Partial separation and interconversion of NADH‐ and NADPH‐linked activities of purified glyceraldehyde 3‐phosphate dehydrogenase from spinach chloroplasts

Pawlizki, K.-H.; Latzko, Erwin

doi:10.1016/0014-5793(74)80747-7

Cited by 49 publications

(10 citation statements)

References 9 publications

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“…Interaction with Sepharose in the absence of salt has been described for a variety of other proteins [27]. Retarded elution of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase has been observed on Bio-Gel by Pawlitzki and Latzko for the spinach enzyme [28] and on Sephadex by TheissSeuberling for the Euglena enzyme [29]. In these publications this has been regarded as evidence for the existence of 79000-M, and 68000-M, enzyme species respectively.…”

Section: Discussionmentioning

confidence: 99%

Glyceraldehyde‐3‐Phosphate Dehydrogenase(NADP) from Sinapis alba L.

Cerff

1978

European Journal of Biochemistry

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Partially purified glyceraldehyde‐3‐phosphate dehydrogenase (NADP) (EC 1.2.1.13) from Sinapis alba seedlings was filtered on Sephadex G‐200 in the presence of NADP(H) and NAD(H). With NADP(H) the enzyme elutes as a dissociated species (Mr∼ 170 000), whereas with NAD(H) it travels as an aggregate in the void volume (Mr≥ 800 000). The NAD‐specific enzyme (EC 1.2.1.12) elutes with an Mr of about 160 000, regardless of whether NADP(H) or NAD(H) is present. If the NADP‐dependent enzyme is first filtered with NADP+ on Sephadex G‐200 and then with NAD+ on Sepharose 6B, only part of the enzyme aggregates during the second filtration. The other part (fraction S) travels as a narrow band slightly ahead of the marker cytochrome c. Refiltration of fraction S with NADP+ and with increasing [NADP+]/[NAD+]ratios on Sepharose 6B demonstrates that this transport retardation is NAD‐dependent and counteracted by NADP. The effect is also eliminated by inorganic cations and is not observed on Sephadex G‐200. Dodecylsulfate/polyacrylamide gel electrophoresis of fraction S before and after affinity chromatography on NADP‐agarose shows that the enzyme is pure after transport retardation on Sepharose 6B. It is active with both NADP and NAD and contains at least three different polypeptide chains with apparent Mr of 39 000, 42 000, and 43 000. The NAD‐specific enzyme is shown to be a tetramer of four 38 000‐Mr subunits. Kinetic investigations show that the binding of NADP(H) and NAD(H) with glyceraldehyde‐3‐phosphate dehydrogenase (NADP) is mutually exclusive. The present data indicate that the NADP‐dependent enzyme, a species structurally distinct from the NAD‐specific enzyme, occurs in two different conformations in the presence of NADP(H) and NAD(H) respectively. The existence of a ‘sticky’ NAD conformer is suggested, which interacts with Sepharose 6B and/or aggregates with other molecules in crude or partially purified extracts.

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Section: Discussionmentioning

confidence: 99%

Glyceraldehyde‐3‐Phosphate Dehydrogenase(NADP) from Sinapis alba L.

Cerff

1978

European Journal of Biochemistry

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“…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) [ We have isolated a chloroplast G3PDH from the green alga, Scenedesmus obliquus [12].…”

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confidence: 96%

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

Clasper

Easterby

Powls

1991

European Journal of Biochemistry

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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.

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“…The activation of P-ribulokinase by DTT has been reported by other workers (1,31) as has activation by DTT plus thioredoxin (31 Table IV. SDS-polyacrylamide gel electrophoresis of the NADPH-dependent glyceraldehyde-P dehydrogenase has been reported to give two distinct subunits with estimated mol wt of 3.6-3.9 x 104 (12,13,20). This evidence suggests that our estimate of 3.1 x 104 for the subunit mol wt is somewhat low.…”

Section: Resultsmentioning

confidence: 38%

“…This retarded peak of activity corresponded to the peak eluting close to fraction 43 in Figure 2. The best estimate of the mol wt of this peak was obtained by gel filtration on a Sephadex G-100 column (2.5 x 36 cm) which gave a value of 3.1 x 104 and indicated that this fraction corresponded to the enzyme subunits whose mol wt have been reported to lie in the range of 3.6 x 104 to 4.3 x I04 (12,13,20).…”

Section: Resultsmentioning

confidence: 99%