Yasutomo Sugimura

Crystal structure of plastocyanin from a green alga, Enteromorpha prolifera☆

The crystal structure of the Cu-containing protein plastocyanin (Mr 10,500) from the green alga Enteromorpha prolifera has been solved by molecular replacement. The structure was refined by constrained-restrained and restrained reciprocal space least-squares techniques. The refined model includes 111 solvent sites. There is evidence for alternate conformers at eight residues. The residual is 0.12 for a data set comprising 74% of all observations accessible at 1.85 A resolution. The beta-sandwich structure of the algal plastocyanin is effectively the same as that of poplar leaf (Populus nigra var. italica) plastocyanin determined at 1.6 A resolution. The sequence homology between the two proteins is 56%. Differences between the contacts in the hydrophobic core create some significant (0.5 to 1.2 A) movements of the polypeptide backbone, resulting in small differences between the orientations and separations of corresponding beta-strands. These differences are most pronounced at the end of the molecule remote from the Cu site. The largest structural differences occur in the single non-beta strand, which includes the sole turn of helix in the molecule: two of the residues in a prominent kink of the poplar plastocyanin backbone are missing from the algal plastocyanin sequence, and there is a significant change in the position of the helical segment in relation to the beta-sandwich. Several other small but significant structural differences can be correlated with intermolecular contacts in the crystals. An intramolecular carboxyl-carboxylate hydrogen bond in the algal plastocyanin may be associated with an unusually high pKa. The dimensions of the Cu site in the two plastocyanins are, within the limits of precision, identical.

Isolation of water-soluble chlorophyll protein from the leaves of Chenopodium album

Abstract 1. 1. A water-soluble, photosensitive chlorophyll protein was obtained from the leaves of Chenopodium album . 2. 2. The substance, purified by ammonium sulfate fractionation and column chromatography, showed a single-peaked sedimentation pattern on ultracentrifugal analysis. 3. 3. The native chlorophyll protein (prepared in the dark) showed absorption bands at 277, 429 and 667 mμ. Irradiation caused a marked change of the spectrum. New peaks appeared at 362, 399, 567 and 743 mμ. 4. 4. The chlorophyll protein contained chlorophylls a and b . The irradiated protein had three additional pigments which were not found in the native one.

Changes in photosynthetic characteristics and photosystem stoichiometries in the lower leaves in rice seedlings

Abstract Changes in the Chl a / b ratio, electron transport, electron carriers, and photosystem stoichiometries were examined in rice leaves in the present study. The Chl a / b ratio is known to decrease gradually from the top to the bottom leaves, indicating a increase in the abundance of LHC II relative to the reaction center complexes of the two photosystems. We used juvenile rice canopy and obtained the following results: (1) the photosynthetic activity and Chl content per leaf area decreased from the top to the bottom leaves, the Chl a / b ratio also declined from 3.7 to 3.0; (2) when determined on the basis of Chl content, C-550 and Cyt f content decreased, but there was no loss of P-700, Consequently, the PS II/PS I ratio significantly decreased; (3) on the basis of mmol Chl, the levels of Cyt f dramatically decreased and, therefore, no loss was observed for whole chain electron transport per Cyt f ; and (4) the percentage abundance of PS IIα increased, but the rate constants of Q A photoreduction and P-700 photooxidation gradually decreased. From these results, we hypothesize that there is a compensatory relationship between the decline in the Chl a / b ratio and that in the PS II/PS I ratio in the lower leaves in rice seedlings.

Effects of light on the photosynthetic apparatus and a novel type of degradation of the photosystem I peripheral antenna complexes under darkness.

