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Biomedical subjects

A Moriyama

Publications and source records attributed to A Moriyama.

At least 91 records · Page 5Linked to original sources
PubMed1985

Efficient purification of human plasma beta 2-microglobulin from the haemodialysate of a patient with chronic renal failure by use of high-performance liquid chromatography.

Human plasma beta 2-microglobulin was isolated in a good yield (more than 80%) from the haemodialysate (blood ultrafiltrate) of a patient with chronic renal failure. The isolation procedure consisted of Sephadex G-100 gel filtration and two steps of high-performance liquid chromatography: reversed-phase high-performance liquid chromatography and gel permeation chromatography. The purified beta 2-microglobulin was homogeneous by sodium dodecyl sulphate polyacrylamide gel electrophoresis and two-dimensional electrophoresis.

Blood Proteins↗
PubMed1985

Purification of Japanese monkey prostate acid protease zymogen and its identification as a pepsinogen C-like zymogen.

A pepsinogen C-like acid protease zymogen was found in Japanese monkey prostate extract and seminal plasma by means of the double immunodiffusion method using rabbit anti-pepsinogen C antiserum, and was purified from the prostate by a combination of ammonium sulfate fractionation, DEAE-Sephacel chromatography, Sephadex G-100 gel filtration, and immunoadsorption to an anti-pepsinogen C column. The zymogen was purified 6,400-fold in a yield of 13.1%. The purified zymogen gave a single band on polyacrylamide gel electrophoresis both in the presence and absence of sodium dodecyl sulfate. The zymogen was converted to the active form by acid treatment at pH 2.8 for 4 h with concurrent reduction of the molecular weight from 41,000 to 36,000. By the double immunodiffusion method, prostate pepsinogen C-like acid protease zymogen, pepsinogen C, lung procathepsin D-II, and their active forms gave a single, fused precipitin line in agar plate with anti-pepsinogen C antiserum, which did not react with cathepsin D and pepsinogen A. Furthermore, the optimal pH of 2.5-3.0, the effect of pepstatin on the activity, and the amino acid compositions were also in good agreement among these three zymogens, showing that they are very similar protease zymogens.

Amino Acids↗
PubMed1985

[Research for the prevalence of scoliosis].

The number of scoliotic patients under treatment have recently showed a marked increase in Japan. In this situation, the author has raised two important questions. Firstly, has the incidence of scoliosis increased in Japan? Secondly, what happened to scoliotic patients who had been left untreated before the establishment of the current therapy. To seek answers for these questions, prevalence studies for all generations were carried out. As to the first question, the prevalence rate of scoliosis in adult from the twenties to forties was lower than in junior high and high school students. From this incidence and other several factors, the author concluded that the incidence of scoliosis in Japan increased recently. As to the second question, questionnaires were sent to each patients subjected to the secondary examination. Since the difference between scoliotic group and control group on the back symptoms did not appear significant, scoliosis and subjective symptoms apparently failed to show a correlation.

Adolescent↗
PubMed1983

Identification of monkey lung procathepsin D-II as a pepsinogen-C-like acid protease zymogen.

Procathepsin D-II (Mr = 37 500) was purified from Japanese monkey lung at pH 7.0, and was shown to be converted to the active form, cathepsin D-II (Mr = 33 000) via an intermediate (Mr = 35 500) upon treatment at pH 3.0 and 14 degrees C. Procathepsin D-II was shown to be the inactive precursor of cathepsin D-II based on the following results: the former was inactive toward heat-denaturated casein at pH 5.4 whereas the latter was active; the former was not inactivated by diazoacetyl-DL-norleucine methyl ester in the presence of Cu2+ ion at pH 6.0 whereas the latter was inactivated rapidly under the same conditions; and the former had no affinity to pepstatin-Sepharose between pH 5 and 7 whereas the latter was adsorbed to it. With a rabbit antiserum against procathepsin D-II, cathepsin D-II, pepsinogen C and pepsin C of Japanese monkey were each found to give a single precipitin line which fused completely with each other on agarose plate. On the other hand, cathepsin D-I purified from the monkey lung, and pepsinogens A (I, II, III-1, III-2 and III-3) obtained from the monkey gastric mucosa failed to precipitate with the antiserum. With the antiserum against the monkey pepsinogen C, the same results were obtained. Further, procathepsin D-II and pepsinogen C were shown to have the same amino-terminal amino acid sequence, Ala-Val-Val-Lys-Val-Pro-Leu-Lys-Lys-Phe-Lys-. All these results indicate a strong similarity of procathepsin D-II and cathepsin D-II to pepsinogen C and pepsin C, respectively.

Amino Acids↗
PubMed1983

Structural study on the active site of porcine pepsin and Rhizopus chinensis acid protease. Spin labeling with diazoketone reagents.

