Paul Sauve

Molecular properties and thioredoxin-mediated activation of spinach chloroplastic NADP-malate dehydrogenase

There is no doubt that light-generated reducing equivalents can be used in C3 plants to perform a number of reactions occurring outside the chloroplasts [1-3]. Since the chloroplastic membrane is impermeable to NADPH and to reduced ferredoxin, it is currently believed that these reduction reactions in the cytoplasm involve the so-called C4dicarboxylic acid shuttle [4]. A specific malate translocator located on the chloroplastic membrane exports, outside the chloroplast, this dicarboxylic acid which is used in the cytosol to perform a number of reduction reactions [3]. NADPH formed during photosynthesis is used to reduce oxaloacetate to malate in the chloroplastic stroma. Chloroplastic malate dehydrogenase plays therefore an important role in the generation of reducing power used in reactions occurring in the cytoplasm. Moreover, crude extracts containing chloroplastic malate dehydrogenase are totally inactive in the dark and become active upon illumination. The effect of light is mimicked by a mixture of dithiothreitol and thioredoxin [5,6]. Surprisingly, chloroplastic NADP-malate dehydrogenase has never been purified to homogeneity. Therefore, nothing is known as to its structure, its reaction mechanism and the molecular bases of photoregulation by thioredoxin. Moreover one totally ignores whether several iso-forms of malate dehydrogenase exist in the chloroplast. The aim of this paper is to describe a purification procedure of chloroplastic NADP-malate dehydrogenase, to report some molecular properties of this enzyme as well as some features of its activation by dithiothreitol and thioredoxin.

Purification and Molecular Properties of a Cell-Wall β-Glucosyltransferase From Soybean Cells Cultured In Vitro

Summary A β-glucosidase has been isolated and purified to apparent homogeneity from soybean cell walls. The enzyme is a glycoprotein with a molecular weight close to 60 000 and a sedimentation constant equal to 4.17. The protein is made up with one polypeptide chain only.

Interactions of pancreatic colipase with taurodeoxycholate–oleate mixtures above the critical micelle concentration

The hydrolysis of insoluble triglycerides by pancreatic lipase requires pre-adsorption of the enzyme to the interface of emulsified particles [I]. This adsorption had been shown to be hindered by physiological concentrations of bile salts and restored by a pancreatic protein cofactor called colipase [2-41. Colipase is supposed to anchor lipase during fat digestion at interfaces coated with bile salts and other amphiphiles [S], thus necessitating specific interactions between the cofactor, the enzyme and the interface. The interactions of colipase with interfaces may be conveniently investigated using isotropic micellar dispersions of amphiphilic lipids which are more readily accessible to physical techniques than turbid emulsions. When applied to one of these systems, ultracentrifugation and UV spectrophotometry demonstrated that colipase formed complexes with pure NaTDC above the CMC and that, for the relatively low cofactor concentrations existing in vivo, the number of bile salt monomers bound to each cofactor molecule was appro~mately that present in a micelle under the same conditions [6,7]. In this type of complex called in what follows ‘the binary complex’, bile salts monomers were shown by small angle neutron scattering not to be distributed randomly over the surface of the protein but to form a micelle-like cluster distinct from and adjacent to colipase [8]. The site in colipase serving for micelle recognition may be assumed to be that insuring bind-

The activation peptide of pancreatic procarboxypeptidase A is the keystone of the bovine procarboxypeptidase A-S6 ternary complex.

In some ruminant species, pancreatic procarboxypeptidase A is the central element of a ternary complex involving two other components, a C-type chymotrypsinogen and an inactive protease E. Although the complex is devoted to protein digestion, the fate of this system upon activation of its constituent subunits has, as yet, not been clearly established. In this paper, the activation peptide of procarboxypeptidase A is shown to play a key role in the association of the three subunits and a model is proposed for the in vivo function of the complex.

Purification and molecular properties of an acid phosphatase from Asclepias curassavica latex

Abstract An acid phosphatase was isolated and purified from Asclepias curassavica latex. This phosphatase is the first to be obtained in the pure state from non-articulated laticifers. The enzyme has a molecular weight close to 27 000 and its amino acid composition was determined. It catalyses the hydrolysis of various phosphorylated compounds, p- nitrophenyl phosphate being the most efficient as found for most other phosphatases. The optimal pH was found to be approx. 6.0 and at this pH the Michaelis constant was 0.69 mM. Phosphate was a competitive inhibitor indicating that it is the last product to be liberated during the reaction course. Pyrophosphate, arsenate, molybdate, cupric and mercuric salts were inhibitory. Calcium, magnesium, potassium and tungsten had no effect. The physiological significance of the presence of this enzyme in the latex was discussed.

Control of Chloroplastic NADP-malate dehydrogenase activity by thioredoxins

Abstract Oxidized (inactive) chloroplastic NADP-malate dehydrogenase is 56-k Da homodimer, the two subunits of which are bound by a disulfide bridge. The enzyme is activated by three distinct chloroplast thioredoxins, thioredoxin m , f A and f B . When reduced by these proteins, the active enzyme is stable, whereas when reduced by dithiothreitol, activity is unstable. This result is apparently the consequence of aggregation of the reduced protein during prolonged incubation with dithiothreitol. At pH-values similar to those in the chloroplast stroma in the dark (pH 7), the reduced enzyme is still active.

Determination of the G + C Content of Two Syntrophus buswellii Strains by Ultracentrifugation Techniques

An anaerobic syntrophic bacterial culture degrading benzoate was isolated from a river sediment. The syntrophic organism was grown in coculture in the presence of a hydrogenotrophic strain,Desulfovibrio fructosovorans orMethanospirillum hungatei. The G+C content of the syntrophic benzoate degrader determined by density gradient ultracentrifugation was similar to that ofSyntrophus buswellii (54.3%). A method ensuring the G+C% determination of syntrophic bacteria is presented.

Analytical ultracentrifugation studies on chloroplastic fructose bisphosphatase

Abstract Analytical ultracentrifugation studies performed on spinach chloroplast fructose bisphosphatase show that the tetrameric oxidized (inactive) or reduced (active) enzyme dissociates into inactive dimers and monomers at alkaline pH. The dissociation process is, at least, partially reversible if the enzyme is dimeric. Moreover, the oxidized inactive tetrameric enzyme is less prone to dissociation into dimers and monomers than the reduced active tetramer. The irreversibility of the dissociation process may be explained by a sulfhydryl-disulfide interchange. Together with the findings from previously published sulfhydryl group titration experiments (J. Pradel et al., Eur. J. Biochem., 113 (1981) 507), the above results suggest that the activation of the oxidized tetramer involves the reduction of two inter-protomeric disulfide bonds.