Polyacetylenes as systematic markers in dicotyledons

In the sequence of superorders Rutiflorae-Santaliflorae-Araliiflorae-Asteriflorae an increase in the mean oxidation state for each series of C_n-polyacetylenes and an extension in the range of the carbon atoms of these polyacetylenes in the direction of smaller numbers are observed. These trends of polyacetylene evolution also seem to be operative at lower hierarchic levels in the family Asteraceae.     

Reconstitution of the two terminal enzymes of the heme biosynthetic pathway into phospholipid vesicles

Purified mouse protoporphyrinogen oxidase (EC 1.3.3.4) and ferrochelatase (EC 4.99.1.1), the two terminal enzymes of the heme biosynthetic pathway, have been reconstituted into phospholipid vesicles, and the kinetics of the enzymes in the reconstituted systems were compared with the values obtained with the free enzymes. The apparent Km for free protoporphyrinogen oxidase in detergent solution is 5.61 +/- 0.62 microM for free protoporphyrinogen. The Km was lower when the enzyme was inserted into phospholipid vesicles (0.78 +/- 0.28 microM) and when both enzyme and substrate were incorporated into phospholipid vesicles (0.61 +/- 0.14 microM). In the presence of cardiolipin, a phospholipid present mainly in the inner mitochondrial membrane, the value of the Km for the substrate decreased 3-fold (0.20 +/- 0.02 microM). For reconstituted ferrochelatase similar kinetic analyses were carried out and it was found that the apparent Km values were only weakly affected by the lipid environment. Studies on the orientation of ferrochelatase demonstrated that approximately 50% of the enzyme in the reconstituted system had the active site located in the inner face of the phospholipid vesicle. This is in contrast to intact mitochondria where the active site is located on the matrix side of the inner mitochondrial membrane. The activation energies for both enzymes were determined for free and reconstituted enzymes. It was found that for both enzymes the activation energies were lower for the reconstituted systems than for the free enzymes.     

Expression of the Major Light-Harvesting Chlorophyll a/b-Protein and Its Import into Thylakoids of Mesophyll and Bundle Sheath Chloroplasts of Maize

Distribution of the major light-harvesting chlorophyll a/b-protein (LHCII) and its mRNA within bundle sheath and mesophyll cells of maize (Zea mays L.) was studied using in situ immunolocalization and hybridization, respectively. In situ hybridization with specific LHCII RNA probes from maize and Lemna gibba definitively shows the presence of high levels of mRNA for LHCII in both bundle sheath cells and mesophyll cells. In situ immuno-localization studies, using an LHCII monoclonal antibody, demonstrate the presence of LHCII polypeptides in chloroplasts of both cell types. The polypeptide composition of LHCII and the amount of LHCII in bundle sheath cells are different from those in mesophyll cells. Both mesophyll and bundle sheath chloroplasts can take up, import and process the in vitro transcribed and translated LHCII precursor protein from L. gibba. Although bundle sheath chloroplasts incorporate LHCII into the pigmented light-harvesting complex, the efficiency is lower than that in mesophyll chloroplasts.     

Effect of osmotic stress on protein turnover in Lemna minor fronds

Evidence is presented that although many proteins from the fronds of Lemna minor L. undergo enhanced degradation during osmotic stress, ribulose-1,5-bisphosphate carboxylase (RuBPCase) is not degraded. Instead RuBPCase is converted in a series of steps to a very high-molecular-weight form. The first step involves the induction of an oxidase system which after 24 h of stress converts RuBPCase to an acidic and catalytically inactive form. Subsequently, the oxidised RuBPCase protein is gradually polymerized to a number of very large aggregates (molecular weight of several million).The conversion of RuBPCase to a high-molecular-weight form appears to be correlated with (i) a reduction in the number of-SH residues and (ii) the susceptibility to in-vitro proteolysis. Indeed, the number of-SH groups per RuBPCase molecule decreases from 89 in the native enzyme to 54 and 22 in the oxidised and polymerized forms, respectively. On the other hand, the oxidised enzyme is more susceptible to in-vitro proteolysis than the native form. However, it is the polymerized form of RuBPCase which is particularly susceptible to in-vitro proteolysis.Western-blotting experiments and anti-ubiquitin antibodies were used to detect the presence of ubiquitin conjugates in extracts from osmotically stressed Lemna fronds. The possible involvement of ubiquitin in the formation of the aggregates is discussed.Abbreviations DTT dithiothreitol - EDTA ethylenediamine-tetraacetic acid - FPLC fast protein liquid chromatography - kDa kilodaltons - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulphonyl fluoride - RuBPCase ribulose bisphosphate carboxylase - SDS sodium dodecyl sulphate - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Mouse protoporphyrinogen oxidase. Kinetic parameters and demonstration of inhibition by bilirubin.

