Prolactin Preparations Research Articles

Point mutation of serine 179 in the human Prolactin (PRL) affects recombinant protein expression, folding and secretion, abolishes PRL nickel (II)-binding and increases heparin binding capacities

Background S179D prolactin (S179D PRL) is a pseudophosphorylated form of human prolactin (PRL) which has inhibitory effects on tumor growth [1] and angiogenesis [2]. The S179D PRL preparations used for these experiments consisted of properly refolded inclusion bodies (IB) from Escherichia coli [3]. Trying to attain a better folded mutant, we used secretion expression based systems. However, single point mutations can affect protein periplasmic expression [4], and secretion from mammalian cells [5]. We observed that upon a mutation of Serine 179 to an Aspartate, expression was nearly abolished when compared with PRL in E. coli periplasm, while the cytoplasmic product was more prone to proteolysis. Using eukaryotic cells we were able to produce preparations comparable to IBs in terms of bioactivity. We also demonstrated that this mutant had a higher affinity for heparin and lower binding capacity towards divalent metals (M (II)).

Anterior pituitary hormones: development of a bioassay leading to the discovery of prolactin.

This essay looks at the historical significance of an APS classic paper that is freely available online: Riddle O, Bates RW, and Dykshorn SW. The preparation, identification and assay of prolactin–a hormone of the anterior pituitary. Am J Physiol 105: 191—216, 1933 ( http://ajplegacy.physiology.org/cgi/reprint/105/1/191 ).

Reversed-phase high-performance liquid chromatography method for the determination of prolactin in bacterial extracts and in its purified form

Reversed-phase high-performance liquid chromatography methodology for the determination of human prolactin (hPRL) in bacterial periplasmic space or in purified preparations has been developed. The technique, based on the high hydrophobicity of the hPRL molecule, allows its separation from the bulk of bacterial proteins. The precision for periplasmic shock fluid analysis was characterized by relative standard variations of 3–7% for intra-day and of 3–25% for inter-day determinations. Accuracy, evaluated by recovery tests, was of the order of 90%, a calibration curve being constructed with the use of a lyophilized osmotic shock fluid extract, which provided a stable, readily prepared internal reference. Sensitivity was of the order of 0.5 μg of hPRL. The methodology developed also provided a tool for comparing the hydrophobicity of glycosylated and non-glycosylated prolactin molecules obtained from several different species and of different preparations of native or biosynthetic human prolactin.

Administration of unmodified prolactin (U-PRL) and a molecular mimic of phosphorylated prolactin (PP-PRL) during rat pregnancy provides evidence that the U-PRL:PP-PRL ratio is crucial to the normal development of pup tissues.

During rat pregnancy initial high concentrations of prolactin (PRL) decline by about day 9, concomitant with an increase in the ratio of unmodified to phosphorylated PRL. The physiological significance of both the decline in total PRL and the change in ratio of the two PRLs is unknown. To test the importance of each, either unmodified PRL (U-PRL) or a molecular mimic of phosphorylated PRL (PP-PRL) were continuously administered to rats throughout pregnancy. A dose of 6 microg/24 h resulted in circulating concentrations of 50 ng/ml of each administered PRL and had little effect on the pregnancy itself. After birth, pups were killed and various tissues examined. In the pup lungs, exposure to additional PP-PRL caused a reduction in epithelial integrity and an increase in apoptosis, whereas exposure to additional U-PRL had beneficial, anti-apoptotic effects. In the heart, PP-PRL caused an apparent developmental delay, whereas U-PRL promoted tissue compaction. In the blood, U-PRL increased the number of mature red blood cells at the expense of white blood cell production. Within the white blood cell population, myelopoiesis was favored at the expense of lymphopoiesis. PP-PRL, in contrast, had a less dramatic influence on the hematopoietic compartment by promoting red blood cell maturation and granulocyte production. In the thymus, exposure to PP-PRL caused accumulation of apoptotic thymocytes in enlarged glands, whereas exposure to U-PRL resulted in smaller thymi. In the spleen, exposure to U-PRL increased cellularity, with the majority of cells belonging to the erythroid series - a finding consistent with increased red blood cells in the circulation. Exposure to PP-PRL was without discernible effect. In all of these tissues, the contrasting effects of the two PRLs indicate that the absolute concentration of PRL is not crucial, but that the ratio of U-PRL to PP-PRL has a profound effect on tissue development. In brown fat, both PRL preparations decreased the number of lipid droplets. This result is therefore probably a consequence of the increase in total PRL. The results of this study attest to the importance of the U-PRL:PP-PRL ratio normally present during pregnancy and have provided clues as to the possible pathogenesis of a variety of neonatal problems.

