Amino acid sequence of a progesterone-binding protein.

Abstract

The amino acid sequence of blastokinin, also called uteroglobin, has been determined by a combined study of both the intact native molecule and the peptide fragments resulting from tryptic and chymotryptic digestions. Sequence analyses performed by automated methods and by sequential digestion with leucine aminopeptidase and carboxypeptidase Y demonstrate that blastokinin is a dimer of identical 69-amino acid subunits held together in parallel orientation by two disulfide bridges at positions 3 and 68. The polypeptide chains are further characterized by the absence of tryptophan residues and by single residues of histidine and tyrosine at positions 8 and 21, respectively. Six of eight amino acids, positions 17-24, near the progesterone binding site of blastokinin contain a hydroxyl group. Knowledge of the chemistry of this receptor site should allow better perspectives of the chemistry of molecules in normal tissues that are dependent on progesterone for growth and development, as -well as compounds that could act as cancer antagonists for endocrine therapy of hormone-dependent tumors. Blastokinin (1), also termed uteroglobin (2), is a low molecular weight protein that was first isolated from the uterine fluid of pregnant rabbits. The synthesis and secretion of this protein have been shown to increase during the first 6 days of pregnancy, accounting for-more. than 50% of the total uterine protein by day 6 (day 0 = coitus) (3-5). The increase in blastokinin/uteroglobin (BK/UG)t levels at a time when endogenous levels of progesterone were rising implied that a correlation may exist between these two events. Several reports demonstrated that exogenous progesterone was capable of inducing the synthesis of this protein (2, 6, 7). The recent finding that BK/UG in turn binds progesterone with high affinity (7-9) implies that BK/UG may serve as a carrier molecule for the same steroid that induces its synthesis. These facts not only make BK/UG unique but they also make a further study of its structure important. In the paper we present the primary structure for BK/UG, its possible physiological role(s), and the significance of knowing its structure as a hormone receptor. amide gel electrophoresis and a single immunoprecipitin band in agar immunodiffusion plates. Prior to chemical analyses, an additional purification step was performed by molecular sieving through a Sephadex G-25 column (1.9 X 95 cm) equilibrated with 10 mM HC1. Elution with the same buffer (flow rate, 2 ml/min) allowed BK/UG to elute with the void volume while salts and other low molecular weight contaminants were still migrating. Peak fractions of BK/UG, identified by absorbancy at 280 nm, were pooled and lyophilized. Four samples, each containing 7-14 mg of BK/UG, were isolated. One sample was digested by trypsin without further treatment. The disulfide bonds of cystine in the other three samples were reduced and aminoethylated (11) prior to digestion by trypsin or chymotrypsin. MATERIALS AND METHODSAmino acid analyses were performed with a Be...

Prolactin as a factor in the uterine response to progesterone in rabbits

“…An elution profile was obtained with a protein assay by the method of Lowry, Rosebrough, Farr & Randall (1951) and by absorption at 280 nm. The fractions of the uteroglobin peak were pooled and concentrated, and Sephadex G-50 gel filtration was used in a similar manner to purify the sample further as described by Popp, Foresman, Wise & Daniel (1978). PAGE was used to check the homogeneity of this sample.…”

Embryo-uterine interactions during early stages of pregnancy in domestic mammals

“…However, in these extra-uterine sites, uteroglobin is not progesterone dependent (Savouret et al, 1980). Its amino acid sequence (Ponstingl et al, 1978 ;Popp et al, 1978) and crystalline structure (Mornon et al, 1980) have been determined ; the gene has been cloned (Chandra et al, 1980). Uteroglobin binds progesterone (Beato, 1977;Fridlansky and Milgrom, 1976) and passes through the blastocyst coverings (Kirchner, 1972).…”

Progesterone dependent uptake of uteroglobin by rabbit endometrium

“…UGL with a molecular weight of about 15,000 (Murray et al 1972;Bullock and Connell 1973;Nieto et al 1977) has fourteen free a-amino plus e-amino groups of lysine (Ponstingl et al 1978;Popp et al 1978). After N-acetylation of UGL about every 500th amino group was substituted by the labelling procedure.…”