Structure-function studies on the human placental insulin receptor
Insulin receptor with high insulin binding and tyrosine kinase activities has been purified from human placenta. Kinetic analysis of {beta} subunit autophosphorylation revealed that {sup 32}P incorporation is linear with respect to receptor concentration, complete after ten minutes, and stable in the presence of a 100-fold excess of unlabeled ATP. The K{sub m} for ATP is 208 {mu}M. The nonhydrolyzable ATP analog 5{prime}({beta},{tau}imido)triphosphate (AMP-PNP) stimulates autophosphorylation of the {beta} subunit, an effect that is evident at ATP concentrations below 1 mM. The stimulatory effect of AMP-PNP is the result of increasing the binding of insulin to the {alpha} subunit, and this reflects itself in a shift to the left of the insulin-dose response curve for autophosphorylation. The same is true for ATP. N-ethylmaleimide (NEM) alkylation of a single cysteine residue in each {beta} subunit inhibits autophosphorylation in a dose- and time-dependent manner. Alkylation is reduced in the presence of ATP and AMP-PNP when the nucleotides are added as Mn{sup 2+} complexes. The presence of insulin has no effect on NEM alkylation. Guanidinium chloride (6 M) does not render additional sulfhydryl groups available for NEM alkylation; however, when receptor is reduced with tributylphosphine and exposed to guanidinium chloride, up to 32 mol of NEM can be incorporated into the {alpha} subunit. In the {beta} subunit, only three mol of NEM are incorporated under the same conditions. Receptor can be reduced to the {alpha}{beta} dimer and subunit stage by varying the amount of tributylphosphine. Dimer is formed when as few as two groups in the {alpha} subunit, and one additional group in the {beta} subunit are alkylated with NEM. Free {beta} subunit is formed when three mol of NEM are incorporated.
- Research Organization:
- Pittsburgh Univ., PA (United States)
- OSTI ID:
- 5604839
- Country of Publication:
- United States
- Language:
- English
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59 BASIC BIOLOGICAL SCIENCES
ALKYLATION
AMINO ACIDS
AMP
ANIMALS
ANTIMITOTIC DRUGS
ATP
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
DAYS LIVING RADIOISOTOPES
DOSE-RESPONSE RELATIONSHIPS
DRUGS
ENZYME ACTIVITY
ENZYMES
FETAL MEMBRANES
HORMONES
HYDROXY ACIDS
IMIDES
INSULIN
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LIGHT NUCLEI
MAMMALS
MAN
MEMBRANE PROTEINS
MEMBRANES
NEM
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PEPTIDE HORMONES
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHORYLATION
PHOSPHOTRANSFERASES
PLACENTA
PRIMATES
PROTEINS
RADIOISOTOPES
RADIOSENSITIZERS
REACTION KINETICS
RECEPTORS
STRUCTURE-ACTIVITY RELATIONSHIPS
TIME DEPENDENCE
TRACER TECHNIQUES
TRANSFERASES
TYROSINE
VERTEBRATES