Masayuki Nara

Synergistic Activation of the Arabidopsis NADPH Oxidase AtrbohD by Ca2+ and Phosphorylation

Plant respiratory burst oxidase homolog (rboh) proteins, which are homologous to the mammalian 91-kDa glycoprotein subunit of the phagocyte oxidase (gp91phox) or NADPH oxidase 2 (NOX2), have been implicated in the production of reactive oxygen species (ROS) both in stress responses and during development. Unlike mammalian gp91phox/NOX2 protein, plant rboh proteins have hydrophilic N-terminal regions containing two EF-hand motifs, suggesting that their activation is dependent on Ca2+. However, the significance of Ca2+ binding to the EF-hand motifs on ROS production has been unclear. By employing a heterologous expression system, we showed that ROS production by Arabidopsis thaliana rbohD (AtrbohD) was induced by ionomycin, which is a Ca2+ ionophore that induces Ca2+ influx into the cell. This activation required a conformational change in the EF-hand region, as a result of Ca2+ binding to the EF-hand motifs. We also showed that AtrbohD was directly phosphorylated in vivo, and that this was enhanced by the protein phosphatase inhibitor calyculin A (CA). Moreover, CA itself induced ROS production and dramatically enhanced the ionomycin-induced ROS production of AtrbohD. Our results suggest that Ca2+ binding and phosphorylation synergistically activate the ROS-producing enzyme activity of AtrbohD.

Infrared studies of interaction between metal ions and Ca2+-binding proteins Marker bands for identifying the types of coordination of the side-chain COO− groups to metal ions in pike parvalbumin (pI = 4.10)

Metal‐ligand interactions in the Ca2+‐binding sites of pike parvalbumin (pI = 4.10) have been examined by Fourier‐transform infrared spectroscopy. The region of the COO− antisymmetric stretch provides useful information on the types of coordination of the COO− groups to the metal ions in the Mg2+‐, Mn2+‐, and Ca2+‐bound forms. In the spectrum of the Ca2+‐bound form, two bands are observed at 1,582 and 1,553 cm−, whereas, in the spectra of the Mg2+‐ and Mn2+‐bound forms, bands are observed only in the region around 1,582 cm−1 and no band is found in the region around 1,553 cm−1. The 1,553‐cm−1 band of the Ca2+‐bound form reflects the bidentate coordination of the COO− groups of both Glu‐62 in the CD site and Glu‐101 in the EF site to the Ca2+ ions, which has been made clear by X‐ray analysis as a feature of the Ca2+‐bound form. Absence of such a band in the spectrum of the Mn2+‐bound form is consistent with the X‐ray structure of this form where both of the two COO− groups are unidentate. These unidentate COO− groups of Glu‐62 and Glu‐101 in the Mn2+‐bound form seem to give rise to a band at 1,577‐1,574 cm−. The spectrum of the Mg2+‐bound form is also consistent with the ‘pseudo‐bridging’ coordination of the COO− group of Glu‐101 reported in the X‐ray structure of a form where the Mg2+ ion occupies only the EF site, and the same spectrum is further indicative of the ‘pseudo‐bridging’ coordination of the COO− group of Glu‐62.

FT-IR study of the Ca2+-binding to bovine α-lactalbumin: Relationships between the type of coordination and characteristics of the bands due to the Asp COO− groups in the Ca2+-binding site

Fourier-transform infrared spectroscopy (FT-IR) was applied to examine relationships between the type of coordination and the COO- antisymmetric and symmetric stretches of the COO- groups in the Ca2+-binding site of bovine alpha-lactalbumin. The peaks at 1593, 1578, 1425, and 1403 cm(-1) were assigned to the COO- groups of Asp-82, 87, and 88 coordinating to Ca2+ in the pseudo bridging mode, according to the results of X-ray crystallography. The bands due to the COO- groups were quite similar to each other between alpha-lactalbumin and EDTA which is the model compound for the pseudo bridging state.Fourier‐transform infrared spectroscopy (FT‐IR) was applied to examine relationships between the type of coordination and the COO− antisymmetric and symmetric stretches of the COO− groups in the Ca2+‐binding site of bovine α‐lactalbumin. The peaks at 1593, 1578, 1425, and 1403 cm−1 were assigned to the COO− groups of Asp‐82, 87, and 88 coordinating to Ca2+ in the pseudo bridging mode, according to the results of X‐ray crystallography. The bands due to the COO− groups were quite similar to each other between α‐lactalbumin and EDTA which is the model compound for the pseudo bridging state.

