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S Lemaire

Publications and source records attributed to S Lemaire.

At least 37 records · Page 2Linked to original sources
PubMed1998

Characterization of the high affinity [3H]nociceptin binding site in membrane preparations of rat heart: correlations with the non-opioid dynorphin binding site.

The binding parameters of [3H]nociceptin were examined in membrane preparations of rat heart and compared with those of [3H]dynorphin A-(1-13) ([3H]Dyn A-(1-13)). Scatchard analysis of [3H]nociceptin binding revealed the presence of two distinct sites: a high affinity (Kd: 583 nM) low capacity (Bmax: 132 pmol/mg protein) site and a low affinity (Kd: 10,316 nM) high capacity (1552 pmol/mg protein) site. Dyn A and related peptides were potent competitors of the binding to the high affinity site with the following rank order of potency: alpha-neo-endorphin > Dyn A-(2-13) = Dyn A-(3-13) > Dyn A-(5-13) > Dyn A-(1-13) > Dyn A > Dyn B > Dyn A-(6-10) >> Dyn A-(1-8). Nociceptin was 6.7 times less potent than Dyn A with a Ki of 4.8 microM as compared with 0.72 microM for Dyn A. The order of potency of the various peptides in inhibiting [3H]nociceptin binding correlated well (r = 0.93) with their ability to complete with the binding of [3H]Dyn A-(1-13) (Dumont and Lemaire, 1993). In addition, the high affinity [3H]nociceptin and non-opioid [3H]Dyn A-(1-13) sites were both sensitive to NaCl (120 mM) and the phospholipase C (PLC) inhibitors, U-73122 and neomycin (100 microM). The binding activities were less affected by the weak PLC inhibitor, U-73343, and no effect was observed with the non-hydrolysable GTP analogs. Gpp(NH)p and GTP-gamma-S. Nociceptin (1-50 microM) was also shown to inhibit the uptake of [3H]noradrenaline ([3H]NA) by cardiac synaptosomal preparations. In spontaneously hypertensive rats (SHR), the potency of nociceptin in inhibiting [3H]NA uptake was increased by 1.6-fold as compared with Wistar Kyoto (WKY) control rats and such effect was accompanied by comparable increased levels of cardiac ORL1 mRNA and [3H]nociceptin high affinity sites. These changes correlated well with the previously observed increased levels of non-opioid cardiac [3H]Dyn A-(1-13) sites in SHR (1.3 times as compared with WKY) and increased potency of Dyn A-(1-13) in inhibiting [3H]NA uptake by cardiac synaptosomes in SHR (2.2-fold as compared with WKY) (Dumont and Lemaire, 1995). The results demonstrate that in rat heart the characteristics of the high affinity, low capacity [3H]nociceptin binding site are similar to those of the non-opioid Dyn binding site. The stimulation of this site by nociceptin, Dyn A or related peptides is more likely to produce a modulation of PLC activity and [3H]NA uptake and may participate to the pathophysiology of hypertension.

Animals↗
PubMed1998

Heart rate as a determinant of L-type Ca2+ channel activity: mechanisms and implication in force-frequency relation.

Early studies in enzymatically isolated animal cardiomyocytes indicated that voltage-gated "L-type" Ca2+ currents (ICaL) can be upregulated following an increase of the frequency of activation. Recently, we evidenced a similar regulation of ICaL in human cardiomyocytes from both left and right ventricles and atria over a physiopathological range of stimulations (between 0.5 and 5 Hz). This regulation, enhanced by the beta-adrenergic stimulation, may be involved in the frequency-dependent potentiation of cardiac contractile force in the human healthy myocardium. We show here that the frequency-dependent regulation of ICaL is controlled by the level of phosphorylation, as well as dephosphorylation, of the Ca2+ channels. It was enhanced following activation of the protein kinase A activated by intracellular cyclic AMP (cAMP). Therefore, we anticipate that all agents stimulating cAMP production will favor this process, which was demonstrated here by activating 5HT-4 receptors using serotonin. Alternatively, it was also enhanced by the phosphatase inhibitor okadaic acid which prevents Ca2+ channels dephosphorylation. Alteration or abnormal modulation by beta-adrenergic receptor stimulation of the frequency-dependent facilitation of ICaL may partly explain the altered force-frequency relation described in heart failure.