Phosphorylation of bovine prolactin eliminates luteotropic activity in the rat

Phosphorylated and nonphosphorylated prolactins were isolated from bovine pituitaries and their luteotropic activity determined in female rats. Three groups of rats in day 1 of diestrus were treated ip twice daily for three days with 0.25 mg of either prolactin preparation or vehicle. Rats were sacrificed each day of treatment. Serum progesterone concentrations of the groups receiving vehicle or phosphorylated prolactin were similar and the vaginal cytology of these animals indicated that phosphorylated bovine prolactin (bPRL) treatment had not prolonged diestrus. Treatment with nonphosphorylated bPRL significantly increased serum progesterone concentration and the vaginal cytology indicated a diestrus prolonged for up to 4 days. Nonphosphorylated and phosphorylated bPRLs were cleared from the blood at similar rates after ip injection. In vitro receptor binding studies demonstrated that phosphorylated bPRL did not bind the ovarian prolactin receptor. Nonphosphorylated, but not phosphorylated, bPRL competed with radiolabeled bovine hormone for occupancy of rat ovarian prolactin receptors. These data are the first to test the activities of phosphorylated bPRL in vivo and indicate; 1) nonphosphorylated bPRL is luteotropic, 2) phosphorylated bPRL is neither luteotropic nor a prolactin receptor agonist or antagonist and 3) phosphorylated bPRL is not dephosphorylated in vivo rapidly enough to provide sufficient biologicallyactive bPRL to maintain luteal function.

Influence of Recombinant Chicken Prolactin on Thyroid Hormone Metabolism in the Chick Embryo

The influence of a recombinant chicken prolactin (rcPRL) preparation on thyroid function was studied in 18- and 19-day-old chicken embryos. Displacement studies on hepatic microsomes indicate that this preparation does not compete with radiolabeled chicken growth hormone (cGH) for hepatic GH-receptor binding. In a first series of experiments rcPRL or immunoaffinity-purified cGH was injected intravenously in 19-day-old chicken embryos. After 2 hr, cGH increased plasma T3 in a dose-dependent way by inhibiting hepatic inner ring type III deiodination (IRD-III) and consequently T3 degradation. Outer ring deiodination (ORD-I) was not influenced confirming previous results. The rcPRL preparation (2 and 10 μg) did not influence plasma T3, but depressed T4and raised hepatic IRD-III activity simultaneously, whereas no influence on hepatic ORD-I activity could be found. In a second experiment on 18-day-old embryos, it could be demonstrated that the effect of 2.5 μg cGH on plasma T3and liver IRD-III lasted up to 6 hr after injection, whereas 2.5 μg cPRL affected plasma T4and liver IRD-III up to 2 hr. Both rcPRL and cGH depressed rT3up to 6 hr, whereas an injection of rcPRL, but not of cGH, elevated plasma concentrations of corticosterone. These results indicate that prolactin may have a role, together with GH, in controlling peripheral thyroid hormone metabolism.

Temporal relationships and IL-2 dependency of prolactin-induced lymphocyte proliferation

Recombinant preparations of human prolactin (hPRL) and interleukin 2 (hIL-2) as well as monoclonal antibodies to these growth factors were used to study the synergistic interaction of PRL and IL-2 in Nb2 rat lymphoma lactogen-dependent cells. It was shown that IL-2 stimulated Nb2 cell proliferation in lactogen-free culture medium. Experiments with short-term exposure to growth factor demonstrated that PRL was required only during the initial 12 h of incubation while IL-2 was mitogenic regardless of the time it was added. Antibody to IL-2 partially inhibited both PRL- and IL-2-induced proliferation whereas antibody to PRL significantly decreased PRL but not IL-2-induced proliferation. These findings suggest that the complete mitogenic effect of PRL on Nb2 cells requires stimulation of IL-2 production.

