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Biomedical subjects

M Niinobe

Publications and source records attributed to M Niinobe.

53 records · Page 3Linked to original sources
PubMed1983

Cytochemical demonstrations of protease in human peripheral blood cells by use of new alpha-naphthyl ester substrates.

The proteases of human leukocytes were cytochemically studied by use of new alpha-naphthyl esters, tosyl-L-lysine-alpha-naphthyl ester (TLNE) and acetyl-L-tyrosine-alpha-naphthyl ester (ATNE). The hydrolytic activities were strong only in neutrophils, with both substrates. They were inhibited completely by DFP and chymostatin, but not by leupeptin and iodoacetate. These results indicate that chymotrypsin-like enzyme(s), capable of hydrolyzing both substrates, exist in neutrophils.

Chymotrypsin↗
PubMed1983

Purification of porcine pancreatic kallikrein using various affinity chromatographies and intestinal absorption of the purified kallikrein.

For the simple purification of porcine pancreas kallikrein, various affinity chromatographies using L-prolyl-L-phenylalanyl-L-arginine (Pro-Phe-Arg-OH), tosyl-L-arginine (Tos-Arg-OH) and acetyl-L-phenylalanyl-L-arginine (Ac-Phe-Arg-OH) as ligands were examined. The purification was performed as follows using the extract from acetone powder of porcine pancreas as starting material; ammonium sulfate fractionation (30-80%), affinity chromatography (repeated) and gel filtration on Sephadex G-75, respectively. With these purification techniques, porcine pancreas kallikrein was purified about 2000-fold, respectively. From the results, these affinity chromatographies were considered to be effective for the purification of porcine pancreas kallikrein. Intestinal absorption of the purified kallikrein was also investigated using a rabbit. After the administration of the purified kallikrein into the intestinal tract, peripheral blood was taken from the ear vein and the plasma was assayed for Pro-Phe-Arg-7-amino-4-methylcoumarinamide hydrolytic activity. The concentration of the kallikrein in the blood was maximal at 3 hr after the administration and the absorption at this time amounted to about 0.07%.

Animals↗
PubMed1982

A sensitive colorimetric assay for thrombin, prothrombin and antithrombin III in human plasma using a new synthetic substrate.

Benzoyl-L-leucyl-L-alanyl-L-arginine-alpha-naphthylester (Bz-Leu-Ala-Arg-NE) was synthesized as a new substrate for use in the assay of thrombin. In the assay alpha-naphthol released by the enzyme reaction was measured colorimetrically. With Bz-Leu-Ala-Arg-NE as substrate, the minimum detectable concentration of human thrombin was 0.0025 U. This assay using Bz-Leu-Ala-Arg-NE is a highly sensitive method for detecting prothrombin, thrombin and antithrombin III in human plasma. Prothrombin could be determined with 0.2 microliter of human plasma using Echis carinatus venom (ECV) as activator. Antithrombin III activity could be determined with 2 microliter of human plasma using human thrombin and heparin as cofactor. A zymogram of human prothrombin was prepared with Bz-Leu-Ala-Arg-NE as substrate. The preparation gave one band (pI 4.9) on polyacrylamide disc gel isoelectrophoresis.

Antithrombin III↗
PubMed1981

Purification and properties of thiol protease inhibitor from rat liver cytosol.

Thiol protease inhibitors were found in the cytosol fractions of various rat tissues. An inhibitor, named cytosol thiol protease inhibitor, was purified from rat liver cytosol by acid treatment and column chromatographies on Sephadex G-50, DEAE-Sephadex and Sephadex G-75. The purified inhibitor gave a single protein band on sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The molecular weight of the inhibitor was found to be 12 400 by gel filtration on Sephadex G-75 and SDS-polyacrylamide gel electrophoresis, and its isoelectric point was found to be 5.04. This inhibitor inhibited rat liver lysosomal cathepsin B, B2, C, H and L and papain, but not cathepsin A or D, trypsin or chymotrypsin. The inhibitor caused noncompetitive inhibition of the hydrolytic activity of cathepsin H on alpha-N-benzoyl-DL-arginine 2-naphthylamide and its Ki value was 4.08 . 10(-8) M. Heat treatment at 80 degrees C for 10 min reduced the activity 40%.