Biomechanical Phenomena↗
PubMed1998

Ca2+ currents in compensated hypertrophy and heart failure.

Transmembrane voltage-gated Ca2+ channels play a central role in the development and control of heart contractility which is modulated by the concentration of free cytosolic calcium ions (Ca2+). Ca2+ channels are closed at the normal membrane resting potential of cardiac cells. During the fast upstroke of the action potential (AP), they are gated into an open state by membrane depolarisation and thereby transduce the electrical signal into a chemical signal. In addition to its contribution to the AP plateau, Ca2+ influx through L-type Ca2+ channels induces a release of Ca2+ ions from the sarcoplasmic reticulum (SR) which initiates contraction. Because of their central role in excitation-contraction (E-C) coupling, L-type Ca2+ channels are a key target to regulate inotropy [1]. The role of T-type Ca2+ channels is more obscure. In addition to a putative part in the rhythmic activity of the heart, they may be implicated at early stages of development and during pathology of contractile tissues [2]. Despite therapeutic advances improving exercise tolerance and survival, congestive heart failure (HF) remains a major problem in cardiovascular medicine. It is a highly lethal disease; half of the mortality being related to ventricular failure whereas sudden death of the other patients is unexpected [3]. Although HF has diverse aetiologies, common abnormalities include hypertrophy, contractile dysfunction and alteration of electrophysiological properties contributing to low cardiac output and sudden death. A significant prolongation of the AP duration with delayed repolarisation has been observed both during compensated hypertrophy (CH) and in end-stage HF caused by dilated cardiomyopathy (Fig. 1A) [4-8]. This lengthening can result from either an increase in inward currents or a decrease in outward currents or both. A reduction of K+ currents has been demonstrated [6,9]. Prolonged Na+/Ca2+ exchange current may also be involved [9]. In contrast, there is a large variability in the results concerning Ca2+ currents (ICa). The purpose of this paper is to review results obtained in various animal models of CH and HF with special emphasis on recent studies in human cells. We focus on: (i) the pathophysiological role of T-type Ca2+ channels, present in some animal models of hypertrophy; (ii) the density and properties of L-type Ca2+ channels and alteration of major physiological regulations of these channels by heart rate and beta-adrenergic receptor stimulation; and (iii) recent advances in the molecular biology of the L-type Ca2+ channel and future directions.

Adrenergic beta-Agonists↗
PubMed1998

T lymphocyte activation results in an increased expression of beta-1,4-galactosyltransferase: phorbol ester induces a similar enhancement in the absence of mitosis.

We previously showed that in vitro activated human T lymphocytes expressed increased amounts of beta-1,6-branched N-linked oligosaccharides (Lemaire S etal. (1994) J Biol Chem269: 8069-74), which have been proposed to participate in the regulation of the immune process. In the present paper, we compared the activity and expression of beta-1,4-galactosyltransferase (GalT), one of the glycosyltransferases involved in the biosynthesis of these beta-1,6-branched N-linked oligosaccharides, before and after in vitro activation of T lymphocytes after a 40h treatment with a mixture of phorbol 12-myristate 13-acetate and Phaseolus vulgaris lectin. After treatment, the enzymatic activity of the GalT was significantly increased and immunoblot experiments performed with a monoclonal antibody to human GalT showed an increased intensity of the GalT band at 49 kDa, attributable to an enhancement of GalT mRNA level, as shown by Northern blots. However, treatment of the same T-lymphocytes by phorbol ester alone, which is unable to induce mitosis, resulted in a comparable increase of the expression of GalT. Moreover, these phorbol ester-treated T lymphocytes, analysed by flow cytometry exhibited a two-fold increase in the expression of GalT. Finally, confocal fluorescence microscopy performed on all T lymphocytes (treated or not) showed that the flow cytometric signal of GalT originates from intracellular, Golgi-associated antigen only since no surface GalT was detected.

Flow Cytometry↗
PubMed1998

Glutathione is implied in the control of 7-ketocholesterol-induced apoptosis, which is associated with radical oxygen species production.

