Prolactin-binding components in rabbit mammary gland: characterization by partial purification and affinity labeling

Journal Article · Sat Jun 01 00:00:00 EDT 1985 · Endocrinology; (United States)

DOI:https://doi.org/10.1210/endo-116-6-2612· OSTI ID:5023703

Katoh, M; Djiane, J; Kelly, P A

The molecular characteristics of the PRL receptor isolated from rabbit mammary gland microsomes were investigated. Two approaches were employed: 1) affinity purification of PRL receptors and direct electrophoretic analysis, and 2) affinity cross-linking of microsomal receptors with (/sup 125/I)ovine PRL ((/sup 125/I)oPRL). PRL receptors were solubilized from mammary microsomes with 3-((3-cholamidopropyl)dimethylammonio)1-propane sulfonate and purified using an oPRL agarose affinity column. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and silver staining of the gel revealed at least nine bands, including a 32,000 mol wt band which was most intensively labeled with /sup 125/I using the chloramine-T method. Covalent labeling of PRL receptors with (/sup 125/I)oPRL was performed using N-hydroxysuccinimidyl-4-azido benzoate, disuccinimidyl suberate, or ethylene glycol bis (succinimidyl succinate). A single band of 59,000 mol wt was produced by all three cross-linkers when sodium dodecylsulfate-polyacrylamide gel electrophoresis was performed under reducing conditions. Assuming 1:1 binding of hormone and binding subunit and by subtracting the mol wt of (/sup 125/I)oPRL, which was estimated from the migration distance on the gel, the mol wt of the binding subunit was calculated as 32,000. In the absence of dithiothreitol during electrophoresis, only one major hormone-receptor complex band was observed. The same mol wt binding components were also detected in microsomal fractions of rabbit kidney, ovary, and adrenal. A slightly higher mol wt binding subunit was observed in rat liver microsomes. Rabbit liver microsomes revealed five (/sup 125/I)oPRL-binding components, three of which were considered to be those of a GH receptor. Moreover, affinity labeling of detergent-solubilized and affinity purified mammary PRL receptors showed a similar major binding subunit.

• OSTI ID: 5023703
• Journal Information: Endocrinology; (United States), Vol. 6
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
LTH
RECEPTORS
BIOCHEMICAL REACTION KINETICS
LABELLING
ADRENAL GLANDS
ELECTROPHORESIS
FEMALES
IN VITRO
IODINE 125
LIQUID COLUMN CHROMATOGRAPHY
LIVER
MAMMARY GLANDS
MICROSOMES
MOLECULAR WEIGHT
OVARIES
RABBITS
STH
ANIMALS
BETA DECAY RADIOISOTOPES
BODY
CELL CONSTITUENTS
CHROMATOGRAPHY
DAYS LIVING RADIOISOTOPES
DIGESTIVE SYSTEM
ELECTRON CAPTURE RADIOISOTOPES
ENDOCRINE GLANDS
FEMALE GENITALS
GLANDS
GONADOTROPINS
GONADS
HORMONES
INTERMEDIATE MASS NUCLEI
IODINE ISOTOPES
ISOTOPES
KINETICS
MAMMALS
NUCLEI
ODD-EVEN NUCLEI
ORGANOIDS
ORGANS
PEPTIDE HORMONES
PITUITARY HORMONES
RADIOISOTOPES
REACTION KINETICS
SEPARATION PROCESSES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques