Biomedical subjects

M Maeshima

Publications and source records attributed to M Maeshima.

At least 37 records · Page 2Linked to original sources

PubMed1998

Molecular cloning of vacuolar H(+)-pyrophosphatase and its developmental expression in growing hypocotyl of mung bean.

Vacuolar proton-translocating inorganic pyrophosphatase and H(+)-ATPase acidify the vacuoles and power the vacuolar secondary active transport systems in plants. Developmental changes in the transcription of the pyrophosphatase in growing hypocotyls of mung bean (Vigna radiata) were investigated. The cDNA clone for the mung bean enzyme contains an uninterrupted open reading frame of 2298 bp, coding for a polypeptide of 766 amino acids. Hypocotyls were divided into elongating and mature regions. RNA analysis revealed that the transcript level of the pyrophosphatase was high in the elongating region of the 3-d-old hypocotyl but was extremely low in the mature region of the 5-d-old hypocotyl. The level of transcript of the 68-kD subunit of H(+)-ATPase also decreased after cell maturation. In the elongating region, the proton-pumping activity of pyrophosphatase on the basis of membrane protein was 3 times higher than that of H(+)-ATPase. After cell maturation, the pyrophosphatase activity decreased to 30% of that in the elongating region. The decline in the pyrophosphatase activity was in parallel with a decrease in the enzyme protein content. These findings indicate that the level of the pyrophosphatase, a main vacuolar proton pump in growing cells, is negatively regulated after cell maturation at the transcriptional level.

Amino Acid Sequence↗

PubMed1998

Genes involved in osmoregulation during turgor-driven cell expansion of developing cotton fibers are differentially regulated.

Cotton (Gossypium hirsutum L.) fibers are single-celled trichomes that synchronously undergo a phase of rapid cell expansion, then a phase including secondary cell wall deposition, and finally maturation. To determine if there is coordinated regulation of gene expression during fiber expansion, we analyzed the expression of components involved in turgor regulation and a cytoskeletal protein by measuring levels of mRNA and protein accumulation and enzyme activity. Fragments of the genes for the plasma membrane proton-translocating ATPase, vacuole-ATPase, proton-translocating pyrophosphatase (PPase), phosphoenolpyruvate carboxylase, major intrinsic protein, and alpha-tubulin were amplified by polymerase chain reaction and used as probes in ribonuclease protection assays of RNA from a fiber developmental series, revealing two discrete patterns of mRNA accumulation. Transcripts of all but the PPase accumulated to highest levels during the period of peak expansion (+12-15 d postanthesis [dpa]), then declined with the onset of secondary cell wall synthesis. The PPase was constitutively expressed through fiber development. Activity of the two proton-translocating-ATPases peaked at +15 dpa, whereas PPase activity peaked at +20 dpa, suggesting that all are involved in the process of cell expansion but with varying roles. Patterns of protein accumulation and enzyme activity for some of the proteins examined suggest posttranslational regulation through fiber development.

Blotting, Western↗

PubMed1997

Analysis of the substrate binding site and carboxyl terminal region of vacuolar H+-pyrophosphatase of mung bean with peptide antibodies.

Vacuolar H+-translocating inorganic pyrophosphatase is a single-protein enzyme and uses a simple substance as an energy donor. Functional domains of the enzyme were investigated by using antibodies specific to peptides corresponding to the putative substrate-binding site (DVGADLVGKVE) in the hydrophilic loop and the carboxyl terminal part. The antibody to the former peptide clearly reacted with the pyrophosphatases of different plant species, and strongly inhibited the hydrolytic activity of the purified enzymes and the proton pumping activity of membrane vesicles. These results indicate that the sequence functions as an actual substrate-binding site and is a common motif. The antibody to the carboxyl terminal part reacted only to the mung bean enzyme, suppressing its hydrolytic and proton pumping activities. The results suggest that the carboxyl terminus is exposed to the cytosol and is close to the catalytic site. H+-Pyrophosphatase hydrolyzed triphosphate and tetraphosphate at low rates. Phytic acid, myo-inositol hexaphosphate, inhibited the enzyme even in the presence of Mg2+. The concentration for 50% inhibition was 0.15 mM. The inhibition of H+-PPase by dicyclohexyldiimide was partly reversed by Mg2+. The catalytic site and the membrane topology of the enzyme are discussed.

Amino Acid Sequence↗

PubMed1997

Accumulation of a glycoprotein that is homologous to a seed storage protein in mung bean hypocotyls at the late stage of tissue elongation.

