Comparison of heat- and pressure-induced unfolding of ribonuclease a: the critical role of Phe46 which appears to belong to a new hydrophobic chain-folding initiation site

To clarify the structural role of Phe46 inside the hydrophobic core of bovine pancreatic ribonuclease A (RNase A), thermal and pressure unfolding of wild-type RNase A and three mutant forms (F46V, F46E, and F46K) were analyzed by fourth-derivative UV absorbance spectroscopy. All the mutants, as well as the wild type, exhibited a two-state transition during both thermal and pressure unfolding, and both T(m) and P(m) decreased markedly when Phe46 was replaced with valine, glutamic acid, or lysine. The strongest effect was on the F46K mutant and the weakest on F46V. Both unfolding processes produced identical blue shifts in the fourth-derivative spectra, indicating that the tyrosine residues are similarly exposed in the temperature- and pressure-induced unfolded states. A comparison of Gibbs free energies determined from the pressure and temperature unfoldings, however, gave DeltaG(p)/DeltaG(t) ratios (r) of 1.7 for the wild type and 0.92 +/- 0.03 for the mutants. Furthermore, the DeltaV value for each mutant was larger than that for the wild type. CD spectra and activity measurements showed no obvious major structural differences in the folded state, indicating that the structures of the Phe46 mutants and wild type differ in the unfolded state. We propose a model in which Phe46 stabilizes the hydrophobic core at the boundary between two structural domains. Mutation of Phe46 decreases protein stability by weakening the unfolding cooperativity between these domains. This essential function of Phe46 in RNase A stability indicates that it belongs to a chain-folding initiation site.     

A comparative study on the hydroperoxide and thiol specificity of the glutathione peroxidase family and selenoprotein P

Glutathione peroxidase catalyzes the reduction of hydrogen peroxide and organic hydroperoxide by glutathione and functions in the protection of cells against oxidative damage. Glutathione peroxidase exists in several forms that differ in their primary structure and localization. We have also shown that selenoprotein P exhibits a glutathione peroxidase-like activity (Saito, Y., Hayashi, T., Tanaka, A., Watanabe, Y., Suzuki, M., Saito, E., and Takahashi, K. (1999) J. Biol. Chem. 274, 2866-2871). To understand the physiological significance of the diversity among these enzymes, a comparative study on the peroxide substrate specificity of three types of ubiquitous glutathione peroxidase (cellular glutathione peroxidase, phospholipid hydroperoxide glutathione peroxidase, and extracellular glutathione peroxidase) and of selenoprotein P purified from human origins was done. The specific activities and kinetic parameters against two hydroperoxides (hydrogen peroxide and phosphatidylcholine hydroperoxide) were determined. We next examined the thiol specificity and found that thioredoxin is the preferred electron donor for selenoprotein P. These four enzymes exhibit different peroxide and thiol specificities and collaborate to protect biological molecules from oxidative stress both inside and outside the cells.     

Developmental alteration of nerve injury induced glial cell line-derived neurotrophic factor (GDNF) receptor expression is crucial for the determination of injured motoneuron fate

Axotomy-induced neuronal death occurs in neonatal motoneurons, but not in adult rat. Here we demonstrated that during the course of postnatal development, nerve injury induced down-regulation of the glial cell line-derived neurotrophic factor (GDNF) receptor GFRalpha1 in axotomized hypoglossal motoneurons of rat are gradually converted to the adult up-regulation pattern of response. The compensatory expression of GFRalpha1 specifically in the injured motoneurons of neonates by adenovirus succeeded in rescuing the injured neurons without an application of growth factors. To the contrary, the nuclear antisense RNA for GFRalpha1 expression accelerates the axotomy-induced neuronal death in pups. These findings suggest that the receptor expression response after nerve injury is critical for the determination of injured motoneuron fate.     

Doxorubicin directly binds to the cardiac-type ryanodine receptor

The clinical use of doxorubicin, an antineoplasmic agent, is limited by its extensive cardiotoxicity which is mediated by the mobilization of intracellular Ca2+ from SR. In order to elucidate the mechanism of Ca2+ release, we analyzed the binding sites of doxorubicin on rabbit cardiac SR (sarcoplasmic reticulum). One of the binding sites was identified as cardiac-type ryanodine receptor (RyR2) which was purified by immunoprecipitation from solubilized cardiac SR in the presence of DTT. Ligand blot analysis revealed the direct binding of doxorubicin to RyR2. The binding of doxorubicin to RyR2 was specific and displaced by caffeine. Both doxorubicin and caffeine enhanced [3H]-ryanodine binding to RyR2 in a Ca2+ dependent manner. These results suggest that there is a doxorubicin binding site on RyR2.     

