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Y Emori

Publications and source records attributed to Y Emori.

At least 109 records · Page 6Linked to original sources
PubMed1989

A second type of rat phosphoinositide-specific phospholipase C containing a src-related sequence not essential for phosphoinositide-hydrolyzing activity.

A fourth type of rat phosphoinositide-specific phospholipase C (PLC IV) has been cloned for cDNA and sequenced. PLC IV is distinct from the other three types of rat PLC (PLC I, II, and III) with respect to primary structure and tissue distribution of its mRNAs. PLC IV contains two homologous regions included commonly in PLC I, II, and III and is most similar to PLC II (identity: 50.2%). PLC IV, in common with PLC II, has a sequence homologous to the N-terminal regulatory domains of nonreceptor tyrosine kinases of the src-family of oncogenes. Using an Escherichia coli expression system, we succeeded in producing active PLC IV in E. coli crude extracts. Various truncation experiments of the PLC IV cDNA revealed that the src-related domain is not necessary for catalytic activity while both domains homologous among PLC I-IV are essential. PLC IV is expressed in various rat tissues and abundant in spleen, suggesting that PLC IV plays a fundamental role in cellular functions such as growth and secretion.

Amino Acid Sequence↗
PubMed1989

An ice nucleation active gene of Erwinia ananas. Sequence similarity to those of Pseudomonas species and regions required for ice nucleation activity.

The ice nucleation active gene, inaA, of Erwinia ananas IN-10 has been sequenced. This gene encodes a protein composed of 1322 amino acid residues. The inaA protein contains a 1120-residue segment consisting of 70 repeats of closely related 16 amino acid motifs (R-domain), which is flanked by N- and C-terminal sequences (N- and C-domains, respectively). Its primary structure is similar to, but not identical with, those of Pseudomonas inaW and inaZ gene products. By truncating the inaA gene to various extents, it was found that deletion of the C-domain resulted in complete loss of the ice nucleation activity, whereas removal of the N-domain led to a moderate decrease in the activity. Complete loss of the activity was also observed when the N-domain plus a large part of the P-domain were deleted. It is suggested that the C-domain is required for the assembly of inaA protein to form a functional ice nucleus.

Amino Acid Sequence↗
PubMed1989

Molecular cloning of a novel mammalian calcium-dependent protease distinct from both m- and mu-types. Specific expression of the mRNA in skeletal muscle.

Two types of calcium-dependent protease with distinct calcium requirements (termed muCANP and mCANP) are known in mammalian tissues. These two isozymes consist of different large (80-kDa) subunits (mu- or m-types) and identical small (30-kDa) subunits. By screening human and rat muscle cDNA libraries with a cDNA probe for the chicken CANP large subunit, which has a structure similar to both the mammalian mu- and m-types, a cDNA clone encoding a novel member of the CANP large subunit family was obtained. The encoded protein (designated "p94") consists of 821 amino acid residues (Mr 94,084) and shows significant sequence homology with both human mu-type (54%) and m-type (51%) large subunits. p94 can be divided into four domains (I-IV) as reported for the CANP large subunit family. Domains II and IV are potential cysteine protease and calcium-binding domains, respectively, and have sequences homologous to the corresponding domains of other CANP large subunits. However, domain I of p94 is significantly different from others. Moreover, p94 contains two unique sequences of 62 and 77 residues in domains II and III, respectively. In contrast to the ubiquitous expression of mu- and m-types, Northern blot analysis revealed that the mRNA for p94 exists only in skeletal muscle with none detected in other tissues including heart muscle and smooth muscles such as intestine.

Amino Acid Sequence↗
PubMed1989

Tissue- and cell type-specific expression of mRNAs for four types of inositol phospholipid-specific phospholipase C.