Animals↗
PubMed1980

Purification and properties of rat stomach kallikrein.

Kallikrein (EC 3.4.21.8) was purified from rat stomach by column chromatography on p-aminobenzamidine-Sepharose, DEAE-Sephadex A-50 and Sephadex G-150 and by isoelectric focusing, measuring its activities to hydrolyse L-prolyl-L-phenylalanyl-L-arginine-4-methyl-coumaryl-7-amide and to release kinin from heat-treated rat plasma. the purified stomach kallikrein showed a single band on polyacrylamide gel electrophoresis at pH 7.0. Its molecular weight was calculated to be 29 000 by gel-filtration on a column of Sephadex G-50. The kallikrein was stable between pH 6-11 and hydrolyzed L-prolyl-L-phenylalanyl-L-arginine-4-methyl-coumaryl-7-amide optimally at pH 11.0. The L-prolyl-L-phenylalanyl-L-arginine-4-methyl-coumaryl-7-amide hydrolyzing activity of rat stomach kallikrein was inhibited by diisopropyl fluorophosphate and Trasylol, but not by trypsin inhibitors from soybean, lima bean and ovomucoid. These properties of rat stomach kallikrein are different from those of partially purified rat plasma kallikrein, but similar to those of glandular kallikreins from other species. From these results, it was concluded that kallikrein is present in rat stomach and that it can be classified as a glandular kallikrein.

Animals↗
PubMed1980

A sensitive colorimetric assay for human urinary kallikrein.

L-Prolyl-L-phenylalanyl-L-arginine-alpha-naphthylester (Pro-Phe-Arg-NE) was synthesized as a new substrate for use in the assay of kallikreins. An assay was developed based on the colorimetric determination of alpha-naphthol released by the enzyme reaction. With Pro-Phe-Arg--NE as substrate, the minimum detectable concentration of human urinary kallikrein, was about 0.001 KU. Thus use of Pro-Phe-Arg-NE provides a highly sensitive method for detection of human urinary kallikrein. Kallikrein could be determined with a 25-microliter sample of human urine. Zymograms of human urinary kallikrein were prepared using Pro-Phe-Arg-NE as substrate. Six bands were separated by polyacrylamide disc gel isoelectrophoresis.

Colorimetry↗
PubMed1980

Purification and properties of aminopeptidase and arylamidase from human liver.

Aminopeptidase and arylamidase from human liver were purified 1,580- and 1,130-fold, respectively, by ammonium sulfate fractionation, TEAE-cellulose and hydroxylapatite column chromatographies, and Sephadex G-200 gel filtration. The purified preparations appeared to be homogeneous on polyacrylamide gel electrophoresis. The molecular weights of aminopeptidase and arylamidase were determined to be 280,000 and 170,000, respectively, by gel filtration on Sephadex G-200. Aminopeptidase showed marked specificities for amino acid amides, such as L-leucineamide, L-phenylalanineamide, and L-methionineamide. On the other hand, arylamidase showed specificities for amino acid beta-naphylamides, such as L-alanyl-beta-naphthylamide and L-leucyl-beta-naphthylamide, but it also hydrolyzed amino acid amides to a considerable extent. Immunological studies using antibodies to purified aminopeptidase and arylamidase showed that the antigenicities of these two enzymes were different. Neuraminidase treatment of the purified arylamidase changed its isoelectric point from 3.25 to 4.95. Various properties (Km values, optimum pH, substrate specificities, and antigenicity) of the arylamidase were not changed by neuraminidase treatment, but the enzyme became thermo-labile.

Aminopeptidases↗
PubMed1980

A sensitive colorimetric assay for various proteases using naphthyl ester derivatives as substrates.