Physiological changes were examined in the amount of a 50-kDa glycoprotein (gp50) that was recovered in a nuclear fraction from hypocotyls of mung bean (Vigna radiata) seedlings. Immunoblot analysis indicated that the glycoprotein was present in hypocotyls and epicotyls from 4- and 5-day-old seedlings but not in hypocotyls from 2-day-old seedlings. The glycoprotein was not detected in leaves or roots. When we divided hypocotyls of 3-day-old seedlings into elongating region (0 to 1.5 cm below the cotyledon) and the mature region, we found gp50 in the mature region only. The results suggest that the 50-kDa glycoprotein is synthesized de novo and accumulates at the late stage during elongation of cells in the hypocotyl. Furthermore, an antibody specific to gp50 reacted with a major 50-kDa protein in cotyledons, which is known as a storage protein in mung bean cotyledon. Eighteen amino acid residues among 22 amino-terminal residues of gp50 were identical to those of the storage protein from cotyledon. A peptide map of the glycoprotein after digestion with V8 protease was similar to that of the storage protein. Overall, our findings suggest that the glycoprotein recovered in the nuclear fraction is an isoform of the seed storage protein that is expressed only in the mature cells of hypocotyls and epicotyls.

Allergens↗

PubMed1997

Changes in H(+)-pumps and a tonoplast intrinsic protein of vacuolar membranes during the development of pear fruit.

Vacuolar H(+)-ATPase (V-ATPase) was purified from pear fruit and antibodies were raised against the subunits of 55 and 33 kDa. Antibodies against mung bean H(+)-pyrophosphatase (V-PPase) and radish VM23, which is a tonoplast intrinsic protein (TIP) and a water channel, cross-reacted with the vacuolar membrane proteins of pear fruit. To clarify the roles of these proteins in development of pear fruit, we determined their levels relative to the total amount of protein by immunoblot analysis. The levels of subunits of the V-ATPase increased with fruit development. By contrast, the level of V-PPase was particularly high at the cell-division stage and remained almost the same at other stages. The changes in the activities of V-ATPase and V-PPase corresponded to those in their protein levels. The ratio of V-PPase activity to V-ATPase activity indicated that V-PPase is a major H(+)-pump of the vacuolar membranes of young fruit and that the contribution of V-ATPase increases with fruit development, finally, V-ATPase becomes the major H(+)-pump during the later stages of fruit development. The level of a protein analogous to VM23 (VM23P) was especially high during the active cell-expansion stage in young fruit, and VM23P might, therefore, play an important role in the rapid expansion of cells as a vacuolar water channel. Our results show that the levels of V-ATPase, V-PPase and VM23P change differently and reflect the roles of the respective protein in the development of pear fruit.

Adenosine Triphosphatases↗

PubMed1996

Cloning and expression of the inorganic pyrophosphatase gene from thermophilic bacterium PS-3.

The thermophilic bacterium PS-3 ppa gene encoding inorganic pyrophosphatase (PPiase) has been cloned and sequenced. The deduced amino acid sequence was exactly the same as that determined on Edman degradation (Ichiba, T., Takenaka, O., Samejima, T. and Hachimori, A., J. Biochem. 108: 572-578, 1990) except that amino acid-70 is threonine instead of proline and two more amino acids, Asn-Lys, are present at the carboxyl terminus. The structural gene containing BamH I and Hind III restriction cleaving sites at the 5' and 3' ends, respectively, was amplified by the PCR method. Escherichia coli JM109 was transformed with a synthetic DNA, and we found that thermophilic ppa was expressed in E. coli, as judged on enzyme assaying, SDS-PAGE and immune assaying.

Amino Acid Sequence↗

PubMed1996

Enhancement of the expression of progesterone receptor on progesterone-treated lymphocytes after immunotherapy in unexplained recurrent spontaneous abortion.

PROBLEM: The immunological mechanism of an effective immunotherapy with paternal lymphocytes for unexplained recurrent spontaneous abortion (RSA) is not yet clear. Previous studies revealed that progesterone plays an important role in maintaining normal pregnancy and lower expression of progesterone receptor (PGR) on lymphocytes was found in RSA. Therefore, it was of interest to investigate whether immunotherapy for RSA would be able to enhance the expression of PGR on lymphocytes of RSA. METHOD: PGR expression on lymphocytes was analyzed with indirect immunofluorescence using flow cytometry. RESULTS: There was no change of PGR expression on PBL of RSA between pre- and post-immunotherapy (P > 0.05), while in the presence of 10.0 micrograms/ml progesterone for 24 h, PGR expressed on PBL on post-immunotherapy was increased significantly as compared with that of pre-immunotherapy in successful cases (P < 0.05) and decreased in abortive cases (P < 0.05). Most PGR was expressed on both CD4+ and CD8+ lymphocyte subsets. In successful cases, CD8+PGR+ subset of post-immunotherapy was found to be increased significantly (P < 0.05) in comparison with that of pre-immunotherapy. CONCLUSION: The data in the present study suggest that immunotherapy for RSA induced a higher expression of PGR on progesterone-treated lymphocytes, which may be involved in successful pregnancy.