The mRNA levels for four types of inositol phospholipid-specific phospholipase C (PLC) in various tissues and cell cultures have been studied by Northern analysis using cDNA probes for PLC isozyme I, II, and III [Sue, P.-G., Ryu, S.H., Moon, K.H., Sue, H.W., and Rhee, S.G. (1988) Proc. Natl. Acad. Sci. USA 85, 5419-5423 and Cell 54, 161-169], and the recently identified isozyme IV. All four types are ubiquitously expressed in rat tissues, but the levels of the mRNAs vary among tissues and cell lines. PLC-I mRNA levels are extremely high in brain and rat C6 glioma cells with lower levels in other tissues tested. PLC-II and -III have a more widespread distribution, with relatively high levels in brain, lung, spleen, thymus, and testis in the case of PLC-II, and in skeletal muscle, spleen, and testis for PLC-III. PLC-II and -III mRNAs were also detected in all cell lines examined except human promyelocytic HL60 cells. PLC-IV mRNA levels are extraordinarily high in spleen and HL60 cells. These results indicate that rat C6 glioma cells, together with most rat tissues, contain all four PLC isozymes. Other cultured cell types examined also contain two or three PLC isozymes except for HL60 cells, which contain only PLC-IV. The concomitant expression of PLC isozymes in cultured cells suggests a diverse function for PLC isozymes in single cells.

Animals↗
PubMed1989

Cloning and sequence analysis of the genomic DNA fragment encoding oryzacystatin.

A genomic DNA clone encoding oryzacystatin (Oc), a cysteine proteinase inhibitor (cystatin) of rice, was isolated from a lambda EMBL3 phage library constructed with Sau3AI partial digests of rice chromosomal DNA, by screening with an oc cDNA as a probe. The restriction map of the isolated DNA fragment was consistent with the pattern of the genomic Southern-blot analysis using a cDNA probe, and consequently, the gene is considered to be a single-copy gene. The oc gene is about 1.4 kb long and composed of three exons and two introns. The first intron (336 bp) intervenes between Ala-38 and Asp-39. The second intron (372 bp) exists in the 3'-noncoding region at the G residue next to the stop codon. S1 nuclease mapping showed the major transcription start point (tsp) at A, 104 bp upstream from the start codon (ATG). Typical CAT and TATA box sequences were found in the 5'-upstream region of the tsp. The nucleotide sequences around the TATA box, the tsp, the start codon, and the stop codon essentially matched the consensus sequences of other higher plant genes. The intron boundaries of the oc gene were quite different from those of the human kininogen-encoding gene and the human salivary cystatin (cystatin S)-encoding gene.

Amino Acid Sequence↗
PubMed1989

Measurement of postoperative intraocular lens tilting and decentration using Scheimpflug images.

A three-dimensional, in vivo method for determining the position of implanted intraocular lenses (IOLs) using an image-processing technique has been developed. Two images of the anterior segment of the eye with an implanted IOL were photographed with a Scheimpflug camera at slit-lamp angles of 90 degrees and 180 degrees. After geometrical correction, the contours of the original images were enhanced using binarization and curve-fitting techniques. These images afforded calculation of the angle of tilt of the IOL optic axis relative to a standard reference line which connected the center of anterior corneal surface curvature with the geometrical center of the pupil. The extent and direction of IOL decentration could also be shown. The IOL image in the posterior chamber was expressed as a wire-frame figure. This method provides several kinds of biometry in pseudophakic eyes.

Anterior Eye Segment↗
PubMed1989

Identification and characterization of inhibitory sequences in four repeating domains of the endogenous inhibitor for calcium-dependent protease.

We reported previously the cDNA cloning of the endogenous inhibitor for calcium-dependent protease (CANP inhibitor, calpastatin) and the expression of its fragments in Escherichia coli. The CANP inhibitor has four internal repeating domains each spanning about 140 amino acid residues. The inhibitory activity arises from these domains which have a well-conserved sequence, TIPPXYR, in their central positions. The inhibitory activities of various fragments expressed in E. coli suggest the involvement of the regions around the well-conserved sequences. In this report, we describe further detailed investigation on the interaction site of the CANP inhibitor with CANP by truncating inhibitor fragments and by using chemically synthesized peptides. The results clearly indicate that the sequence around the well-conserved sequence, TIPPXYR, is an interaction site. A peptide as short as 23 amino acid residues retained inhibitory activity, but a 9-residue peptide corresponding to the conserved sequence, VTIPPKYRE had none. The inhibitory sequence is suggested as LGXKDREXTIPPXYRXLL. The analysis of the competition between an inhibitor peptide and an irreversible inhibitor, E-64 for the reaction with the active site suggests no involvement of the active site cysteine residue of CANP in the inhibitory interaction between CANP and the CANP inhibitor. The high specificity of the CANP inhibitor to CANP arises from its interaction with residues other than the active site cysteine residue, possibly the subsite for substrate-binding of CANP.

