Dopamine transporter; solubilization and characterization of ( sup 3 H) GBR-12935 binding in canine caudate

Miscellaneous · Fri Jan 01 00:00:00 EST 1988

OSTI ID:7195179

Sallee, F R

The dopamine (DA) transporter protein, as indexed by ({sup 3}H)GBR-12935 binding, was solubilized from canine striatal membranes with the detergent digitonin. This solubilized protein retained the same pharmacological characteristics as membrane attached uptake sites. The binding of ({sup 3}H)GBR-12935 to solubilized preparations was specific, saturable and reversible with an equilibrium dissociation constant of approximately 3 nM and a maximum ligand binding (B{sub max}) of 3.4 pmol/mg protein. ({sup 3}H)GBR-12935 also bound to solubilized sites in a sodium-independent manner with a K{sub D} of approximately 6 nM and a B{sub max} of 1.2 {plus minus} 0.2 pmol/mg protein. Dopamine uptake inhibitors and substrates of DA uptake inhibited ({sup 3}H)GBR-12935 binding in a stereoselective and concentration dependent manner. For these compounds rank order of potency for inhibition of ({sup 3}H)GBR-12935 binding correlated with their potency for inhibition of dopamine uptake. K{sub D} values for DA uptake inhibitors in solubilized preparations correlated with those obtained on ({sup 3}H)GBR-12935 binding in the native state. The dopamine transporter appears to be a transmembrane glycoprotein by virtue of its absorption and specific elution from wheat germ agglutinin (WGA)-lectin column. Solubilization of the putative dopamine transporter with full retention of binding activity now allows for the purification and biochemical characterization of this important membrane protein.

• Research Organization: Pittsburgh Univ., PA (USA)
• OSTI ID: 7195179
• Resource Relation: Other Information: Thesis (Ph.D)
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
DOPAMINE
BIOCHEMICAL REACTION KINETICS
TRITIUM COMPOUNDS
TRACER TECHNIQUES
DOGS
EXPERIMENTAL DATA
GLYCOPROTEINS
AMINES
ANIMALS
AROMATICS
AUTONOMIC NERVOUS SYSTEM AGENTS
CARDIOTONICS
CARDIOVASCULAR AGENTS
DATA
DRUGS
HYDROGEN COMPOUNDS
HYDROXY COMPOUNDS
INFORMATION
ISOTOPE APPLICATIONS
KINETICS
MAMMALS
NEUROREGULATORS
NUMERICAL DATA
ORGANIC COMPOUNDS
PHENOLS
POLYPHENOLS
PROTEINS
REACTION KINETICS
SYMPATHOMIMETICS
VERTEBRATES
550201* - Biochemistry- Tracer Techniques