[Independence of special forms of chloeophyll a and chlorophyll holochromes]. / Indépendance des formes spectrales de chlorophylle a et des holochromes chlorophylliens

Zea mays L. seedlings were cultivated for 10 days with submission to 4 s illumination periods interspersed with dark periods varying in length from 30 min to 6 h depending on the lot analyzed. The results show that, for the case in which the dark periods were shorter than 1 h, the relative proportions of different spectroscopic chlorophyll forms (maxima at 662, 670, 677.5, and 684 nm) were constant. For longer durations of darkness between illuminations, the relative proportion of the form Da670 increases, while that of Ca684 diminishes with the length of darkness; to a lesser extent, the relative proportion of Ca662 increases and a form Ca692 disappears. A scheme is proposed to explain the evolution of the relative proportions of the different spectral forms. The different chlorophyll holochromes present in the chloroplasts were also analysed. If the dark period was longer than 1 h, chlorophyll was associated with peptide chains of molecular weights 21 000 and 29 000. If the dark period was shorter than 1 h chlorophyll was associated with four peptide chains of molecular weights 21 000, 25 000, 29 000 and 70 000. The results taken together demonstrate that a given spectral chlorophyll a form cannot be associated with a definite chlorophyll holochrome.

Structure and morphogenesis of photosynthetic membranes. I. Is there a single basic peptide within Zea mays thylakoids?

We have previously given evidence for a physical heterogeneity among proteins from photosynthetic membranes of maize chloroplast [1]. About fifteen components have now been identified, differing in their molecular weights and also in their varying abilities to bind protochlorophyllide and chlorophyll [2]. In this paper, we show that all these proteins have very similar amino acid compositions. A detailed chemical comparison is made between, on one hand the mixture of all proteins and on the other the smallest component of molecular weight 10 000. All the features analysed (percent of non-proteic material, partial peptide maps, N and Cterminal ends and content of linked sugars) are the same in both cases. We conclude from these experiments that it is likely that all components are generated with the same basic peptide unit. This assertion seems to be confirmed by the fact that, after mild alkaline treatment, all the components are converted into the smallest one, as seen by electrophoresis. Thus, within the photosynthesizing membranes, there appears to be a basic structural protein unit, present in a form that is more or less "polymerized". This may account for most of the particular, and sometimes disturbing, properties so far described for this system. It is likely that such a peptide, or a similar one, would provide a framework for all photosynthesizing structures.

[Photosynthetic activity in the absence of CPl and CP2 pigmentary complexes (author's transl)]. / Des activités photosynthétiques sans les complexes pigmentaires CP1 et CP2

Various photochemical activities were tested on chloroplasts of Zea mays that received 4 s of light every 4 h during the culture period. Photosystem I and Photosystem II were functioning, as well as the photosynthetic electron transport. These chloroplasts exhibited upon sodium dodecyl sulphate gel electrophoresis neither Complex 1 (Mr 70 000) generally associated with Photosystem I nor Complex 2 Mr 25 000) generally associated with Photosystem II. Chlorophyll is indeed attached to polypeptides of molecular weight 21 000 and 29 000. These results lead us to question the functional role of chloroplast protein-pigment complexes observed by sodium dodecyl sulphate gel electrophoresis.

Effect of DNase on the activity of neutrophil elastase, cathepsin G and proteinase 3 in the presence of DNA.

It has been shown previously that DNA binds and inhibits neutrophil elastase (NE). Here we demonstrate that DNA has a better affinity for neutrophil cathepsin G (cat G) than for NE and is a better inhibitor of cat G than of NE. DNase-generated <0.5 kb DNA fragments inhibit NE and cat G as potently as full length DNA. This rationalises our observation that administration of DNase to cystic fibrosis patients does not enhance the NE and cat G activity of their lung secretions. Neutrophil proteinase 3 is not inhibited by DNA and might thus be the most harmful proteinase in inflammatory lung diseases.

Effect of various denaturing treatments on the structure and activity of a purified CP1 complex.