Amino Acid Sequence↗
PubMed1988

Purification of two distinct types of phosphoinositide-specific phospholipase C from rat liver. Enzymological and structural studies.

Two kinds of phosphoinositide-specific phospholipase C (PLC) were purified from rat liver by acid precipitation and several steps of column chromatography. About 50% of the activity could be precipitated when the pH of the liver homogenate was lowered to pH 4.7. The redissolved precipitate yielded two peaks, PLC I and PLC II, in an Affi-gel Blue column, and each was further purified to homogeneity by three sequential h.p.l.c. steps, which were different for the two enzymes. The purified PLC I and PLC II had estimated Mr values of 140,000 and 71,000 respectively on SDS/polyacrylamide-gel electrophoresis. Both enzymes hydrolysed phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) in a Ca2+- and pH-dependent manner. PLC I was most active at 10 microM- and 0.1 mM-Ca2+ for hydrolysis of PI and PIP2 respectively, whereas PLC II showed the highest activity at 5 mM- and 10 microM-Ca2+ for that of PI and PIP2 respectively. The optimal pH of the two enzymes also differed with substrates or Ca2+ concentration, in the range pH 5.0-6.0. Hydrolysis of phosphoinositides by these enzymes was completely inhibited by Hg2+ and was affected by other bivalent cations. From data obtained by peptide mapping and partial amino acid sequencing, it was clarified that PLC I and PLC II had distinct structures. Moreover, partial amino acid sequences of three proteolytic fragments of PLC I completely coincided with those of PLC-148 [Stahl, Ferenz, Kelleher, Kriz & Knopf (1988) Nature (London) 332, 269-272].

Amino Acid Sequence↗
PubMed1988

Molecular cloning and characterization of cDNAs coding for apo-polysialoglycoprotein of rainbow trout eggs. Multiple mRNA species transcribed from multiple genes contain diverged numbers of exact 39-base (13-amino acid) repeats.

Polysialoglycoprotein (PSGP) of unfertilized eggs of rainbow trout (Salmo gairdneri) consists of tandem repeats (about 25) of a glycotridecapeptide, Asp-Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Gly (* denotes the attachment site of a polysialoglycan chain) (Kitajima, K., Inoue, Y., and Inoue, S. (1986) J. Biol. Chem. 261, 5262-5269). By using oligodeoxynucleotide probes based on the above sequence, we isolated a genomic clone for apoPSGP which contains 39-base pair repeats (5'-GACGACGCCACCTCTGAAGCT-GCGACCGGCCCGTCTGGC-3') encoding the tridecapeptide. Using a fragment of this genomic DNA as a probe, we next screened a cDNA library constructed with mRNA from immature ovaries of rainbow trout. Nucleotide sequencing analyses of cDNA clones thus obtained revealed that apoPSGP is encoded by multiple mRNA species consisting of diverged numbers (6-32) of the 39-base repeat encoding the tridecapeptide unit and homologous 5'- and 3'-bordering regions. The encoded protein consists of three distinct regions: the N-region consisting of a putative signal peptide and a pro-peptide, the R-region containing diverged numbers of the tandem repeat of 13-amino acid residues, and the C-region with six amino acid residues. Southern blot analysis showed that multiple mRNAs are transcribed from multiple genes for apoPSGP containing diverged numbers of the 39-base pair repeat. Thus, the genes for apoPSGP constitute a multigene family. Expression of the mRNAs is stage and organ specific, i.e. they are expressed only in immature ovaries and not in mature ovaries or in any other organ.

Amino Acid Sequence↗
PubMed1988

Molecular cloning of the cDNA for the large subunit of the high-Ca2+-requiring form of human Ca2+-activated neutral protease.

