The solution conformation of plastocyanin from the green alga Scenedesmus obliquus has been determined from distance and dihedral angle constraints derived by nuclear magnetic resonance (NMR) spectroscopy. Structures were generated with distance geometry and restrained molecular dynamics calculations. A novel molecular replacement method was also used with the same NMR constraints to generate solution structures of S. obliquus plastocyanin from the x-ray structure of the homologous poplar protein. Scenedesmus obliquus plastocyanin in solution adopts a beta-barrel structure. The backbone conformation is well defined and is similar overall to that of poplar plastocyanin in the crystalline state. The distinctive acidic region of the higher plant plastocyanins, which functions as a binding site for electron transfer proteins and inorganic complexes, differs in both shape and charge in S. obliquus plastocyanin.
The in vitro secretion of ecdysteroids from the prothoracic glands of last instar larvae of Spodoptera littorulis was detected and analysed by HPLC-RIA. The primary product was identified as 3-dehydroecdysone (= 82%), with lesser amounts of ecdysone (= 18%). Interconversion of ecdysone and 3-dehydroecdysone by prothoracic glands was not detectable.3-Dehydroecdysone 3P-reductase activity was demonstrated in the haemolymph. Ecdysone, the endproduct, was characterised by reverse-phase and adsorption HPLC, chemical transformation into ecdysone 2, 3-acetonide, and mass spectrometry. The conditions for optimal activity were determined. The enzyme requires NADPH or NADH as cofactor and K,,, values for NADPH and NADH were determined to be 0.94 pM and 22.8 pM, respectively. Investigation of the kinetic properties of the enzyme, using either NADPH or NADH as cofactor, revealed that it exhibits maximal activity at low 3-dehydroecdysone substrate concentrations, with a drastic inhibition of activity at higher concentrations (> 5 pM). The results suggest that the 3-dehydroecdysone 3P-reductase has a high-affinity (low K,) binding site for 3-dehydroecdysone substrate, together with a lower-affinity inhibition site.The 3P-reductase enzyme was purified to homogeneity using a combination of poly(ethy1ene glycol) 6000 precipitation and successive FPLC fractionation on Mono-Q, phenyl Superose (twice), and hydroxyapatite columns. The native enzyme was shown to be a monomer with molecular mass of 36 kDa by SDSPAGE and gel-filtration chromatography. Furthermore, the activity of the enzyme during the last larval instar was found to reach a peak prior to that of the haemolymph ecdysteroid titre, supporting a role for the enzyme in development.Keywords: 3-dehydroecdysone 3P-reductase; ecdysone ; prothoracic gland; Spodoptera littoralis.In immature stages of insects, the prothoracic glands are the primary source of ecdysteroid (moulting hormone), generally ecdysone (Scheme 1, I) in most species. However, recently it has been shown that in most lepidopteran species studied, the major product of the glands is 3-dehydroecdysone (Scheme 1, U), together with varying proportions of ecdysone [l, 21. After release from the prothoracic glands, the 3-dehydroecdysone is reduced to ecdysone by an NAD(P)H-linked 3-dehydroecdysteroid 3p-reductase in the haemolymph [2-41. The level of reductase activity in different insect species would appear to be related to the proportion of 3-dehydroecdysone secreted by the prothoracicThe exact details of ecdysteroid biosynthesis are not fully elucidated, although the early and late stages are the best understood [5-81. There is substantial evidence for the intermediacy of 2,22,25-trideoxyecdysone (5p-ketodiol ; Scheme 1, 111) in the biosynthesis of ecdysone [5,8, 91. This labelled substrate is effi-
A homogeneous multimeric protein isolated from the green alga, Scenedesmus ohliquus, has both latent phosphoribulokinase activity and glyceraldehyde-3-phosphate dehydrogenase activity. The glyceraldehyde-3-phosphate dehydrogenase was active with both NADPH and NADH, but predominantly with NADH. Incubation with 20 mM dithiothreitol and 1 mM NADPH promoted the coactivation of phosphoribulokinase and NADPHdependent glyceraldehyde-3-phosphate dehydrogenase, accompanied by a decrease in the glyceraldehyde-3-phosphate dehydrogenase activity linked to NADH. The multimeric enzyme had a Mr of 560000 and was of apparent subunit composition 8G6R. R represents a subunit of M , 42000 conferring phosphoribulokinase activity and G a subunit of 39000 responsible for the