Spinach CP1 complex, purified as previously described [16], was submitted to various dissociating treatments. Chaotropic agents, like urea and thiocyanate salts, remained without effect on the structure and photooxidation of the complex, just SDS at very high concentrations was able to dissociate the chlorophyll from the polypeptides and to abolish the photoreaction. Proteolytic enzymes have no more action on the apparent structure and activity of CP1, but some of them do cleave the large polypeptides (65 kD) into smaller ones, as observed after pigments dissociation. This last result might be an important step in the search for a smaller active P700 protein complex.

Evidence for a light-harvesting chlorophyll a-protein complex in a chlorophyll b-less barley mutant.

Chloroplasts of a chlorophyll (Chl) b-less barley mutant were solubilized with digitonin and fractionated by polyacrylamide gel electrophoresis with sodium deoxycholate in the running buffer. By this procedure, in contrast to using sodium dodecylsulfate (SDS) for solubilization, a Chl a-protein analogous to the major light-harvesting Chl a-b protein complex from wildtype chloroplasts was recovered. This mutant Chl a-protein comprises about fifty percent of the total Chl a, and is very similar in carotenoid, amino acid, protein and polypeptide composition to the major wildtype antenna Chl a-b protein. The only major differences we have found is its instability in the presence of SDS and sensitivity to protease action. Even with deoxycholate, the mutant Chl a complex often dissociates during electrophoresis into two green bands. The lack of Chl b appears to affect the normal organization of Chl a and protein in such a way as to render the complex more unstable.

Contribution to the structural characterization of eucaryotic PSI reaction centre-I. Critical analysis of the polypeptidic composition of different P700 enriched fractions.

In thylakoid membranes, several peptides of high MW† are present which may interfere with the study of CP1's components. Modifying Cleveland's technique [7] for limited proteolysis, we have characterized the polypeptides found in the 60 kD region. Some may result from incomplete washing of the CF1 while others come from the CP1; indeed, this chlorophyll protein complex, which has a higher MW (above 100 kD), very often undergoes a dissociation into smaller components of about 60 KD MW.Analysis of the protein content of different preparations commonly used to obtain PSI reaction centre enriched fractions has been performed. The α and ß subunits of CF1 are among the main contaminants of most of these preparations. A further purification step is described which can be applied to all these preparations, but numerous peptides are still present in the active fractions. It is most unlikely that all these polypeptides are required for the primary photochemical event, and this emphasizes the necessity to find a new simple method to purify PSI reaction centres.

On the specific binding of protochlorophyllide and chlorophyll to different peptide chains.

By means of labelling experiments with δ-[(3)H]aminolevulinic acid (ALA) and SDS acrylamide gel electrophoresis the following results were obtained with plantlets of Zea mays L.: Protochlorophyllide is associated with two peptide chains of the molecular weights (MW) 21000 and 29000, thus forming two protochlorophyllide holochromes. Chlorophyll may be associated with four peptide chains of MW 21000, 25000, 29000 and 70000, forming four chlorophyll holochromes. When plantlets are transfrrred to light, protochlorophyllide is converted to chlorophyll but this remains associated with the peptide chains MW 21000 and 29000; in addition, chlorophyll is synthesized from unmetabolized ALA and associated with peptide chains MW 25000 and 70000. These conversions occur both in dark-grown plantlets and in green plantlets transferred to darkness and then again exposed to light, but in the latter considerably faster than in the former. The results suggest the existence of two pathways for the formation of chlorophyll holochromes, one requiring a dark period followed by light, the other occurring without a dark phase. Light may not only be required for photoconversion of protochlorophyllide to chlorophyll, but may also be involved in the regulation of the formation of the protein-pigment complexes.

A single step separation of PS 1, PS 2 and chlorophyll-antenna particles from spinach chloroplasts.

After solubilization of photosynthetic membranes by digitonin, three main protein pigment complexes were isolated by electrophoresis with deoxycholate as detergent.The band with the slowest mobility, fraction 1, had PS 1 activity and was devoid of PS 2 activity. This fraction was four times enriched in P700 when compared with chloroplasts. Fraction 1 had little chl b, a long wavelength absorption maximum in the red, a maximum of low temperature emission fluorescence at 730nm, and a circular dichroism spectrum characteristic of PS 1 enriched fraction.Fraction 2 exhibited a PS 2 activity and no PS 1 activity. It was enriched five times in PS 2 reaction centre and had little chl b and carotenoids. The absorption maximum was at 674 nm and the low temperature fluorescence emission maximum was at 700 nm. Fraction 2 might be useful PS 2 enriched particle because of the great stability of this fraction with regard to photochemical activity and also rapidity and simplicity of its preparation.Fraction 3, which had the fastest migration, was devoid of photochemical activities; It was rich in chl b and had the fluorescence and the circular dichroism spectrum characteristic of an antenna complex.