In a number of experimental systems, inhibition of apoptosis by antioxidants has led to the production of radical oxygen species (ROS) in certain apoptotic forms of cell death. Since antioxidant therapies can reduce vascular dysfunctions in hypercholesterolemic patients who frequently have increased plasma levels of oxysterols constituting potent inducers of apoptosis, we speculate that oxysterol-induced apoptosis could involve oxidative stress. Here, we tested the protective effects of the aminothiols glutathione (GSH) and N-acetylcysteine (NAC), which are two potent antioxidants, on apoptosis induced by 7-ketocholesterol in U937 cells, and we present evidence indicating that oxidative processes are involved in 7-ketocholesterol-induced cell death. Thus, GSH and NAC prevented phenomenona linked to apoptosis such as reduction of cell growth, increase cellular permeability to propidium iodide, and occurrence of nuclear condensation and/or fragmentation, and they delayed internucleosomal DNA fragmentation. In addition, cell treatment with GSH impaired cytochrome c release into the cytosol and degradation of caspase-8 occurring during cell death. During 7-ketocholesterol-induced apoptosis, we also observed a rapid decrease in cellular GSH content, oxidation of polyunsaturated fatty acids, and a production of ROS by flow cytometry with the use of the dye 2', 7'-dichlorofluorescin-diacetate; both phenomena were inhibited by GSH. Prevention of cell death by GSH and NAC does not seem to be a general rule since these antioxidants impaired etoposide (but not cycloheximide) -induced apoptosis. Taken together, our data demonstrate that GSH is implied in the control of 7-ketocholesterol-induced apoptosis associated with the production of ROS.

Acetylcysteine↗
PubMed1997

Induction of apoptosis and of interleukin-1beta secretion by 7beta-hydroxycholesterol and 7-ketocholesterol: partial inhibition by Bcl-2 overexpression.

The oxysterols, 7beta-hydroxycholesterol and 7-ketocholesterol, are involved in the cytotoxicity of oxidized LDL. To elucidate their molecular mechanisms, the human promonocytic leukemia cells U937 and U4 were used. U4 cells overexpressing Bcl-2 were obtained by transfection of U937 cells. 7Beta-hydroxycholesterol and 7-ketocholesterol induced nuclear condensation and/or fragmentation, internucleosomal DNA fragmentation, and IL-1beta secretion, which were partially inhibited by Bcl-2 overexpression. These findings underline that these oxysterols could constitute major risk factors in atherosclerosis by their cytotoxicity and their ability to induce IL-1beta release which might favor the recruitment of immunocompetent cells in the atherosclerotic plaque.

Apoptosis↗
PubMed1997

Cloning and expression of human 5-HT4S receptors. Effect of receptor density on their coupling to adenylyl cyclase.

We have isolated a cDNA encoding the 5-HT4S receptor by RT-PCR on poly (A)+ RNA from both human heart and brain. The sequence homology with the rat and mouse 5-HT4 receptors was high: 93.8% of identity in the amino acid sequence. None of the 24 amino acid substitutions observed between rat and human receptors are at positions likely to modify their pharmacology. Comparing the pharmacological properties of six agonists and five antagonists on rat and human 5-HT4S receptors revealed no significant differences. We have analyzed the behavior of renzapride, a full and a partial agonist on mouse colliculi neurons and human heart biological responses respectively. The coupling efficiency of renzapride was two-fold lower than that of 5-HT for the stimulation of 5-HT4S receptors transfected in two different cell lines (LLC-PK1 and COS-7), but increasing the receptor density suppressed the partial agonist effect of renzapride.

Adenylyl Cyclases↗
PubMed1997

Cysteine-153 is required for redox regulation of pea chloroplast fructose-1,6-bisphosphatase.

Chloroplastic fructose-1,6-bisphosphatases are redox regulatory enzymes which are activated by the ferredoxin thioredoxin system via the reduction/isomerization of a critical disulfide bridge. All chloroplastic sequences contain seven cysteine residues, four of which are located in, or close to, an amino acid insertion region of approximately 17 amino acids. In order to gain more information on the nature of the regulatory site, five cysteine residues (Cys49, Cys153, Cys173, Cys178 and Cys190) have been modified individually into serine residues by site-directed mutagenesis. While mutations C173S and C178S strongly affected the redox regulatory properties of the enzyme, the most striking effect was observed with the C153S mutant which became permanently active and redox independent. On the other hand, the C190S mutant retained most of the properties of the wild-type enzyme (except that it could now also be partially activated by the NADPH/NTR/thioredoxin h system). Finally, the C49S mutant is essentially identical to the wild-type enzyme. These results are discussed in the light of recent crystallographic data obtained on spinach FBPase [Villeret et al. (1995) Biochemistry 34, 4299-4306].

