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I Hara-Nishimura

Publications and source records attributed to I Hara-Nishimura.

51 records · Page 3Linked to original sources
PubMed1993

Amino acid sequence surrounding the retinal-binding site in retinochrome of the squid, Todarodes pacificus.

Squid (Todarodes pacificus) retinochrome was reduced to N-retinyl protein with borane dimethylamine and cleaved by CNBr. The retinyl peptide was then isolated by chromatography while being monitored for absorbances at 215 and 330 nm, and the N-terminal amino acid sequence was determined to be Ser-Lys-Thr-Gly-X-Ala-Leu-Phe-Pro. This sequence was the same that we had observed at the 7th transmembrane domain of retinochrome whose structure was reported previously. During Edman degradation of the retinyl peptide, the yield of the PTH-lysine at the second cycle was lower than those of the other PTH-amino acids, proving that the lysine residue forms a Schiff's base with retinal (Lys-275 in retinochrome). The amino acid sequence surrounding the retinal-binding lysine in retinochrome greatly differed from those in a variety of known visual pigments. This fact would be associated with the difference in the photoisomerization of chromophore between retinochrome and rhodopsin. The protein structure of retinochrome is also compared with that of rhodopsin in Todarodes.

Amino Acid Sequence↗
PubMed1993

Cloning and nucleotide sequence of cDNA for rhodopsin of the squid Todarodes pacificus.

A cDNA for rhodopsin was isolated from a library constructed from poly(A)+RNA of the squid (Todarodes pacificus) retina. One positive clone with the longest insert of cDNA (3.1 kb) was selected by employing a PCR-amplified cDNA fragment as a probe. The nucleotide sequence of the cDNA revealed a single open reading frame of 1,344 bp encoding a polypeptide (M(r)49,833), which covered a complete sequence for the squid opsin. This clone had a very long 3'-non-coding region (1.7 kb) including multiple polyadenylation signals, AATAAA, resembling the clones for Todarodes retinochrome and retinal-binding protein (RALBP). The analysis of hydropathicity demonstrated the presence of seven transmembrane spanning domains, and a possible retinal-binding site, Lys-305, was found in the 7th domain. Todarodes rhodopsin contained characteristic sequences of PPQGY repeated in the C-terminal region, as reported in Loligo and octopus rhodopsins. Structural comparison of those cephalopod rhodopsins is also discussed.

Amino Acid Sequence↗
PubMed1993

Molecular characterization of a vacuolar processing enzyme related to a putative cysteine proteinase of Schistosoma mansoni.

Proproteins of various vacuolar proteins are post-translationally processed into mature forms by the action of a unique vacuolar processing enzyme. If such a processing enzyme is transported to vacuoles together with proprotein substrates, the enzyme must be a latent form. Immunocytochemical localization of a vacuolar processing enzyme, a 37-kD cysteine proteinase, in the endosperm of maturing castor bean seeds places the enzyme in the vacuolar matrix, where a variety of proproteins is also present. To characterize a molecular structure of vacuolar processing enzyme, we isolated a cDNA for the enzyme. Deduced primary structure of a 55-kD precursor is 33% identical to a putative cysteine proteinase of the human parasite Schistosoma mansoni. The precursor is composed of a signal peptide, a 37-kD active processing enzyme domain, and a propeptide fragment. Although the precursor expressed in Escherichia coli has no vacuolar processing activity, a 36-kD immunopositive protein expressed in E. coli is active. These results suggest that the activation of the vacuolar processing enzyme requires proteolytic cleavage of a 14-kD C-terminal propeptide fragment of the precursor.

Amino Acid Sequence↗
PubMed1993

Cloning and sequencing of cDNA for glycolate oxidase from pumpkin cotyledons and northern blot analysis.

