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N Muto

Publications and source records attributed to N Muto.

At least 91 records · Page 5Linked to original sources
PubMed1986

Stereochemistry of estrogen biosynthesis by a reconstituted aromatase cytochrome P-450 preparation from human placenta.

According to the literature, the multistep reaction mechanism of estrogen biosynthesis proceeds with stereospecific loss of the equatorial 1 beta-, and axial 2 beta-protons. These results were deduced from experiments carried out, either with crude microsomes, or at best with impure enzyme extracts. However, when [1,2- 3H]4-androstene-3,17-dione of known absolute 3H-label distribution was incubated with a reconstituted enzyme system, consisting of homogeneous NADPH-cytochrome P-450 reductase and highly purified aromatase, we obtained results that can only be logically explained by a trans- and antiparallel elimination reaction of both the axially oriented C-2 beta-, and C-1-alpha protons. We further demonstrate that the reconstituted enzyme has an aromatase activity optimum at pH 7.2, and an apparent Km of 0.66 microM for NADPH and of 0.24 microM for 4-androstene-3,17-dione. Also, the enzyme requires 3 nmoles of NADPH for each nmole of estrogen that is formed.

Androstenedione↗
PubMed1986

Purification and reconstitution properties of human placental aromatase. A cytochrome P-450-type monooxygenase.

The hemoprotein component of human placental aromatase (estrogen synthetase) has been purified to a high degree of homogeneity by a combination of affinity and adsorption chromatography on aminohexyl-Sepharose, concanavalin-A-Sepharose, and hydroxyapatite. The monomeric form of the enzyme has an Mr of 55000 +/- 1000 as estimated by sodium dodecyl sulfate gel electrophoresis. Its absolute spectrum shows a high-spin Soret band at 394 nm while its reduced, CO-difference spectrum has a maximum at 447 +/- 1 nm. Full reconstitution of aromatase activity was obtained when it was recombined with a homogeneous preparation of the higher-Mr form of either human placental, or bovine hepatic NADPH-cytochrome P-450 reductase. Critical factors for purification of the very unstable, membrane-bound hemoprotein with good retention of activity were, besides the chromatographic sequence, the use of the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps) during the solubilization, and the stabilizing effect of the aromatase substrate, 4-androstene-3,17-dione, throughout the procedure. In the presence of NADPH, the reconstituted enzyme system smoothly aromatizes 19-oxoandrostenedione, 19-hydroxyandrostenedione and androstenedione in this order of reactivity. The same reconstituted system also aromatized testosterone, but it was inactive towards 19-norandrostenedione. Known cytochrome P-450 inhibitors decreased its activity. We conclude: (a) the terminal oxidase of human placental aromatase is indeed a cytochrome P-450-type monooxygenase; (b) the multistep aromatization reaction of C19 androstenes is catalyzed by a single enzyme; (c) aromatization of 19-norsteroids reported by other authors must be due to a different aromatase. Experimental data obtained with the reconstituted enzyme are fully compatible with the concept of a reaction mechanism for the aromatization sequence involving an all-trans, antiparallel elimination of the 19-methyl group, the 2 beta proton and the 1 alpha proton, rather than the 1 beta proton, as generally assumed.

Adult↗
PubMed1986

Purification and properties of human placental NADPH-cytochrome P-450 reductase.

Human placental NADPH-cytochrome P-450 reductase (EC 1.6.2.4) was purified to electrophoretic homogeneity in two chromatographic steps with a high retention of bioactivity. After solubilization with 1% sodium cholate in a protective medium containing 20% glycerol, 10 microM 4-androstene-3,17-dione, 1 mM dithiothreitol, and 0.2 mM EDTA, a 35-60% ammonium sulfate precipitate was prepared. The crude protein mixture was then applied to a 2',5'-ADP-Sepharose 4B affinity column, followed by high-performance anion-exchange chromatography (Pharmacia Mono-Q column). Two forms of the reductase were isolated. One was eluted at higher salt concentration and had a relative mass (Mr) of 79 kdaltons (kDa) as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance gel permeation chromatography. A smaller size reductase with a Mr of 70 kDa, eluting at lower salt concentration, was also formed by trypsinolysis of the 79-kDa reductase. It must therefore be regarded as a proteolytic artifact. The absolute spectra in the visible region of the two reductases were identical with maxima at 376 and 452 nm, typical of a flavoprotein. They also had the same specific activity of 24.7 +/- 0.7 mumol/min per milligram protein towards cytochrome c. However, only the 79-kDa reductase showed aromatase-reconstitution activity. The homogeneity of these reductases was further confirmed by the appearance of a single peak when subjected to gradient, reversed-phase high-performance liquid chromatography. According to its amino acid composition, the 79-kDa reductase is a highly acidic and hydrophobic protein, composed of 695 residues.

Amino Acids↗
PubMed1985

A comparative study of two kinds of cathepsin D-type proteinases from rat gastric mucosa.

