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T Chikuma

Publications and source records attributed to T Chikuma.

31 records · Page 2Linked to original sources
PubMed1993

High-performance liquid chromatographic determination of PZ-peptidase activity.

A rapid and sensitive assay method for the determination of PZ-peptidase activity is reported. This method is based on the monitoring of the absorption at 320 nm of 4-phenylazobenzyloxycarbonyl-L-Pro-L-Leu (PZ-Pro-Leu), enzymatically formed from the substrate 4-phenylazobenzyloxycarbonyl-L-Pro-L-Leu-Gly-L-Pro-D-Arg (PZ-peptide), after separation by high-performance liquid chromatography using a C18 reversed-phase column by isocratic elution. This method is sensitive enough to measure PZ-Pro-Leu at levels as low as 10 pmol, yields highly reproducible results and requires less than 5.5 min per sample for separation and determination. The optimum pH for PZ-peptidase activity was 7.5-8.0. The Km and Vmax values were 166.7 microM and 5.35 pmol/min.microgram protein, respectively, with the use of enzyme extract obtained from mouse whole brain. The approximate molecular mass of this enzyme was estimated to be 64,000 by gel filtration. PZ-peptidase activity was strongly inhibited by Zn2+, Cu2+ and p-chloromercuriphenylsulphonic acid. By using this method, PZ-peptidase activity could be readily detected in a single mouse pituitary gland. Among the tissues examined in various mouse brain regions, the highest specific activity of the enzyme was found in cerebral cortex. The sensitivity and selectivity of this method will aid in efforts to examine the physiological role of this peptidase.

Amino Acid Sequence↗
PubMed1992

Processing of adrenocorticotropin by two proteases in bovine intermediate lobe secretory vesicle membranes. A distinct acidic, tetrabasic residue-specific calcium-activated serine protease and a PC2-like enzyme.

Adrenocorticotropin (ACTH) is cleaved at the tetrabasic residue site, in pituitary intermediate lobe secretory vesicles, to yield ACTH1-17 and corticotropin-like intermediate lobe peptide (CLIP). ACTH1-17 is then converted to alpha-melanocyte-stimulating hormone (N-AcACTH1-13NH2) by first removing the Lys15-Lys16-Arg17 residues, followed by amidation of the COOH terminus and acetylation of the NH2 terminus. Bovine intermediate lobe secretory vesicle membranes were screened for proteolytic enzyme activity that will cleave the tetrabasic residues of ACTH. Two activities with pH optima of 5.0-6.0 and 7.5-8.0 were detected. The acidic, ACTH-converting enzyme cleaved ACTH1-39 at the tetrabasic residues between the Arg17-Arg18 bond to yield ACTH1-17 and CLIP, but did not cleave paired basic residues of pro-opiomelanocortin. This enzyme activity was characterized as a Ca(2+)-activated serine protease with unique specificity for the tetrabasic residues of ACTH1-39. The neutral activity preferentially generated ACTH1-17 and to a small extent ACTH1-16 from ACTH1-39 and ACTH1-24. This enzyme activity was Ca(2+)-dependent but was not inhibited by serine or aspartic protease inhibitors. The neutral activity was significantly immunodepleted by antiserum raised against bovine PC2/PC3, and together with specificity studies, suggests that the enzyme is a PC2-like serine protease. The pH optimum, distinct specificity for tetrabasic residues, and subcellular localization of the acidic ACTH-converting enzyme indicate a function of this enzyme in the in vivo conversion of ACTH1-39 to alpha-melanocyte-stimulating hormone in intermediate lobe secretory vesicles which have an acidic internal pH.

Adrenocorticotropic Hormone↗
PubMed1992

Secretion of peptidylglycine alpha-amidating monooxygenase (PAM) from rat salivary glands.

Peptidylglycine alpha-amidating monooxygenase (PAM) is a regulating enzyme to synthesize the biologically active hormones having carboxy-terminal amide. In the present study we investigated secretion of the enzyme from rat saliva. Property of PAM in the saliva was similar to that in the submandibular gland. Both enzymes showed similar pH optimum at 5.0 and optimal ascorbic acid concentration at 2.5 mM. But molecular size of PAM in the saliva was 75 kDa in the gel permeation chromatography on Superose 12 column, while the size in the submandibular gland was 25 kDa. After the treatment with trypsin, PAM in the saliva was converted to a small size molecule, which is similar to the size in rat submandibular gland. These and other data indicate that a native molecular size of PAM is secreted into saliva and plays some physiological roles.

Aging↗
PubMed1991

A colorimetric assay for measuring peptidylglycine alpha-amidating monooxygenase using high-performance liquid chromatography.

