No abstract

In vivo pulse-chase labeling of rabbit jejunum loops was used in conjunction with subcellular fractionation and quantitative immunoprecipitation to determine whether or not the newly synthesized aminopeptidase N transits through the basolateral membrane before it reaches the apical brush border, its final localization. The kinetics of the arrival of the newly synthesized enzyme in the Golgi complex, basolateral and brush border membrane fractions strongly suggest that on leaving the Golgi aminopeptidase N is transiently integrated into the basolateral domain before reaching the brush border.

Activation by light of spinach fructose‐1,6‐bisphosphatase is mimicked by dithiothreitol. This process of activation by dithiothreitol implies the specific reduction of two disulfide bridges and a conformation change of the enzyme that makes eight sulfhydryl groups available. The activated enzyme has an apparent allosteric kinetic behavior with respect to both fructose‐1,6‐bisphosphate and magnesium.

Thioredoxin f has been isolated and purified to homogeneity from spinach chloroplasts. The protein is a dimer which dissociates in two apparently identical halves when ionic strength is raised. The dimer has a molecular weight of 16000. In its oxidized state, chloroplastic thioredoxin f has no sulfhydryl group directly available, even after denaturation of the protein. When reduced by dithiothreitol, four -SH groups become available on the dimer: Taken together, these results show that there exists one disulfide bridge per monomer. Reduction by dithiothreitol results in the breaking of two disulfide bridges and the appearance of four sulfhydryl groups.Monomeric thioredoxin f does not result in a significant activation of fructose 1,6-bisphosphatase. Full activation is obtained in presence of the dimeric protein. This is understandable since activation of fructose bisphosphatase necessitates reduction of two disulfide bridges of the enzyme.Full activation of fructose bisphosphatase is obtained in the presence of either an excess of dimeric thioredoxin f, or in the presence of dithiothreitol. Moreover a large excess of oxidized thioredoxin deactivates fructose bisphosphatase. These results suggest that there exists an equilibrium rTh + oFruPzase + oTh + rFruPzase where rTh and oTh stand for reduced and oxidized thioredoxin, oFruPzase and rFruPlase for oxidized and reduced fructose bisphosphatase. Moreover, this equilibrium must be strongly shifted towards the left.NADPH in presence of NADP+-reductase may reduce thioredoxin, and conversely reduced thioredoxin may reduce NADP' if the reductase is present. These results together with the existence of the above equilibrium provides a tentative scheme of fructose bisphosphatase inactivation in vivo in the dark. When the light has been turned off, electrons are transferred from thioredoxin to NADP' via NADP+-reductase. Thioredoxin becomes oxidized and then deactivates fructose bisphosphatase.It is now firmly established that COz fixation by green plants is controlled by light [l -31. This control seems to be effected thanks to several factors, such as the level of substrates and cofactors [4], the local Hc and cation concentration [5] and the light-dependent activation of enzymes belonging to the reductive pentose phosphate cycle [6]. The most extensively studied of these enzymes is, without doubt, chloroplastic fructose 1,6-bisphosphatase [7 -91. This enzyme is a tetramer made up of apparently identical subunits. Dithiothreitol can mimic the effect of light and can activate the enzyme in the dark. It has recently been shown that this activation is due to the reduction of two strategic disulfide bridges of the tetrameric enzyme, followed by a conformation change of the protein that can be monitored by spectrofluorimetry [lo].