Search PubMed⌕ Search

Biomedical subjects

K Murakami

Publications and source records attributed to K Murakami.

At least 829 records · Page 46Linked to original sources
PubMed1991

[Health examination in school age children].

Within recent years, various medical examinations have been performed on school age children. The purpose of the school examination is early detection of disease, but those with abnormal results should receive pertinent care (electrocardiogram, phonocardiogram, complete blood count, total cholesterol, and urinalysis). Therefore we analyzed the results obtained by questionnaire method on the management of the school age children with abnormal examination results.

Adolescent↗
PubMed1990

Structure and expression of mouse furin, a yeast Kex2-related protease. Lack of processing of coexpressed prorenin in GH4C1 cells.

We have cloned and sequenced a mouse cDNA encoding the 793-residue amino acid sequence of furin, which is a protein homologous to the yeast Kex2 protease. The entire sequence is 94% identical to that of human furin, and it contains the 289-residue sequence of the subtilisin-like catalytic domain. Within this region, 99, 64, and 53% of the amino acids are identical to those of human furin, human PC2 (the other mammalian Kex2-like protein), and yeast Kex2, respectively. It has been proposed that furin is a mammalian prohormone processing enzyme which cleaves precursors at paired basic amino acids, based on the fact that the Kex2 protease is responsible for processing of alpha-mating factor and killer toxin precursors at dibasic sites. However, Northern blot analysis has revealed that a furin mRNA transcript is present in all tested mouse tissues and culture cell lines, including those known not to process prohormones. Moreover, when furin and a prohormone, prorenin, have been coexpressed in mammalian cells by DNA transfection, no processing has been observed. These observations fail to show a role for furin, a Kex2-like mammalian protease, in prohormone processing.

Amino Acid Sequence↗
PubMed1990

Substrate specificity of prorenin converting enzyme of mouse submandibular gland. Analysis using site-directed mutagenesis.

Renin is produced from a larger, inactive precursor, prorenin, through endoproteolytic cleavage at paired basic amino acids. Recently, we have purified and characterized an enzyme, which catalyzes the endoproteolytic process, from mouse submandibular gland. The enzyme, named prorenin converting enzyme, specifically cleaves the peptide bond on the COOH-side of the Arg residue at the Lys-Arg pair of mouse Ren 2 prorenin, but does not cleave mouse Ren 1 and human prorenins. In this study, by synthesizing a series of mutant mouse prorenins using site-directed mutagenesis and the Xenopus oocyte expression system, we have investigated the role of the basic pair as the recognition signal for the enzyme as well as the determinant of the substrate specificity. The results indicate that the basic amino acid at the COOH-side but not at the NH2-side of the basic pair of Ren 2 prorenin is essential for processing directed by prorenin converting enzyme, and that the Arg residue at the COOH-side is more preferable for processing than the Lys. The results also demonstrated that the presence of a Pro residue next to the Lys-Arg pair prevents the processing of Ren 1 prorenin.

Amino Acid Sequence↗
PubMed1990

A simple enzyme-linked immunoassay of human carbonic anhydrase I for the study of developing erythroid cells.

A simple and rapid enzyme-linked immunoassay for human erythrocyte carbonic anhydrase isozyme I was developed. The assay was found to be sensitive enough for the detection of nanogram amounts of the enzyme in incubation mixtures. The first incubation with anti-human carbonic anhydrase I IgG was carried out for 6 hours at room temperature. The second incubation of the enzyme was carried out in the presence of goat plasma C1q coupled with peroxidase for 1 h at room temperature. The enzymatic reaction was performed for 30 min using 2,2'-azino-di(3-ethyl-benzthiazoline sulfonate) as a substrate, and absorbance at 414 nm was recorded. Carbonic anhydrase I was assayed on the range of 1 to 200 ng/ml using this method. The levels of carbonic anhydrase I in K562 cells induced by erythropoietin and in other blood cells were determined. This enzyme-linked immunoassay has application for the study of developing erythroid cells.

