PMID- 2841089
OWN - NLM
STAT- MEDLINE
DCOM- 19880921
LR  - 20071114
IS  - 0013-7227 (Print)
IS  - 0013-7227 (Linking)
VI  - 123
IP  - 3
DP  - 1988 Sep
TI  - Enzyme binding-inhibiting assay for iodothyronine 5'-monodeiodinase (5'-MD) and 
      its application to isolation of complementary deoxyribonucleic acid clones for 
      the 5'-MD in rat liver.
PG  - 1264-73
AB  - To identify and/or quantify type I T4 5'-monodeiodinase (5'-MD) immunologically, 
      polyclonal antibodies were produced by immunization of rabbits with solubilized 
      microsomal proteins (SMP) from rat liver. Pilot studies showed that the antibody 
      binds to, but does not neutralize, rat liver enzyme. We have employed the 
      polyclonal antibody to develop a 5'-MD enzyme binding-inhibiting assay (MBIA). 
      For this purpose, active, inactive, or synthetic 5'-MD was preincubated with 
      rabbit antibody and removed by Staphylococcus aureus protein-A (Staph-A). The 
      5'-MD-binding sites that were left on the Staph-A-bound rabbit antibody were 
      assayed by adding active 5'-MD in fresh liver SMP. After centrifugation of 
      Staph-A, the unbound 5'-MD enzyme activity was measured in the supernatant using 
      [125I]rT3 in the presence of dithiothreitol. Incubation with 3.2 +/- 0.9 
      micrograms (mean +/- SEM; n = 4) rat liver SMP inhibited the binding of active 
      5'-MD to protein-A-bound antibody by 50%. Based on doses with similar 50% binding 
      inhibitory activity, liver SMP were 2.3 times more potent than liver microsomes 
      and 19 times more potent than liver homogenate; liver nuclei and mitochondria 
      showed little or no inhibition of the binding of 5'-MD to antibody. The relative 
      5'-MD content of liver, kidney, pituitary, placenta, and cerebral cortex based on 
      relative potency in the MBIA approximated 100:25:8:3:3. The threshold of the MBIA 
      approximated 0.9 micrograms liver SMP/tube. The coefficient of variation 
      approximated 2% within an assay and 6% between assays. To obtain a cDNA clone for 
      5'-MD we used the SMP antibody to screen a rat liver lambda gt11 cDNA expression 
      library. Of 16 positive (antibody-reactive) clones that were isolated, the fusion 
      proteins from only 2 (no. 23 and 54) inhibited the binding of 5'-MD in rat liver 
      SMP to protein-A-bound rabbit antibody in a dose-dependent manner. Western blot 
      analysis showed that the molecular size of liver protein encoded by clones 23 and 
      54 approximates 31K. Sequence analysis showed that clone 23 insert is 804 
      basepairs long, contains a single long open-reading frame, and is identical to 
      clone 54. Southern blot analysis showed that clones 23 and 54 did not 
      cross-hybridize DNA from other SMP antibody-positive clones. Northern blot 
      analysis using clone 23 insert cDNA as the probe showed a hybridization band 
      corresponding to a mRNA approximating 2.8 kilobases.(ABSTRACT TRUNCATED AT 400 
      WORDS)
FAU - Boado, R J
AU  - Boado RJ
AD  - Department of Medicine, University of California School of Medicine, Los Angeles 
      90024.
FAU - Chopra, I J
AU  - Chopra IJ
FAU - Flink, I L
AU  - Flink IL
FAU - Campbell, D A
AU  - Campbell DA
LA  - eng
SI  - GENBANK/M21476
SI  - GENBANK/M21831
GR  - 1F05-TW-03618-01/TW/FIC NIH HHS/United States
GR  - AM-16155/AM/NIADDK NIH HHS/United States
GR  - RR-5354/RR/NCRR NIH HHS/United States
PT  - Journal Article
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - Endocrinology
JT  - Endocrinology
JID - 0375040
RN  - 9007-49-2 (DNA)
RN  - EC 1.11.1.8 (Iodide Peroxidase)
RN  - EC 3.1.21.- (DNA Restriction Enzymes)
SB  - IM
MH  - Amino Acid Sequence
MH  - Animals
MH  - Bacteriophage lambda/genetics
MH  - Base Sequence
MH  - Cloning, Molecular
MH  - DNA/genetics/*isolation & purification
MH  - DNA Restriction Enzymes
MH  - Escherichia coli/genetics
MH  - Hyperthyroidism/enzymology
MH  - Immunochemistry
MH  - Iodide Peroxidase/antagonists & inhibitors/*genetics/isolation & purification
MH  - Liver/*enzymology
MH  - Microsomes/enzymology
MH  - Molecular Sequence Data
MH  - Nucleic Acid Hybridization
MH  - Organ Specificity
MH  - Rats
MH  - Reference Values
MH  - Thyroidectomy
EDAT- 1988/09/01 00:00
MHDA- 1988/09/01 00:01
CRDT- 1988/09/01 00:00
PHST- 1988/09/01 00:00 [pubmed]
PHST- 1988/09/01 00:01 [medline]
PHST- 1988/09/01 00:00 [entrez]
AID - 10.1210/endo-123-3-1264 [doi]
PST - ppublish
SO  - Endocrinology. 1988 Sep;123(3):1264-73. doi: 10.1210/endo-123-3-1264.