Contribution to the structural characterization of eucaryotic PSI reaction centre - II. Characterization of a highly purified photoactive SDS-CP1 complex.

Under precise conditions, SDS PAGE† allows purification of a photoactive P700-chla-protein complex from eucaryotic cells. The yield of P700 recovery is close to 100%. A total protein content equivalent to about 140 kD for one mole of P700 has been estimated by chemical analysis, and electrophoresis revealed the presence of two peptidic chains with MWs close to 65 kD. Photochemical and structural properties of this complex are given and compared with those of other complexes previously isolated.

Kinetic mechanism of the inhibition of cathepsin G by alpha 1-antichymotrypsin and alpha 1-proteinase inhibitor.

Uncontrolled proteolysis due to cathepsin G (cat G) may cause severe pathological disorders. Cat G is inhibited by alpha 1-antichymotrypsin (ACT) and alpha 1-proteinase inhibitor (alpha 1PI), two members of the serpin superfamily of proteins. To see whether these two inhibitors play a physiological proteolysis-preventing function, we have made a detailed kinetic investigation of their reaction with cat G. The kinetics of inhibition of cat G in the presence of inhibitor and substrate evidenced a two-step inhibition mechanism: E + I EI EI. The cat G/ACT interaction is described by Ki = 6.2 x 10(-)8 M and k2 = 2.8 x 10(-)2 s-1, while the cat G/alpha 1PI association is governed by Ki = 8.1 x 10(-)7 M and k2 = 5.5 x 10(-)2 s-1. The reliability of these kinetic constants was checked using a number of experiments which all gave consistent results: (i) both EI complexes were found to be enzymatically inactive, (ii) the Ki values were determined directly using initial velocity experiments of cat G-catalyzed hydrolysis of substrate in the presence of inhibitor, (iii) the second-order rate constants k2/Ki were measured using second-order inhibition experiments in the absence of substrate, and (iv) the ratio of the two second-order rate constants was determined by measuring the partition of cat G between the two fluorescently labeled serpins. Since the plasma concentrations of ACT and alpha 1PI are much higher than their Ki values, cat G released from neutrophils will be fully taken up as rapidly forming EI complexes, that is, 70% with ACT and 30% with alpha 1PI. Both ACT and alpha 1PI are thus physiological cat G inhibitors whose inhibitory potential does not depend on the formation of the stable inhibitory species EI characteristic of serpins. Such an in vivo inhibition mechanism might take place with other serpin/proteinase systems.

Heparin accelerates the inhibition of cathepsin G by mucus proteinase inhibitor: potent effect of O-butyrylated heparin.

Heparin tightly binds cathepsin G and so protects the enzyme from inhibition by alpha1-antichymotrypsin, alpha1-proteinase inhibitor and eglin c, three proteins which do not bind heparin [Ermolieff J., Boudier C., Laine A., Meyer B. and Bieth J.G. (1994) J. Biol. Chem. 269, 29502-29508]. Here we show that heparin no longer protects cathepsin G from inhibition when the enzyme is reacted with mucus proteinase inhibitor (MPI), a heparin-binding protein. Heparin fragments of Mr=4500 and 8100 and O-butyrylated heparin of Mr=8000 form tight complexes with cathepsin G (Kd=0.5-2.2 nM) and MPI (Kd=0. 4-0.8 muM) and accelerate the MPI-promoted inhibition of cathepsin G by a factor of 17-26. They also accelerate the inhibition of neutrophil elastase and pancreatic chymotrypsin. The rate acceleration is due to the binding of heparin to MPI. Butyrylation of heparin slightly decreases its affinity for cathepsin G and MPI but sharply decreases the ionic interactions between the positively charged proteins and the negatively charged polyanion. The butyrylated heparin derivative is the best rate accelerator: it increases the rate constant for the MPI-induced inhibition of cathepsin G and elastase by factors of 26 and 23, respectively. This, together with the fact that it has a good bioavailability and a very low anticoagulant activity, suggests that it might be an adjuvant of MPI-based therapy of cystic fibrosis.

