Monoclonal antibody-directed radioimmunoassay detects cytochrome P-450 in human placenta and lymphocytes

Science ◽  
1985 ◽  
Vol 228 (4698) ◽  
pp. 490-492 ◽  
Author(s):  
B. Song ◽  
H. Gelboin ◽  
S. Park ◽  
G. Tsokos ◽  
F. Friedman
Keyword(s):  
Monoclonal Antibody ◽  
Human Placenta ◽  
Cytochrome P 450

PURIFICATION AND CHARACTERIZATION OF REACTIVE AND NON-REACTIVE PLASMINOGEN ACTIVATOR INHIBITOR-1 (PAI-1) FROM HUMAN PLACENTA

1987 ◽  
Author(s):  
M Philips ◽  
A G Juul ◽  
S Thorsen ◽  
J Selmer ◽  
L Thim
Keyword(s):  
Monoclonal Antibody ◽  
Plasminogen Activator ◽  
Human Placenta ◽  
Solid Phase ◽  
Plasminogen Activator Inhibitor ◽  
Tissue Type ◽  
Single Chain ◽  
Plasminogen Activator Inhibitor 1 ◽  
Sds Page ◽  
Pai 1

Reactive and non-reactive forms of PAI-1 have been identified in various biological materials. The structural differences between these forms remain to be determined.A monoclonal antibody specific for a non-reactive PAI-1 and a monoclonal antibody reacting with both the reactive and nonreactive form of the inhibitor were obtained by immunization with a tissue-type plasminogen activator (t-PA)-PAI-1 complex (Philips et al., Thromb Haemostas 1986; 55:213-7). These antibodies were used for the isolation of reactive and non-reactive PAI-1 by solid-phase immunoadsorption from extracts of human placenta. The inhibitor preparations were further purified by HPLC. Reactive and non-reactive PAI-1 both migrated with a Mr ∼ 52,000 when analyzed by SDS-PAGE. Furthermore, the two inhibitor forms were indistinguishable by N-terminal sequence analysis. Two N-terminal sequences were found in about equal ammounts for both the reactive and non-reactive PAI-1. They were Ser-Ala-Val-His-His-Pro-Pro- and a two residues shorter sequence (Val-His-His-Pro-Pro-). These sequences are in agreement with the published cDNA sequence of PAI-1 and shows that the inhibitor is N-terminally heterogeneously processed. The second order rate constant (ki) for the reaction between reactive PAI-1 and single-chain t-PA was about 6 106 M-1s-1. Treatment with 4 M guanidinium-HCl partially converted the non-reactive PAI-1 to a reactive form exhibiting a similar k1 for inhibition of single-chain t-PA. SDS-PAGE showed that the t-PA-PAI-1 complex could be dissociated by 1,5 M NH4OH/ 39 mM SDS resulting in the release of a PAI-1 with approximately the same Mr as native PAI-1. This indicates either that t-PA does not cleave the inhibitor or that it cleaves a peptide bond close to the C-terminus.In conclusion a non-reactive and a reactive form of PAI-1 can be purified from placenta. The two forms are distinguishable by monoclonal antibodies but they show similar Mr′ls and the same N-terminal sequences.

Immunocytochemical localization of cytochrome P-450 in hepatic and extra-hepatic tissues of the rat with a monoclonal antibody against cytochrome P-450 c

1986 ◽  
Vol 35 (24) ◽  
pp. 4543-4554 ◽  
Author(s):  
John R. Foster ◽  
Clifford R. Elcombe ◽  
Alan R. Boobis ◽  
Donald S. Davies ◽  
Dorothea Sesardic ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Immunocytochemical Localization ◽  
Cytochrome P 450

scholarly journals Characterization of mitochondrial cytochromes P-450 from pig kidney and liver catalysing 26-hydroxylation of 25-hydroxyvitamin D3 and C27 steroids

1991 ◽  
Vol 276 (2) ◽  
pp. 427-432 ◽  
Author(s):  
T Bergman ◽  
H Postlind
Keyword(s):  
Monoclonal Antibody ◽  
Protein Band ◽  
Similar Species ◽  
Mitochondrial Cytochrome ◽  
Pig Liver ◽  
Kidney Mitochondria ◽  
Pig Kidney ◽  
Sds Page ◽  
Liver And Kidney ◽  
Cytochrome P 450

The properties of cytochrome P-450 from pig kidney mitochondria, catalysing 26-hydroxylation of 25-hydroxyvitamin D3 and C27 steroids [Postlind & Wikvall (1989) Biochem. Biophys. Res. Commun. 159, 1135-1140; Postlind (1990) Biochem. Biophys. Res. Commun. 168, 261-266], were compared with those of a 26-hydroxylating cytochrome P-450 from pig liver mitochondria. The liver enzyme was purified to a cytochrome P-450 content of 7.4 nmol/mg of protein and showed only one protein band with an apparent Mr of 53,000 upon SDS/PAGE. The cytochrome P-450 catalysed 26-hydroxylation of 25-hydroxyvitamin D3, cholesterol and 5 beta-cholestane-3 alpha, 7 alpha-diol at rates of 361, 1090 and 2065 pmol/min per nmol of cytochrome P-450. A monoclonal antibody against the purified liver mitochondrial cytochrome P-450 26-hydroxylase (cytochrome P-450(26] was prepared. After coupling to Sepharose, the antibody was able to bind to cytochrome P-450(26) from liver as well as from kidney mitochondria and to immunoprecipitate the 26-hydroxylase activity towards 25-hydroxyvitamin D3 and cholesterol when assayed in a reconstituted system. After SDS/PAGE and immunoblotting with the antibody, the cytochrome P-450(26) was detected in the purified liver and kidney preparations. These results indicate that similar species of cytochrome P-450 catalyse 26-hydroxylation of 25-hydroxyvitamin D3 and C27 steroids in liver and kidney mitochondria. The results with the monoclonal antibody together with the finding that cholesterol competitively inhibits the 26-hydroxylation of 25-hydroxyvitamin D3 further indicate that 26-hydroxylation of 25-hydroxyvitamin D3 and cholesterol is catalysed by the same species of cytochrome P-450 in each tissue. The N-terminal amino acid sequence of cytochrome P-450(26) in kidney mitochondria resembled that of pig kidney microsomal 25-hydroxylase active in 25-hydroxylation of vitamin D3 and C27 steroids, whereas the sequence of pig liver mitochondrial cytochrome P-450(26) differed from those of rabbit and rat liver mitochondrial 26-hydroxylases as well as from those of other hitherto isolated mammalian cytochromes P-450.

Amino-terminal sequence analysis of six cytochrome P-450 isozymes purified by monoclonal antibody directed immunopurification

1984 ◽  
Vol 123 (3) ◽  
pp. 1201-1208 ◽  
Author(s):  
Kuo-Chi Cheng ◽  
Henry C. Krutzsch ◽  
Sang S. Park ◽  
Preston H. Grantham ◽  
Harry V. Gelboin ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Sequence Analysis ◽  
Terminal Sequence ◽  
Amino Terminal ◽  
Cytochrome P 450 ◽  
Amino Terminal Sequence
Sign in / Sign up

Export Citation Format

Share Document