A cDNA clone for glycolate oxidase (EC 1.1.3.1) was isolated by an immunochemical method from a cDNA expression library constructed from poly(A)+-RNA of green pumpkin cotyledons. The analysis of in vitro transcription-translation products of the cDNA insert revealed that the cDNA clone contained the complete coding region for glycolate oxidase. The entire insert of the cDNA was 1,440 nucleotides in length and encoded 367 amino acid residues, equivalent to a molecular mass of 40,353 daltons. The amino acid sequence of the C-terminal tripeptide was Pro-Arg-Leu, which is slightly different from the proposed signal for targeting to microbodies, Ser-Lys/Arg/His-Leu. Characteristic hydrophilic domains observed in the C-terminal regions of most microbody proteins were found in the deduced sequence of glycolate oxidase by hydropathy analysis. Immunoblot analysis showed that the amount of glycolate oxidase was low in dark-grown cotyledons and increased during greening of pumpkin cotyledons. Northern blot analysis showed that the probe could hybridize with a single 1.5-kb species of mRNA from pumpkin cotyledons and that the amount of the hybridizable mRNA increased dramatically during greening of the cotyledons. This observation indicates that the induction of glycolate oxidase during greening of the cotyledons is due to an increase in the level of the mRNA.

Alcohol Oxidoreductases↗
PubMed1991

A unique vacuolar processing enzyme responsible for conversion of several proprotein precursors into the mature forms.

Proprotein precursors of vacuolar components are transported from the endoplasmic reticulum into vacuoles, where they are proteolytically processed into their mature forms. However, the processing mechanism in plant vacuoles is very obscure. Characterization of a purified processing enzyme is required to determine whether a single enzyme is responsible for processing many vacuolar proteins with a large variability of molecular structure. If this is true, how can it recognize the numerous varieties of processing sites? We have now purified a processing enzyme (Mr = 37,000) from castor bean seeds. Our results show that the purified enzyme can process 3 different proproteins isolated from either the endoplasmic reticulum or transport vesicles in cotyledon cells to produce the mature forms of these proteins which are found at different suborganellar locations in the vacuole: the 2S protein found in the soluble matrix, the 11S globulin found in the insoluble crystalloid and the 51 kDa protein associated with the membrane. Thus a single vacuolar processing enzyme is capable of converting several proprotein precursors into their respective mature forms.

Amino Acid Sequence↗
PubMed1991

Pumpkin malate synthase. Cloning and sequencing of the cDNA and northern blot analysis.

A cDNA clone encoding the glyoxysomal malate synthase (EC 4.1.3.2) was identified by immunoscreening of a cDNA expression library constructed from poly(A)-rich RNA of etiolated pumpkin cotyledons. Determination of the DNA sequence of the 1979-nucleotide cDNA revealed a 1698-nucleotide open reading frame that encodes a polypeptide of 64632 Da. The identification of the cDNA for malate synthase was confirmed by matching three sequences obtained by peptide-sequence analyses of fragments generated by acid treatment of the purified enzyme. Northern blot analysis revealed that the probe hybridized to a single 2.3-kb species of mRNA species from etiolated pumpkin cotyledons which was not present in green pumpkin cotyledons. In a comparison of deduced amino acid sequences, pumpkin malate synthase was found to exhibit 83% and 48% similarity to the malate synthases from rape and Escherichia coli, respectively. Based on the amino acid sequence similarity and the hydropathy profiles of these three malate synthases, the signal for targeting the enzyme to microbodies is discussed.

Amino Acid Sequence↗
PubMed1990

Cloning and nucleotide sequence of cDNA for retinochrome, retinal photoisomerase from the squid retina.

The Rhodopsin-retinochrome system is essential for the visual photoreception of molluscs. cDNA coding for retinochrome of the squid (Todarodes pacificus) was cloned and the nucleotide sequence has been determined. The sequence (2.1 kb) covers the whole coding region of 903 bp. The deduced primary sequence suggests that retinochrome contains seven transmembrane spanning domains. The homology with bovine rhodopsin and the possible retinal binding site are also discussed.

