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S Okuyama

Publications and source records attributed to S Okuyama.

At least 19 recordsLinked to original sources
PubMed2006

Dietary gamma-aminobutyric acid affects the brain protein synthesis rate in young rats.

The purpose of this study was to determine whether the gamma-aminobutyric acid (GABA) affects the rate of brain protein synthesis in male rats. Two experiments were done on five or three groups of young rats (5 wk) given the diets containing 20% casein administrated 0 mg, 25 mg, 50 mg, 100 mg or 200 mg/100 g body weight GABA dissolved in saline by oral gavage for 1 day (d) (Experiment 1), and given the diets contained 0%, 0.25% or 0.5% GABA added to the 20% casein diet (Experiment 2) for 10 d. The plasma concentration of growth hormone (GH) was the highest in rats administrated 50 mg and 100 mg/100 g body weight GABA. The concentration of serum GABA increased significantly with the supplementation groups. The fractional (Ks) rates of protein synthesis in brain regions, liver and gastrocnemius muscle increased significantly with the 20% casein + 0.25% GABA diet and still more 20% casein + 0.5% GABA compared with the 20% casein diet. In brain regions, liver and gastrocnemius muscle, the RNA activity [g protein synthesized/(g RNA . d)] significantly correlated with the fractional rate of protein synthesis. The RNA concentration (mg RNA/g protein) was not related to the fractional rate of protein synthesis in any organ. Our results suggest that the treatment of GABA to young male rats are likely to increase the concentrations of plasma GH and the rate of protein synthesis in the brain, and that RNA activity is at least partly related to the fractional rate of brain protein synthesis.

Aging↗
PubMed2001

Effect of ACTH on the imipramine- and desipramine-induced decrease in duration of immobility time as measured in a rat forced swimming test.

In a rat forced swimming test (FS), we examined the effect of repeated injections of ACTH (adrenocorticotropic hormone) for 14 days on the decreased duration of immobility time produced by imipramine and desipramine. Both imipramine (15 and 30 mg/kg, p.o.) and desipramine (15 and 30 mg/kg, p.o.) significantly decreased the duration of immobility time in the FS. On the other hand, ACTH (100 microg/kg, i.p.) alone did not affect the duration of immobility time in FS. When ACTH (100 microg/kg, i.p.) was injected for 14 days before the 15-min swim session, it counteracted the decreased duration of immobility time induced by both imipramine and desipramine. Thus, ACTH seems to play a key role in decreasing the duration of immobility time of antidepressants in this test.

Adrenocorticotropic Hormone↗
PubMed2001

Repeated treatment with imipramine, fluvoxamine and tranylcypromine decreases the number of escape failures by activating dopaminergic systems in a rat learned helplessness test.

Chronic administration of antidepressants has been shown to reduce the number of escape failures in the rat learned helplessness test (LH). In the present study we investigated the role of D1, D2 and D3 receptors in mediating this effect. In our first series of experiments, we demonstrated that SKF38393, D1 receptor agonist, in a dose of 2.5 mg/kg (i.p.) and quinpirole, D2 receptor agonist in a dose of 0.5 mg/kg (i.p.), significantly decreased the number of escape failures in LH, and these were reversed by SCH23390 (0.015 mg/kg), D1 receptor antagonist, and by sulpiride (25 mg/kg), D2 receptor antagonist, respectively. In contrast, 7-OH-DPAT, a D3 receptor agonist, in a dose of 10 mg/kg (i.p.) did not affect the number of escape failures in LH. In a second series of experiments, we showed that eight days of repeated treatment with imipramine (10 mg/kg, p.o.), fluvoxamine (1.25 mg/kg, p.o.) and tranylcypromine (1.25 mg/kg, p.o.) significantly decreased the number of escape failures in LH. The decrease in escape failures seen with use of imipramine and tranylcypromine was reversed by sulpiride in LH, but not by SCH23390. On the other hand, the effect of fluvoxamine was reversed by both SCH23390 and sulpiride. These findings indicate that stimulation of D1 and D2 receptors decreased the number of escape failures in LH, respectively. Thus, D2 and/or D1 receptors are probably involved in the decreased number of escape failures in case of repeated treatment with antidepressants in LH.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
PubMed2001

Multiple pathways of sigma(1) receptor ligand uptakes into primary cultured neuronal cells.