A nearly full-length cDNA clone for the large subunit of high-Ca2+-requiring Ca2+-activated neutral protease (mCANP) from human tissues has been isolated. The deduced protein, determined for the first time as an mCANP, has essentially the same structural features as those revealed previously for the large subunits of the low-Ca2+-requiring form (muCANP) [Aoki, K., Imajoh, S., Ohno, S., Emori, Y., Koike, M., Kosaki, G., & Suzuki, K. (1986) FEBS Lett. 205, 313-317] and chicken CANP [Ohno, S., Emori, Y., Imajoh, S., Kawasaki, H., Kisaragi, M., & Suzuki, K. (1984) Nature (London) 312, 566-570]. Namely, the protein, comprising 700 amino acid residues, is characterized by four domains, containing a cysteine protease like domain and a Ca2+-binding domain. The overall amino acid sequence similarities of the mCANP large subunit with those of human muCANP and chicken CANP are 62% and 66%, respectively. These values are slightly lower than that observed between muCANP and chicken CANP (70%). Local sequence similarities vary with the domain, 73-78% in the cysteine protease like domain and 48-65% in the Ca2+-binding domain. These results suggest that CANPs with different Ca2+ sensitivities share a common evolutionary origin and that their regulatory mechanisms are similar except for the Ca2+ concentrations required for activation.

Amino Acid Sequence↗
PubMed1988

The NH2-terminal 21 amino acid residues are not essential for the papain-inhibitory activity of oryzacystatin, a member of the cystatin superfamily. Expression of oryzacystatin cDNA and its truncated fragments in Escherichia coli.

Oryzacystatin, a proteinaceous cysteine proteinase inhibitor (cystatin) in rice, is comprised of 102 residues (Met1-Ala102) (Abe, K., Emori, Y., Kondo, H., Suzuki, K., and Arai, S. (1987) J. Biol. Chem. 262, 16793-16797). We constructed an expression plasmid containing a full length oryzacystatin cDNA at the multi-cloning site of pUC18 and produced a lacZ'-oryzacystatin fusion protein in Escherichia coli. The partially purified expressed protein efficiently inhibits papain activity assayed using N-benzoyl-DL-arginine-2-naphthylamide as a substrate. We also constructed expression plasmids lacking the 5'- and 3'-regions of cDNAs that encode NH2- and COOH-terminally truncated oryzacystatins. An N-truncated oryzacystatin lacking Gly5 and retaining Gln53-Val54-Val55-Ala56-Gly57 inhibited papain as efficiently as the full length oryzacystatin, although both Gly5 and Gln53-Gly57 (oryzacystatin numbering) are conserved among members of most cystatin superfamilies. However, another N-truncated oryzacystatin lacking the NH2-terminal 38 residues was almost completely inactive. On the other hand, a COOH-terminally truncated oryzacystatin lacking the COOH-terminal 11 residues possesses potent papain-inhibitory activity, whereas another COOH-terminally truncated oryzacystatin lacking 35 residues shows much less inhibitory activity, although it retains the two well conserved features Gly5 and Gln53-Gly57. These results indicate that the NH2-terminal 21 residues containing Gly5 and the COOH-terminal 11 residues are not essential, suggesting that a portion of the polypeptide segment containing Gln53-Gly57 is necessary for oryzacystatin to elicite its papain-inhibitory activity efficiently.

Amino Acid Sequence↗
PubMed1988

Expression of human leukotriene A4 hydrolase cDNA in Escherichia coli.

The cDNA clone encoding human leukotriene A4 hydrolase was inserted into a vector pUC9 and expressed in Escherichia coli as a fusion protein containing the first 10 amino acid residues derived from a vector. The leukotriene A4 hydrolase activity was recovered in the soluble fraction of the transformants. The purified enzyme showed kinetic properties similar to the native enzyme, including inactivation by the substrate and sulfhydryl-modifying reagents. The results demonstrate that a protein with an Mr of 70,000 was expressed in Escherichia coli with a full enzyme activity and structural fidelity. Acquisition of the expression system makes it feasible to elucidate the reaction mechanism of the enzyme.