A convenient and highly sensitive colorimetric assay for various proteases, such as trypsin, chymotrypsin, plasmin, thrombin, and urokinase is described. The substrates used are alpha-naphthyl ester derivatives of N alpha-tosyl-L-lysine, N alpha-acetylglycyl-L-ination of alpha-naphthol released from them. Use of these alpha-naphthyl ester derivatives made the method more sensitive than the use of the corresponding methyl or ethyl ester derivatives. The minimum detectable concentrations of trypsin, chymotrypsin, plasmin, thrombin and urokinase in this method were about 0.002 micrograms, 0.01 microgram, 0.002 CU, 0.01 IU, and 2 IU, respectively. The Km values of trypsin and thrombin for TLNE were 0.11 mM and 0.15 mM while those for TLME were 2.5 mM and 6.7 mM, respectively; the Km values of chymotrypsin for ATNE and ATEE were 0.18 mM and 0.7 mM, respectively; and the Km values of urokinase for AGLNE and AGLME were 0.17 mM and 4 mM, respectively. Zymograms of various proteases were easy to prepare using these alpha-naphthyl ester substrates, and zymograms of trypsin and chymotrypsin were made with TLNE and ATNE, respectively, as substrates.

Chymotrypsin↗
PubMed1980

Comparison of proteases in normal and metaplastic human stomach mucosa.

The proteases in normal and metaplastic human stomach mucosa were examined by preparing zymograms of tissue extracts with tosyl-L-lysine-alpha-naphthyl ester and L-prolyl-L-phenylalanyl-L-arginine-alpha-naphthyl ester as substrates. With both substrates, the zymogram of metaplastic mucosa showed an additional band (Rf 0.64) not present with normal mucosa. This band was also observed with extracts of normal small intestine and cancerous stomach tissue. It disappeared on treatment of the preparations with leupeptin, but not with esterastin.

Gastric Mucosa↗
PubMed1979

Rat stomach kallikrein: its purification and properties.

From rat stomach, kallikrein was purified by chromatographies on columns of p-aminobenzamidine-Sepharose, DEAE-Sephadex A-50 and Sephadex G-150 and by isoelectric focusing, measuring its activities to hydrolyse prolylphenylalanyl-arginine-4-methyl-coumarine amide (Pro-Phe-Arg-MCA) and to release kinin from rat heated-plasma. The purified stomach kallikrein showed a single band on Disc electrophoresis at pH 7.0. The molecular weight of the kallikrein was calculated to be 29,000 by gel-filtration on a column of Sephadex G-50. The kallikrein was stable between pH 6 and 11 and hydrolysed Pro-Phe-Arg-MCA optimally at pH 11.0. The Pro-Phe-Arg-MCA hydrolysing activity of rat stomach kallikrein was inhibited by DFP and Trasylol, but not by trypsin inhibitors from soyabean, limabean and ovomucoid. These properties of rat stomach kallikrein was clearly distinguishable from those of partially purified rat plasma kallikrein, but similar properties to other glandular kallikreins from other species. From these results, it was concluded that kallikrein is present in rat stomach, which can be classified into glandular kallikrein.

Animals↗
PubMed1975

Aminopeptidases and arylamidases in normal and cancer tissues in humans.

Multiforms of aminopeptidases and arylamidases in normal human liver, stomach, lung, ileum, colon, rectum, and kidney, and cancer tissue from human liver, stomach, and lung were separated by triethylaminoethyl cellulose column chromatography. The aminopeptidases and arylamidases were solubilized from human tissues by treatment with bromelain, and their column chromatograms on triethylaminoethyl-cellulose gave different patterns of multiforms of enzymes in these tissues. The fractions of enzymes separated specificities toward L-leucyl-beta-naphthylamide, L-leucinamide, L-methioninamide, L-phenylalaninamide, and L-alaninamide. The activity of aminopeptidase toward L-leucinamide and of arylamidase toward L-leucyl-beta-naphthylamide was higher in human stomach cancer tissue and lower in hepatic cancer tissue than in normal stomach and liver, respectively. In lung cancer tissue, the activity of aminopeptidase toward L-leucinamide was abnormally low, while the activity of arylamidase toward L-leucyl-beta-napthylamide was similar to that in normal lung. The substrate specificities or patterns of the multiforms of these enzymes in cancer tissue from human liver, stomach, and lung were shown to differ from those of normal liver, stomach, and lung, respectively, by triethylaminoethyl cellulose column chromatography.

Alanine↗