Although many antipsychotics have affinities for sigma receptors, the transportation pathway of exogenous sigma(1) receptor ligands to intracellular type-1 sigma receptors are not fully understood. In this study, sigma(1) receptor ligand uptakes were studied using primary cultured neuronal cells. [(3)H](+)-pentazocine and [(3)H](R)-(+)-1-(4-chlorophenyl)-3-[4-(2-methoxyethyl)piperazin-1-yl]methyl-2-pyrrolidinone L-tartrate (MS-377), used as a selective sigma(1) receptor ligands, were taken up in a time-, energy- and temperature-dependent manner, suggesting that active transport mechanisms were involved in their uptakes. sigma(1) receptor ligands taken up into primary cultured neuronal cells were not restricted to agonists, but also concerned antagonists. The uptakes of these ligands were mainly Na(+)-independent. Kinetic analysis of [(3)H](+)-pentazocine and [(3)H]MS-377 uptake showed K(m) values (microM) of 0.27 and 0.32, and V(max) values (pmol/mg protein/min) of 17.4 and 9.4, respectively. Although both ligands were incorporated, the pharmacological properties of these two ligands were different. Uptake of [(3)H](+)-pentazocine was inhibited in the range 0.4-7.1 microM by all the sigma(1) receptor ligands used, including N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylamine monohydrochloride (NE-100), a selective sigma(1) receptor ligand. In contrast, the inhibition of [(3)H]MS-377 uptake was potently inhibited by haloperidol, characterized by supersensitivity (IC(50), approximately 2 nM) and was inhibited by NE-100 with low sensitivity (IC(50), 4.5 microM). Moreover, kinetic analysis revealed that NE-100 inhibited [(3)H]MS-377 uptake in a noncompetitive manner, suggesting that NE-100 acted at a site different from the uptake sites of [(3)H]MS-377. These findings suggest that there are at least two uptake pathways for sigma(1) receptor ligands in primary cultured neuronal cells (i.e. a haloperidol-sensitive pathway and another, unclear, pathway). In addition, pretreatment of cells with a calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7), a myosin light chain kinase inhibitor, 1-(5-chloronaphthalene-1-sulfonyl)homopiperazine (ML-9), or microsomal Ca(2+)-ATPase inhibitors resulted in a reduction of the amount of sigma receptor ligand uptake. These findings suggest that the Ca(2+) pump on the endoplasmic reticulum and/or calmodulin-related events might be involved in the regulation of the uptake of sigma receptor ligands into primary neuronal cells.

Animals↗
PubMed2001

Neuropharmacological profiles of a novel atypical antipsychotic, NRA0562, in rats.

Neuropharmacological profiles of 5-(2-[4-(6-fluoro-1H-indole-3-yl) piperidine-1-yl] ethyl)-4-(4-fluorophenyl) thiazole-2-carboxylic acid amide (NRA0562) in rats were examined using electrophysiological and immunohistochemical methods. The firing rates of the substantia nigra pars compacta (A9) and the ventral tegmental area (A10) dopamine neurons were inhibited by methamphetamine (1 mg/kg, i.v.). NRA0562 dose-dependently reversed the inhibitory effects of methamphetamine on A9 and on A10 dopamine neurons. NRA0562 was more potent to reverse the inhibitory effects of methamphetamine on A10 (ED(50)=0.3 mg/kg) than on A9 (ED(50)=0.9 mg/kg) dopamine neurons. NRA0562 produced significant increases in Fos-like immunoreactivity in both the nucleus accumbens and the dorsolateral striatum. The difference between the number of Fos-like immunoreactivity produced by NRA0562 in the nucleus accumbens vs. dorsolateral striatum, referred to as the atypical index, was positive. Similar results could be observed with risperidone, an atypical antipsychotic. These results suggest that NRA0562 may have the atypical antipsychotic activities seen with risperidone, but without the liability of motor side effects typical of classical antipsychotics.

Action Potentials↗
PubMed2001

Localization and characterization of a short isoform of the corticotropin-releasing factor receptor type 2alpha (CRF(2)alpha-tr) in the rat brain.

