Abstract
Abstract A receptor for prolactin and other lactogenic hormones (human growth hormone and placental lactogens) has been solubilized by 1% (v/v) Triton X-100 from a crude particulate membrane fraction isolated from pregnant rabbit mammary glands. The receptor remained in solution after centrifugation at 200,000 x g for 2 hours. Triton X-100 at concentrations higher than 0.01% (v/v) affected the physical properties of 125I-prolactin, whereas 125I-human growth hormone (hGH) was unaffected and, therefore, the latter has been used in studies of binding to the prolactin receptor in situations where 125I-prolactin could not be used. It was possible to use 125I-hGH for binding studies because this hormone has the same lactogenic potency as prolactin does in the rabbit and the prolactin receptor cannot discriminate between these two hormones. When an aliquot of soluble receptor protein was incubated with 125I-hGH, the hormone-receptor complex was detected in the void volume of Sephadex G-100 column or, more rapidly, by precipitation with polyethylene glycol (12.5%, w/v). The soluble receptor retained the specificity of binding noted in the particulate fraction. However, Scatchard analysis demonstrated that the affinity of the soluble receptor (Ka = 16 x 109 m-1) for the hormone was 5 times greater than that of the particulate receptor (Ka = 3 x 109 m-1). The soluble prolactin receptor has been purified approximately 1500-fold by affinity chromatography using hGH-coupled N-hydroxysuccinimide ester of 3,3'-diaminodipropylaminosuccinyl agarose (Affi-Gel 10, Bio-Rad Laboratories). The receptor was labile in the presence of many of the commonly used dissociating reagents such as sodium thiocyanate, guanidine hydrochloride, and acetic acid. However, magnesium chloride (5 m) proved to be most effective for the elution of the receptor from the affinity adsorbent. About 0.5 mg of partially purified receptor protein could be obtained from 100 g of mammary gland. By gel filtration on Sepharose 6B the molecular weight of the receptor was found to be approximately 220,000. Polyacrylamide gel electrophoresis (gel concentration 7.5%, w/v; pH 8.9) of the partially purified receptor revealed several protein bands. Upon assaying the material eluted from gel slices, the receptor activity coincided with one or possibly two of the major protein bands at Rf 0.12. These studies suggest that it is feasible to utilize the soluble receptor for the development of a very sensitive radioreceptor assay for prolactin and, further, to obtain a highly purified receptor in sufficient quantity to facilitate studies on its physiological, biochemical, and immunological properties.
Highlights
We describe the solubilization of the prolactin receptor from the rabbit mammary gland and partial purification of the receptor by affinity chromatography
Between 30 and 65% of the total protein in the particulate membrane fraction is solubilized with 1% Triton X-100 under the conditions employed
It became apparent that 1251-prolactin when fractionated on Sephadex G-100 column in the presence of detergent, emerged close to the void volume (Fig. 1, top), whereas in the absence of detergent, the 1251-ovine prolactin (oPRL) eluted at a position about twice the void volume where “normal” prolactin with a molecular weight of 23,000 is found. ‘%I-human growth hormone (hGH), on the other hand, is unaffected by Triton X-100 (Fig. 1, bottom)
Summary
A receptor for prolactin and other lactogenic hormones (human growth hormone and placental lactogens) has been solubilized by 1% (v/v) Triton X-100 from a crude particulate membrane fraction isolated from pregnant rabbit mammary glands. Purified receptors obtained from 3 to 5 of such experiments were pooled and concentrated in an Amicon cell with UM-10 filter which retains molecules having molecular weights larger than 10,000 This concentrated solution contained protein concentration of 100 to 300 pg per ml which could be measured accrately by the procedure of Lowry et al (18) or by the more sensitive method using fluorescamine (19). V,) where V. is the elution volume corresponding to the peak concentration of the solute (marker proteins were measured by the method of Lowry et al (18)), Vo is the void volume of the column as determined by the appearance of blue dextran (monitored by absorbance at 660 nm), and VI is the total liquid volume as determined by Na12SI. The relative mobility (RF) of receptor activity and protein bands were compared
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