Cloning, Molecular↗
PubMed1988

All four repeating domains of the endogenous inhibitor for calcium-dependent protease independently retain inhibitory activity. Expression of the cDNA fragments in Escherichia coli.

We have already determined the primary structure of the endogenous inhibitor for calcium-dependent protease (CANP inhibitor, calpastatin) from the cDNA sequence and revealed that the CANP inhibitor contains four internally repeating units which could be responsible for its multiple reactive sites (Emori, Y., Kawasaki, H., Imajoh, S., Imahori, K., and Suzuki, K. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 3590-3594). Restriction fragments of the cDNA corresponding to each of the four domains (encoding 104-156 amino acid residues of the total 718 residues) were subcloned into the multicloning site of pUC9 or pUC18 in a direction and frame matched to the lacZ' open reading frame of the vector. Under the lac operator-promoter system, we succeeded in producing truncated fragments of the CANP inhibitor in Escherichia coli. The CANP inhibitor fragments were partially purified, and the inhibitory activities toward calcium-dependent protease (CANP) were examined. All fragments containing well conserved regions of about 30 amino acid residues (domains I-IV) located in the middle of the four units exhibited the inhibitory activity. However, their inhibitory activities varied considerably. Further truncation experiments revealed that small fragments containing 30-70 amino acid residues of the CANP inhibitor still retained inhibitory activity. From these experimental results the following conclusions can be drawn: 1) each of the four repeating units of the CANP inhibitor (about 140 amino acid residues) is a real functional unit and can inhibit CANP activity independently; and 2) domains corresponding to well conserved sequences of about 30 amino acid residues containing a consensus Thr-Ile-Pro-Pro-X-Tyr-Arg sequence are essential for the inhibitory activity, and the bordering regions are important for its modulation.

Amino Acid Sequence↗
PubMed1988

Regulation of activity of calcium activated neutral protease.

Various lines of evidence suggest that calcium dependent protease (CANP, calpain) exists in the cytosol as an inactive proenzyme which is converted to an active form by autolysis. During autolysis only the N-terminal regions of both subunits of proCANP are modified. About 20 and 90 residues are removed from the large and small subunits, respectively. The N-terminal region (domain I) of the large subunit modified during autolysis precedes the protease domain and corresponds to the propeptides of various cysteine proteinases. Analyses of the autocatalytic activation of CANP in the presence of plasma membranes reveal that proCANP translocates to the membrane in the presence of microM Ca2+ and is activated at the membrane. The CANP inhibitor and Ca2+ are the most important factors for the regulation of CANP activity. The primary translation product of the mRNA for rabbit liver CANP inhibitor contains four internal repeats. Structural analyses of the liver and erythrocyte inhibitors reveal that they contain four and three repeats, respectively. The repeating unit was identified as the functional unit of the inhibitor and each unit inhibits one mole of CANP. On the basis of these results, an activation mechanism for proCANP at the membrane was proposed. The native enzyme, which has been called CANP or calpain, should now be called proCANP or calpainogen. CANP and calpain should be used for the autolyzed active form.

Animals↗
PubMed1988

Carboxyl-terminal truncation and site-directed mutagenesis of the EF hand structure-domain of the small subunit of rabbit calcium-dependent protease.

A mutant of the small subunit of rabbit calcium-dependent protease lacking the amino-terminal one-fourth produced in Escherichia coli could associate with the native large subunit to exert protease activity. Deletion of a few carboxyl-terminal residues of this variant small subunit caused a significant decrease in the protease activity after reconstitution with the native large subunit. Loss of the fourth EF hand loop region by further truncation of the variant small subunit made interaction with the large subunit impossible. The calcium binding assay revealed that the fourth EF hand structure of the rabbit small subunit, which has been previously demonstrated to possess two calcium-binding sites, can bind calcium ions. Furthermore it was established by site-directed mutagenesis that the first EF hand structure, in addition to the fourth one, is capable of binding calcium ions. Replacement of amino acids in the EF hand structure affected interaction with the native large subunit or the calcium sensitivity of the reconstituted product. These findings indicate that the EF hand structure-domain of the small subunit is essential for the full protease activity.

Amino Acid Sequence↗