We have recently isolated a cDNA encoding a short isoform of the corticotropin-releasing factor (CRF) receptor subtype, referred to as CRF(2)alpha-tr, from the rat amygdala. The present study determined the localization of the truncated receptor mRNA in the rat brain by in situ hybridization histochemistry. The results showed significant levels of hybridization in the lateral septum, central nucleus of the amygdala, cortico-amygdaloid nucleus, ventromedial nucleus of the hypothalamus (VMH), and frontal cortex. In the physiological study, antidepressive drugs increased the expression of CRF(2)alpha-tr mRNA and the total binding activity to CRF in the rat amygdala. These findings suggest that CRF(2)alpha-tr may regulate endogeneous CRF release in the amygdala.

Amino Acid Sequence↗
PubMed2001

Virol A, a toxic trans-polyacetylenic alcohol of Cicuta virosa, selectively inhibits the GABA-induced Cl(-) current in acutely dissociated rat hippocampal CA1 neurons.

The effects of virol A (VA), a toxic component of Cicuta virosa (water hemlock), on the GABA-induced Cl(-) current (I(GABA)) in acutely dissociated rat hippocampal CA1 neurons were investigated using whole-cell patch-clamp techniques. VA reversibly reduced I(GABA) and the muscimol (Mus)-induced current (I(Mus)) in a concentration-dependent manner. The IC(50) values for VA against I(GABA) and I(Mus) were 9.6x10(-7) and 9.8x10(-7) M, respectively. VA shifted the EC(50) value of I(GABA) from 6.5x10(-6) to 2.1x10(-5) M, whereas it had no effect on the maximum response, thereby suggesting that VA inhibited I(GABA) in a competitive manner. VA had no apparent effect on current-voltage relationships for I(GABA), thus indicating the lack of voltage-dependency. On the other hand, application of VA (10(-6) M) did not additionally reduce the I(GABA) suppressed by >10(-5) M picrotoxin. VA but not bicuculline accelerated the decay phase of I(GABA), as was seen with picrotoxin. Moreover, pre-application of 10(-5) M VA reduced I(GABA). VA did not inhibit that induced by glycine (10(-4) M). These results indicate that VA inhibits I(GABA) by acting both on the GABA agonist site and on the Cl(-) channel of the GABA(A) receptor-channel complex. VA is a structurally novel type of compound that selectively inhibits the GABA(A) receptor-Cl(-) channel complexes in mammalian central nervous system neurons.

Animals↗
PubMed2001

Synthesis and structure-affinity relationships of 4-(5-Aryl-1,2,3,6-tetrahydropyridino)pyrimidine derivatives as corticotropin-releasing factor(1) receptor antagonists.

Recently, various non-peptide corticotropin-releasing factor(1) (CRF(1)) receptor antagonists have been reported. Structure-affinity relationships (SARs) of non-peptide CRF(1) antagonists suggest that such antagonists can be constructed of three units: a hydrophobic unit (Up-Area), a proton accepting unit (Central-Area), and an aromatic unit (Down-Area). We previously presented 4-aryl-1,2,3,6-tetrahydropyridinopyrimidine derivatives including potent CRF receptor ligands 1a and 1b and proposed that the 4-aryl-1,2,3,6-tetrahydropyridino moiety might be useful as a substituent in the Up-Area. Our interest shifted to 5-aryl-1,2,3,6-tetrahydropyridinopyrimidine derivatives 2, among which compound 2m (CRA0165) had highest affinity for CRF(1) receptors (IC(50)=11nM). We report here the design, synthesis and SARs of derivatives 2.

Animals↗
PubMed2001

Chemical modification of aryl-1,2,3,6-tetrahydropyridinopyrimidine derivative to discover corticotropin-releasing factor(1) receptor antagonists: aryl-1,2,3,6-tetrahydropyridino-purine, -3H-1,2,3-triazolo[4,5-d)pyrimidine, -purin-8-one, and -7H-pyrrolo[2,3-d]pyrimidine derivatives.

Structure-affinity relationships (SARs) of non-peptide CRF(1) antagonists suggest that such antagonists can be constructed of three units: a hydrophobic unit (Up-Area), a proton accepting unit (Central-Area), and an aromatic unit (Down-Area). Recently, various non-peptide corticotropin-releasing factor(1) (CRF(1)) receptor antagonists obtained by modification of the Central-Area have been reported. In contrast, we modified the Up-Area and presented 4- or 5-aryl-1,2,3,6-tetrahydropyridinopyrimidine derivatives including potent CRF receptor ligands 1a-c, and proposed that the 4- or 5-aryl-1,2,3,6-tetrahydropyridino moiety might be useful as a substituent in the Up-Area. Our interest shifted to the chemical modification in which the pyrimidine ring of 1a-c was replaced by other heterocycles, purine ring of 2, 3H-1,2,3-triazolo[4,5-d]pyrimidine ring of 3, purin-8-one ring of 4 and 7H-pyrrolo[2,3-d]pyrimidine ring of 5. Among them, 5-aryl-1,2,3,6-tetrahydropyridinopurine compound 6j (CRA0186) had the highest affinity for CRF(1) receptors (IC(50)=20nM). We report here the synthesis and SARs of derivatives 6-9.