Amino Acid Sequence↗
PubMed1997

Nonexocytotic noradrenaline release from rat cardiac synaptosomal-mitochondrial fractions.

Nonexocytotic noradrenaline (NA) release was examined in rat cardiac synaptosomal-mitochondrial fractions prelabeled with [3H]NA (300 nM; 1 h at 37 degrees C). Ischemic conditions (1 mM iodoacetate + 2 mM NaCN; 15 min at 37 degrees C) evoked a Ca(2+)-independent release of [3H]NA from isolated synaptosomes, which represented 33.4% of total content, whereas the release evoked by 56 mM K+ was Ca2+ dependent and represented 5.8% of total content. Tyramine, phencyclidine (PCP), and rimcazole also caused important Ca(2+)-independent releases of [3H]NA (from 12 to 45% of total content) with median effective concentrations (EC50s) of 6.8, 182, and 41.8 microM, respectively. The release responses evoked by ischemic conditions, tyramine, PCP, and rimcazole were mimicked by the delta-receptor ligand, 1,3-ditolyl guanidine (DTG), and blocked by the uptake 1 inhibitor, desipramine (100 microM). The delta 1-receptors ligands, (+)-3-hydroxyphenyl-N-(1-propyl)piperidine ((+)-3-PPP) and (+)N-allylnormetazocine [(+)SKF-10047], were potent blockers of the release of [3H]NA evoked by ischemic conditions but not by PCP or rimcazole. These data indicate that ischemic conditions and PCP/delta 2-receptor ligands induce carrier-mediated NA efflux from cardiac sympathetic nerve terminals, whereas delta 1-receptor ligands produce marked inhibition of the ischemic response.

Animals↗
PubMed1997

Design of potent dynorphin A-(1-9) analogues devoid of supraspinal motor effects in mice.

Four analogues of dynorphin (Dyn) A-(1-9) incorporating D-Leu in position 8 alone or in combination with the nonhydrolysable psi [CS-NH] thiopeptide bond surrogate between positions 6 and 7 were tested in vitro for their ability to compete with the binding of selective kappa, mu, and delta opioid ligands, using membrane preparations of guinea pig cerebellum (kappa) and rat brain (mu and delta), for their ability to block the electrically induced contractions of the guinea pig ileum, and for their in vivo antinociceptive (writhing test) and motor (motor dysfunction assay) activities in mice. [D-Leu8]Dyn A-(1-9) displayed an affinity and a selectivity for the kappa opioid receptor that were comparable with those of Dyn A-(1-9). The potencies of [D-Leu8]Dyn A-(1-9) in the guinea pig ileum, writhing, and motor dysfunction assays were markedly enhanced (8-12 fold) compared with those of Dyn A-(1-9). [6 psi 7(CS-NH),D-Leu8]Dyn A-(1-9), [Lys6, 6 psi 7(CS-NH),D-Leu8] Dyn A-(1-9), and [Leu6, 6 psi 7(CS-NH), D-Leu8]Dyn A-(1-9) were somewhat less potent than [D-Leu8]Dyn A-(1-9) in all opioid assays. However, the thiopeptides were more potent analgesics than Dyn A-(1-9)(ED50 of 29.5, 23.9, and 15.5 nmol/mouse, respectively, compared with 90.7 nmol/mouse for Dyn A-(1-9)) and caused little or no motor impairment at analgesic doses.

2-Amino-5-phosphonovalerate↗
PubMed1997

N-methyl-D-aspartate antagonist activity of alpha- and beta-sulfallorphans.

