Immunochemical characterization and measurement of neuronal type nitric oxide synthase in human neuroblastoma NB-OK-1 cell using novel anti-synthetic peptide antibody and specific immunoassay system.
We developed a sensitive and specific immunoassay system for human neuronal nitric oxide synthase (hnNOS) using synthetic hnNOS(998-1024) peptide and anti-hnNOS(998-1024) antibody. The novel antibody and radioimmunoassay system revealed a typical nNOS protein in human neuroblastoma NB-OK-1 cell (160 kDa, 180 fmol/10(6) cells). The kinetic parameters of the enzyme were K(m)=4.88 microM and V(max)=4.34 pmol/min/mg protein for L-arginine. On incubation of NB-OK-1 cell for 24 h, betamethasone phosphate decreased both nNOS-immunoreactivity (nNOS-IR) and enzymatic activity in the cell dose-dependently. On the other hand, pituitary adenylate cyclase activating polypeptide(1-38) (PACAP38) increased both nNOS-IR and enzymatic activity at concentrations of 10(-10) and 10(-9) M, but inversely decreased both at 10(-7) M. These suggest the positive and negative implications of endogenous NO in proliferation and differentiation of the cell, which support mitogenic activity of NO generated by nNOS in the cell. The present findings also provided evidence that the quantitative change of nNOS protein controls the integrated activity of the enzyme in the cell and, in turn, substantiate the validity and reliability of the present immunoassay system for hnNOS and its practical usefulness.
Arakawa
Yukio
Y
Laboratory of Pharmaceutical Sciences, Osaka University Graduate School of Medicine, Suita, Japan. arakawa@drug.med.osaka-u.ac.jp
Takao
Etsuko
E
Hirotani
Yoshihiko
Y
Kato
Ikuo
I
Li
Jun
J
Yanaihara
Noboru
N
Yanaihara
Chizuko
C
Iwanaga
Toshihiko
T
Kurokawa
Nobuo
N
D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D001623:Betamethasone / Q000031:analogs & derivatives* / Q000494:pharmacology; D015153:Blotting, Western; D002455:Cell Division; D006801:Humans; D007150:Immunohistochemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D009447:Neuroblastoma / Q000201:enzymology* / Q000378:metabolism / Q000473:pathology; D009479:Neuropeptides / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000032:analysis / Q000276:immunology* / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D011863:Radioimmunoassay / Q000379:methods*; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
C495401:ADCYAP1 protein, human; D000906:Antibodies; D009479:Neuropeptides; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D009569:Nitric Oxide; C028994:betamethasone sodium phosphate; D001623:Betamethasone; C496320:NOS1 protein, human; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I