Affinity Labels↗
PubMed2001

Availability of learned helplessness test as a model of depression compared to a forced swimming test in rats.

This study was designed to evaluate the antidepressant activity of various antidepressants using the learned helplessness test (LH) or the forced swimming test (FS) in rats. Repeated treatment of the tricyclic antidepressants imipramine (10 mg/kg, p.o.), clomipramine (0.625 mg/kg, p.o.), amitriptyline (10 mg/kg, p.o.) and amoxapine (20 mg/kg, p.o.) reduced the number of escape failures in the LH group, respectively. Repeated treatment of an atypical antidepressant, mianserin (2.5 and 5 mg/kg, p.o.), and one of the selective serotonin reuptake inhibitors (SSRI), fluvoxamine (1.25 mg/kg, p.o.), also reduced the number of escape failures in the LH group. In the FS, repeated treatment of imipramine (5, 10 mg/kg, p.o.), amitriptyline (5, 10 mg/kg, p.o.) and mianserin (10 mg/kg) significantly decreased the duration of immobility time. On the other hand, repeated treatment of amoxapine (5-20 mg/kg), clomipramine (0.1325-1.25 mg/kg, p.o.) and fluvoxamine (0.3125-1.25 mg/kg, p.o.) failed to decrease the duration of immobility time in the FS group. In conclusion, these results suggest that the LH group is sensitive to agents with a variety of antidepressant properties compared to the FS group in rats.

Amoxapine↗
PubMed2000

Synthesis, SARs, and pharmacological characterization of 2-amino-3 or 6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid derivatives as potent, selective, and orally active group II metabotropic glutamate receptor agonists.

(+)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (4, LY354740), a highly selective and orally active group II metabotropic glutamate receptor (mGluR) agonist, has increased interest in the study of group II mGluRs. Our interest focused on a conformationally constrained form of compound 4, because it appeared that the rigid form resulted in not only selectivity for group II mGluR but was orally active. Therefore, we introduced a fluorine atom to compound 4, based on the molecular size (close resemblance to hydrogen atom) and electronegativity (effects on the electron distribution in the molecule) of this atom and carbon-fluorine bond energy. Compound (+)-7 (MGS0008), the best compound among 3-fluoro derivatives 7-10, retained the agonist activity of compound 4 for mGluR2 and mGluR3 ((+)-7: EC(50) = 29.4 +/- 3.3 nM and 45.4 +/- 8.4 nM for mGluR2 and mGluR3, respectively; 4: EC(50) = 18.3 +/- 1.6 nM and 62.8 +/- 12 nM for mGluR2 and mGluR3, respectively) and increased the oral activity of compound 4 ((+)-7: ED(50) = 5.1 mg/kg and 0.26 mg/kg for phencyclidine (PCP)-induced hyperactivity and PCP-induced head-weaving behavior, respectively; 4: ED(50) = >100 mg/kg and 3.0 mg/kg for PCP-induced hyperactivity and PCP-induced head-weaving behavior, respectively). In addition, a compound [(3)H]-(+)-7 binding study using mGluR2 or 3 expressed in CHO cells was successful ((+)-7: K(i) = 47.7 +/- 17 nM and 65.9 +/- 7.1 nM for mGluR2 and mGluR3, respectively; 4: K(i) = 23.4 +/- 7.1 nM and 53.5 +/- 13 nM for mGluR2 and mGluR3, respectively). On the basis of a successful result of compound 7, we focused on the introduction of a fluorine atom on the C6 position of compound 4. (1R,2S,5R, 6R)-2-amino-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid ((-)-11) exhibited a high degree of agonist activity for group II mGluRs equal to that of compound 4 or 7 ((-)-11: K(i) = 16.6 +/- 5.6 and 80.9 +/- 31 nM for mGluR2 and mGluR3, respectively). Our interest shifted to modification on CH(2) at C4 position of compound 11, since replacement of the CH(2) group with either an oxygen atom or sulfur atom yielded compound 5 or 6, resulting in increased agonist activity. We selected a carbonyl group instead of CH(2) at the C4 position of compound 11. The carbonyl group might slightly change the relative conformation of three functional groups, the amino group and two carboxylic acids, which have important roles in mediating the interaction between group II mGluRs and their ligand, compared with the CH(2) group of 4, oxygen atom of 5, and sulfur atom of 6. (1R,2S,5S,6S)-2-Amino-6-fluoro-4-oxobicyclo[3.1. 0]hexane-2,6-dicarboxylic acid monohydrate ((+)-14, MGS0028) exhibited a remarkably high degree of agonist activity for mGluR2 (K(i) = 0.570 +/- 0.10 nM) and mGluR3 (K(i) = 2.07 +/- 0.40 nM) expressed in CHO cells but not mGluR4, 6, 7, 1a, or 5 expressed in CHO cells (K(i) = >100 000 nM). Furthermore, compound (+)-14 strongly inhibited phencyclidine (PCP)-induced head-weaving behavior (ED(50) = 0.090 microg/kg) and hyperactivity (ED(50) = 0.30 mg/kg) in rats. Thus, (+)-7 and (+)-14 are potent, selective, and orally active group II mGluR agonists and might be useful not only for exploring the functions of mGluRs but in the treatment of schizophrenia.