Comparative studies of genetic diversity in kenaf (Hibiscus cannabinus L.) varieties based on analysis of agronomic and RAPD data

Kenaf (Hibiscus cannabinus L.) is a fiber crop classified in the genus Hibiscus (Malvaceae), and has a great potential for its multipurpose utilization, in addition to its traditional usage. Varietal identification of kenaf is always problematic and knowledge on genetic diversity of kenaf varieties is also limited, which significantly hindered our effective utilization and conservation of the valuable kenaf germplasm. In order to find a proper method for identifying kenaf varieties and studying their variation, morpho-agronomic characters and random amplified polymorphic DNA (RAPD) markers were analyzed among 14 kenaf varieties commonly used in Japan. Data from morphological analysis showed that the included kenaf varieties could be divided into three major groups. The characters, such as middle stem diameter, whole stalk weight, and days to 50% flowering, are highly responsible for the variation of the kenaf varieties, but it is difficult to identify individual varieties merely by the morpho-agronomic characters. On the other hand, clearly separation of the kenaf varieties was achieved based on the RAPD variation patterns. Genetic relationship of the kenaf varieties can also be traced through the analysis of RAPD and morph-agronomic variation. It is concluded from the present study that RAPD analysis is an effective tool in identifying of kenaf varieties and determining their genetic relationships, particularly when combined with the analysis of morpho-agronomic characters.     

Substrate regulation of calcium binding in Ca2+-ATPase molecules of the sarcoplasmic reticulum. I. Effect of ATP

The effect of ATP on calcium binding of the Ca2+-ATPase of the sarcoplasmic reticulum has not been clarified. By comparing the calcium dependence of the ATPase activity and of phosphorylation of the ATPase molecules with that of calcium binding in the absence of ATP, we show the existence of two types of regulatory site of the enzyme molecules at which ATP binding variously improves the calcium binding performance of the molecules depending on the aggregation state of the molecules and pH; the two regulatory sites bind ATP at submillimolar (0.25 mm) and millimolar (5 mm) ATP, respectively. The results are discussed based on a model of two conformational variants (A and B forms) of the chemically equivalent ATPase molecules (Nakamura, J., and Furukohri, T. (1994) J. Biol. Chem. 269, 30818-30821). For example, in the sarcoplasmic reticulum membrane at pH 7.40, submillimolar ATP converted the calcium binding manner of the A form from noncooperative (Hill number (n(H)) of approximately 1) to cooperative (n(H) approximately 2), concurrent with a decrease in the apparent calcium affinity (K(0.5)) from 2-6 to 0.1-0.3 microm. The binding of the A form became almost the same as that of the B form (n(H) approximately 2, K(0.5) approximately 0.2 microm), which was not affected by ATP. Millimolar ATP further decreased the K(0.5) of the cooperative binding of the two forms to approximately 0.05 microm. Regulation of the calcium binding performance by ATP is discussed in terms of monomeric and oligomeric pathway models.     

Molecular cloning and immunohistochemical localization of ubiquitin C-Terminal hydrolase expressed in testis of a teleost,the Nile Tilapia,Oreochromis niloticus

We previously produced four monoclonal antibodies to testicular proteins of a teleost, the Nile tilapia. One of the monoclonal antibodies, TAT(Testicular Antigen of Tilapia)-10, recognizes a Mr=27,000 protein (27 kD protein), which is present in A and early B type spermatogonia, spermatids, and spermatozoa in testis. In order to clarify the function of this protein, molecular cloning was conducted. The cDNA for the 27 kD protein contains a complete open reading frame encoding 220 amino acid residues. The predicted amino acid sequence of the 27 kD protein was homologous to those of the ubiquitin carboxy-terminal hydrolases (UCH) reported in mammals. The measurement of the ubiquitin-releasing activity of the recombinant 27 kD protein revealed that the protein is the active form of UCH. Northern blot analysis showed that the UCH mRNA was expressed in ovary and brain in addition to the testis. Immunohistochemical study showed that, in brain, UCH was localized especially on the olfactory organ including the olfactory bulb and olfactory epithelium in olfactory rosetta, suggesting the involvement of the protein in chemoreceptive function. In the Tilapia ovary, UCH localized especially in pre-vitellogenic oocytes, suggesting that the enzyme activity could be important in oocyte growth. This is the first report for the cDNA cloning and cellular localization of UCH in fish. J. Exp. Zool. 293:368-383, 2002.