Properties to two high‐molecular‐mass forms of glyceraldehyde‐3‐phosphate dehydrogenase from spinach leaf, one of which also possesses latent phosphoribulokinase activity

Abstract

Two high-Mr forms of chloroplast glyceraldehyde-3-phosphate dehydrogenase from spinach leaf can be separated by DEAE-cellulose chromatography. One form, the high-M, glyceraldehyde-3-phosphate dehydrogenase, resembles an enzyme previously described [Yonuschot, G. R. This complex is composed not only of subunits A (39.5 kDa) and B (41.5 kDa) characteristic of the high-M, glyceraldehyde-3-phosphate dehydrogenase, but also of a third subunit, R (40.5 kDa) comigrating with that from the active phosphoribulokinase of spinach.Incubation of the complex with dithiothreitol markedly stimulated both its phosphoribulokinase and NADPH-dependent dehydrogenase activities. This dithiothreitol-induced activation was accompanied by depolymerisation to give two predominantly NADPH-linked tetrameric glyceraldehyde-3-phosphate dehydrogenases (the homotetramer, A4, and the heterotetramer, A2B2) as well as the active dimeric phosphoribulokinase. Incubation of the high-M, glyceraldehyde-3-phosphate dehydrogenase with dithiothreitol promoted complete depolymerisation yielding only the hetero te tramer (Az B 2 ) . Possible structures suggested for the glyceraldehyde-3-phosphate dehydrogenaselphosphoribulokinase complex are (AzB2)zA4R2 or (A2B2)(A4),R2.Spinach chloroplast glyceraldehyde-3-phosphate dehydrogenase (G3PDH) has been isolated in a high-M, form by several workers [l -61. However there is disagreement on the coenzyme dependence of the enzyme. In some cases it has been reported to be predominantly active with NADP [l, 2, 51, whereas in others NAD is the preferred coenzyme [3, 41. In the presence of NADP the high-M, enzyme dissociated into low-M, forms predominantly active with NADP [4,6,7]. This could be reversed by NAD which promoted the reassociation of the low-M, forms. The chloroplast enzyme has been shown by SDSjPAGE to consist of two similar but distinct subunits in all species examined [8]. For the spinach enzyme, M , of 37 and 43 kDa 151, 38 and 42 kDa [8], and 39 and 42 kDa [9] have been reported for the A and B subunits respectively. The low-M, chloroplast G3PDH, formed by NADP-induced depolymerisation of the high-M, form, has been shown to be a mixture of two isoenzymes, a homotetramer (A4) and a heterotetramer (A,B2) [lo, 111.

CP12 provides a new mode of light regulation of Calvin cycle activity in higher plants

“…4A) demonstrates that only NADPH can stimulate Ru-1,5-bP formation, whereas NAD and NADP inhibit PRK activity. These results, taken together with the data presented and discussed above, suggest that the spinach PRK͞ GAPDH complex, published by Powls and coworkers (8), is identical with our PRK͞CP12͞GAPDH complex. Because CP12 is not simply detectable by gel electrophoresis of chloroplast extracts and protein staining, and nothing was known about the existence of CP12 at the time they isolated the complex, they might have failed to notice it.…”

“…Powls and coworkers have described 560-kDa protein complexes in the chloroplast stroma of the green alga, Scenedesmus obliquus (25,26) and spinach (8), both having latent PRK and GAPDH activities. Incubation of these complexes with DTT and NADPH induced depolymerization and marked stimulation of the latent enzyme activities.…”

“…Another mode of regulating enzyme activity in the cycle was shown for GAPDH, which, depending on its substrate, 1,3-bisphosphoglycerate, or NADP(H) and ATP, can reversibly dissociate from the less active hexadecameric A8B8 form to the highly active A2B2 tetramer (6,7). GAPDH was also shown to be associated with PRK in a 600-kDa protein complex, made up by the A2B2 and the A4 forms of GAPDH, together with one PRK dimer (8). Further data suggest that GAPDH and PRK could be associated in a complex together with Rubisco (9).…”

Modulation, via Protein-Protein Interactions, of Glyceraldehyde-3-phosphate Dehydrogenase Activity through Redox Phosphoribulokinase Regulation

“…We have shown (14) that oxidized PRK from C. reinhardtii may have some activity when it is associated with GAPDH, contrary to common belief, but no direct evidence of the regulatory disulfide bridge between Cys 16 and Cys 55 present in oxidized PRK, was given. We have now used alkylation of the so-called oxidized complex to show that there is one disulfide bridge in the PRK monomer.…”

“…In particular, thioredoxin (39,40) reduces the disulfide between Cys 55 and Cys 16 of inactive oxidized spinach PRK (41,42).…”