A novel type of degradation of photosystem I peripheral antenna complexes has been observed in rice leaves under darkness in the present study. Photosynthesis, chlorophyll content, the chlorophyll a/b ratio, and relative amounts of ribulose-1,5-bisphosphate carboxylase/oxygenase decrease during dark treatment. The levels of photosystem II reaction-center complex and cytochrome f on the basis of units of chlorophyll also decline rapidly under darkness. In contrast, the levels of photosystem I reaction-center complex remain stable under darkness for six days. Low-temperature fluorescence emission spectra ascribed to photosystem I antennae clearly show a blue shift. A similar shift is also observed in the photosystem I complexes resolved with dodecyl maltoside-polyacrylamide gel electrophoresis. Moreover, polypeptide analysis of the thylakoids and photosystem I complexes isolated from the green gels shows that some polypeptides originating from photosystem I peripheral antenna complexes disappear during the dark treatment. A curve-fitting method also displays remarkable changes in the chlorophyll components between the light and dark treatments. It is likely that these results indicate the disconnection/disassembly of the photosystem I antenna as well as the photosystem II complexes induced by dark treatment. Moreover, these findings also imply the existence of different degradation mechanisms for the photosystem I and II complexes.

A novel protein induced in cortical epileptic focus of rat cerebrum: a possible linkage to epileptogenesis.

To precisely evaluate a protein-related mechanism of epileptogenesis, we quantitatively analyzed the 70-kDa protein (namely, P70), a specific protein found in the cobalt-induced epileptic focus, and examined its effect on the electrocorticogram (ECoG) and cortical neurons in cerebral slices and its immunocytochemical localization in rats. Cobalt-induced cortical epileptogenic cortex exhibited a marked induction of P70. Its initiation time was ahead of the generation of epileptogenic activities. The anticonvulsant phenytoin (PHT) attenuated the cobalt-induced epileptogenic activities, but failed to suppress protein induction. Injection of this protein into the motor region of normal rat cerebral cortex elicited an epileptic ECoG and behavioral seizures. It also caused epileptiform activity with paroxysmal depolarization shifts in cortical neurons. These epileptogenic phenomena elicited by P70 were abolished by prior treatment with PHT or phenobarbital. Immunocytochemical analysis with an antiserum against P70 revealed that the reactivity was confined to pyramidal cells only in the region of the focus and was mainly localized on somatic, dendritic, and nuclear membranes and microtubles. These findings suggest that P70 may be linked to epileptogenesis.

CHANGES IN PHOTOSYNTHETIC APPARATUS IN THE JUVENILE RICE CANOPY AND A POSSIBLE FUNCTION OF PHOTOSYSTEM I IN THE BOTTOM LEAVES

Abstract Changes in the photosynthetic apparatus and relative antenna sizes of photosystem (PS) I and PS II were measured in the rice canopy. We used juvenile rice seedlings to examine light utilization and its absorption in the bottom leaves and obtained the following results: (1) When referred to chlorophyll (Chl), levels of the electrochromic shift at 550 nm and cytochrome ƒ decreased from the sixth to the third leaves, but there was no loss of pigment (P)-700. As a consequence, the PS II/PS I ratio significantly decreased from 1.5 in the sixth leaves to 0.9 in the third leaves. (2) The electron transport capacity in the sixth leaves was 1.5-times larger than that in the third leaves. (3) The levels of cytochrome b6 referred to Chl were almost constant from top to bottom. (4) The photosynthetic performance of the leaf decreased concomitant with the depth, whereas the respiration was slightly increased. From these results, we hypothesize that there are maintenance mechanisms when the imbalances of light absorption and electron transport capacity occur in the bottom leaves.

A specific 70 kDa protein induced in the epileptogenic cortex of rats elicits bursting activity and inactivation of potassium current in snail neurons

The capability of a specific 70 kDa (P70) protein produced in cobalt-induced epileptogenic cortex of rat cerebrum in promoting epileptic bursting activity was examined in snail neurons, using the voltage clamp method in combination with the pressure injection technique. Injection of P70 into the neurons elicited bursting activity with paroxysmal depolarization shift together with a reduction in the delayed outward current which was suppressed by tetraethylammonium. These changes of membrane properties induced by P70 were dose-dependently inhibited by prior treatment with the anticonvulsant phenytoin. These findings indicate that P70 causes an inactivation of potassium conductance which may generate epileptic bursting activity.