To investigate the active site structures of porcine pepsin and Rhizopus chinensis acid protease (RAP), spin label techniques were applied for these enzymes. Comparison of spin labeled porcine pepsin and RAP suggested that the active site cleft of porcine pepsin was narrower at the top, but wider at the bottom than that of RAP. Addition of pepstatin restricted the motion of the labeled nitroxide radicals. Under alkaline conditions, the enzymes changed their conformation discontinuously and irreversibly to open the active site clefts and to lose the binding ability for pepstatin. The denaturation points of both the enzymes were determined to be pH 6.2.

Animals↗
PubMed1983

Porcine liver succinyltrialanine p-nitroanilide hydrolytic enzyme. Its purification and characterization as a post-proline cleaving enzyme.

Succinyltrialanine p-nitroanilide(STANA)-hydrolytic enzyme was purified 5,200-fold from porcine liver soluble fraction with a yield of 75% by ammonium sulfate fractionation and chromatographies on DEAE-Sephacel, Sephadex G-150, and hydroxylapatite columns. The purified enzyme was homogeneous as judged by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate (SDS). The pI of the enzyme was 4.9 by dis gel electrofocusing and the molecular weight was calculated to be 72,000 by gel filtration on a Sephadex G-150 column and 74,000 by SDS-polyacrylamide gel electrophoresis. Acidic amino acids amounted to 17.2% of the total amino acid residues, and the basic ones, 12.9%. No hexosamine was detected. The STANA-hydrolytic enzyme showed maximal activity at pH 7.4 against succinyltrialanine p-nitroanilide and at pH 6.5 against succinyl-Gly-Pro-4-methylcoumaryl 7-amide (MCA), and was stable between pH 6 and 7 in the presence of dithiothreitol. This enzyme hydrolyzed succinyl-Gly-Pro-Leu-Gly-Pro-MCA, succinyl-Gly-Pro-MCA, succinyl-Ala-Pro-Ala-MCA, and several proline-containing natural peptides in addition to succinyltrialanine p-nitroanilide, but was unable to hydrolyze the substrates of aminopeptidases, dipeptidylaminopeptidase IV, trypsin, and chymotrypsin. Elastatinal and chymostatin were effective inhibitors and their IC50 values were 8.7 micrograms/ml and 18.2 micrograms/ml, respectively. The enzyme was completely inhibited by 10(-7) M p-chloromercuribenzoic acid (pCMB), 10(-7) M p-chloromercuriphenylsulfonic acid (pCMPS), and 10(-4) M diisopropyl phosphofluoridate (DFP), but not by 1 mM E-64, which is known as an inhibitor specific to thiol proteinase. The enzyme was easily inactivated by agitation in a Vortex mixer, and its activity was recovered by the addition of thiol compounds such as dithiothreitol, 2-mercaptoethanol and cysteine. The effects of inhibitors and thiol compounds were substantially identical when the enzyme activity was measured with either succinyltrialanine p-nitroanilide or succinyl-Gly-Pro-MCA as a substrate. These results indicate that the STANA-hydrolytic enzyme in the liver soluble fraction is a post-proline cleaving enzyme [EC 3.4.21.26].

Amino Acids↗
PubMed1983

Purification and characterization of pepsinogens and pepsins from Asiatic black bear, and amino acid sequence determination of the NH2-terminal 60 residues of the major pepsinogen.

Five pepsinogens were purified to homogeneity from the gastric mucosa of Asiatic black bear and termed pepsinogens I-1, I-2, II-1, II-2, and III. Pepsinogen II-1 was the major component and accounted for more than half of the total pepsinogens. Their molecular weights were estimated to be 40,000 for pepsinogens I-1 and I-2, 38,000 for pepsinogens II-1 and II-2, and 42,000 for pepsinogen III. They resembled each other in amino acid composition, except that pepsinogens I-1 and I-2 contained larger numbers of basic residues than the others. Pepsinogen III was a glycoprotein containing about 3.7% carbohydrate. Each was activated to the corresponding pepsin and their enzymatic characteristics were investigated. The optimal pH against hemoglobin was about 2.2 for pepsin I-1, and about 2.5 for pepsins II-1, II-2, and III. Each pepsin was inhibited by pepstatin as well as porcine pepsin and also by diazoacetyl-DL-norleucine methyl ester, 1,2-epoxy-3-(p-nitrophenoxy)-propane, and p-bromophenacyl bromide. Each pepsin could hydrolyze N-acetyl-L-phenylalanyl-3,5-diiodo-L-tyrosine, but the specific activity was much lower than that of porcine pepsin. Activation peptides corresponding to residues 1-43, 1-25, and 26-43 were isolated from an activation mixture of pepsinogen II-1. The amino acid sequences of these peptides and of the NH2-terminal portions of pepsinogen II-1 and pepsin II-1 were determined, resulting in the complete NH2-terminal 60-residue sequence of pepsinogen II-1.

Amino Acid Sequence↗
PubMed1980

Cathepsins D from rhesus monkey lung. Purification and characterization.