Antibodies, Monoclonal↗
PubMed1990

Isolation of anti-endothelin receptor monoclonal antibodies for use in receptor characterization.

Monoclonal antibodies reactive with endothelin (ET) receptors have been prepared by immunization of mice with rat lung membranes. Of four clones isolated, three clones preferentially recognized 32,000-dalton ET receptor and the other has a higher affinity for the 45,000-dalton receptor. The binding of 125I-ET-1 to detergent-solubilized ET receptors which were adsorbed to the antibodies was displaced by increasing concentrations of unlabeled ET isopeptides. These results demonstrate that the four clones specific for the receptor have the potential to be a useful tool in the characterization of ET receptors.

Animals↗
PubMed1990

Identification and localization of two triad junctional foot protein isoforms in mature avian fast twitch skeletal muscle.

We report evidence for two foot protein isoforms in chicken pectoral muscle. (i) Two polypeptides with molecular masses of approximately 500 kDa copurify with [3H]ryanodine binding. (ii) Both polypeptides are associated with oligomeric proteins similar in size to the mammalian skeletal muscle foot protein. (iii) The polypeptides are shown to be unique by limited proteolysis. (iv) By using isoform-specific antibodies, the polypeptides are shown to be subunits of different [3H]ryanodine-binding proteins. Using immunolabeling techniques, we have localized these proteins in chicken breast muscle by both light and electron microscopy. (v) From immunofluorescent light microscopy of longitudinal sections, it was determined that both ryanodine-binding protein isoforms exhibit identical repetitive punctate distributions near the Z-lines. (vi) In serial cross-sections both proteins have similar distributions in the same fibers. (vii) Both proteins were found to be associated with the terminal cisternae of the sarcoplasmic reticulum by immunoelectron microscopy. Based on their localization to the triadic junction, their large size and their ability to bind [3H]ryanodine, these proteins are identified as foot proteins. In conclusion, two distinct homo-oligomeric foot proteins coexist in avian fast twitch skeletal muscle. We have termed these proteins, alpha and beta foot proteins.

Alkaloids↗
PubMed1990

The influence of glycosylation on the fate of renin expressed in Xenopus oocytes.

It has been recently reported that, in Xenopus oocytes injected with the mRNA for human renin, this secretory renal glycoprotein acquires phosphomannosyl residues on its asparagine-linked oligosaccharide chains, remains intracellular and undergoes a proteolytic cleavage which removes the prosegment. To understand the influence of glycosylation on the fate of renin in Xenopus oocytes and whether it is specific for human renin, we have expressed human renin and mouse Ren1 renin, which are glycosylated at two and three selected asparagine residues, respectively, and mouse Ren2 renin, which is not glycosylated, in Xenopus oocytes. The majority of human and Ren1 renins remained intracellular and underwent proteolytic cleavage, whereas mouse Ren2 renin was secreted efficiently. When human and Ren1 renins were expressed in oocytes treated with tunicamycin, both were secreted efficiently. A mutant of human renin, which had amino-acid substitutions at both glycosylation sites, was also secreted efficiently, whereas that mutated at one of the two sites was not. These results indicate that the majority of all of the glycosylated renin molecules remain intracellular and undergo proteolytic cleavage, probably due to the acquisition of phosphomannosyl residues, and the human renin remains intracellular if it is only glycosylated at one of the two sites.

Amino Acid Sequence↗
PubMed1990

Effects of propeptide deletion on human renin secretion from mouse pituitary AtT-20 cells.

To study the role of the N-terminal propeptide in the secretory process of renin, mouse pituitary AtT-20 cells were transfected with expression plasmids of human preprorenin and a mutant deleted of its propeptide. The transfectant of the native construct secreted inactive prorenin and active renin, and renin secretion was stimulated by a secretagogue, 8-Br-cAMP. On the contrary, the transfectant of the deleted construct secreted only active renin, whose release was also stimulated by the secretagogue. The amount of renin molecule secreted from the latter transfectant was lower than that from the former one, although a significant amount of fully active renin could be produced. These results suggest that the propeptide plays an important role in the secretory process of renin, probably folding and/or stabilizing the renin molecule, but it does not contain the signal for intracellular sorting to target renin to secretory granules.