Infrared study of human serum very-low-density and low-density lipoproteins. Implication of esterified lipid CO stretching bands for characterizing lipoproteins

Fourier-transform infrared (FT-IR) spectroscopy was applied to examine human serum very-low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) in aqueous solution and in solid film for characterizing lipid components. On the basis of the FT-IR second-derivative spectra for standard samples of triglycerides, cholesterol esters and phospholipids, it was found that the band at 1746 cm(-1) for VLDL and the band at 1738 cm(-1) for LDL were mainly due to the unsaturated triglycerides and unsaturated cholesterol esters, respectively. The implications of ester C=O stretching bands are discussed.

Experimental alteration of molybdenite: evaluation of the Re–Os system, infrared spectroscopic profile and polytype

Experiments have been carried out to clarify the effect of alteration on Re–Os system, near infrared (NIR)–infrared (IR) spectroscopic characteristics and polytype of a natural molybdenite mineral (MoS2). The molybdenite sample was placed in H2O and various media of 0.1 mol/L NaCl, NaHCO3, CaCl2, and AlCl3 solutions, and heated in a sealed quartz tube at a temperature of 180°C for 20 d. The unaltered and altered samples were subsequently used for analysis of Re and Os, NIR microscopic observation, and NIR–IR spectroscopy, and microfocus X-ray diffraction (XRD). Molybdenites subjected to NaCl and NaHCO3 solutions give younger Re–Os ages than that of the original unaltered molybdenite. No significant changes in d spacing and width of micro-XRD patterns can be found in these altered molybdenite, indicating the possibility of Re–Os fractionation without significant structural conversion of molybdenite mineral. These results strongly suggest that the Re–Os system in molybdenite would be frequently disturbed if it has experienced alteration, because alteration by the low salinity (<1%), low temperature (∼180°C) hydrothermal solution containing NaCl and/or CO2 is commonly found in the natural environment. We maintain, therefore, that one set of analyses of Re and Os in a sample is not enough to determine whether the obtained Re–Os age has been affected by postdepositional alteration, but systematic replicate analyses are indispensable. Additionally, pulverizing all the collected molybdenite in a sample might give misleading results because portions, which have been altered and experienced Re–Os fractionation, may possibly mix into the undisturbed sample and be homogenized. The molybdenite used for the experiment was originally opaque under NIR light. Infrared microscopic and spectroscopic profiles show that some parts of the molybdenite subjected to CaCl2 and AlCl3 solutions become transparent to NIR. Increased NIR transmittance is possibly attributed to the removal of the impurity band in molybdenite. It was also found in this study, however, that change in IR profile does not correlate with the Re–Os fractionation and, therefore, IR measurement solely is not useful to detect disturbance of Re–Os systematics of molybdenite in alteration.

Coordination to divalent cations by calcium-binding proteins studied by FTIR spectroscopy.

We review the Fourier-transform infrared (FTIR) spectroscopy of side-chain COO(-) groups of Ca(2+)-binding proteins: parvalbumins, bovine calmodulin, akazara scallop troponin C and related calcium binding proteins and peptide analogues. The COO(-) stretching vibration modes can be used to identify the coordination modes of COO(-) groups of Ca(2+)-binding proteins to metal ions: bidentate, unidentate, and pseudo-bridging. FTIR spectroscopy demonstrates that the coordination structure of Mg(2+) is distinctly different from that of Ca(2+) in the Ca(2+)-binding site in solution. The interpretation of COO(-) stretches is ensured on the basis of the spectra of calcium-binding peptide analogues. The implication of COO(-) stretches is discussed for Ca(2+)-binding proteins. This article is part of a Special Issue entitled: FTIR in membrane proteins and peptide studies.

Fourier transform infrared spectroscopic analysis of the intact zona pellucida of the mammalian egg : Changes in the secondary structure of bovine zona pellucida proteins during fertilization