The penultimate step of haem biosynthesis, the oxidation of protoporphyrinogen to protoporphyrin, was examined with purified murine hepatic protoporphyrinogen oxidase (EC 1.3.3.4) in detergent solution. The kinetic parameters for the two-substrate (protoporphyrinogen and oxygen) reaction were determined. The limiting Km for protoporphyrinogen when oxygen is saturating is 6.6 microM, whereas the Km for oxygen with saturating concentrations of protoporphyrinogen is 125 microM. The kcat. for the overall reaction is 447 h-1. The ratio of kcat. to the Km for protoporphyrinogen is approx. 20-fold greater than the kcat./Km,O2 ratio. The ratio of protoporphyrin formed to dioxygen consumed is 1:3. Ubiquinone-6, ubiquinone-10 and dicoumarol stimulate protoporphyrinogen oxidase activity at low concentrations (less than 15 microM), whereas coenzyme Q0 and menadione show no activation at these concentrations. Above 30 microM, all five quinones inhibit the enzyme activity. FAD does not significantly affect the activity of the enzyme. Bilirubin, a product of haem catabolism, is shown to be a competitive inhibitor of the penultimate enzyme of the haem-biosynthetic pathway, protoporphyrinogen oxidase, with a calculated Ki of 25 microM. The terminal enzyme of haem-biosynthetic pathway, namely ferrochelatase, is not inhibited by bilirubin at concentrations over double the Ki value for the oxidase. In contrast with other enzymic systems, the toxicity of bilirubin is not reversed by binding to albumin.     

Metabolic adaptation of the renal carbohydrate metabolism. I. Effects of starvation on the gluconeogenic and glycolytic fluxes in the proximal and distal renal tubules

Summary The influence of starvation on renal carbohydrate metabolism was studied in the proximal and distal fragments of the nephron. Starvation induced a double and opposite adaptation mechanism in both fractions of the renal tubule. In renal proximal tubules, the gluconeogenic flux was stimulated progressively during a period of 48 hours of starvation (2.15 fold), due, in part, to a significant increase in the fructose 1,6-bisphosphatase and phosphoenolpyruvate carboxykinase activities although with different characteristics. Fructose 1,6-bisphosphatase activity from this tubular fragment increased only at subsaturating subtrate concentration (68%) which involved a significant decrease in the Km (35%) for fructose 1,6-bisphosphate while there was no change in Vmax. This behaviour clearly indicates that it is related to modifications in the activity of the preexistent enzyme in the cell. Proximal phosphoenolpyruvate carboxykinase activity increased proportionally at both substrate concentrations (86 and 89% respectively) which brought about changes in Vmax without changes in Kin, all of which are in accordance with variations in the cellular levels of the enzyme. In the renal distal tubules, the glycolytic capacity drastically decreased throughout the starvation time. At 48 hours 65% of inhibition was shown. We have found a short term regulation of phosphofructokinase activity by starvation which involves an increase in Km (2.2 fold) without changes in Vmax, as a result of these kinetic changes, an inactivation of phosphofructokinase was detected at subsaturating concentration of fructose 6-phosphate. On the contrary, this nutritional state did not modify the kinetic behaviour of renal pyruvate kinase. Finally, neither proximal glycolytic nor distal gluconeogenic capacities and related enzymes activities were changed during starvation.     

Synthesis of the biologically active β-subunit of human nerve growth factor in Escherichia coli

The gene(NGFB) encoding the β subunit of mature human nerve growth factor (hNGFB) was subcloned into the pJLA503 expression vector under the control of bacteriophage promoters p_R and p_L, and expressed in Escherichia coli. The recombinant protein represented approximately 3% of the total cellular protein. Biologically active hNGFB was solubilized (0.2% total NGFB) and purified by cation-exchange chromatography and it yielded two bands on polyacrylamide-gel electrophoresis under nonreducing conditions, corresponding to the monomeric (14 kDa) and homodimeric (26.5 kDa) forms of the molecule. Both hNGFB forms were immunopositive on Western blots with rabbit anti-NGFB antibodies; however, following additional purification, only the species corresponding to the hNGFB homodimer was biologically active on cultured chicken dorsal root ganglion neurons. These results demonstrate the feasibility of synthesizing the biologically active form of hNGFB in E. coli.