Abortion, Habitual↗

PubMed1993

Molecular cloning of cDNA for vacuolar membrane proton-translocating inorganic pyrophosphatase in Hordeum vulgare.

We obtained a cDNA clone(PP10) for the vacuolar membrane proton-translocating inorganic pyrophosphatase from barley roots by immunoscreening. The nucleotide sequence contained a 2308 bp open reading frame capable of coding for a polypeptide with 761 amino acids (M(r) 79,841). The polypeptide is highly hydrophobic and 12 membrane-spanning regions are deduced from the hydropathic evaluation. The characteristic cluster of the basic and acidic residues is observed in the hydrophilic segment. A consensus sequence with the dicyclohexyl-carbodiimide binding subunits of FOF1-type and vacuolar-type H(+)-ATPase is also observed in the membrane-spanning domain 5. Comparison of the deduced amino acid sequence with that of pyrophosphatase cDNA from Arabidopsis thaliana revealed 85.8% homology.

Amino Acid Sequence↗

PubMed1993

The nuclear gene for subunit Vc of sweet potato cytochrome c oxidase.

We have cloned the gene for subunit Vc of sweet potato cytochrome c oxidase using its cDNA as a probe. There is a single intron, located one nucleotide upstream of the start of the open reading frame. No typical TATA-box sequence is present in the region upstream of the site of initiation of transcription in the cloned genomic DNA, suggesting that an alternative sequence may function in control of the expression of this gene. The nuclear genome of sweet potato contains only a single species of gene for subunit Vc.

Amino Acid Sequence↗

PubMed1992

Mechanism of the Decline in Vacuolar H -ATPase Activity in Mung Bean Hypocotyls during Chilling.

The mechanism responsible for the decrease in the activity of vacuolar H(+) -ATPase during chilling was investigated in seedlings of mung bean (Vigna radiata). After chilling at 0 degrees C for 3 d, the activity of vacuolar H(+) -ATPase, calculated on the basis of membrane protein, decreased to 47% of the original value. Of the nine subunits of the ATPase, the specific contents of at least six subunits, of 68, 57, 44, 38, 37, and 32 kD, decreased in vacuolar membranes after chilling, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These subunits were released by treatment with chaotropic anions such as thiocyanate. The level of the 16-kD subunit did not change. Immunoblot analyses showed the decrease in the levels of the subunits of 68, 57, and 32 kD. Furthermore, the specific activity of the ATPase purified from chilled hypocotyls was two-thirds of that of the enzyme from nonchilled seedlings, and the enzyme from chilled tissue retained only a small amount of the 32-kD subunit. These results suggest that a selective release of the peripheral subunits of the ATPase from the membrane and a partial degradation of the ATPase complex may occur in vivo during chilling.

Journal Article↗

PubMed1992

Characterization of the major integral protein of vacuolar membrane.

The vacuolar membrane of radish (Raphanus sativus) taproot contained a large quantity of a protein of 23 kilodaltons that accounted for more than 25% of the total membrane proteins. The protein, tentatively named VM 23, was purified and characterized. VM 23 tends to aggregate at high temperature even in the presence of 1% sodium dodecyl sulfate. The apparent molecular size of VM 23 was estimated to be about 400 kilodaltons by polyacrylamide gel electrophoresis in the presence of 0.1% Triton X-100. VM 23 was partially extracted from the vacuolar membranes with chloroform:methanol, indicating its high hydrophobicity. The hydrophobic carboxyl modifier N,N'-dicyclohexylcarbodiimide bound covalently to VM 23. The results suggest that VM 23 may act as a secondary transport system coupled with the proton transport. The antibody against radish VM 23 reacted with the major proteins in the vacuolar membranes of mung bean (Vigna radiata) and castor bean (Ricinus communis) hypocotyls and pumpkin (Cucurbita moschata) epicotyl, but not with that of sugar beet (Beta vulgaris) taproot. VM 23 comigrated with vacuolar H(+)-pyrophosphatase on sucrose density gradient centrifugation after sonication of membranes, indicating that it is associated with the vacuolar membrane.