The zona pellucida is the acellular transparent envelope surrounding the mammalian oocyte. An analysis of the changes in the structures of zona pellucida proteins is essential for understanding the molecular mechanisms underlying the important physiological roles of the zona during fertilization and prelmplantatlon. The hardening of the zona caused by the structural changes during fertilization is generally accepted to be responsible for blocking polyspermy. In this study, we analyzed changes in the secondary structure of the zona during fertilization by Fourier transform infrared (FTIR) spectroscopy and transmission electron microscopy. The predominance of β-sheet structure in porcine ovarian egg zona proteins in water was ascertained using FTIR spectra. α-Helix structure was also present. The attenuated total reflection (ATR)-FTIR spectrum of intact, unsolubilized porcine zonae pellucidae from ovarian eggs Indicated that the zona proteins in the native zona pellucida also have β-structure as the main constituent. Attenuated total reflectlon-FTIR spectroscopy of intact bovine zona pellucida obtained from ovarian and fertilized eggs at the blastocyst stage revealed that the β-structure content Increased during fertilization. Furthermore, a reduction of the thickness of the zona during fertilization was observed using transmission electron microscopy. Therefore, the change in the zona architecture that causes hardening of the zona during fertilization is accompanied by changes in the secondary structure of the zona proteins.

Prostaglandin E2 Increases Both Osteoblastic and Osteoclastic Activity in the Scales and Participates in Calcium Metabolism in Goldfish

Using our original in vitro assay system with goldfish scales, we examined the direct effect of prostaglandin E2 (PGE2) on osteoclasts and osteoblasts in teleosts. In this assay system, we measured the activity of alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) as respective indicators of each activity in osteoblasts and osteoclasts. ALP activity in scales significantly increased following treatment at high concentration of PGE2 (10-7 and 10-6 M) over 6 hrs of incubation. At 18 hrs of incubation, ALP activity also significantly increased in the PGE2 (10-9 to 10-6 M)-treated scale. In the case of osteoclasts, TRAP activity tended to increase at 6 hrs of incubation, and then significantly increased at 18 hrs of incubation by PGE2(10-7 to 10-6 M) treatment. At 18 hrs of incubation, the mRNA expression of osteoclastic markers (TRAP and cathepsin K) and receptor activator of the NF-&kgr;B ligand (RANKL), an activating factor of osteoclasts expressed in osteoblasts, increased in PGE2 treated-scales. Thus, PGE2 acts on osteoblasts, and then increases the osteoclastic activity in the scales of goldfish as it does in the bone of mammals. In an in vivo experiment, plasma calcium levels and scale TRAP and ALP activities in the PGE2-injencted goldfish increased significantly. We conclude that, in teleosts, PGE2 activates both osteoblasts and osteoclasts and participates in calcium metabolism.

Fourier-transform infrared spectroscopic study of Ca2+-binding to osteocalcin.

Osteocalcin, the gamma-carboxyglutamic acid-containing protein, which is the most abundant noncollagenous protein of bone and dentin, is considered to play roles in bone formation and remodeling. It is unclear how the gamma-carboxyglutamic acid side-chains in osteocalcin coordinate to Ca2+, since the X-ray structure of osteocalcin is not available. Interactions between Ca2+ and the gamma-carboxyglutamic acid side-chains in osteocalcin were investigated by Fourier-transform infrared spectroscopy. In the region of the antisymmetric stretches, the loss of intensity at 1574 cm(-1) and gain of intensity at 1600 cm(-1) were observed due to Ca2+-binding to osteocalcin. The spectral changes indicate that the gamma-carboxyglutamic acid side-chains in osteocalcin coordinate to Ca- in the malonate chelation mode, where a Ca2+ interacts with two oxygen atoms, one from each of the two COO- groups of a single gamma-carboxyglutamic acid residue. Addition of Ca2+ does not cause any spectral change in the spectra of decarboxylated osteocalcin since the gamma-carboxyglutamic acid residues are converted to the glutamic acid residues by chemical modification.

A pulsed field gradient NMR study of the aggregation and hydration of parvalbumin

Pulsed field gradient NMR is a convenient alternative to traditional methods for measuring diffusion of biological macromolecules. In the present study, pulsed field gradient NMR was used to study the effects of calcium binding and hydration on carp parvalbumin. Carp parvalbumin is known to undergo large changes in tertiary structure with calcium loading. The diffusion coefficient is a sensitive guide to changes in molecular shape and in the present study the large changes in tertiary structure were clearly reflected in the measured diffusion coefficient upon calcium loading. The (monomeric) calcium-loaded form had a diffusion coefficient of 1.4 x 10(-10) m(2) s(-1) at 298 K, which conforms with the structure being a nearly spherical prolate ellipsoid from X-ray studies. The calcium-free form had a significantly lower diffusion coefficient of 1.1 x 10(-10) m(2) s(-1). The simplest explanation consistent with the change in diffusion coefficient is that the parvalbumin molecules form dimers upon the removal of Ca(2+) at the protein concentration studied (1 mM).