Development of homologous radioimmunoassays for equine growth hormone and equine prolactin and their application to the detection of circulating levels of hormone in horse plasma

Highly purified and well-characterised preparations of equine prolactin and growth hormone from equine pituitary glands were employed to set up highly sensitive and specific homologous radioimmunoassays (RIA) for the measurement of hormone in horse plasma. The limit of sensitivity of the GH RIA was 1.2 ng/ml with mean intra- and inter-assay coefficients of variation (CV) of 6.6 and 10%, respectively. The sensitivity of the equine prolactin (ePRL) RIA was 0.5 ng/ml with mean intra and inter-assay CV of 9.1 and 15.6%, respectively. Dose-response curves of a crude pituitary gland extract and plasma samples collected from a mare and foal were parallel to the standards and the PRL RIA was clinically validated by administration of thyrotropin-releasing hormone (TRH). Plasma samples taken at 15 min intervals over 24 h from lactating mares gave 24 h mean GH values in the range 5.5 to 7.95 ng/ml. Large intermittent elevations of GH activity were detected. The mean 24 h PRL concentrations were between 3.2-10.4 ng/ml in the lactating animals, with higher concentrations earlier in lactation. Long episodic bursts of PRL were detected.

Prolactin-Brain Interactions and Reproductive Function

SYNOPSIS. There is mounting evidence that prolactin influences reproductive function in several vertebrate species via direct action on the central nervous system. In most instances, however, the basic properties of these prolactin-induced alterations in brain function, the neurochemical mechanisms underlying these effects, and the identity of the prolactinlike molecules that might normally promote these changes have yet to be adequately characterized. Several properties of the prolactin-brain relationship that have emerged from our work in the ring dove ( Streptopelia risoria ) are described in this paper and discussed in relation to similar work in other species. Receptor binding studies using radiolabelled mammalian prolactin preparations consistently indicate the existence of saturable, high affinity binding sites for prolactin in the brains of several vertebrate species. The apparent concentration of these sites in the diencephalon is corroborated by recent mapping studies in rabbits, ring doves, and three song birds in which in vitro autoradiography and densitometry was employed. Nevertheless, direct comparisons among the three songbird species suggest some differences in the precise distribution and/or concentration of prolactin binding sites within the preoptic-hypothalamiccontinuum that may relate to species differences in the display of prolactin- related incubation behavior and parental activities. Although definitive evidence is currently lacking, it would appear that blood-borne prolactin gains access to binding sites in the central nervous system and that a receptor-mediated, blood-to-cerebrospinal fluid transport process in the choroid plexus may be a major conduit for such uptake. Recent findings from several vertebrate species also suggest that the brain may synthesize one or more prolactin-like molecules that could conceivably interact with these binding sites. Direct actions of prolactin and related hormones on brain mechanisms underlying reproductive processes are suggested by the profound dose-dependent suppression of gonadotropin secretion and gonadal activity in ring doves by intracerebroventricular administration of prolactin or growth hormone at doses that are below those required for significant peripheral actions. A major challenge for future investigation will be to identify the brain site(s) at which these and other effects of prolactin are exerted and to determine how blood-borne prolactin of pituitary origin, prolactin-like molecules of brain origin, and growth hormone-like molecules interact with each other and with other neurochemical systems to promote these types of changes under normal physiological conditions.

Inhibition of recombinant human growth hormone-induced and prolactin-induced activation of neutrophils by octreotide.

