Purification and Partial Kinetic and Physical Characterization of Two Chloroplast-Localized NADP-Specific Glutamate Dehydrogenase Isoenzymes and Their Preferential Accumulation in Chlorella sorokiniana Cells Cultured at Low or High Ammonium Levels

Abstract

Two ammonium-inducible, chloroplast-localized NADP-specific glutamate dehydrogenase isoenzymes were purified to homogeneity from Chlorella sorokiniana. These isoenzymes were homopolymers of either a-or a-subunits with molecular weights of 55,500 or 53,000, respectively.The a-isoenzyme was preferentially induced at low ammonium concentrations (2 millimolar or lower), whereas only the f,-isoenzyme accumulated after cells were fully induced (120 minutes) at high ammonium concentrations (29 millimolar 53,000 (4, 8, 10, 11, 17-19, 22 In a recent study, Prunkard et al. (18) compared the kinetics of induction of NADP-GDH catalytic activity with the accumulation ofNADP-GDH antigen induced in cells under different cultural conditions. The apparent mol wt of the catalytically active NADP-GDH holoenzyme forms were estimated by use of an enzyme activity stain after native polyacrylamide slab-gel electrophoresis. The NADP-GDH antigens were examined after native and SDS slab-gel electrophoresis by use ofa Western blot/ immunodetection procedure. Native gel electrophoresis of extracts, from cells harvested during a 3 h induction time-course in continuous light in 29 mM ammonium medium, revealed that the number of electrophoretic forms of NADP-GDH holoenzyme(s) that were catalytically-active (and antigenic) increased from several forms, at the earliest induction times, to seven forms (Mr = 290,000-360,000) by the end of the 3 h induction period. When these same cell extracts were examined by SDS gel electrophoresis, two NADP-GDH antigens were detected with mol wt of 55,500 and 53,000. The 53,000 D NADP-GDH antigen increased continuously throughout the 3 h induction period. The 55,500 D antigen increased in concentration only during the first 1.5 h of the induction period and decreased in concentration thereafter. In fact, this larger antigenic species was barely detectable by the end of the 3 h induction period and essentially was absent from cells induced for 6 h. The 55,500 D antigen was shown to be associated only with NADP-GDH holoenzyme-size proteins. Therefore, it was assumed to be a subunit of NADP-GDH holoenzyme(s). The 55,500 D and 53,000 D antigens were designated as being a-and ,B-subunits of NADP-GDH, respectively. Depending upon the cultural conditions (i.e. autotrophic versus heterotrophic; light versus dark) and the length of the induction periods, a wide variation was observed in the a:,3 subunit ratio and in the number and sizes of the NADP-GDH holoenzymes.Tischner (23)

Partial characterization of enzymes of nitrogen metabolism in Chlorella autotrophica Shihira & Krauss

“…There have been reports of a similar inducible NADPH-GDH in several microorganisms (Tischner & Lorenzen, 1980;Everest & Syrett, 1983;Martin, Msatef & Botton, 1983;Ahmad & Hellebust, 19866;Bascomb & Schmidt, 1987;Jennings, 1989;Ahmad et al, 1990;Schwartz, Kusnan & Fock, 1991). Interestingly, in the green alga Stichococcus bacillaris (Everest & Syrett, 1983 ;Ahmad & Hellebust, 1986ft) NADPH-GDH exhibits a normal rate response curve for ammonium concentration with a single Kv alue of 1-2 mM, whereas in another green alga, Chlorella sorokiniana (Tischner, 1984;Bascomb & Schmidt, 1987), The NADPH-GDH exhibits dual kinetics with respect to ammonium concentration, showing a low K^^ of < 5 mM and a high K,,, of > 20 mM. It has been suggested that the high Kv alue for ammonium in the kinetic measurements of NADPH-GDH in C. sorokiniana is associated with post-translational modifications of the low K^.ê nzyme that occur when the culture ages or when the concentration of ammonium in the medium is high (Prunkard et al, 1986).…”

“…Furthermore, inhibition of GS by methionine sulphoximine (MSX) has little effect on the assimilation of ammonium by C. autotrophica (Ahmad & Hellebust 1985a, 1988a. Interestingly, NADPH-GDH in Chlorella sorokiniana shows a complex pattern of accumulation and also an alteration of its affinity for ammonium under different growth conditions (Tischner & Lorenzen, 1980;Tischner, 1984;Bascomb & Schmidt, 1987). It would therefore be interesting to examine the kinetic properties of NADPH-GDH from C autotrophica under various growth conditions.…”

Different Rates of Synthesis and Degradation of Two Chloroplastic Ammonium-Inducible NADP-Specific Glutamate Dehydrogenase Isoenzymes during Induction and Deinduction in Chlorella sorokiniana Cells

“…The same size precursor-protein is synthesized in vitro by poly(A)+RNA isolated from cells accumulating a-and (3-isoenzymes or only the (3-isoenzyme (2, 1 1). Cell extracts from C. sorokiniana have been shown (2) to process in vitro the 58,500 D precursor-protein(s) to a-or ,8-subunits with mol wt of 55,500 and 53,000, respectively. The a-and (3-subunits have been shown to be in active holoenzymes (homopolymers) localized in the chloroplast (2,11).…”

“…an amino acid), utilized during protein synthesis, might protect the NADP-GDH from degradation. When (2,3,10,11,13,20) from this laboratory, have been summarized and formulated into a working model/hypothesis for the regulation of expression of the gene(s) encoding the NADP-GDH isoenzymes in C. sorokiniana (Fig. 6).…”

“…Tischner (16) used an enzyme kinetic approach to show that extracts, from C. sorokiniana cells cultured in 5 mm ammonium medium, contained forms of NADP-GDH with high and low affinities for ammonium. These forms probably correspond to the a-and fl-isoenzymes purified and characterized by Bascomb and Schmidt (2).…”