Protein identification of purified particles isolated from spinach thylakoids by deoxycholate electrophoresis.

Three thylakoid complexes were isolated by deoxycholate preparative electrophoresis. The protein composition of each fraction was analyzed by SDS analytical electrophoresis. No protein of the PS 1 enriched fraction (fraction 1) was found in the PS 2 enriched fraction (fraction 2) and inversely. The antenna complex (fraction 3) did not have any contamination by proteins of fraction 1 or fraction 2. Fraction 1 was mainly composed of the CP1, the reaction center complex of the PS1, and by low molecular weight proteins, previously found in other PS 1 preparations. Tentative assignments of these proteins are presented; among them are iron sulfur proteins. After analytical SDS electrophoresis of fraction 2, the reaction center complex was dissociated. Nevertheless three proteins of 50 kD, 42 kD and 35 kD were assigned to this complex. Fraction 2 contained also the three cytochromes of the thylakoid membranes: cyt f, cyt b6, cyt b559. Fraction 3 was exclusively composed of one protein pigment complex, CP2.

Inhibition of neutrophil serine proteinases by suramin.

Suramin, a hexasulfonated naphtylurea recently used as an anti-tumor drug, is a potent inhibitor of human neutrophil elastase, cathepsin G, and proteinase 3. The complexes it forms with these enzymes are partially active on synthetic substrates, but full inhibition takes place when elastase activity is measured with fibrous elastin or when cathepsin G activity is measured using platelet aggregation. One molecule of elastase binds four molecules of suramin with a Ki of 2 x 10(-7) M as determined by enzyme inhibition or intrinsic fluorescence enhancement of suramin. The binding curves show no sign of cooperativity or anticooperativity. The Ki for the complexes with cathepsin G and proteinase 3 are 8 x 10(-8) and 5 x 10(-7) M, respectively. Ionic strength increases the Ki of the elastase-suramin complex in a way that suggests that four of the six sulfonate groups of suramin form ionic interactions with basic residues of the enzyme and that at saturation almost all arginines of elastase form salt bridges with suramin. The neutrophil proteinase-inhibitory activity of suramin might be used to prevent tissue destruction and thrombus formation in diseases where massive infiltration and activation of neutrophils take place.

DNA strongly impairs the inhibition of cathepsin G by alpha(1)-antichymotrypsin and alpha(1)-proteinase inhibitor.

This paper explores the possibility that neutrophil-derived DNA interferes with the inhibition of neutrophil cathepsin G (cat G) and proteinase 3 by the lung antiproteinases alpha(1)-proteinase inhibitor (alpha(1)PI), alpha(1)-antichymotrypsin (ACT), and mucus proteinase inhibitor (MPI). A 30-base pair DNA fragment ((30bp)DNA), used as a model of DNA, tightly binds cat G (K(d), 8.5 nM) but does not react with proteinase 3, alpha(1)PI, ACT, and MPI at physiological ionic strength. The polynucleotide is a partial noncompetitive inhibitor of cat G whose K(i) is close to K(d). ACT and alpha(1)PI are slow binding inhibitors of the cat G-(30bp)DNA complex whose second-order rate constants of inhibition are 2300 M(-1) s(-1) and 21 M(-1) s(-1), respectively, which represents a 195-fold and a 3190-fold rate deceleration. DNA thus renders cat G virtually resistant to inhibition by these irreversible serpins. On the other hand, (30bp)DNA has little or no effect on the reversible inhibition of cat G by MPI or chymostatin or on the irreversible inhibition of cat G by carbobenzoxy-Gly-Leu-Phe-chloromethylketone. The polynucleotide neither inhibits proteinase 3 nor affects its rate of inhibition by alpha(1)PI. These findings suggest that cat G may cause lung tissue destruction despite the presence of antiproteinases.