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Y Ichihara

Publications and source records attributed to Y Ichihara.

At least 73 records · Page 4Linked to original sources
PubMed1986

Nucleotide sequence of a nearly full-length cDNA coding for pepsinogen of rat gastric mucosa.

A nearly full-length rat pepsinogen cDNA was isolated from a rat gastric mucosa cDNA library and its nucleotide sequence was determined. The cDNA comprises 1370 base pairs (bp), including the 5'-non-coding region (60 bp), the coding nucleotide sequence (1176 bp) and the 3'-non-coding region (131 bp). The predicted amino acid sequence of rat prepepsinogen (392 residues) contains a 16-residue signal sequence followed by the pepsionogen moiety of 376 residues. Rat pepsinogen has an amino acid composition characteristic of C-type pepsinogens and is much more homologous in amino acid sequence with C-type pepsinogens than A-type pepsinogens. These results indicate that the major form of rat pepsinogen can be classified as a C type pepsinogen.

Amino Acid Sequence↗
PubMed1986

Close linkage of human chromosomal pepsinogen A genes.

We have obtained a clone containing two pepsinogen A genes in a single insert by screening a recombinant cosmid library for human genomic DNA. Restriction endonuclease mappings of this cloned DNA showed that these two genes are very similar, but distinct in structure, and that they are closely linked to one another in the human chromosome DNA. The close arrangement of the genes with very similar structures could facilitate the homologous recombination or the unequal crossing-over which accounts for high frequency of haplotype variation in copy number of pepsinogen A genes as reported by Taggart et al.

Cloning, Molecular↗
PubMed1985

Isolation of human, swine, and rat prepepsinogens and calf preprochymosin, and determination of the primary structures of their NH2-terminal signal sequences.

The total RNAs were extracted from human, swine, rat, and calf gastric mucosae, and translated in vitro in the presence of radiolabeled amino acids using a wheat germ cell-free system. Upon sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the translation products, a protein band with a molecular weight of about 43,000 was obtained in each case as one of the major products. These products could be specifically immunoprecipitated with a corresponding anti-pepsinogen or anti-chymosin antiserum. Radiosequence analysis of these translation products purified by SDS-polyacrylamide gel electrophoresis showed that each of them is a precursor form, i.e., prepepsinogen or preprochymosin, having an amino-terminal extension peptide (signal sequence) comprising 15 (human and swine) or 16 (rat and calf) amino acid residues. The primary structures of these signal sequences were determined to be as follows: (Sequence: see text). These signal sequences share common characteristics with those of other pre-secretory proteins, i.e., the presence of positive charges in the NH2-terminal region, hydrophobic amino acid clusters in the interior part, and amino acids with short side chains at the site of cleavage by the signal peptidase.

Amino Acid Sequence↗
PubMed1983

Primary structure of human pepsinogen gene.

A recombinant clone, which covers the pepsinogen gene in a single insert, has been isolated by screening a library of human genomic DNA, using a swine pepsinogen cDNA as a probe. Sequence analysis of coding DNA segments of the clone revealed that the pepsinogen gene occupies approximately 9.4-kilobase pairs of the genomic DNA and is separated into nine exons by eight introns of various lengths. The predicted amino acid sequence of human pepsinogen consists of 373 residues and is 82% homologous with that of swine pepsinogen. In addition, the predicted sequence contained a single sequence of 15 amino acid residues at the NH2 terminus, showing that the protein is synthesized as prepepsinogen. The structure of the gene, in which two homologous sequences including the two active site aspartyl residues of pepsin are present in different coding segments, is in support of the view that the pepsinogen gene evolved by duplication of a shorter ancestral gene.

Amino Acid Sequence↗
PubMed1983

Purification and characterization of a calcium-activated neutral protease from monkey cardiac muscle.

A calcium-activated neutral protease (CANP) was purified from monkey cardiac muscle by a method involving column chromatography on DEAE-cellulose, Sepharose CL-6B, DEAE-Sephacel, organomercurial-Sepharose 4B, and Sephadex G-150 in succession. This protease required both millimolar concentration of Ca2+ and the SH-group for activation, and it was maximally active around pH 8.0. It was strongly inhibited by thiol protease inhibitors such as iodoacetic acid, antipain, leupeptin, and epoxysuccinic acid derivatives. The molecular weight of this protease was estimated to be 110,000 by gel filtration. Upon nondenaturing electrophoresis the purified protease gave two bands, both of which were active at millimolar concentration of Ca2+, indicating the existence of two forms of the protease. The less acidic band (form I CANP) contained two components with molecular weights of 74,000 and 28,000 and the more acidic one (form II CANP) contained components with molecular weights of 74,000 and 26,000. The protease was synergistically activated by Mn2+ and Ca2+ at a concentration where Mn2+ or Ca2+ alone was not effective. In the presence of millimolar level of Ca2+, limited autolysis reduced the Ca2+-requirement of this protease. The proteolysis of myofibrils by this protease resulted in the production of a component with a molecular weight of 30,000 as well as various other higher and lower molecular weight peptide fragments.

Animals↗
PubMed1982

Occurrence and some properties of membrane-bound neutral proteinase in the microsomal fraction of rat skeletal muscle.