glyceraldehyde-3-phosphate dehydrogenase activity. On SDS-PAGE the Mr-42000 subunit comigrates with the subunit of the active form of phosphoribulokinase whereas that of M,-39 000 corresponds to that of NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase.The multimeric enzyme had a szo,w of 14.2 S. Following activation with dithiothreitol and NADPH, sedimenting boundaries of 7.4 S and 4.4 S were formed due to the depolymerization of the multimeric protein to NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase (4 G) and active phosphoribulokinase (2 R). It has been possible to isolate these two enzymes from the activated preparation by DEAE-cellulose chromatography.Prolonged activation of the multimeric protein by dithiothreitol in the absence of nucleotide produced a single sedimenting boundary of 4.6 S, representing a mixture of the active form of phosphoribulokinase and an inactive dimeric form of glyceraldehyde-3-phosphate dehydrogenase.Algal thioredoxin, in the presence of 1 mM dithiothreitol and 1 mM NADPH, stimulated the depolymerization of the multimeric protein with resulting coactivation of phosphoribulokinase and NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase. Light-induced depolymerization of the multimeric protein, mediated by reduced thioredoxin, is postulated as the mechanism of light activation in vivo. Consistent with such a postulate is the presence of high concentrations of the active forms of phosphoribulokinase and NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase in extracts from photoheterotrophically grown algae. By contrast, in extracts from the dark-grown algae the multimeric enzyme predominates.There is now considerable evidence that the activities of several enzymes of the Calvin cycle are regulated by light [l]. The first enzyme demonstrated to be activated in vivo by light was NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase [2]. The effect of light was shown to be reversed by a subsequent period of darkness. The stimulatory effect of light was also observed in isolated spinach chloroplasts [3 -41. Incubation of the isolated chloroplast enzyme with a number of effector molecules (e.g. NADPH and ATP) stimulated the NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase activity [5 -71. The effect o...
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.
In the midgut cytosol of Lepidoptera, ecdysteroids undergo inactivation by transformation via the 3-dehydro derivative to the corresponding 3-epiecdysteroid (3 alpha-hydroxy) and by phosphate conjugation. The oxygen-dependent oxidase catalyses formation of 3-dehydroecdysteroid, which can be reduced either irreversibly by 3-dehydroecdysone 3 alpha-reductase to 3-epiecdysteroid, or by 3-dehydroecdysone 3 beta-reductase back to the initial ecdysteroid. Furthermore, these ecdysteroids undergo further inactivation by phosphorylation. These ecdysteroid transformations have been investigated in last instar larvae of the cotton leafworm, Spodoptera littoralis. The products of the phosphorylation have been characterized as predominantly ecdysteroid 2-phosphate accompanied by smaller amounts of the corresponding 22-phosphate. The phosphotransferases require Mg2+ and ATP. Whereas the 3-dehydroecdysone 3 alpha-reductase has a clear preference for NADPH rather than NADH, the corresponding 3 beta-reductase markedly favours NADH. The physiological significance of the latter enzyme is unclear. The profiles of the various enzymic activities in dialysed midgut cytosol supplemented with appropriate cofactors were determined throughout the last larval instar. All activities were detectable throughout the instar, but the respective enzymes exhibited maxima at different times. Ecdysone oxidase showed a peak early in the instar, with 3-dehydroecdysone 3 alpha-reductase increasing to a peak as the former activity declined. The 3-dehydroecdysone 3 beta-reductase exhibited peak activity late in the instar, a profile similar to that observed for the corresponding haemolymph enzyme involved in reduction of the 3-dehydroecdysone product of the prothoracic glands to ecdysone. Thus, the significance of the midgut 3 beta-reductase may be related to production of active hormone. Both ecydsteroid 22- and 2-phosphotransferases showed high activities early in the instar and then declined. The physiological significance of the profiles for the ecdysone oxidase, the 3-dehydroecdysone 3 alpha-reductase and phosphotransferases is unclear.
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