In many peptide hormones and neuropeptides, the carboxy-terminal alpha-amide structure is essential in eliciting biological activity. In the present study, a rapid and sensitive assay method for the determination of peptidylglycine alpha-amidating monooxygenase (PAM) activity has been reported. This method is based on the monitoring of the absorption at 460 nm of 4-dimethylaminoazobenzene-4'-sulfonyl-Gly-L-Phe-NH2 (Dabsyl-Gly-Phe-NH2), enzymatically formed from the substrate 4-dimethylaminoazobenzene-4'-sulfonyl-Gly-L-Phe-Gly, after separation by high-performance liquid chromatography (HPLC) using a C-18 reversed-phase column by isocratic elution. This method is sensitive enough to measure Dabsyl-Gly-Phe-NH2 at concentrations as low as 1 pmol and yield highly reproducible results and requires less than 5 min per sample for separation and quantitation. The concentrations of copper and ascorbic acid required for maximal enzyme activity were 1 microM and 2 mM, respectively. The pH optimum for PAM activity was 5.0 to 5.5. The Km and Vmax values were respectively 3.5 microM and 100 pmol/micrograms/h with the use of enzyme extract obtained from bovine pituitary. By using this method, PAM activity could be readily detected in a single rat saliva. The sensitivity of this assay method will also aid in the effort to examine the regulation of in vivo PAM activity.

Amino Acid Sequence↗
PubMed1991

Prenatal processing of pro-opiomelanocortin in the brain and pituitary of mouse embryos.

The processing of pro-opiomelanocortin (POMC) to ACTH- (adrenocorticotropin), MSH- (melanotropin) and endorphin-related peptides was studied in mouse embryos with the ultimate aim of determining the role of the POMC-related peptides in early development especially in the CNS. Mouse embryos at gestational days 10.5, 11.5, 12.5 and 14.5 were analyzed for POMC-derived peptides by SDS-PAGE, HPLC and radioimmunoassay using antisera specific for various regions of the prohormone. At embryonic day 10.5 (E 10.5) the prohormone was the major product detected. At E 11.5, POMC was processed to ACTH(1-39), des-acetyl alpha-MSH and beta-endorphin(1-31) and beta-endorphin(1-27). The amounts of these peptides increased at E 12.5, and at E 14.5. At E 14.5, there was a major increase in ACTH(1-39) and beta-endorphin(1-31) peptides. This was attributed to the large increase of corticotrophs in anterior pituitary at this stage. Des-acetyl alpha-MSH levels, however, were similar at E 12.5 and E 14.5 and the peptide was confined mainly to the central nervous system. gamma-MSH was not detected until E 16.5 in the brain. No alpha-MSH or acetylated beta-endorphin was detected between E 11.5 and E 14.5. Thus in early embryonic development, POMC is processed to des-acetyl alpha-MSH, beta-endorphin(1-31), beta-endorphin(1-27) and gamma-MSH in the brain, and primarily to ACTH(1-39) and beta-endorphin(1-31) in the anterior pituitary. Some differences exist in the forms of POMC-derived peptides found in embryonic versus adult brain and pituitary. The embryonic forms of the peptides may be significant in playing a role during development.

Adrenocorticotropic Hormone↗
PubMed1990

Anterograde axonal transport of peptidylglycine alpha-amidating monooxygenase in rat sciatic nerves.

Axonal transport of peptidylglycine alpha-amidating monooxygenase (PAM) activity was studied in rat sciatic nerves from 12 to 120 h after double ligations. The anterograde axonal transport increased and reached a plateau between 48 and 72 h and then decreased. The flow rate was 100 mm/day, and the molecular mass of the active entity was 70 kDa, which was determined by gel filtration. In contrast, there was no evidence for significant retrograde axonal transport. Anterograde axonal transport of immunoreactive cholecystokinin, a carboxy-terminal-amidated putative neuropeptide, was also found. These results suggest that PAM is transported by a rapid axonal flow and may play a role as a processing enzyme during transport or in the terminals of rat sciatic nerves.

Animals↗
PubMed1990

Purification and properties of dipeptidyl peptidase IV from human urine.

Dipeptidyl peptidase IV (DPP-IV) (EC 3.4.14.5) has been purified from normal human urine using immunoaffinity chromatography. The purified enzyme was homogeneous as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular mass of urinary DPP-IV was estimated to be 280 kDa by gel filtration. The Km value for the hydrolysis of (Gly-L-Pro)-4-methylcoumaryl-7-amide tosylate was calculated to be 1.25 +/- 0.25 X 10(-4) M; the pH optimum and pI were 8.7 and 6.5, respectively. These properties were the same as those of renal DPP-IV. Both renal and this urinary DPP-IV were inhibited by diisopropylfluorophosphate and activated by phospholipid. From these results we suppose that urinary DPP-IV may be derived from the kidney rather than from the blood

Adult↗
PubMed1987

Effect of chlorpromazine on PZ-peptidase and several other peptidase activities in cloned osteoblastic cells (MC3T3-E1).