Animals↗
PubMed1990

Structure and expression of the human angiotensinogen gene. Identification of a unique and highly active promoter.

We have isolated the human angiotensinogen gene from a genomic library and determined the exon-intron junction sequences. The gene is 12 kilobases long and consists of five exons interrupted by four introns, as a single copy in the human genome. Of particular interest are the positions of the introns in the human angiotensinogen gene which are identical to those in the highly homologous human alpha 1-antitrypsin and alpha 1-antichymotrypsin genes, as well as rat and mouse angiotensinogen genes. Northern blot analysis showed that human hepatoma cells (HepG2) produce a large amount of angiotensinogen mRNA but not human glioma cells (T98G). To assay the promoter activity, the 1.3-kilobase genomic fragment containing the 5'-flanking region, first exon, and a part of first intron at positions -1222 to +44 was fused upstream to the chloramphenicol acetyltransferase gene, then transfected into HepG2 and T98G cells. The gene sequence was active only in HepG2 cells, suggesting the presence of a functional promoter. Analysis of deletion mutants demonstrated that the 76-base pairs region from -32 to +44 containing the TATA box and first exon is the minimal promoter, whose activity is as high as that of the SV40 enhancer-promoter. Since the basal expression of the human angiotensinogen gene is much higher in HepG2 than T98G cells, these results may reflect cell-specific differences in the gene transcription.

Amino Acid Sequence↗
PubMed1990

Modification of glycophorin A during oxidation of erythrocyte membrane.

Human erythrocyte ghosts were oxidized with tert-butyl hydroperoxide and subsequently treated with tritiated borohydride to label the membrane proteins modified during the membrane oxidation. From the ghosts, oxidized-and-tritiated glycophorin A was isolated and characterized. No intermolecular cross-links were observed as analyzed by sodium dodecylsulfate gel electrophoresis. But, the number of lysine residues was significantly reduced and susceptibility to proteinases such as trypsin, chymotrypsin and pronase was lower than that of control glycophorin A. Trypsinization of the oxidized-and-tritiated glycophorin A gave insoluble and soluble trypsin fragments. After dansylation, N-terminal amino acids of the trypsin-fragments were determined. Dansyl amino acids from the insoluble trypsin fragments were not identical with those from control insoluble counterparts in the membrane-spanning region of glycophorin A molecule. Fractionation by gel filtration of dansyl-soluble trypsin fragments, and the N-terminal amino acid analysis of the fractionated peptides indicated that the peptides derived from the glycosylated region located in the outside of the membrane matrix were identical with those from control soluble counterparts. The results suggest that the glycosylated outside region of glycophorin A was modified only slightly but the hydrophobic membrane-spanning region was extensively modified during membrane oxidation, most likely by oxidized lipids.

Amino Acids↗
PubMed1990

A processing enzyme for prorenin in mouse submandibular gland. Purification and characterization.

Renin is produced from an inactive precursor, prorenin, through proteolytic cleavage at paired basic amino acid residues. In this study, an enzyme which specifically cleaves mouse Ren 2 prorenin at the paired basic residues has been purified from mouse submandibular gland by CM-Toyopearl chromatography, antipain-Sepharose chromatography, and isoelectric focusing. This enzyme, named prorenin converting enzyme, consists of two polypeptide chains of 17 and 10 kDa. The enzyme has an isoelectric point of 9.5-9.8, and its pH optimum is between 7.5 and 8.5. It specifically cleaves the peptide bond on the carboxyl side of the Arg at the Lys-Arg pair of mouse Ren 2 prorenin to yield mature renin but does not cleave mouse Ren 1 and human prorenins. Studies on the effects of inhibitors indicate that this enzyme is a serine protease that differs from the enzymes processing other prohormones at paired basic amino acid residues.