Amino Acid Sequence↗
PubMed1988

Nucleotide sequence of cloned cDNA coding for pumpkin 11-S globulin beta subunit.

cDNA coding for preproglobulin beta, a precursor protein of 11-S globulin beta subunit, was cloned and the nucleotide sequence has been determined. The sequence covers the whole coding region (1440 base pairs) with 5' and 3' noncoding region (30 and 214 base pairs, respectively). The deduced amino acid sequence of preproglobulin beta consists of a 21-amino-acid N-terminal signal peptide, preceding the acidic gamma polypeptide region (275 amino acids) and the subsequent basic delta region (184 amino acids). The site for post-translational cleavage of the precursor polypeptide to make the gamma and delta chains is estimated to be located between the asparagine-glycine residues. The N-terminal amino acid of the gamma chain of mature 11-S globulin beta subunit was reported to be blocked by 5-oxoproline (pyroglutamic acid) [Ohmiya et al. (1980) Plant Cell Physiol. 21, 157-167]. It was shown that the blocked N-terminal amino acid is coded as a glutamine residue. The derived amino acid sequence was also compared with those of precursor proteins of other 11-S globulins such as soybean glycinin, cotton beta globulin, pea legumin and rape 11-S globulin by dot matrix analysis.

Amino Acid Sequence↗
PubMed1987

Proglobulin processing enzyme in vacuoles isolated from developing pumpkin cotyledons.

The enzymic conversion of proglobulin to globulin catalyzed by the extracts of vacuoles isolated from developing pumpkin (Cucurbita sp. cv Kurokawa Amakuri Nankin) cotyledons was investigated. The endoplasmic reticulum fraction isolated from the developing cotyledons pulselabeled with [(35)S]methionine was shown to contain mainly the radiolabeled proglobulin, which was used as a substrate for assaying the proteolytic processing in vitro. The vacuolar extracts catalyzed the proteolytic processing of the proglobulin molecule to produce globulin containing two kinds of polypeptide chains, gamma and delta. The pH optimum for the vacuole-mediated conversion was at pH 5.0. The proteolytic processing of proglobulin by the vacuolar extracts was inhibited in the presence of various thiol reagents, e.g. p-chloromercuribenzoate, N-ethylmaleimide, iodoacetic acid, Hg(2+), and Cu(2+), but not phenylmethylsulfonyl fluoride, EDTA, o-phenanthroline, leupeptin, antipain, pepstatin, chymostatin, or pumpkin trypsin inhibitor, and was activated in the presence of dithiothreitol and cysteine, indicating that the processing enzyme is a thiol protease. The suborganellar fractionation of the vacuoles showed that the processing activity was localized in the matrix fraction, but not in the membrane or crystalloid fractions. During the seed development, the enzyme was shown to increase, exhibiting the maximal activity at the late developmental stage. The matrix fraction of the protein bodies isolated from the dry castor bean (Ricinus communis) exhibited the processing activity toward the pumpkin proglobulin molecules in the same manner as that by the matrix fraction of pumpkin vacuoles.

Journal Article↗
PubMed1987

[Protein body].

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Cell Fractionation↗
PubMed1985

Biosynthesis and Intracellular Transport of 11S Globulin in Developing Pumpkin Cotyledons.

In vitro studies to explore the biosynthesis of 11S globulin developing cotyledons of pumpkin (Cucurbita sp.) demonstrated that 11S globulin is synthesized on membrane-bound polysomes. M(r) of the translation products (preproglobulin) synthesized by the poly(A)(+)-RNA isolated from developing cotyledons were determined to be 64,000 and 59,000, which are larger than those of the mature globulin subunit (62,000 and 57,000). Preproglobulin is then cotranslationally processed by cleavage of the signal peptide to produce proglobulin. In vivo pulse-chase experiments showed the sequential transformation of the single-chain proglobulin to mature globulin subunit (disulfide-linked doublet polypeptides) indicating posttranslational modification of the proglobulin.Subcellular fractionation of the pulse-chased intact cotyledons showed that the [(35)S]methionine label is detectable in proglobulin in rough endoplasmic reticulum shortly after the pulse label. With time, the labeled proteins move into other cellular fractions: proglobulin in the density = 1.24 grams per cubic centimeter fractions after 30 minutes and mature globulin subunit associated with protein bodies after 1 to 2 hours. The distribution of proglobulin in sucrose density gradients did not correspond with those of catalase (microbody marker) or fumarase (mitochondria marker). An accumulation of proglobulin occurred in the density = 1.24 grams per cubic centimeter fractions, whereas the mature globulin was scarcely detectable in this fraction. In contrast, proglobulin was not detected by immunochemical blotting analysis in the protein bodies prepared under the mild conditions from cotyledon protoplasts. The results suggest that the d = 1.24 grams per cubic centimeter fractions are engaged in the translocation of proglobulin into the protein bodies.

