of GGPDH, while a higher accumulation of Zn2+ in the control-grown cells could be one of the factors responsible for lowered G6PDH activity.