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Biomedical subjects

K Fukasawa

Publications and source records attributed to K Fukasawa.

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

Depth of side-chain pocket in the S2 subsite of dipeptidyl peptidase IV.

Kinetic studies of pig kidney dipeptidyl peptidase IV (dipeptidyl-peptide hydrolase, EC 3.4.14.5) were carried out using substrates possessing a side-chain of different length at the P2 position (or amino-terminal position in this case) such as Lys-, Arg-, Phe-, Met-, Ser-, His-, Glu- and Gly-Pro-pNA. The hydrolytic coefficient (Kcat/Km) has determined in the order Met- greater than Glu- greater than Ser- greater than His- greater than Phe- greater than Lys- greater than Gly- greater than Arg-, indicating a gradual increase with elongation of the side-chain from 0.03 to 0.60 nm followed by a decline when side-chain length approached 0.70 nm. Thus, the most probable depth of the side-chain pocket at the S2 subsite of the enzyme is proposed to be 0.50-0.60nm.

Animals↗
PubMed1985

A sensitive and specific assay for dipeptidyl-aminopeptidase II in serum and tissues by liquid chromatography-fluorometry.

A highly sensitive and specific method for the assay of dipeptidyl-aminopeptidase II (DAP II) in crude enzyme preparations such as serum and tissue homogenates has been established by using a newly synthesized fluorogenic substrate, 7-Lys-Ala-4-methylcoumarinamide. The enzymatically formed 7-amino-4-methylcoumarin was determined by high-performance liquid chromatography with fluorescence detection. The activities of other aminopeptidases in human serum and rat brain homogenates were completely inhibited by o-phenanthroline without any effect on DAP II activity to permit specific determination of DAP II. The limit of sensitivity for DAP II activity was about 300 fmol/30 min. DAP II activity was found to be increased in sera from cancer patients, in contrast to the decrease in serum DAP IV activity. DAP II activity was found to be unequally distributed in rat brain regions, and the highest activity was found in the hypothalamus.

Animals↗
PubMed1984

Chemical modification of dipeptidyl peptidase iv: involvement of an essential tryptophan residue at the substrate binding site.

Inactivation of pig kidney dipeptidyl peptidase IV (EC 3.4.14.5) by photosensitization in the presence of methylene blue at pH 7.5 was observed to have pseudo-first-order kinetics. During the process, until over 95% inactivation was achieved, the histidine and tryptophan residues were decreased from 14.0 to 2.7 and 12.6 to 7.1, respectively, per 94,000-Da subunit, without any detectable changes in other photosensitive amino acids. Modification of four histidine residues per subunit using diethylpyrocarbonate resulted in only 30% inactivation of the enzyme, while N-bromosuccinimide almost completely inactivated the enzyme with the modification of only one tryptophan residue per subunit, as determined by absorption spectrophotometry at 280 nm. The protective action of the substrate and inhibitors such as Ala-Pro-Ala and Pro-Pro against the modification of tryptophan residues with N-bromosuccinimide was observed both fluorometrically and by measurement of activity. On the basis of these results it is suggested that one of the tryptophan residues in the enzyme subunit is essential for the functioning of the substrate binding site of pig kidney dipeptidyl peptidase IV.

Animals↗
PubMed1984

Similarity between alkaline phosphatases from bovine dental pulp and liver.

Specific antibody against purified alkaline phosphatase from bovine dental pulp was raised in a rabbit. Immunological analyses by Ouchterlony double-gel diffusion and quantitative immunoprecipitation showed that there was neither a detectable antigenic difference between liver and pulp alkaline phosphatases nor a detectable cross-reaction with the intestinal enzyme.

Alkaline Phosphatase↗
PubMed1984

The effect of chronic isoproterenol administration on the plasma membrane of acinar cells in rat submandibular gland.

The effect of chronic administration of isoproterenol on the plasma membrane of acinar cells in the rat submandibular gland was studied by biochemical and immunohistochemical methods, using dipeptidyl peptidase (DPP) IV as a marker enzyme. Chronic isoproterenol treatment led to a marked reduction of DPP IV activity as well as of its immunoreactivity in the gland. Its effect progressively increased with the age of the animals. The present study showed that chronic treatment with isoproterenol caused a qualitative alteration of the plasma membrane of the luminal surface in acinar cells of the rat submandibular gland, and that the effect of the drug on the plasma membrane was dependent on the developmental stage of the acinar cells of the gland.