Journal Article↗

PubMed1991

Immunological cross-reactivity between proton-pumping inorganic pyrophosphatases of widely phylogenic separated species.

Immunological cross-reactivity among three types of inorganic pyrophosphatases, that is, the proton pumping inorganic pyrophosphate synthase (H(+)-PPi synthase) and the soluble inorganic pyrophosphatase, both from Rhodospirillum rubrum, and the vacuolar membrane inorganic pyrophosphatase (H(+)-PPase) from mung bean (Vigna radiata), were examined by means of immunoblot analyses. Antibodies raised against the mung bean H(+)-PPase cross-reacted with the H(+)-PPi synthase from R. rubrum but not with the soluble PPase from R. rubrum. N,N'-dicyclohexylcarbodiimide (DCCD), which inhibits both synthesis and hydrolysis of PPi catalysed by purified and chromatophore H(+)-PPi synthase, binds to the enzyme as shown by fluorography of [14C]DCCD labelling. These results suggest that the R. rubrum H(+)-PPase share close structural similarities with the vacuolar H(+)-PPase from Mung bean.

Blotting, Western↗

PubMed1991

A vacuolar ATPase and pyrophosphatase in Acetabularia acetabulum.

Vacuole-rich fractions were isolated from Acetabularia acetabulum by Ficoll step gradient centrifugation. The tonoplast-rich vesicles showed ATP-dependent and pyrophosphate-dependent H(+)-transport activities. ATP-dependent H(+)-transport and ATPase activity were both inhibited by the addition of a specific inhibitor of vacuolar ATPase, bafilomycin B1. A 66 kDa polypeptide present in the preparation cross-reacted with the anti-IgG fractions against the alpha and beta subunits of Halobacterium halobium ATPase and with the antibody against the A subunit (68 kDa subunit) of mung bean vacuolar ATPase. A 56 kDa polypeptide present in the vacuole preparation showed cross-reactivity with the antibody against the B subunit (57 kDa) of mung bean vacuolar ATPase but not with the anti-beta subunit of H. halobium ATPase. A 73 kDa polypeptide cross-reacted with the antibody against inorganic pyrophosphatase of mung bean vacuoles. These results suggest that vacuolar membrane of A. acetabulum equipped energy transducing systems similar to those found in other plant vacuoles.

Acetabularia↗

PubMed1991

Dimeric structure of H(+)-translocating pyrophosphatase from pumpkin vacuolar membranes.

Vacuolar membrane H(+)-translocating pyrophosphatase (H(+)-PPase) was purified from pumpkin seedlings. Its enzymatic properties including molecular size of constituting polypeptide (75 kDa) were very similar to those of mung bean H(+)-PPase [(1989) J. Biol. Chem. 264, 20068-20073]. The native, functional molecular size of the pumpkin H(+)-PPase was estimated to be 135-139 kDa from gel permeation HPLC of the purified enzyme in the presence of detergent and from radiation inactivation of the enzyme in vacuolar membranes. It is concluded that native, functional pumpkin H(+)-PPase, and also probably H(+)-PPases from other plants, is a dimer of 75 kDa subunits.

Biological Transport, Active↗

PubMed1991

H(+)-translocating inorganic pyrophosphatase of plant vacuoles. Inhibition by Ca2+, stabilization by Mg2+ and immunological comparison with other inorganic pyrophosphatases.

The effects of divalent cations, especially Ca2+ and Mg2+, on the proton-translocating inorganic pyrophosphatase purified from mung bean vacuoles were investigated to compare the enzyme with other pyrophosphatases. The pyrophosphatase was irreversibly inactivated by incubation in the absence of Mg2+. The removal of Mg2+ from the enzyme increased susceptibility to proteolysis by trypsin. Vacuolar pyrophosphatase required free Mg2+ as an essential cofactor (K0.5 = 42 microM). Binding of Mg2+ stabilizes and activates the enzyme. The formation of MgPPi is also an important role of magnesium ion. Apparent Km of the enzyme for MgPPi was about 130 microM. CaCl2 decreased the enzyme activity to less than 60% at 40 microM, and the inhibition was reversed by EGTA. Pyrophosphatase activity was measured under different conditions of Mg2+ and Ca2+ concentrations at pH 7.2. The rate of inhibition depended on the concentration of CaPPi, and the approximate Ki for CaPPi was 17 microM. A high concentration of free Ca2+ did not inhibit the enzyme at a low concentration of CaPPi. It appears that for Ca2+, at least, the inhibitory form is the Ca2(+)-PPi complex. Cd2+, Co2+ and Cu2+ also inhibited the enzyme. The antibody against the vacuolar pyrophosphatase did not react with rat liver mitochondrial or yeast cytosolic pyrophosphatases. Also, the antibody to the yeast enzyme did not react with the vacuolar enzyme. Thus, the catalytic properties of the vacuolar pyrophosphatase, such as Mg2+ requirement and sensitivity to Ca2+, are common to the other pyrophosphatases, but the vacuolar enzyme differs from them in subunit mass and immunoreactivity.