Amino Acid Sequence↗
PubMed1990

An isozyme of hexokinase specific for the human red blood cell (HKR)

The hexokinase (HK) of the human red blood cell (RBC) was separated into two distinct major isozymes by fast protein liquid chromatography using a linear salt gradient on a MonoQ column. The first isozyme (HKI) eluted as a sharp peak at the same position as HKI of human liver. The second isozyme eluted between HKI and HKII of human white blood cells, and it appeared to be unique to the RBC (it was designated HKR). From a gel filtration column, HKR eluted before HKI, suggesting that it was larger than HKI by several kilodaltons. In a mitochondria-enriched fraction from human reticulocytes, no HKR was found; thus, HKR was not a mitochondrial enzyme. Despite these differences in chromatographic behavior, size, and mitochondrial binding, both forms behaved kinetically as HKI. RBC from normal blood contained HKI and HKR at an equal activity, but in reticulocyte-rich RBC, HKR dominated. When RBC of increasing age was separated by buoyant density ultracentrifugation, the total HK activity decayed in a biphasic manner, with half-lives respectively of approximately 15 and approximately 51 days. When isolated by MonoQ column from each age-separated fraction, HKR was the major form in the youngest RBC, and decreased rapidly with cell age, with a t 1/2 of approximately 10 days, representing a negligible activity in the oldest RBC. Instead, HKI was relatively stable through the entire life span of the RBC, with a t 1/2 of approximately 66 days. Thus, HKR appears to be an RBC-specific isozyme that is predominant in the reticulocyte and is then rapidly degraded. During maturation of the RBC, the fast decay of HKR contributes to the early sharp decline of HK activity and the slow decay of HKI to the later gradual decline.

Animals↗
PubMed1990

Affinity labelling of endothelin receptor and characterization of solubilized endothelin-endothelin-receptor complex.

Chick cardiac membranes were affinity labelled by cross-linking to membrane-bound 125I-endothelin-1 with disuccinimidyl tartarate. SDS/PAGE and autoradiographic analysis of the 125I-endothelin-1-labelled material in the presence or absence of 2-mercaptoethanol revealed one major labelled band, corresponding to a molecular mass of 53 kDa, whose appearance was dose-dependently inhibited by the addition of unlabelled endothelin-1 (1-100 nM). Subtracting the molecular mass of 125I-endothelin-1 and disuccinimidyl tartarate, the binding protein appeared to have a molecular mass of 50 kDa. To investigate further the molecular properties of endothelin receptor, the 125I-endothelin-1-endothelin-receptor complex was solubilized from chick cardiac membranes using the detergent digitonin. Sucrose gradient sedimentation of the solubilized complex indicated a sedimentation coefficient of 13 S, whereas the complex of (+)-[3H]PN200-110, a dihydropyridine derivative, and dihydropyridine-sensitive Ca2+ channels sedimented at 22 S. A monoclonal antibody raised against dihydropyridine-sensitive Ca2+ channels from the chick brain did not immunoprecipitate the 125I-endothelin-1-endothelin-receptor complex. These data suggest that endothelin receptor is clearly distinct from dihydropyridine-sensitive Ca2+ channels and endothelin has its own specific 50-kDa receptor.

Affinity Labels↗
PubMed1990

Renin immunohistochemistry in the adrenal gland of the mouse fetus and neonate.