Administration, Oral↗
PubMed2000

Pain threshold, learning and formation of brain edema in mice lacking the angiotensin II type 2 receptor.

The main biological role of angiotensin II type 2 receptor (AT2) has not been established. We made use of targeted disruption of the mouse AT2 gene to examine the functional role of the AT2 receptor in the central nervous system (CNS). We have previously shown that AT2-deficient mice displayed anxiety-like behavior in comparisons with wild-type mice. In the present study, we analyzed the pain threshold, learning behavior and brain edema formation using the tail-flick test, the tail-pinch test, the passive avoidance task and cold injury, respectively. In the passive avoidance task and cold injury, no differences were found between wild-type mice and AT2-deficient mice. In contrast, the pain threshold was significantly lower in AT2-deficient mice, compared with findings in wild-type mice. The immunohistochemical distribution of beta-endorphin in the brain was analyzed quantitatively in AT2-deficient mice and wild-type mice, using microphotometry. The fluorescence intensity of beta-endorphin in the arcuate nucleus of the medial basal hypothalamus (ARC) was significantly lower in AT2-deficient mice, compared with findings in wild-type mice. We found that the AT2 receptor does not influence learning behavior and brain edema formation. As AT2-deficient mice have increased sensitivity to pain and decreased levels of brain beta-endorphin, AT2 receptors may perhaps mediate regulation of the pain threshold.

Animals↗
PubMed2000

Modification of glial-neuronal cell interactions prevents photoreceptor apoptosis during light-induced retinal degeneration.

Prolonged or high-intensity exposure to visible light leads to photoreceptor cell death. In this study, we demonstrate a novel pathway of light-induced photoreceptor apoptosis involving the low-affinity neurotrophin receptor p75 (p75NTR). Retinal degeneration upregulated both p75NTR and the high-affinity neurotrophin receptor TrkC in different parts of Müller glial cells. Exogenous neurotrophin-3 (NT-3) increased, but nerve growth factor (NGF) decreased basic fibroblast growth factor (bFGF) production in Müller cells, which can directly rescue photoreceptor apoptosis. Blockade of p75NTR prevented bFGF reduction and resulted in both structural and functional photoreceptor survival in vivo. Furthermore, the absence of p75NTR significantly prevented light-induced photoreceptor apoptosis. These observations implicate glial cells in the determination of neural cell survival, and suggest functional glial-neuronal cell interactions as new therapeutic targets for neurodegeneration.

Animals↗
PubMed2000

Design, synthesis and structure-affinity relationships of 4-methylidenepiperidine and 4-aryl-1,2,3,6-tetrahydropyridine derivatives as corticotropin-releasing factor1 receptor antagonists.