Animals↗

PubMed1991

Reduced high harmonic power spectra in patients with old myocardial infarction.

Acceleration of blood flow ejected from the ventricle into the aorta and the hydraulic power state of blood flow in the aorta may be affected by asynchronous or locally depressed contraction of the left ventricle as well as by arterial impedance. In order to evaluate the effect of an infarcted heart on the hydraulic power state of blood flow, the aortic input impedance spectrum and the hydraulic power spectrum were calculated from simultaneous recordings of pulsatile aortic pressure and flow velocity at the aortic root in 6 normal controls (group 1), 6 patients with old myocardial infarction (OMI) without aneurysm (group 2), and 6 patients of OMI with aneurysm (group 3). The ratios (Rh) of power in high harmonics to that of fundamental harmonics, that is, Rh = (E2 + E3 + ...E10)/E1, where E represents hydraulic power and the numbers represent harmonic numbers, were 0.64 +/- 0.17, 0.23 +/- 0.09, and 0.22 +/- 0.10 in groups 1, 2, and respectively (p less than 0.001 between groups 1 and 2; p less than 0.001, between groups 1 and 3). As the acceleration of ejecting flow of blood by the left ventricle is inversely related with arterial impedance, Rh will be also inversely related with arterial impedance. Then, the product of Rh and the characteristic impedance (Zc) would be an indicator of the left ventricular contractility. In fact, the products were 64 +/- 15, 32 +/- 14, and 31 +/- 13 dyne.sec.cm-5 in groups 1, 2, and 3 respectively (p less than 0.005, between groups 1 and 2; p less than 0.005, between groups 1 and 3). These results suggest that asynchronous or locally depressed contractions of the left ventricular wall are closely related to depressed Rh and further to depressed values of the products of Rh and Zc. It is concluded that analysis of the power spectrum of blood flow in the root of the aorta provides information of the left ventricular contractility in connection with arterial impedance.

Blood Flow Velocity↗

PubMed1990

Molecular cloning of a cDNA for the smallest nuclear-encoded subunit of sweet potato cytochrome c oxidase. Analysis with the cDNA of the structure and import into mitochondria of the subunit.

A cDNA clone for the subunit Vc, the smallest nuclear-encoded subunit, of sweet potato cytochrome c oxidase was isolated. Nucleotide sequence analysis showed that the subunit is synthesized as a precursor from which only the amino-terminal amino acid, methionine, residue is removed to form the mature subunit. The mature subunit consists of 63 amino acid residues which are arranged so as to form one hydrophobic cluster lying between two hydrophilic clusters; such an arrangement is also seen with some nuclear-encoded small subunits of the enzymes from other eukaryotes. The labelled precursor protein was synthesized with the cDNA by in vitro transcription (SP6 system) and translation (wheat germ system). This precursor was imported into isolated sweet potato tuberous root mitochondria without any significant processing, the import being dependent on membrane potential.

Amino Acid Sequence↗

PubMed1990

Oligomeric structure of H(+)-translocating inorganic pyrophosphatase of plant vacuoles.

The topography and oligomeric structure of the vacuolar membrane-bound inorganic pyrophosphatase (73,000 daltons) of mung bean were studied. When the vacuolar membranes were treated with thiocyanate or sodium carbonate which are known to remove the peripheral membrane proteins, the enzyme could not be detected in the solubilized fraction by the specific antibody. The apparent molecular size of the enzyme was estimated to be about 480 kDa by polyacrylamide gel electrophoresis in the presence of Triton X-100. Crosslinking treatment of the pyrophosphatase with dimethyl suberimidate produced a complex corresponding to the dimer. The rate of PPi hydrolysis showed a sigmoidal relationship to substrate concentration with a Hill coefficient of 2.5. These results suggest that the vacuolar pyrophosphatase is an integral membrane protein and functions as an oligomer, probably a dimer.

Carbonates↗