The reductive pentose phosphate cycle for photosynthetic CO2 assimilation: enzyme modulation.

The reductive pentose phosphate cycle (Benson-Calvin cycle) is the main biochemical pathway for the conversion of atmospheric CO2 to organic compounds. Two unique systems that link light-triggered events in thylakoid membranes with enzyme regulation are located in the soluble portion of chloroplasts (stroma): the ferredoxin-thioredoxin system and ribulose 1,5-bisphosphate carboxylase/oxygenase-Activase (Rubisco-Activase). The ferredoxin-thioredoxin system (ferredoxin, ferredoxin-thioredoxin reductase, and thioredoxin) transforms native (inactive) glyceraldehyde-3-P dehydrogenase, fructose-1,6-bisphosphatase, sedoheptulose-1,7-bisphosphatase, and phosphoribulokinase to catalytically competent forms. However, the comparison of enzymes reveals the absence of common amino acid sequences for the action of reduced thioredoxin. Thiol/disulfide exchanges appear as the underlying mechanism, but chloroplast metabolites and target domains make the activation process peculiar for each enzyme. On the other hand, Rubisco-Activase facilitates the combination of CO2 with a specific epsilon-amino group of ribulose 1,5-bisphosphate carboxylase/oxygenase and the subsequent stabilization of the carbamylated enzyme by Mg2+, in a reaction that depends on ATP and ribulose 1,5-bisphosphate. Most of these studies were carried out in homogeneous solutions; nevertheless, a growing body of evidence indicates that several enzymes of the cycle associate either with thylakoid membranes or with other proteins yielding supra-molecular complexes in the chloroplast.

A procedure for the generation and the purification of Escherichia coli thioredoxins with variable N-terminal sequences.

We have developed a rapid and simple procedure for the production and the purification of Escherichia coli thioredoxins containing additional amino acid residues at the N-terminus. By the polymerase chain reaction, the complete gene encoding for E. coli thioredoxin was modified and amplified with the addition at its 5' end of a BamHI cloning site and a triplet coding for an arginine residue instead of the initiator methionine codon, whereas at the 3' end the stop codon was followed by an EcoRI cloning site. The synthetic DNA was ligated into the BamHI/EcoRI site of the vector plasmid pGEX-2T, and the novel plasmid [pFTG] was used for the transformation of E. coli cells. Following induction and cell disruption, a protein composed of Schistosoma japonicum glutathione S-transferase and E. coli thioredoxin was obtained in soluble form and purified by affinity chromatography on agarose columns bearing immobilized glutathione. This procedure yielded 50 mg of homogeneous fusion protein per liter of culture media. Digestion of the chimeric thioredoxin with bovine plasma thrombin followed by an additional chromatography on glutathione-agarose gave a protein that contained the entire sequence of E. coli thioredoxin and three additional amino acid residues [G-S-R-] at the N-terminal side. The structural characteristics and the protein disulfide oxidoreductase activity of this recombinant protein, in terms of variations of emission fluorescence and reduction of insulin disulfide bonds, respectively, were essentially identical to those of its counterpart obtained from wild-type cells by conventional techniques of proteins purification.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of the visible spectrum of reflected light from muscle tissue in rats breathing varying amounts of oxygen in nitrogen or perfused with saline.

One of the most important problems in the treatment of injuries caused by high velocity missiles is to find the borderline between viable and non-viable muscle tissue. Technical aids using spectrophotographic principles are designed. The present investigation was performed in order to study the absorption of incident light in muscle tissue spectrographically and to compare vital muscle tissue supplied with blood of varying degrees of oxygen saturation in rats. Transmission of light through transparencies was analysed spectrographically. Transmission of muscle tissue without blood is responsible for approximately 60% of the total transmission. Rats breathing 100% oxygen differ in transmission significantly from the control group (breathing air) within the wavelength region of 430--570 nm. Rats breathing 10% oxygen show no significant difference to the control group.

Optical properties of damaged and undamaged muscle tissue studied in high-energy missile wounds.

Modern surgical treatment of wounds caused by high-energy traumata includes the excision of all non-viable muscle tissue. To judge the transition zone between viable and non-viable tissue correctly, however, it takes a highly qualified surgeon, experienced in high-energy trauma surgery. Less skilled surgeons are tending to leave some non-viable tissue behind in the wound, which often leads to infection and re-operation and causes, among other things, extended suffering and hospitalization for the patient. Among the visible and palpable properties serving as criteria for the surgeon is that the non-viable tissue is dark purple, whereas the viable tissue is of lighter red colours. The perception of colour is depending on the optical properties of a surface and on the composition of the light illuminating it. A pilot study has shown that the colour contrast between viable and non-viable muscle tissue can be enhanced by using colour filters. In this investigation optical properties for muscle tissue in different degrees of viability have been scrutinized. It seems to be possible to find a clinically applicable method to help the surgeon in his work.

Interspecies cross-reactivity of a monoclonal antibody directed against wheat chloroplast fructose-1,6-bisphosphatase.

The primary structure of several chloroplast fructose-1,6-bisphosphatase (CFBPase) was deduced from DNA sequences, but only spinach, pea and rapeseed enzymes have been characterized structurally. We analyzed whether CFBPases from different phylogenetic origin contain a common epitope. To this end a DNA fragment of 1200 base pairs encoding 338 amino acid residues of wheat CFBPase (38 kDa) was cloned in the expression plasmid pGEX-1 in frame with the gene coding for glutathione S-transferase (GT) of Schistosoma japonicun (26.5 kDa). Upon transformation of Escherichia coli and induction with isopropyl-beta-D-thiogalactopyranoside, centrifugation of the lysate partitioned 10% of the fusion protein in the supernatant fraction and the remaining 90% in the precipitate. The expected 65 kDa protein was purified from both the soluble and the particulate fraction by affinity chromatography on columns of glutathione-agarose. This fusion protein was successfully used to produce a monoclonal antibody that specifically recognized the CFBPase region of the fusion protein but not the GT moiety. Moreover, the monoclonal antibody immunoreacted not only with polypeptides (ca. 40 kDa) present in leaf crude extracts of other varieties of wheat (Triticum spelta, T. aestivum and T. durum), but also with homogeneous preparations of the spinach (Spinacia oleracea) and rapeseed (Brassica napus) enzymes. Thus, the cross reaction of this monoclonal antibody with counterparts from different plant species indicates the persistency of a common epitope through biological evolution.