The localization of cathepsin D-like acid proteinase in the rat stomach and other tissues was studied, and its biochemical properties were compared with those of rat gastric cathepsin D (EC 3.4.23.5). Cathepsin D-like acid proteinase existed overwhelmingly in the mucosal layer and was hardly detected in the gastric juice. Its subcellular distribution profile was very similar to that of acid phosphatase, but not to that of pepsinogen. This proteinase-like enzyme activity was also found in rat splenic extract. These results strongly suggest that the proteinase is a lysosomal enzyme. In addition, cathepsin D-like acid proteinase demonstrated an in vitro transition of molecular species during storage at -30 degrees C. Although this molecular change was distinctive in ion-exchange column chromatography and susceptibility to some enzyme inhibitors, it was not accompanied by a significant decrease in molecular weight. To compare cathepsin D-like acid proteinase with ordinary cathepsin D, gastric cathepsin D was newly purified to apparent homogeneity in polyacrylamide gel electrophoresis. Its biochemical properties demonstrate that this is a true cathepsin D in rat gastric mucosa. Moreover, this cathepsin D activity was not abolished by treatment with antiserum specific to cathepsin D-like acid proteinase or pepsinogen. From these results, we can conclude that the proteinase is a lysosomal acid proteinase different from newly purified gastric cathepsin D.

Animals↗
PubMed1985

Purification of oestrogen synthetase by high-performance liquid chromatography. Two membrane-bound enzymes from the human placenta.

Human placental NADPH-cytochrome P-450 reductase, obtained by 2',5'-ADP-Sepharose affinity chromatography, was separated into two reductase-active peaks on a Pharmacia Mono-Q column. By this short, two-step chromatographic procedure, the two reductases were obtained in a homogeneous state with high retention of activity and in over 900-fold purification. Aromatase-reconstituting activity was present only in the higher-molecular-weight reductase (79 000 D), not in the smaller, 70 000 D reductase, which turned out to be a proteolysis product of the former. Both proteins were eluted as a single peak in reversed-phase high-performance liquid chromatography on a Protesil-diphenyl column. Similar results were obtained with bovine hepatic NADPH-cytochrome P-450 reductases. On the other hand, starting from a reductase-free preparation, we have obtained by high-performance ion-exchange chromatography and high-performance size-exclusion chromatography, only partial purification of the aromatase cytochrome P-450, which showed the following values: aromatase activity, 3.995 nmol/min/mg protein (60-fold purification); cytochrome P-450 content, 1.376 nmol/mg protein (23-fold purification); molecular weight, 165 000 D (estimated as an aggregate by size-exclusion chromatography). Although complete purification of the aromatase component has yet to be accomplished, our results suggest that high-performance ion-exchange chromatography on a Mono-Q column is very useful for the purification of acidic, membrane-bound enzymes with good retention of activity.

Ammonium Sulfate↗
PubMed1985

Effects of gastrointestinal hormones and their related compounds on gastric motility in the rat.

We examined the effects of certain gastrointestinal hormones on gastric motility using rat stomach preparatios in vivo. Changes of water level caused by the movement of the stomach which was filled with saline were recorded. Single injections of cholecystokinin (1, 2 and 4 micrograms/kg) induced relaxation of the stomach. Single injections of bombesin in low doses (below 0.2 microgram/kg) induced relaxation and in high doses (over 0.2 micrograms/kg) contraction after brief relaxation. Single injections of neurotensin (1, 2, 4 and 8 micrograms/kg), somatostatin (5, 10 and 20 micrograms/kg) and substance P (1, 2, 4 and 8 micrograms/kg) induced relaxation followed by contraction, but their dose-response relations were obscure. Infusions of neurotensin (1, 5 and 25 micrograms/kg/h) and somatostatin (2.5 and 5 micrograms/kg/h) enhanced the stomach tension, whereas substance P (1, 5 and 25 micrograms/kg/h) reduced it. Single injections and infusions of neurotensin, somatostatin or substance P showed different effects on gastric motility. On the other hand, Met-enkephalin (1, 10 and 100 micrograms/kg) and porcine motilin (1, 10 and 100 micrograms/kg) did not affect gastric motility in our rat stomach preparations. These results suggest that some gastrointestinal hormones take part in stomach movements.

Animals↗
PubMed1984

The N-terminal sequence of rat pepsinogen.

The amino-acid sequence of 96 residues in the N-terminal region of rat pepsinogen I was determined and the first 46 residues were found to constitute the activation peptide segment. There was high degree of homology between the activation segments of rat pepsinogen and some pepsinogens A (pig, cow, Japanese monkey and human). However, the number of residues substituted between rat and the other pepsinogens were considerably larger than those among pepsinogens A. In the N-terminal 24 residues of active pepsin, homology (88%) between rat pepsin and human gastricsin was higher than that (50%) between rat pepsin and pepsin A from human or pig. This strongly suggests that rat pepsin should be classified as pepsin C.

Amino Acid Sequence↗
PubMed1984

Takaokamycin, a new peptide antibiotic produced by Streptomyces sp.