Growth hormone, prolactin and somatostatin are polypeptide hormones of the neuroendocrine and peripheral nervous systems. In vitro, these have opposing effects on cells of the immune system. We compared the effects of these peptides on activation of neutrophils using a recombinant preparation of human growth hormone, human prolactin and octreotide, a long acting analog of somatostatin. In the absence of growth hormone, octreotide did not affect either neutrophil locomotion or respiratory burst. Octreotide, however, significantly antagonized growth hormone-induced activation of neutrophils for enhanced respiratory burst as well as growth hormone-induced inhibition of stimulated migration. As the effect of growth hormone on neutrophils is mediated by the prolactin receptor, its inhibition by octreotide was also tested using prolactin as priming agent. Data indicate comparable effects of octreotide on priming of neutrophils by prolactin. The effect of octreotide was dose-dependent and appeared to be selective, as activation of neutrophil respiration burst by gamma-interferon, and inhibition of stimulated migration by tumor necrosis factor-alpha were unaffected by octreotide. The present study suggests that octreotide may act on neutrophils directly by antagonizing growth hormone or prolactin at the cellular level.

International Standards for human prolactin: calibration by international collaborative study.

Three ampouled preparations of purified human prolactin were assessed by 20 laboratories in eight countries for their suitability to serve as International Standards for the estimation of human prolactin in serum. Bioassays (pigeon crop sac assays and NB2 cell assays) were carried out in two laboratories, radioreceptor assays by one laboratory and radioimmunoassays by 17 laboratories. By physicochemical analysis the preparations appeared similar. Each preparation contained small amounts of contaminants and/or prolactin variants. No major differences among the three preparations were detected by immunoassay although, in one radioreceptor assay system, one of the preparations was found to differ from the other two. On the basis of all the available information, the Expert Committee on Biological Standardization of the World Health Organization (ECBS) in 1986 established the preparation in ampoules coded 83/562 as the Second International Standard for Prolactin and in October 1988 established the preparation in ampoules coded 84/500 as the Third International Standard for Prolactin. A value of 0.053 IU (53 mIU) prolactin activity/ampoule was assigned to both the Second and Third IS on the basis that this unitage would, insofar as possible, maintain continuity of the IU defined by the First International Reference Preparation of Prolactin, human, for Immunoassay (coded 75/504).

Fluid transport across human fetal membranes affected by human amniotic fluid prolactin: an in vitro study.

The aim of the study was to investigate the effects of human amniotic fluid (AF) prolactin (PRL) on the transfer of fluids across human fetal membranes, detected in vitro by weight change. We have developed a system, consisting of an inner compartment (4 ml), suspended in an outer compartment (40 ml), and separated from it by a 2 cm2 circular piece of fresh human fetal membrane (amnion, chorion and adhering decidua). The weight of the inner compartment was continuously registered with an electronic precision balance. Osmolality changes or hydrostatic pressure differences did not affect the rate of mass transfer through the membranes. Potassium cyanide significantly influenced the mass transfer in favour of the applied hydrostatic pressure difference (+2 cm H2O to the amniotic side) or osmotic pressure difference (amniotic side 265 mosm/kg/decidual side 285 mosm/kg). The PRL preparations used were human and ovine pituitary PRL, as well as three fractions isolated from human AF by fractionated ammonium sulphate and ethanol precipitations, followed by Sephacryl chromatography. All PRL preparations were tested in physiologic concentrations (0.5 to 5 micrograms/ml). Only the two largest AF-PRL variants significantly disturbed the balance of fluid transfer across the membranes when added to the fetal (amniotic) side of the membrane. This resulted in a net increase in fetomaternal transfer of 120 to 180 microliters. This effect could be repeated and lasted for at least 25 minutes. Using an antibody against hPRL the effect was completely blocked. Neither AF-PRL added to the maternal (decidual) side of the membrane nor oPRL or human pituitary PRL added to the amniotic or decidual side changed the rate of mass transfer across the membranes.

Purification and characterisation of prolactin from sheep and buffalo pituitaries

A study of the problem of structural variants of proteins and their relative contribution to the expressed immunological and biological activity has been initiated using sheep and buffalo prolactins as models. The feasibility of obtaining immunologically and biologically active prolactin in high yields from the discarded ’acid pellet’ of sheep and buffalo pituitaries has been demonstrated. This permits use of the same batch of glands for purifying lutropin, follitropin and prolactin as side fractions. The major component in preparations of buffalo prolactin has a molecular size of 24 kDa. The preparations were active in a radioligand binding inhibition assay and in a rat liver based radioreceptor assay. Charge and size isomers of sheep prolactin and buffalo prolactin have been observed. The reference sheep prolactin did not, in preliminary work, give any indication of being glycosylated. However radioactive sulphate was found to be incorporated into prolactin-rich fractions of sheep and buffalo pituitariesin vitro. By physico-chemical and immunochemical criteria the [35S]-labelled material was similar to standard reference prolactin. The structural implications of sulphation have been probed.