Journal Article↗
PubMed1985

Translocation of photosynthates into vacuoles in spinach leaf protoplasts.

A method was developed for the isolation of vacuoles from the mesophyll protoplasts of spinach leaf, employing the discontinuous Ficoll density gradient centrifugation technique. Isolated vacuole preparations were judged to be free from other organellar fractions based on the assays of marker enzyme activities of individual organelles.Using this isolation method, a time-dependent translocation of (14)C-labeled photosynthates into vacuoles was determined. In contrast to a significant transport of (14)C organic acids such as malate and citrate within 10 to 15 minutes (14)C neutral sugars and amino acids were barely transported into vacuoles during 40 minutes incubation, in spite of the fact that a relatively large amount of these compounds are found in the vacuoles. It was also found that a majority of [(14)C]sucrose remains in the cytosol, apparently not actively moving into the vacuoles. Overall results appear to suggest that vacuoles are not actively engaged in photosynthetic carbon metabolism in spinach leaf protoplasts.

Journal Article↗
PubMed1984

Possible roles of calcium and calmodulin in the biosynthesis and secretion of alpha-amylase in rice seed scutellar epithelium.

The scutellar epithelial cells of rice (Oryza sativa L. cv Kimmazé) seeds actively secrete alpha-amylase in an early stage of germination. Employing an in vivo system of freshly dissected scutellar tissues, effect of Ca(2+) on the biosynthesis and the secretion of alpha-amylase have been studied. The maximum biosynthetic rate was saturated at about 0.5 mm external Ca(2+) concentrations, whereas the secretion continued to increase to concentrations above 10 mm Ca(2+). In the presence of 1 mm Ca(2+), 0.01 mum A-23187 significantly increased both the biosynthesis and the secretion of alpha-amylase.A cation-specific requirement for Ca(2+) was apparent, since both biosynthesis and extracellular secretion of alpha-amylase were inhibited by 0.1 mm EGTA but were increased above basal rate only with Ca(2+) and Sr(2+); K(+), Mg(2+), and Ba(2+) being ineffective.La(3+) and ruthenium red (selective inhibitors of [Ca(2+) + Mg(2+)]-ATPase) were found to profoundly inhibit the secretion of alpha-amylase. A calmodulin antagonist, W-7, also inhibited the secretion of alpha-amylase at concentrations where the enzyme synthesis was not much affected. Overall data indicate that Ca(2+) movement and secretion of alpha-amylase are tightly linked and it is likely that they are regulated by the cytoplasmic Ca(2+) concentration under possible control by calmodulin.

Journal Article↗
PubMed1982

Suborganellar localization of proteinase catalyzing the limited hydrolysis of pumpkin globulin.

Protein bodies were prepared from the cotyledons of pumpkin (Cucurbita sp.) seeds by employing a nonaqueous isolation method. Both light micrographic examination and the marker enzyme assays have shown that the isolated protein bodies were intact and contamination with other cell organelles or cytoplasmic components was negligible. A proteolytic enzyme catalyzing the limited hydrolysis of carboxymethylated gamma' chain of globulin was found to be present in the protein bodies. The specific activity in the protein body (18 units per milligram protein) was higher than that in the whole cell extract (13 units per milligram protein), indicating that the limited proteolytic enzyme was localized in the protein body.After lysis of the protein bodies using hypotonic buffer solution, the suborganellar components (matrix, membranes, and crystalloids) were separated by sucrose density gradient centrifugation. The crystalloid was composed of only globulin, a major seed protein. The major proteins of matrix and membrane fractions were shown to have mol wt of approximately 10,000. About 90% of the limited proteolytic activity was found in the matrix region.

Journal Article↗