Radioreceptor assay for lactogenic hormones based on membranes from rat mammary tumour.

A highly sensitive radioreceptor assay (RRA) for human prolactin (hPRL) based on membrane preparations obtained from chemically induced rat mammary tumour is described. The binding of 125I-labelled, highly purified pituitary human prolactin was specific for lactogenic hormones and depending on time, temperature, and concentration of receptor protein. Optimal specific receptor binding (18-20%) was obtained by incubation at 21 degrees C for 18 h. The prolactin receptor was shown to have a single "class" of binding sites with an affinity constant (Ka) of 6.0 X 10(10) mol-1. The binding capacity was 8-33 fmol/mg membrane protein. The sensitivity of the radioreceptor assay was 0.5 ng/ml ovine prolactin (NIH-PS-10) or 0.84 ng/ml human prolactin (NIH-VLS-4). The receptor binding activity of various purified prolactin preparations from different species was comparable to the biological hormone activities, indicating that this in vitro assay system measures values which are biologically relevant.

Polymorphism of human pituitary lutropin (LH). Effect of the seven isohormones on mouse Leydig cell functions.

The effect of the 7 different molecular forms of human pituitary lutropin (LH) on mouse Leydig cells as a model of a target organ were studied. The hormone functions have been characterized by receptor binding, cAMP accumulation and testosterone production. One important finding was the similar intrinsic in vitro biological activity for all isohormones. Quantitatively, however, the potencies of the 7 hormone forms did not correlate with the activities obtained by radioimmunoassay: there was a dramatic decrease of receptor binding activity and biological activity compared to immunoactivity from the more alkaline to the more acidic LH isohormones.

Polymorphism of human pituitary lutropin (LH). Isolation and partial characterization of seven isohormones.

The complete microheterogeneous system of human pituitary lutropin was demonstrated by gel isoelectric focusing. In addition 7 LH isohormones could be isolated by preparative column focusing and characterized with respect to physicochemical, biological and immunological properties. The in vivo biopotencies ranged from 4.50-11.50 IU/mg, the in vitro bioactivity was from 1.20-10.10 IU/mg, and the immunological activity was from 3.10-7.55 IU/mg. The sialic acid content was found to be 1.8-3.2%. Treatment with neuraminidase resulted in a shift of all bands to the alkaline region, however the 7 LH forms were still present.

Biological and immunological potencies of lutropin (LH) in human serum: comparative studies using different standard preparations.

The mouse leydig cell in vitro-bioassay for LH, first reported by van Damme, Robertson and Diczfalusy (1974) was modified and applied to female and male serum. Non-parallelism of dose-response curves between serial dilutions of individual male as well as female sera and LH standards was caused by damage to incubated interstitial cells in the presence of human serum. The extent of cell damage- paralleled by an inhibition of testosterone production - was a characteristic of individual human sera rather than a general protein effect. This inhibition could be completely avoided by mild heating of the serum for 15 min at 50 degree C prior to the assay. Using this pretreatment, reliable LH values were obtained for normal males, cycling females and postmenopausal women. Biological LH measurements were compared with RIA-LH potencies. The following LH value were obtained by both methods (mean +/- sd) in terms of mlU 2nd IRP HMG/ml serum: males (n = 35), BIO: 23.3 +/- 9.0, IMMUNO: 11.0 +/- 4.3; cycling females (n = 30), BIO: 30.9 +/- 14.6, IMMUNO: 17.4 +/- 5.9; postmenopausal women (n = 12), BIO: 324 +/- 138, IMMUNO: 117 +/- 43. It could be shown that the use of different reference preparations caused great differences in the ratios of biological to immunological potencies e.g. for male serum 0.9, female serum 0.7-5.6 and postmenopausal female serum 1.1 - 8.8. Irrespective of the standard used, significant differences between the B:l ratios of male, female and postmenopausal female sera were found.

Isolation of highly purified sex hormone binding globulin (SHBG): evidence for microheterogeneity.

Highly purified sex hormone binding globulin (SHBG) was isolated in milligram amounts from a human serum fraction (Cohn IV-4). The final preparation was homogeneous by the criteria of polyacrylamide-gel electrophoresis. Immunological evidence for purity could be given by double diffusion according to Ouchterlony. However, following gel isoelectric focusing highly purified SHBG displayed four different bands, as could be demonstrated by staining as well as by a photoscan of the [3H]5alpha-dihydrotestosterone-SHBG complex. After incubation with neuraminidase the microheterogeneity of SHBG disappeared and the asialo-SHBG showed only one band.

A low molecular weight substance obtained from serum which has luteinizing hormone like activity ("mini LH").

A low molecular weight substance of 1000-2000 daltons has been isolated in dialysates of normal human serum which, on testing in a radio-immunoassay, a radioligand receptorassay and an in-vitro bioassay exhibited LH activity comparable to the range of LH activity found in normal serum. The substance was heat labile. Its reactivity in a RIA suggests peptide nature. No "mini LH" activity could be derived from LH-free serum samples of hypophysectomized patients.

Existence of big and little forms of luteinizing hormone in human serum.

Serum fractions from normal subjects obtained by gel chromatography have been investigated using three different assay systems: radioimmunoassay (RIA), radioligand receptor assay (RRA), and testosterone production assay (TPA). The bulk of immunoassayable and "bioassayable" LH-activity was found in two fractions differing widely in their molecular size. The slower moving component, designated as "little" LH, migrated identical to the radioiodinated pituitary hormone (LER 960) with a molecular weight of about 30,000, while "big" LH appeared in an elution volume consistent with a molecular weight range between 140,000 and 180,000. Concordance was seen between the LH-activities measured in all three assay systems. The RRA/RIA ratio varied between 1.6 and 8.9, the RRA/TPA ratio was close to unity. Treatment with 6 M urea and 0.1% mercaptoethanol and also, exposure to different pH values and salt concentrations did not change the elution position of the two LH components. Also, "big" and "little" LH appeared unaltered after re-filtration and no conversion each other could be found. In another experiment injection of gonadotrophin releasing hormone (Gn-RH) into a male induced a profound shift of LH towards the low molecular weight species. Kinetic uptake studies with "big" and "little" LH using RRA showed identical affinities to the receptor preparation. Ion exchange chromatography of serum, however, did not give two LH components, indicating no major differences in charge properties. This finding could be confirmed by preparative gel isoelectric focusing. The RRA potencies following gel filtration were in good agreement with that applied to the column, however, the immunological activities exceeded that of loaded by a factor 3-4. A new aspect of serum LH heterogeneity is the finding of a low molecular substance (mol. weight approximately 1000) in the outer dialysate of serum, which has LH like activity in all three assay systems.