Development of radioimmunoassay for eel growth hormone.

A highly specific and homologous radioimmunoassay (RIA) for the measurement of growth hormone (GH) in the plasma and the pituitary of the eel was developed using a rabbit antiserum to eel GH. Two forms of eel GH: eGHI and II, were purified from the media from organ-cultures of pituitaries of the Japanese eel Anguilla japonica. An antiserum against eGH I, which does not distinguish between the two forms, was used to measure total GH levels. Pituitary extracts from the Japanese eel and the European eel A. anguilla exhibited displacement curves parallel to the eGHI standard, whereas extracts from chum salmon, rainbow trout, Japanese charr, carp, goldfish and tilapia pituitaries showed no cross-reactivity. GH or prolactin preparations from sheep, whale, chum salmon and tilapia also did not cross-react with the antibody. RIA sensitivity was 0.2ng/ml of plasma when 50μl of plasma were employed. Most of the plasma GH levels were low or undetectable in starved eels either in fresh water or in seawater, or in fed eels in fresh water. On the other hand, plasma samples from seawater-adapted male eels receiving injections of human chorionic gonadotropin showed high GH levels. Apparent plasma half-life of eGH I injected intravascularly through a cannula into the pneumogastric artery was about 15 min.

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.

Development and validation of a salmon growth hormone radioimmunoassay

A highly specific and sensitive radioimmunoassay (RIA) for the measurement of plasma and pituitary growth hormone (GH) levels in salmonid fishes was developed using an antiserum raised in rabbit against chum salmon ( Oncorhynchus keta) GH (sGH). Pituitary extracts and plasma from chum, coho, masu, and amago salmon, and from rainbow trout and Japanese charr, all exhibited displacement curves parallel to the sGH standard. Samples from the eel, carp, goldfish, and tilapia, as well as plasma from hypophysectomized chum salmon and rainbow trout, all showed negligible cross-reactivity. None of the mammalian or teleostean GH or prolactin preparations tested cross-reacted with the antibody in the assay system. RIA sensitivity was 0.6 ng sGH/ml of plasma when 100 μl of plasma was employed. Intra- and interassay coefficients of variation were 3.9 and 4.1%, respectively. Plasma GH levels of the mature chum salmon caught in Otsuchi Bay were highly variable, especially in females (20.2 ± 8.2 ng/ml) as compared with males (16.0 ± 1.1 ng/ml), and there was no significant change after transfer to fresh water. Whereas there was no change in plasma GH levels in males kept in seawater, the levels in females increased with time in close correlation with the increase in plasma chloride.

Isolation and properties of sea turtle ( Chelonia mydas) pituitary prolactin

Sea turtle prolactin (PRL) was isolated in a highly purified state from sea turtle pituitary side fractions obtained from other studies and some of its biological, chemical, and immunological properties were determined. Sea turtle PRL is a protein of 22–24 kDa [sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis]. Its sole amino terminal amino acid residue is leucine. The amino acid composition of sea turtle PRL is similar to ovine PRL and is characterized by a high content of aspartic acid (20 residues), glutamic acid (34 residues), serine (19 residues), and leucine (24 residues). It possesses three disulfide bonds and 2 tryptophan residues which is also characteristic of many species of PRL. As with PRLs of other species, it displayed multiple stained bands in disc gel electrophoresis, at pH 8.3. Biologically, sea turtle PRL was active in pigeon crop-sac assay but the doseresponse characteristics were nonparallel when compared to ovine PRL. An antiserum against sea turtle PRL was raised in rabbit and a homologous radioimmunoassay was developed with a sensitivity of 2.8 ng for sea turtle PRL. Sea turtle gonadotropins did not cross-react, but sea turtle growth hormone showed a 5% cross-reactivity. Pituitary extracts from other species of turtles displayed parallel inhibition curves to the sea turtle PRL. Extracts and prolactin preparations from several birds, snakes, alligator, and marsupials cross-reacted, but in a nonparallel fashion. Bullfrog pituitary extract and Tilapia PRL showed no cross-reaction at high doses. Several purified mammalian PRLs (pig, sheep, human, horse, dog) showed minimal or no ability to cross-react in the RIA.