A new peptide antibiotic named takaokamycin was isolated from a fermentation broth of Streptomyces sp. AC-1978, a soil isolate. It exhibits antibacterial activity against some Gram-positive bacteria. The molecular weight was found to be 1,130 on the basis of elemental analysis, FD-mass spectrum and 1H and 13C NMR. Acid hydrolysate of takaokamycin contains isoleucine, threonine and unidentified amino acids.

Animals↗
PubMed1983

Purification and properties of a cathepsin D-like acid proteinase from rat gastric mucosa.

A unique acid proteinase was purified from rat gastric mucosa by DEAE-Sepharose CL-6B chromatography, gel filtration and isoelectric focusing. The purified enzyme was homogeneous by polyacrylamide disc gel electrophoresis. The molecular weight of the enzyme was estimated to be 86000 by gel filtration on a Sephadex G-100 column. In SDS-polyacrylamide gel electrophoresis, the enzyme showed a single band corresponding to a molecular weight of 42000. So it was concluded that the native enzyme is composed of two identical monomeric units. The enzyme is a glycoprotein and its isoelectric point was determined to be 4.33. The enzyme hydrolyzed bovine hemoglobin with maximum activity at pH 3.0, but it showed considerably lower activity on bovine serum albumin or casein than on bovine hemoglobin. The enzyme was strongly inhibited by pepstatin, but not by urea or thiol-reactive inhibitors. These results lead us to conclude that this enzyme is a cathepsin D-like acid proteinase.

Amino Acids↗
PubMed1982

Effects of pentagastrin, histamine, carbamylcholine and catecholamines on gastric secretion, motility and emptying in the rat.

We developed a simplified method for the simultaneous measurement of gastric secretion and gastric motility in the rat. By using this method, we investigated the actions of pentagastrin, histamine, carbamylcholine and catecholamines on the stomach. Carbamylcholine stimulated acid secretion and induced contraction of the stomach, but norepinephrine tended to inhibit acid secretion and induced relaxation of the stomach. Histamine induced relaxation first and then contraction after 2-5 min. Pentagastrin induced a relaxation that did not show dose dependence, but tachyphylaxis. Isoproterenol, which stimulated acid secretion in our experiments, induced relaxation of the stomach in a dose-dependent manner. We also investigated the relationship between gastric motility and gastric emptying. Carbamylcholine caused an enhancement of gastric emptying, but isoproterenol caused its suppression. Pentagastrin, histamine and norepinephrine did not affect gastric emptying. As shown in the results of our experiments, carbamylcholine caused stimulation of acid secretion, contraction of the stomach, and enhancement of gastric emptying. However, other secretagogues did not always induce contraction of the stomach or increase gastric emptying.

Animals↗
PubMed1982

Genetic variation of pepsinogen 1 in the rat.

A pepsinogen (Pg 1) polymorphism was observed in gastric mucosal samples of Wistar rats by polyacrylamide slab gel electrophoresis. Three phenotypes were observed: S (a slow-moving band), F (a fast-moving band), and FS (both bands). Pg 1 variation was controlled by a pair of codominant alleles at a single autosomal locus. Interstrain variation of Pg 1 was studied in five strains of laboratory rats. Genetic variation of Pg 1 was detected only in the Wistar strain.

Alleles↗
PubMed1981

Studies on neplanocin A, new antitumor antibiotic. I. Producing organism, isolation and characterization.

Neplanocin A. C11H13N5O3, is a novel carbocyclic analog of adenosine with cyclopentene. It was isolated from the culture filtrate of Ampullariella regularis A11079 by means of ion-exchange, carbon, silica gel adsorption, or partition chromatography. Neplanocin A forms crystals, and is stable at acidic or alkaline pH. Neplanocin A has cytotoxicity against L5178Y cells in culture and showed a remarkable effect on the life prolongation of mice infected with L1210 leukemia.

Adenosine↗
PubMed1980

Biochemical and immunological characterizations of rat pepsinogens and pepsins.

Biochemical and immunological properties of two kinds of pepsinogens isolated from the gastric mucosal extracts of adult Wistar rats were studied. Their activated enzymes were prepared from the zymogens using a DEAE-Sepharose CL-6B column. The isoelectric points of pepsinogens I and II were estimated to be 3.90 and 3.75, respectively, by isoelectric focusing, and those of pepsins I and II to be 3.60 and 3.45, respectively. Amino acid compositions of the two pepsinogens or pepsins were strikingly similar to each other and neither pepsinogen I nor II contained organic phosphate. The biochemical properties of rat preparations compared with porcine pepsinogens A and C and pepsins A [EC 3.4.23.1] and C [EC 3.4.23.3] showed that rat pepsinogens and pepsins resembled porcine pepsinogen C and pepsin C, respectively. Pepsinogens I and II were demonstrated to share a similar immunogenic molecular structure by double diffusion analysis and Laurell immunoelectrophoresis. Rabbit antipepsinogen I serum cross-reacted with the mouse preparation but did not with the rabbit and porcine preparations. The possibility of the genetically controlled occurrence of pepsinogens I and II in the rat is discussed.

Amino Acids↗