Spectroscopic characterization and kinetic studies of a novel plastocyanin from the green alga Ulva pertusa

Abstract A novel plastocyanin from the green alga Ulva pertusa was isolated and characterized. The electronic absorption and the electron paramagnetic resonance spectroscopic properties of Ulva plastocyanin showed features characteristic of the usual plastocyanins reported so far. However, the resonance Raman spectrum on excitation at 607 nm indicated the Raman band of Cu-SCys at 413 cm−1; this is at a lower frequency than the corresponding Raman band of higher plant plastocyanins. Electron-transfer reactions were investigated with [Fe(CN)6]3− and [Co(phen)3]3+ complexes. The electron-transfer rate constant of Ulva plastocyanin was determined to be (1.18 ± 0.06) × 105 M−1 s−1 for the reaction with [Fe(CN)6]3− at pH 7.5, and the intramolecular electron-transfer rate constant and equilibrium constant for complex formation for the reaction with [Co(phen)3]3+ were evaluated to be 7.7 ± 0.6 s−1 and (4.2 ± 0.4) × 102 M−1 respectively. The electron transfer rate constant for the reaction with [Fe(CN)6]3− at pH 7.5 is two times larger than the values obtained from the other higher plant plastocyanins. The kinetic behavior suggested that the structure is slightly different from those of other plastocyanins. It has been reported that the electron-transfer reaction of plastocyanin is inhibited by the protonation of the active site histidine (His87 in poplar plastocyanin) at acidic pH. The dependence on pH of the rate constant for the reaction with [Fe(CN)6]3− was investigated. The acid-dissociation constant accompanying the electron-transfer reaction was determined to be pKH = 5.8. Second-order rate constants for the reduction of plastocyanin by cytochrome c were determined to be (1.71 ± 0.04) × 106 M−1 s−1 at I = 0.1 M (NaCl), pH 7.0 (20 mM Tris—HCl buffer). The saturation kinetic behavior for the reaction was not observed even at the lower ionic strength (20 mM Tris—HCl).

Preliminary crystallographic data for plastocyanins from an alga (Enteromorpha prolifera) and from cucumber (Cucumis sativus)

The plastocyanins from a green alga (Enteromorpha prolifera) and cucumber (Cucumis sativus) have been crystallized. Crystal data are as follows: E. prolifera plastocyanin, space group I4, a = b = 53.9 A, c = 59.4 A, Z = 8; C. sativus plastocyanin, space group P4(1) (or P4(3) ), a = b = 66.7 A, c = 46.0 A, Z = 8. Accordingly, the asymmetric units of the crystals contain one and two molecules, respectively.

Studies on algal cytochromes II. Some physical and chemical properties of further purified Petalonia cytochrome c-553

A cytochrome c-553 was isolated from a brown alga, Petalonia fascia, and its physical and chemical properties were investigated. At liquid nitrogen temperature, the α-, β- and γ-band of this cytochrome shifted 1-2 nm to the shorter wavelength in comparison with those at room temperature. The α-band split into two peaks, 551.5 (major band) and 546.5 nm (minor band), at the low temperature. The cytochrome in high concentration showed a shoulder at 695 nm in its oxidized state, suggesting a methionine residue to be the sixth ligand of heme iron. The molecular weight was estimated to be about 10,000 containing one mole of heme c based on analyses of gel filtration, sodium dodecyl sulfate acrylamide gel electrophoresis, amino acid composition and iron content. The isoelectric points of the ferro- and ferricytochromes were estimated to be at pH 4.1 and 4.3, respectively, by the isoelectric focusing method. The amino acid composition of this cytochrome was Lys6, Arg1, Asp15, Thr5, Ser6, Glu11, Pro2, Gly7, Ala7, Cys2, Val6, Met3, Ile6, Leu3, Tyr1, Phe3, Trp1, with a total of 86 residues. The amino- and carboxylterminal sequences and four chymotryptic peptide sequences were compared with those of other cytochromes c to show that these cytochromes were homologous to one another.