Age Factors↗
PubMed1984

[Serum tissue polypeptide antigen (TPA) in patients with urogenital cancer].

We measured the serum TPA level in 260 patients with benign urogenital diseases, 189 patients with urogenital cancer and 72 healthy blood donors by using a radio-immunoassay kit in order to evaluate its usefulness as an indicator for the presence of cancer. TPA value (mean +/- 2SD) in healthy blood donors was in the range of 36.8-110.8 units per liter, so that values higher than 110 U/L were considered to be abnormal. Significantly higher serum TPA levels were observed in patients with non-treated cancers, compared to healthy blood donors. But elevated serum TPA levels were also observed in the patients with benign diseases, especially in cases of benign prostatic hyperplasia with urinary tract infection. Consequently, it is considered that the serum TPA test is very useful for detecting urogenital diseases, but not for screening urogenital cancer. In cases of urogenital cancer, serum TPA levels elevated significantly by recurrence or recrudescence of the disease. Therefore, a good correlation was established between serum TPA and the efficiency of a given therapy in patients with prostatic cancer and bladder cancer.

Alkaline Phosphatase↗
PubMed1983

Characterization of a soluble form of dipeptidyl peptidase IV from pig liver.

Soluble dipeptidyl peptidase IV (EC 3.4.14.5) was purified from the 100,000 X g supernatant fraction of pig liver homogenate. The purified enzyme had the same properties as, and immunological identity with, the membrane-bound enzyme which was described previously. However, the purified enzyme had a pattern of molecular heterogeneity different from the membrane-bound enzyme; this was shown by isoelectric focusing. Carbohydrate analysis revealed that the soluble enzyme contained glucose, which is not found in the membrane-bound one, and less fucose, mannose, and sialic acid than the latter. From these results, we conclude that the soluble form of dipeptidyl peptidase IV in pig liver is closely related to the membrane-bound enzyme, but is not simply a proteolytically solubilized product of it.

Amino Acids↗
PubMed1983

Purification and properties of dipeptidyl peptidase II from rat kidney.

Dipeptidyl peptidase II (EC 3.4.14.2) from rat kidney was purified to a specific activity of 66.2 mumol/min per mg protein by a series of column chromatographic techniques. The purified enzyme was apparently homogeneous as judged by disc gel electrophoresis. Gel filtration on a calibrated column indicated an apparent molecular weight of 130 000 for the native enzyme. Gel electrophoresis of the denatured enzyme in the presence of sodium dodecyl sulfate in a constant acrylamide concentration resulted in the appearance of a single component for which a molecular weight of 64 000 was calculated. The pH optima for dipeptidyl arylamides and for tripeptides were pH 5.5 and 4.5, respectively, and the isoelectric point was 4.8. Substrate specificity studies indicated that the purified enzyme hydrolyzes specifically N-terminal X-alanine or X-proline from their respective arylamides and from tri- or tetrapeptides, but not from pentapeptides.

Animals↗
PubMed1983

Purification and characterization of two novel arginine aminopeptidases from Streptococcus mitis ATCC 9811.

Two novel aminopeptidases (I and II) which have specificity for amino-terminal arginine residues and strong sensitivity to divalent cations were purified from Streptococcus mitis ATCC 9811 by a procedure that involved treatment with a lytic enzyme for bacterial cell walls, followed by a series of chromatographies. Enzyme I was obtained as a homogeneous protein as judged by polyacrylamide gel electrophoresis and had a specific activity of 484.8 units per mg protein using L-arginine-2-naphthylamide as substrate; its Km value was 2.6 X 10(-5) M. The molecular weight was estimated to be 62,000, and its isoelectric point was pH 4.4. Enzyme II was purified to a specific activity of 128.0 units per mg protein and had a Km value of 3.8 X 10(-5) M. The molecular weight was estimated to be 360,000, and its isoelectric point was pH 5.7. The pH optima of enzymes I and II were 8.6 and 7.6, respectively. Both enzymes were inactivated by sulfhydryl reagents and metal ions but were markedly activated by EDTA. The chloride ion had an inhibitory rather than a stimulatory effect on the activity of both enzymes. Substrate specificity studies indicated that both the enzymes specifically hydrolyze N-terminal arginine residues from a-aminoacyl 2-naphthylamides and peptides, but they could not attack the L-arginyl-L-prolyl-peptide.