Two types of cathepsin D (cathepsins D-I and D-II) were purified from rhesus monkey lung to homogeneity as judged from disc gel electrophoresis. Cathepsin D-I was purified about 2,000-fold with a 5.1% yield while cathepsin D-II was purified about 2,300-fold with a 14.3% yield. Both cathepsins D were rich in the lysosome fraction of the lung, but appeared to be present in part extracellularly. Both showed a molecular weight of about 35,000 on Sephadex G-100 chromatography, and about 41,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cathepsin D-I showed the maximal activity on bovine hemoglobin and albumin at pH 3.4 and 4.0, respectively. It was most stable in the pH range of 5 to 7, but was rather unstable outside this pH range. Cathepsin D-II was quite similar in properties to that from Japanese monkey lung (Moriyama, A. & Takahashi, K. (1978) J. Biochem. 83, 441-451), and was remarkably stable in the pH range of 1-9. Under the conditions used, it retained at least 80% of the original activity when incubated at 37 degrees C for 20 h in this pH range. This stability seems to allow cathepsin D-II to be fairly active even at pH 1.0. Both cathepsins D acted on protein substrates fairly similarly and hydrolyzed hemoglobin most rapidly among the proteins tested. They did not hydrolyze N-acetyl-L-phenylalanyl-3,5-diiodotyrosine. Upon incubation with the oxidized B-chain of insulin, both cathepsins D hydrolyzed the Ala-Leu, Leu-Tyr, Tyr-Leu, Phe-Phe, and Phe-Tyr bonds at both pH 3.0 and 5.0. In addition, cathepsin D-II hydrolyzed the Leu-Val and Tyr-Thr bonds at pH 3.0 and the Val-Asn bond at pH 5.0. Both cathepsins D were inactivated by acid protease-specific inhibitors such as pepstatin, 1,2-epoxy-3-(p-nitrophenoxy)propane, p-bromophenacyl bromide, and diazoacetyl-DL-norleucine methyl ester, although cathepsin D-II was much less susceptible to these reagents except p-bromophenacyl bromide.

Amino Acids↗
PubMed1978

The structure and function of acid proteases. VIII. Purification and characterization of cathepsins D from Japanese monkey lung.

Two kinds of cathepsin D were found in Japanese monkey lung and were named cathepsins D-I and D-II. Cathepsin D-I was partially purified by ammonium sulfate fractionation and DEAE-cellulose column chromatography. It had properties common to other ordinary cathepsins D in terms of the elution position from a DEAE-cellulose column at pH 8.0, the pH-dependence of activity toward acid-denatured hemoglobin, and the molecular weight of 35,000 as determined by Sephadex G-100 gel filtration. On the other hand, cathepsin D-II was purified about 1,000-fold by a combination of ammonium sulfate fractionation and column chromatographies on DEAE-cellulose and Sephadex G-100. It was a very acidic protein as judged from its elution position from a DEAE-cellulose column at pH 8.0, and the high mobility toward the anode on disc gel electrophoresis at pH 8.6. Its molecular weight was determined to be 35,000 by Sephadex G-100 gel filtration and 39,000 by SDS-polyacrylamide gel electrophoresis. It was optimally active at pH 2.8 against acid-denatured hemoglobin as a substrate, showing 80% of the optimal activity at pH 1.0, and almost no activity above pH 4.0. This pH-profile of activity was similar to that of monkey pepsin C (gastricsin). It did not hydrolyze N-acetyl-L-phenylalanyl-3,5-diiodo-L-tyrosine, a synthetic substrate for pepsin, but was inhibited by a series of pepsin inhibitors such as pepstatin, 1,2-epoxy-3-(p-nitrophenoxy)propane, p-bromophenacyl bromide, and diazoacetyl-DL-norleucine methyl ester, although the diazo reagent was a rather weak inhibitor of the enzyme. The amino acid composition of cathepsin D-II was found to be fairly different from those of other cathepsins D. However, it showed a striking resemblance to that of Japanese monkey pepsinogen C, suggesting some evolutionary relationship between them.

Acetophenones↗
PubMed1977

Studies of antitumor agents. 1. Resolution of racemic 1-(tetrahydro-2-furanyl)-k-fluorouracil into the R and S isomers and examination of the biological activities of the isomers.

1-(Tetrahydro-2-furanyl)-5-fluorouracil (Thf-FU), which is named Ftorafur or FT-207 and is used clinically as an antitumor agent, was conveniently synthesized by condensation of the trimethylsilyl derivative of 5-fluorouracil with 2-acetoxytetrahydrofuran using NaI as a catalyst. This optically inactive Thf-FU was resolved into optically active (R)-(+)- and (S)-(-)-Thf-FU in high optical purity and excellent yield by formation of diastereoisomers with brucine. 13C NMR data were obtained on Thf-FU and related compounds and the antibacterial activities and in vivo antitumor activities of these isomers were tested. The degradations of these isomers to 5-fluorouracil by liver microsomes were also examined. No significant differences were found in any of these properties of these isomers.

Animals↗