Takahide Tsuchiya

Tyrosine phosphorylation in plant bending.

The phosphorylation of the amino acid tyrosine in animal proteins acts as an on–off switch in numerous pathways that regulate growth, differentiation and oncogenesis. In the unicellular slime mould Dictyostelium , tyrosine-phosphorylation of actin controls cell-shape changes and spore activity, but the significance of protein-tyrosine phosphorylation in plants is unknown. Here we show that actin in the contact-sensitive plant Mimosa pudica L. is heavily tyrosine-phosphorylated and that changes in the extent of phosphorylation correlate with the degree of bending of the plants petioles. We propose that tyrosine-phosphorylation of actin controls movements in plants.

Water channel activities of Mimosa pudica plasma membrane intrinsic proteins are regulated by direct interaction and phosphorylation

cDNAs encoding aquaporins PIP1;1, PIP2;1, and TIP1;1 were isolated from Mimosa pudica (Mp) cDNA library. MpPIP1;1 exhibited no water channel activity; however, it facilitated the water channel activity of MpPIP2;1 in a phosphorylation‐dependent manner. Mutagenesis analysis revealed that Ser‐131 of MpPIP1;1 was phosphorylated by PKA and that cooperative regulation of the water channel activity of MpPIP2;1 was regulated by phosphorylation of Ser‐131 of MpPIP1;1. Immunoprecipitation analysis revealed that MpPIP1;1 binds directly to MpPIP2;1 in a phosphorylation‐independent manner, suggesting that phosphorylation of Ser‐131 of MpPIP1;1 is involved in regulation of the structure of the channel complex with MpMIP2;1 and thereby affects water channel activity.

Alzheimer's disease amyloid β-clipping enzyme (APP secretase): Identification, purification, and characterization of the enzyme

Alzheimers disease (AD) is the most frequent cause of dementia, although no genetic abnormality has been identified. Recent studies have elucidated the molecular defect in AD, including the abnormal deposition of amyloid beta peptide (beta/A4) in senile plaques of affected individuals. Normal brain contains the enzyme, APP secretase, which cleaves inside the beta/A4 portion of the precursor protein (APP); abnormal processing of APP occurs in AD brain. Until now, no evidence has been provided that APP secretase is an intracellular proteinase. We have now prepared two synthetic substrates of APP secretase, both of which contain the cleavage point and are much more sensitive than substrates previously available to identify APP secretase. Using these substrates, we found an intracellular proteinase that has APP secretase activity. This proteinase has been identified as cathepsin B.

Antimicrobial action of achacin is mediated by L-amino acid oxidase activity

Achacin is an antibacterial glycoprotein purified from the mucus of the giant snail, Achatina fulica Férussac, as a humoral defense factor. We showed that achacin has L‐amino acid oxidase activity and can generate cytotoxic H2O2; however, the concentration of H2O2 was not sufficient to kill bacteria. The antibacterial activity of achacin was inhibited by various H2O2 scavengers. Immunochemical analysis revealed that achacin was preferentially bound to growth‐phase bacteria, accounting for the important role in growth‐phase‐dependent antibacterial activity of achacin. Achacin may act as an important defense molecule against invading bacteria.

Distinct kinetics of subunit autolysis in mammalian m-calpain activation

Subunit autolysis of mammalian m‐calpain upon activation was examined in kinetic terms using a set of antibodies recognizing different portions of the protease. Activation of m‐calpain by calcium resulted in no apparent autolysis in the large catalytic subunit, whereas the small regulatory subunit underwent immediate autolysis followed by substrate proteolysis. This profile of subunit autolysis is distinct from that of the other ubiquitous isozyme, μ‐calpain, in which autolysis of the large subunit and then of the small subunit precedes substrate proteolysis under the normal conditions. The activation state of m‐calpain thus is not reflected by the large subunit autolysis. The mode and role of autolysis may vary among calpain isozymes.