Resolved equatorial (alpha) and axial (beta) forms of S-allylmorphinans, alpha-sulfallorphan and beta-sulfallorphan, were tested for their ability to compete with the binding of phencyclidine and sigma receptor ligands to mouse brain membranes and to antagonize N-methyl-D-aspartate (NMDA)-induced convulsions in mice. alpha- and beta-sulfallorphans displayed distinct binding affinities for phencyclidine and sigma sites, inhibiting the binding of [3H]-(5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten++ +-5, 10-imine ([3H]MK-801) with Ki values of 2.32 and 0.13 microM and that of [3H](+)-pentazocine with Ki values of 1.97 and 1.61 microM, respectively. Intracerebroventricular administration of these compounds in mice caused dose-dependent inhibitions of NMDA-induced convulsions, but did not affect convulsions induced by (R,S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), kainic acid and bicuculline. alpha- and beta-sulfallorphans blocked the convulsive activity of NMDA (1 nmol/mouse; intracerebroventricular) with ED50 values of 0.48 and 0.015 nmol/mouse, as compared with 0.55, 0.039 and 0.013 nmol/mouse for dextrorphan, MK-801 and (+/-)3-(2-carboxypiperazine-4yl)propyl-1-proprionic acid, respectively. The structurally related compound, dextrallorphan, significantly but less potently blocked NMDA-induced convulsions (ED60, 2.68 nmol/mouse). At the protective doses, alpha- and beta-sulfallorphans markedly reduced NMDA- and AMPA-induced mortality without inducing locomotion and falling behavior. These results indicate that alpha- and beta-sulfallorphans are potent and selective NMDA antagonists devoid of motor side effects at protective doses.

Animals↗
PubMed1997

Nonopioid motor effects of dynorphin A and related peptides: structure dependence and role of the N-methyl-D-aspartate receptor.

Dynorphin (Dyn) A and related opioid and nonopioid peptides were tested for their ability to produce motor effects in mice. Central (intracerebroventricular) administration of Dyn A in mice produced marked motor effects characterized by wild running, jumping, circling and/or barrel rolling with an ED50 value of 14.32 (95% confidence limits, 10.09-20.32) nmol/mouse. The order of potency of the various Dyn A-related peptides and fragments in producing motor effects was Dyn A approximately Dyn A-(1-13) > [Ala1]Dyn A-(1-13) approximately Dyn A-(2-13) > alpha-Neo-End > Dyn A-(1-8) approximately Dyn B approximately Dyn A-(2-8) >>> Dyn A-(3-8). Dyn A-(1- 5) (or Leu-Enk) and Dyn A-(6-10) displayed no motor effect at doses up to 100 nmol/mouse. The potencies of Dyn A and Dyn A-(2-13) were not affected by preadministration of naloxone (5 mg/kg s.c.), but the motor effects of Dyn A-(1-13) (20 nmol/mouse i.c.v.) were significantly reduced by coadministration of low doses (0.2-0.6 nmol/mouse) of the N-methyl-D-aspartate (NMDA) receptor antagonists dextrorphan, MK-801 and CPP. Dyn A was also a potent inhibitor of the binding of the phencyclidine receptor ligand, [3H]MK-801, to rat brain membranes, with a Ki value of 0.41 microM. However, the order of potency of the various Dyn A-related peptides and fragments in inhibiting [3H]MK-801 binding did not correlate with their ability to produce motor effects. On the other hand, Dyn A and related peptides produced a significant potentiation of the binding of the competitive NMDA antagonist [3H]CGP-39653 to rat brain membranes, an effect that correlated well (r = 0.91) with their potency in producing motor effects. These results indicate that the nonopioid motor effects of Dyn A and related peptides are structure dependent, with Dyn A-(2-8) being the minimal core peptide for motor activity. In addition, these effects most likely involve the participation of the excitatory amino acid binding domain on the NMDA receptor complex.

2-Amino-5-phosphonovalerate↗
PubMed1996

Interactions of dynorphin A and related peptides with cardiac ouabain binding sites.

The effect of dynorphin A (Dyn A) and related peptides on the binding of [3H]ouabain was examined in rat cardiac sarcolemma. Scatchard analysis of [3H]ouabain binding revealed the existence of two distinct sites: a high affinity (Kd: 20.4 nM) low capacity (Bmax: 1.55 pmol/mg protein) site and a low affinity (Kd: 3695 nM) high capacity (Bmax: 39.3 pmol/mg protein) site. Dyn A-(1-13) (10 microM) interacted selectively with the low affinity [3H]ouabain binding site causing a significant decrease in the Bmax (from 39.3 to 22.2 pmol/mg protein) without altering the Kd. Dyn A-(1-13) inhibited the binding of [3H]ouabain (500 nM) with an IC50 of 2.9 microM and a maximal inhibition of 77% of the specific binding activity. The non-opioid fragments Dyn A-(2-13), Dyn A-(3-13) and Dyn A-(6-10) displayed 36%, 32% and 14% of the potency of Dyn A-(1-13) respectively; whereas 100 microM of Dyn A-(1-8), Leu-enkephalin (Leu-Enk) and selective ligands for kappa (U-50, 488H) mu [(D-Ala2-Me-Phe4.Glyol5]Enk) and delta [(D-Ser2.Thr6]Leu-Enk) opioid receptors caused little or no inhibition of [3H]ouabain binding. The relative potency of various analogs and fragments of Dyn A in inhibiting the binding of [3H]ouabain correlated well (r = 0.89-0.90) with their potency in inhibiting the binding of [3H]Dyn A-(1-13) to non-opioid Dyn sites (Dumont and Lemaire, 1996) and to block [3H]NA uptake by cardiac synaptosomes (Dumont and Lemaire, 1995). In spontaneously hypertensive rats. [3H]ouabain binding displayed a 3.8-fold enhanced sensitivity to the action of Dyn A-(1-13) an effect that correlated with the enhanced ability of the peptide to inhibit Na+/K+-ATPase and [3H]NA uptake. The results indicate that Dyn A and related peptides may modulate cardiac functions through a non-opioid interaction with the low affinity ouabain binding site.