Aminopeptidases↗
PubMed1982

Purification and properties of bovine dental-pulp alkaline-phosphatase.

Alkaline phosphatase (E.C.3.1.3.1.) from unerupted bovine pulp was extracted from the microsomal fraction with eta-butanol and purified 77-fold, using DEAE-cellulose chromatography, Sephadex G-200 gel-filtration and concanavalin-A affinity chromatography, to a final specific activity of 92.3 units/mg protein. Affinity chromatography confirmed the glycoprotein nature of the enzyme. The pH optimum for the purified enzyme was 10.0 with rho-nitrophenylphosphate, and 8.7 with phosphoserine. The apparent Km was estimated to be 0.7 mM, using rho-nitrophenylphosphate in glycine-NaOH buffer, pH 10.0. The enzyme was markedly inhibited by EDTA, bromotetramisole and homoarginine but was insensitive to phenylalanine, and therefore resembled the alkaline phosphatase of liver and bone, but not that of intestine and placenta. No protein phosphatase activity towards dentine phosphoprotein and phosvitin was observed.

Alkaline Phosphatase↗
PubMed1982

Serum dipeptidyl peptidase (DPP) IV activities in oral cancer patients.

Dipeptidyl peptidase (DPP) IV activities in normal human sera and in the sera of patients with malignant tumors of the oral region were assayed. Patients with oral squamous cell carcinoma had significantly lower enzyme activity than that of normal controls (p less than 0.001). Patients with advanced carcinoma showed a more significant reduction of serum enzyme activity. On the other hand, in oral squamous cell carcinoma patients with hepatic dysfunction, the serum enzyme activity was significantly increased (p less than 0.02).

Adult↗
PubMed1981

Comparison of dipeptidyl peptidase IV prepared from pig liver and kidney.

Dipeptidyl peptidase IV (dipeptidylpeptide hydrolase, EC 3.4.14.-) has been purified from the microsomal fraction of pig liver, using an immunoaffinity chromatography, and its properties compared with those of the enzyme purified from pig kidney. The amino acid compositions of both enzymes were similar. The same kinds of carbohydrates were found in both enzymes, but there were differences in the molar concentrations of individual sugars. The liver enzyme had greater concentrations of mannose, fucose and sialic acid than the kidney enzyme, while the concentrations of galactose and glucosamine were greater in the kidney enzyme. The carbohydrates accounted for approx. 18.3 and 22.7% of the weight of the kidney and liver enzymes, respectively. The pH optima, molecular weights, substrate specificities and Km values of the two enzymes and the effects of diisopropylfluorophosphate on their activities were nearly identical. The liver enzyme was heat- and pH-sensitive, but not attacked by proteinases.

Amino Acids↗
PubMed1981

Immunohistochemical localization of dipeptidyl aminopeptidase (DAP) IV in the rat submandibular gland during postnatal development.

The localization of dipeptidyl aminopeptidase (DAP) IV in the rat submandibular gland during postnatal development was studied immunohistochemically using the unlabeled antibody enzyme method. Cryostat sections of rat submandibular glands were examined in animals 1 to 50 days old. In the first postnatal week, faint immunoreaction was detected in the apical region of the cytoplasm of terminal tubules. During the second postnatal week the reaction was gradually restricted to the luminal region of terminal tubules or developing acinar cells, and its intensity increased. The most striking change of the enzyme localization was observed in 15-day-old rats. At this time, DAP IV was confined to the luminal and lateral membranes of differentiated acinar cells and to the luminal membranes of intercalated and striated duct cells. This localization pattern was similar to that of the adult animal. DAP IV activity in the gland was also measured biochemically during the postnatal development. The results were in agreement with those of the immunohistochemical study. These results suggest that the localization of DAP IV is closely related to the postnatal differentiation and proliferation of acinar cells.

Aging↗