Cleaving of disulfide bridges and apparent molecular weight of human prolactin variants as revealed by immunoperoxidase electrophoresis

Five highly purified preparations of human pituitary prolactin, widely used in radioimmunoassays, were analyzed by immunoperoxidase electrophoresis with rabbit antisera raised against ovine and rat prolactin. When unreduced, the prolactin content of these preparations was separated into one major prolactin-like immunoreactive band of M r 23,000, and four fainter immunoreactive bands of M r's 16,000, 22,000, 25,000, and 45,000. Increasing the 2-mercaptoethanol concentration modified the relative proportions and the M r of immunoreactive bands so that, after extensive reduction, only two immunoreactive bands were separated, one major band of M r 25,000 and one fainter band of M r 17,000. A direct relationship existed between the load of prolactin submitted to electrophoresis in the 0.25 to 75 ng range and the optical density of the separated bands.

Rat growth hormone (GH) but not prolactin (PRL) induces both GH and PRL receptors in female rat liver.

This study was designed to elucidate which hormone is responsible for the induction of GH and PRL receptors in rat liver. Intact female rats were implanted with osmotic minipumps delivering rat GH (rGH) or ovine GH (oGH) or PRL at various rates from 75 to 800 micrograms/day for 7 days, and binding of radioiodinated bovine GH or ovine PRL (oPRL) tracer was measured on liver microsomal membranes. MgCl2 treatment was used to remove bound hormones from receptors before tracer binding. Infusion of rGH resulted in a significant increase (P less than 0.001) in both GH and PRL binding, the effect being maximal (2.5- to 3-fold for both ligands) at rGH infusion rates from 150 to 400 micrograms/day. Serum rGH levels were elevated 3- to 5-fold in these animals, but somatomedin-C concentrations were not higher than in controls. MgCl2 treatment showed that GH, but not PRL, binding sites in rGH-treated animals were significantly occupied by administered hormone. Analysis of competitive binding curves indicated that receptors for both GH and PRL increased in concentration without changes in binding affinity. In contrast to the rGH effect, oGH infusion from 75 to 400 micrograms/day failed in two experiments to consistently alter either bovine GH or oPRL binding sites. This was not explained by the potency of the preparation at the somatogenic receptor; oGH was in fact more potent than rGH. The effects of rat PRL and oPRL infusion on receptor levels were also assessed. In contrast to previous reports, neither preparation caused induction of either PRL or GH binding sites. oPRL decreased PRL binding by 30-40% when infused between 200 and 400 micrograms/day, whereas rat PRL had a less consistent effect. MgCl2 stripping of membranes suggested that administered PRL preparations did not significantly occupy PRL receptors. GH receptors were unaffected in any PRL-treated group. It is concluded that in intact female rats, rGH regulates the concentration of both GH and PRL receptors. The slight down-regulation of PRL receptors resulting from PRL infusion casts further doubt on the concept that PRL induces its own hepatic receptors.

Glycosylated ovine prolactin.

A modification of prolactin, in which the asparagine at position 31 carries a carbohydrate unit, was isolated from ovine pituitary glands. Sequence and amino acid analyses identified the point of linkage of the carbohydrate. Glucosamine was found in acid hydrolysates, an indication that the carbohydrate is attached through N-acetylglucosamine. The glycosylated prolactin binds to concanavalin A and lentil lectin and is eluted with methyl alpha-D-mannopyranoside. During gel electrophoresis in sodium dodecyl sulfate, the glycosylated hormone migrates as a Mr 25,000 protein; prolactin has a Mr of 23,000. The modified prolactin had a potency of 20 international units/mg, approximately equal to 60% the potency of a reference prolactin preparation when measured by the pigeon crop sac assay. In a radioimmunoassay, the glycosylated form had only 34% the immunoreactivity of ovine prolactin.