Animals↗
PubMed1995

Corrigendum to 'the pentadecapeptide [Ser1]histogranin impairs passive avoidance learning in mice' [Eur. J. Pharmacol. 283 (1995) 251-254].

The peptides, histogranin and [Ser1]histogranin, were recently shown to modulate NMDA receptor function. In the present study, the effects of intracerebroventricular (i.c.v.) administration of [Ser1]histogranin and of the histogranin receptor antagonist, histogranin-(1-10), were examined on step-down type passive avoidance learning in mice. [Ser1]Histogranin (30-60 nmol) impaired retention, after post-training administration, but not when it was administered just prior to the retention assay. Histogranin-(1-10) (60 nmol) facilitated learning during training, without affecting retention. Co-administration of histogranin-(1-10) with [Ser1]histogranin (60 nmol each) led to a significant prevention of [Ser1]histogranin-induced learning impairment. These results indicate that [Ser1]histogranin impairs passive avoidance learning according to the pattern of NMDA receptor antagonists and involving specific histogranin sites.

Animals↗
PubMed1995

The pentadecapeptide [Ser1]histogranin impairs passive avoidance learning in mice.

The peptides, histogranin and [Ser1]histogranin, were recently shown to modulate NMDA receptor function. In the present study, the effects of intracerebroventricular (i.c.v.) administration of [Ser1]histogranin and of the histogranin receptor antagonist, histogranin-(1-10), were examined on step-down type passive avoidance learning in mice. [Ser1]Histogranin (30-60 nmol) impaired retention, after post-training administration, but not when it was administered just prior to the retention assay. Histogranin-(1-10) (60 nmol) facilitated learning during training, without affecting retention. Co-administration of histogranin-(1-10) with [Ser1]histogranin (60 nmol each) led to a significant prevention of [Ser1]histogranin-induced learning impairment. These results indicate that [Ser1]histogranin impairs passive avoidance learning according to the pattern of NMDA receptor antagonists and involving specific histogranin sites.

Animals↗
PubMed1995

High-level expression of recombinant pea chloroplast fructose-1,6-bisphosphatase and mutagenesis of its regulatory site.

The cDNA fragment coding for mature chloroplast pea fructose-1,6-bisphosphatase [Fru(1,6)P2ase] was introduced by PCR into the expression vector pET-3d resulting in the construction pET-FBP. After transformation of BL21 (DE3) Escherichia coli cells by the pET-FBP plasmid and induction with isopropyl thio-beta-D-galactoside, high-level expression of the recombinant enzyme was achieved. The protein could be purified in three days by a simple procedure which includes heat treatment, ammonium sulfate fractionation, DEAE Sephacel and ACA 44 chromatographies with a yield of 20 mg/l culture. In every respect, the recombinant enzyme was similar to plant chloroplast Fru(1,6)P2ase and, in particular, its reactivity with Mg2+ and redox regulatory properties were conserved. In a second series of experiments based on three-dimensional modeling of the chloroplast protein and sequence alignments, two cysteine residues of the recombinant enzyme (Cys173 and Cys178) were mutated into serine residues. An active enzyme, which did not respond to thiol reagents and to light activation, was obtained, confirming the putative regulatory role of the insertional sequence characteristic of the chloroplast enzyme.

Amino Acid Sequence↗