The effect of chlorpromazine (CPZ) on the degradation of collagen and non-collagenous peptides in clonal osteoblastic MC3T3-E1 cells was investigated by measuring the activities of PZ-peptidase, collagenase-like peptidase (CL-peptidase), dipeptidyl-aminopeptidase (DAP), leucine aminopeptidase (LAP) (EC 3.4.11.1), and post-proline cleaving enzyme (PPCE) (EC 3.4.21.26). CPZ increased PZ-peptidase and CL-peptidase activities in a dose-related fashion, but it had no effect on LAP and PPCE activities in the cells. CPZ (10 micrograms/ml) enhanced the specific activities of PZ-peptidase, CL-peptidase, and DAP for 72 hr after the start of CPZ stimulation; in particular, about a 3.3-fold increase of PZ-peptidase activity was observed at 12 hr of culture. Furthermore, other phenothiazine derivatives specifically enhanced the PZ-peptidase, CL-peptidase, and DAP activities as well as CPZ. Since PZ-peptidase, CL-peptidase, and DAP, involved in the degradation of collagen peptides, were induced significantly by CPZ (and/or other phenothiazine derivatives) in comparison with LAP and PPCE, involved in the degradation of non-collagenous peptides, these results show that CPZ specifically stimulated collagen catabolism by inducing the collagen-catabolizing enzymes. In addition, CPZ specifically inhibited collagen synthesis in clonal osteoblasts.

Aminopeptidases↗
PubMed1985

Highly sensitive assay for PZ-peptidase activity by high-performance liquid chromatography.

A rapid and highly sensitive assay method for measuring PZ-peptidase activity in newborn rat brain is described. The method is based on monitoring the absorption at 320 nm of PZ-Pro-Leu enzymatically formed from the substrate, PZ-L-Pro-L-Leu-Gly-L-Pro-D-Arg, after separation by high-performance liquid chromatography using a reversed-phase column. This method is sensitive enough to measure PZ-Pro-Leu at concentrations as low as 5 pmol, and is able to make the column ready for the next injection within 10 min after the preceding injection. By using this method, PZ-peptidase activity was discovered in clonal osteoblastic cells derived from newborn mouse calvaria.

Animals↗
PubMed1985

Properties of Suc-GPLGP-MCAase and dipeptidyl-aminopeptidase in mouse calvaria-derived osteoblastic cells (MC3T3-E1).

In this study the properties of Suc-GPLGP-MCAase and dipeptidyl-aminopeptidase (DAP) in clone MC3T3-E1 cells which have osteoblastic ability were examined. The Suc-GPLGP-MCAase was the most active at pH 8.0 and its molecular weight was about 65,000 as judged by gel filtration method. The enzyme activity was increased by some metal ions such as Mn+2, Ca+2, and Mg+2 but inhibited by Zn+2 and Cu+2. EDTA increased the enzyme activity to 23-fold. The enzyme activity was slightly inhibited by thiol inhibitors, N-ethylmaleimide, and p-chloromercuribenzoic acid by 16% and 27%, respectively. The DAP has an optimum pH at 7.5, and a molecular weight of about 100,000. The enzyme was completely inhibited by diisopropylfuorophosphate, but not by N-ethylmaleimide, iodoacetic acid, p-chloromercuribenzoic acid, phenylmethylsulfonyl fluoride, EDTA, and several metals. These results show that Suc-GPLGP-MCAase and DAP in MC3T3-E1 cells have different novel properties compared with these enzymes in the peripheral tissues.

Animals↗
PubMed1985

Effect of prostaglandin E2 on PZ-peptidase and several other peptidase activities in a clonal osteoblast-like cell line derived from newborn mouse calvaria.

The effects of prostaglandin E2(PGE2) on the degradation of collagen and non-collagenous peptides in clonal osteoblastic MC3T3-E1 cells were investigated by using highly sensitive assay methods for PZ-peptidase, collagenase-like peptidase (CL-peptidase), dipeptidyl-aminopeptidase (DAP), leucine aminopeptidase (LAP), and post-proline cleaving enzyme (PPCE). PGE2, at concentrations of 0.1 to 4.0 micrograms/ml, doubled the PZ-peptidase and CL-peptidase activities in the cells on 24 h culturing in a dose-dependent manner. PGE2, at a concentration of 2.0 micrograms/ml, enhanced the specific activities of PZ-peptidase, CL-peptidase, DAP, LAP, and PPCE for 75 h after the start of PGE2 stimulation. The time dependent changes in PZ-peptidase and CL-peptidase activities showed similar patterns, and 3- and 2-fold increases were seen after 48 h, respectively. The protein and DNA contents gradually increased after addition of PGE2. Since the PZ-peptidase and CL-peptidase, involved in degradation of collagen peptides, were significantly induced by PGE2 in comparison with LAP and PPCE, involved in the degradation of non-collagenous peptides, these results show that PGE2 specifically stimulates induction of collagen catabolizing enzymes in clonal osteoblasts.

Animals↗
PubMed1984

Effect of epidermal growth factor on dipeptidyl-aminopeptidase and collagenase-like peptidase activities in cloned osteoblastic cells.

The effect of epidermal growth factor (EGF) on collagen degradation in clonal osteoblastic MC3T3-E1 cells was investigated by measuring the activities of dipeptidyl-aminopeptidase (DAP) and collagenase-like peptidase (CL-peptidase). EGF at concentrations of 2 to 50 ng/ml markedly increased DAP and CL-peptidase activities in the cells. The same concentrations of this factor significantly decreased the cellular hydroxyproline content. Since DAP and CL-peptidase are thought to be enzymes involved in collagen degradation, these results suggest that a physiological concentration of EGF stimulates collagen catabolism in osteoblasts.

Animals↗