The development of renin-containing cells in fetal and neonatal adrenal glands of the mouse was studied using immunohistochemistry. On days 13-14 of gestation, immunoreactivity for renin was first observed in a few cortical cells of the gland, appearing as small patchy or granular reaction products in the perikaryon. The mitotic configurations of the cells demonstrating immunoreactivity were noted. On day 16 of gestation, a number of intensively immunoreactive cells were distributed in the aortal side of the cortical zone. On day 18 of gestation, and day 1 postparturition, a small number of potent immunoreactive cells were still found in the cortical area. Immunoreactivity of the cytoplasm was observed in the cells, some showing an intensive reaction and others possessing numerous tiny granules just below the cell membrane. On days 3, 5, and 7 after birth, no renin-containing cells were found in the adrenal gland. The ratio of the numbers of renin-positive cells in certain areas to the numbers in the entire cortical area was significantly increased on day 16 of gestation, but there was no sexual difference in the ratios. The ratios were decreased subsequently until day 1 after birth. The possible significance of renin synthesis in specific adrenal cells in fetal life is discussed with respect to an important involvement of angiotensin II in the morphogenesis of the adrenal gland of the mouse.

Adrenal Glands↗
PubMed1990

Purified presequence binding factor (PBF) forms an import-competent complex with a purified mitochondrial precursor protein.

In vitro mitochondrial import of the purified precursor form (pOTC) of rat ornithine carbamoyltransferase (OTC) is stimulated by a cytosolic factor(s) contained in rabbit reticulocyte lysate. A protein factor that binds to pOTC but not to mature OTC and was named presequence binding factor or PBF, was purified 91,000-fold from the lysate by affinity chromatography using pOTC-bound Sepharose, DEAE-5PW HPLC and sucrose gradient centrifugation. The purified PBF migrated as a single polypeptide of 50,000 daltons on SDS-PAGE. On sucrose gradients, urea-denatured pOTC sedimented to the bottom, whereas PBF sedimented with an S20,w value of 5.5S. When pOTC and PBF were centrifuged together, both polypeptides sedimented as a complex of 7.1S. Formation of the pOTC-PBF complex was inhibited by micromolar concentrations of the synthetic presequence of pOTC and those of other mitochondrial precursor proteins. The purified PBF markedly stimulated the import of purified or in vitro synthesized pOTC into the mitochondria. PBF-stimulated pOTC import was further enhanced by a 70 kd heat shock protein (hsp 70) purified from yeast; the hsp70 alone had little effect. Thus, PBF binds to the presequence portion of the precursors and may hold them in a transport-competent form in cooperation with hsp70.

Animals↗
PubMed1990

Protein modeling of human prorenin using the molecular dynamics method.

To study the activation-inactivation mechanism of the renin zymogen, prorenin, a tertiary structural model of human prorenin was constructed using computer graphics and molecular dynamics calculations, based on the pepsinogen structure. This prorenin model shows that the folded prosegment polypeptide can fit into the substrate binding cleft of the renin moiety. The three positively charged residues, Arg10, Arg15, and Arg20, in the prosegment make salt bridges with Asp225, Glu331, and Asp60, respectively, in renin. Arg43, which is in the processing site, forms salt bridges with the catalytic residues of Asp81 and Asp269. These ionic interactions between the prosegment and the renin may contribute to keeping the prorenin structure as an inactive form.

Amino Acid Sequence↗
PubMed1990

Shift of anaerobic to aerobic metabolism in the rats acclimatized to hypoxia.

1. Metabolic acclimatization by repeated exposure to a simulated altitude of 4000, 5000 and 6000 m for 2 hr per day throughout 2 to 11 days was evaluated by the increased formation of ketone bodies as a marker of fatty acid oxidation and the decreased production of lactate and uric acid, the indicators of anaerobic metabolism in rats exposed to an altitude of 8000 m. 2. Pre-exposure of rats to an altitude of 5000 m and over caused an acclimatization to hypoxia. The rise of the altitude to which rats were pre-exposed reduced the period until the acquisition of metabolic acclimatization. 3. Acclimatized rats showed an increased activity of mitochondrial glutamate dehydrogenase without changes in glycolytic enzyme activity in skeletal muscle, heart and liver. 4. Acclimatization to high altitude hypoxia is concluded to involve a shift of the anaerobic glycolysis to aerobic metabolism by the increase in the oxidative enzymes.

Acclimatization↗