Membrane-bound neutral proteinase was found in the microsomal fraction of rat skeletal muscle as assayed with heat-denatured casein as a substrate. The enzyme was solubilized from 1 M KC1-washed microsomal fraction by 1% sodium cholate containing 0.1 M NaCl, and partially purified by chromatography on a column of Sepharose CL-6B in the presence of 0.5% sodium cholate and 0.1 M NaCl. The enzyme was eluted from the Sepharose column as a single but rather broad peak at a position corresponding to a molecular weight of about 190,000. The pH optimum for hydrolysis of heat-denatured casein was about 8.0. It was inhibited to significant extents by various reagents including diisopropyl phosphorofluoridate, phenylmethanesulfonyl fluoride, N alpha-tosyl-L-phenylalanine chloromethyl ketone, N alpha-tosyl-L-lysine chloromethyl ketone, p-chloromercuriphenyl sulfonate, chymostatin, EDTA, EGTA, and o-phenanthroline. This inhibition profile suggests that the present muscle proteinase is a mixture of proteinases, such as serine proteinase and a metallo-proteinase similar to those occurring in the microsomal membranes of liver and kidney (or small intestine), respectively. Among urea-denatured proteins tested as substrates, calf thymus histone was hydrolyzed most rapidly, followed by protamine, hemoglobin, and casein.

Animals↗
PubMed1982

A simple and sensitive proteinase assay using Sepharose 4B-coupled fluorescamine-labeled casein as a substrate.

A simple and sensitive method for proteinase assay was developed, which uses fluorescamine-labeled casein-Sepharose 4B as a substrate. Casein-Sepharose 4B was prepared most effectively by coupling casein to cyanogen bromide-activated Sepharose 4B at pH 10.0. Fluorescamine-labeled casein-Sepharose 4B was then prepared by mixing fluorescamine and casein-Sepharose 4B suspension at pH 8.0 and used for the assay as a substrate after removal of the excess reagent and/or its hydrolysis products. The assay can be done by simple measuring the fluorescence (excitation at 390 nm and emission at 475 nm) of the filtrate of the assay mixture after incubation of the substrate with enzyme solution. This method is suited for the assay of proteinases active at neutral to slightly alkaline pH values, and the activity of 3 ng of trypsin or 10 ng of alpha-chymotrypsin can be determined with reasonable accuracy. This method is therefore almost as sensitive as those using radioisotope-labeled proteins as substrates.

Animals↗
PubMed1982

Rat gastric prepepsinogen: in vitro synthesis and partial amino-terminal signal sequence.

The major translation product of rat gastric mucosa RNA in a wheat germ cell-free system was identified as prepepsinogen by electrophoretic analysis of its immunoprecipitate on sodium dodecyl sulfate (SDS)-polyacrylamide gels and amino-terminal sequence determination. The translation product containing radioactive amino acids, purified by SDS-polyacrylamide gel electrophoresis, was shown to have an amino-terminal extension peptide comprising 16 amino acid residues. A partial amino acid sequence of this extension peptide is as follows: Met-X-X-Met-Val-Val-X-Leu-Leu-X-Leu-X-Leu-Leu-X-X-pepsinogen.

Amino Acid Sequence↗
PubMed1981

Molecular cloning of complementary DNA to swine pepsinogen mRNA.

Poly(A)-containing RNA was isolated from total RNA of swine gastric mucosa by oligo(dT)-cellulose chromatography, and was translated in a wheat germ cell-free system. Analysis of the translation products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that pepsinogen was a major translation product and was synthesized as two different molecular forms with apparent molecular weights of 45,000 and 43,000. The pepsinogen translated in the in vitro translation system had no autocatalytic activity. The pepsinogen mRNA was further purified by sucrose density gradient centrifugation, where the mRNA activity for pepsinogen was located around 15 S. At this stage, the translation product of the pooled fractions appeared to be almost exclusively pepsinogen. The peptide maps of the pepsinogen which was translated in vitro and digested by alpha-chymotrypsin and by Staphylococcus aureus V8 protease were nearly identical with the corresponding peptide maps of standard pepsinogen. The double-stranded complementary DNA which had been synthesized from the partially purified mRNA by avian myeloblastosis virus reverse transcriptase was cloned in Escherichia coli chi 1776, using plasmid pBR322 as a cloning vector. A colony carrying pepsinogen cDNA sequence was identified by in situ colony hybridization using the cDNA synthesized from the partially purified mRNA as a probe and further by a positive hybridization-translation assay. One of the recombinant clones (pSPcA1) had an insert of about 850 base pairs, and the nucleotide sequence analysis revealed that pSPcA1 codes for swine pepsinogen.

Animals↗
PubMed1981

Comparison of some characteristics of membrane-bound neutral proteinase activity in the microsomal fractions of rat kidney and small intestine.

Some characteristics of the membrane-bound neutral proteinase activity in the microsomal fractions of rat kidney and small intestine were compared, using heat-denatured casein as a substrate. The proteinases of both kidney and small intestine showed maximal activity between pH 8.0 and 8.5, and were strongly inhibited by EDTA, o-phenanthroline, p-chloromercuriphenyl sulfonate, dithiothreitol, and chymostatin. Phosphoramidon and other reagents tested, including deoxycholate and bestatin, which strongly inhibited the contaminating aminopeptidase activity, were without marked effect on the neutral proteinase activity. Among urea-denatured proteins tested as substrates, casein, histone, and hemoglobin were hydrolyzed rapidly by both proteinase preparations. Fibrinogen was a good substrate for the kidney enzyme whereas it was not hydrolyzed well by the small intestine proteinase. On the other hand, serum albumin was hydrolyzed well by the small intestine proteinase, but not by the kidney proteinase. These results indicate that the neutral proteinase activity of the microsomal membrane fractions is largely due to metalloproteinases, which are quite similar, but not identical, in the kidney and small intestine.

Animals↗