Thermal stability of fish myosin

Abstract 1. 1. Thermal stability of fish myosin has been studied by using differential scanning calorimetry (DSC) and circular dichroism (CD). 2. 2. The temperature range of the sharp decrease in α-helical content agreed very closely with that of the endothermic peaks. 3. 3. There was a high correlation between the enthalpy of denaturation (ΔH) and the decreasing quantity in α-helicity (Δh). 4. 4. The structure of fish myosins was much more unstable than that of rabbit. 5. 5. The instability of fish myosins was reflected in its rod moiety.

Purification and characterization of an antibacterial factor from snail mucus

The antibacterial factor from the body surface of the African giant snail, Achatina fulica Férussac, was isolated by DEAE-Toyopearl 650M ion exchange chromatography. The isolated preparation exhibited highly positive antibacterial activity both for the Gram-positive bacteria, Bacillus subtilis and Staphylococcus aureus and for the Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa, but it lost such activity when heated at 75 degrees C for 5 min. The antibacterial factor of the snail mucus was a glycoprotein whose molecular weight (MW) was about 160,000. It was composed of two subunits of MW 70,000-80,000.

Purification and characterization of two metalloproteinases from squid mantle muscle, myosinase I and myosinase II

Metalloproteinases, myosinase I and myosinase II, that hydrolyze the heavy chain of myosin, were purified from squid mantle muscle. Myosinase I does not hydrolyze other muscle proteins, casein, haemoglobin, or MCA-substrates, while II hydrolyzes tropomyosin. Both myosinase I and myosinase II gave a single protein band on SDS-PAGE with a molecular mass of 16 and 20 kDa, respectively. Their activities were inhibited by EDTA and 1,10-phenanthroline, and II was also inhibited by EGTA. They could be reactivated with some divalent cations, I was especially reactivated with Co2+ and II especially with Zn2+. The optimum pH of both activities was 7.0; the optimum temperature for both was 40 degrees C. Myosinase I hydrolyzes myosin heavy chains to produce 130 and 90 kDa fragments. The N-terminal amino-acid sequence of the 90 kDa fragment indicates that myosinase I splits the myosin heavy chain between Ala-1161 and Thr-1162 in subfragment 2. Myosinase II hydrolyzes myosin heavy chain to produce 158 and 65 kDa fragments, and it splits between Glu-1381 and Thr-1382 in LMM. Myosinases I and II are most likely related to the metabolism of myosin in vivo.

Biochemical and immunohistochemical characterization of Mimosa annexin

To characterize the biochemical properties of plant annexin, we isolated annexin from Mimosa pudica L. and analyzed the biochemical properties conserved between Mimosa annexin and animal annexins, e.g. the ability to bind phospholipid and F-actin in the presence of calcium. We show that Mimosa annexin is distributed in a wide variety of tissues. Immunoblot analysis also revealed that the amount of annexin is developmentally regulated. To identify novel functions of Mimosa annexin, we examined the pattern of distribution and the regulation of its expression in the pulvinus. The amount of annexin in the pulvinus increased at night and was sensitive to abscisic acid; however, there was no detectable induction of annexin by cold or mechanical stimulus. Annexin distribution in the cell periphery during the daytime was changed to a cytoplasmic distribution at night, indicating that Mimosa annexin may contribute to the nyctinastic movement in the pulvinus.

Molecular evolution and diversification of snake toxin genes, revealed by analysis of intron sequences.

The genes encoding erabutoxin (short chain neurotoxin) isoforms (Ea, Eb, and Ec), LsIII (long chain neurotoxin) and a novel long chain neurotoxin pseudogene were cloned from a Laticauda semifasciata genomic library. Short and long chain neurotoxin genes were also cloned from the genome of Laticauda laticaudata, a closely related species of L. semifasciata, by PCR. A putative matrix attached region (MAR) sequence was found in the intron I of the LsIII gene. Comparative analysis of 11 structurally relevant snake toxin genes (three-finger-structure toxins) revealed the molecular evolution of these toxins. Three-finger-structure toxin genes diverged from a common ancestor through two types of evolutionary pathways (long and short types), early in the course of evolution. At a later stage of evolution in each gene, the accumulation of mutations in the exons, especially exon II, by accelerated evolution may have caused the increased diversification in their functions. It was also revealed that the putative MAR sequence found in the LsIII gene was integrated into the gene after the species-level divergence.