Recently, various non-peptide corticotropin-releasing factor1 (CRF1) receptor antagonists have been reported. Structure-affinity relationships (SARs) of non-peptide CRF antagonists suggest that such antagonists can be constructed of three units: a hydrophobic unit (Up-Area), a proton accepting unit (Central-Area), and an aromatic unit (Down-Area). Our interest focused on the Up-Area in deriving the novel methylidenepiperidine derivatives 8-10 and 4-aryl-1,2,3,6-tetrahydropyridine derivatives 11-13 as non-peptide CRF1 receptor antagonists. Compounds 8a and 11a had moderate affinity for CRF1 receptor, but compounds 9, 10, 12 and 13 did not exhibit CRF1 receptor affinity. Modification of derivatives 11 afforded compounds 11i (CRA1001) and 11x (CRA1000), which had high affinity and selectivity for CRF1 receptors with potent anxiolytic-like and antidepressant-like properties in some experimental animal models. These findings suggest that the hydrophonic unit (Up-Area) may be useful for design of CRF1 antagonists. We report here the design, synthesis and SARs of the derivatives 8 and 11 and isosteres 9, 10, 12 and 13.

Animals↗
PubMed2000

Activation of an effector immediate-early gene arc by methamphetamine.

As immediate-early genes (IEGs) are thought to play a critical role in mediating stimulus-induced neural plasticity, IEG response induced by methamphetamine (METH) has been characterized to define the changes in gene expression that may underlie its long-lasting behavioral effects. Although activation of several transcription factor IEGs has been described, little is known about effector IEGs. Here, we have examined whether METH administration affects expression of an effector IEG arc (activity-regulated, cytoskeleton-associated) that encodes a protein with homology to spectrin. Using in situ hybridization, we observed that METH caused a rapid and transient dose-dependent increase in arc mRNA level in the striatum and cortex that was abolished by pretreatment with the specific dopamine D1 receptor antagonist SCH-23390 but not by an atypical neuroleptic clozapine. METH induced arc mRNA in layers IV and VI of the cortex which dopamine receptor are localized to. These results suggest that D1 receptors are coupled to activation of arc gene, which may be involved in functional or structural alterations underlying neural plasticity triggered by METH.

AIDS-Related Complex↗
PubMed1999

Synthesis and SAR of 1-alkyl-2-phenylethylamine derivatives designed from N,N-dipropyl-4-methoxy-3-(2-phenylethoxy)phenylethylamine to discover sigma(1) ligands.

The synthesis and structure-activity relationships (SAR) of 1-alkyl-2-phenylethylamine derivatives 5-8 designed from N, N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylamine hydrochloride (1, NE-100) are presented. The SAR between compound 1 and 1-alkyl-2-phenylethylamine derivatives suggested that the alkyl group on the 1-position carbon of 2-[4-methoxy-3-(2-phenylethyl)phenyl]ethylamine derivatives played the role of one of the propyl groups on the aminic nitrogen of compound 1. (-)-N-Propyl-1-butyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylam ine hydrochloride ((-)-6d, NE-537) and (-)-N-propyl-1-(3-methybutyl)-2-[4-methoxy-3-(2-phenylethoxy )phenyl]e thylamine hydrochloride ((-)-6i, NE-535), typical compounds in this series, have potent and selective sigma(1) affinity.

Alkylation↗
PubMed1999

Cloning, expression of the psbU gene, and functional studies of the recombinant 12-kDa protein of photosystem II from a red alga Cyanidium caldarium.

The encoding extrinsic 12-kDa protein of oxygen-evolving PS II complex from a red alga, Cyanidium caldarium, was cloned and sequenced by means of PCR and a rapid amplification of cDNA ends (RACE) procedure. The gene encodes a putative polypeptide of 154 amino acids with a calculated molecular mass of 16,714 Da. The full sequence of the protein includes two characteristic transit peptides, one for transfer across the chloroplast envelope and another for targeting into the thylakoid lumen. This indicates that the protein is encoded in the nuclear genome. The mature protein consists of 93 amino acids with a calculated molecular mass of 10,513 Da. The cloned gene was successfully expressed in Escherichia coli and the resulting protein was purified, reconstituted to CaCl2-washed PS II complex together with the other extrinsic proteins of 33 and 20 kDa and cyt c-550. The recombinant 12-kDa protein bound completely with the PSII complex, which resulted in a restoration of oxygen evolution equal to the level achieved by binding of the native 12-kDa protein.

Amino Acid Sequence↗