Prolactin peptide (pPRL) induces anti-prolactin antibodies, ROS and cortisol but suppresses specific immune responses in rainbow trout.

Prolactin has several immune functions in fish however, the effects on innate and specific components of rainbow trout immunity are currently unknown. Therefore in this study, prolactin peptide (pPRL) injection in rainbow trout generated anti-PRL antibodies that were confirmed through Western blot assays of fish brain tissue extract. At the same time, this group of fish was immunized with a viral antigen (VP2) and the specific antibody titer generated by the rainbow trout was subsequently determined, as well as the sero-neutralizing capacity of the antibodies. Interestingly, this group of fish (pPRL-VP2) generated approximately 150% less antibodies compared with fish immunized only with the viral antigen (VP2), and pPRL-VP2 fish increased their cortisol level by 4 times compared to the control. Additionally, through qPCR assay, we determined that the pPRL-VP2 fish group decreased pro-inflammatory transcript expression, and the serum of these (pPRL-VP2) fish stimulated ROS production in untreated fish leukocytes, a phenomenon that was blocked by the pharmacological cortisol receptor inhibitor (RU486). Collectively, this is the first report that indicates that pPRL could modulate both components of immunity in rainbow trout.

In silico prediction of prolactin molecules as a tool for equine genomics reproduction.

The prolactin hormone is involved in several biological functions, although its main role resides on reproduction. As it interferes on fertility changes, studies focused on human health have established a linkage of this hormone to fertility losses. Regarding animal research, there is still a lack of information about the structure of prolactin. In case of horse breeding, prolactin has a particular influence; once there is an individualization of these animals and equines are known for presenting several reproductive disorders. As there is no molecular structure available for the prolactin hormone and receptor, we performed several bioinformatics analyses through prediction and refinement softwares, as well as manual modifications. Aiming to elucidate the first computational structure of both molecules and analyse structural and functional aspects related to these proteins, here we provide the first known equine model for prolactin and prolactin receptor, which obtained high global quality scores in diverse software's for quality assessment. QMEAN overall score obtained for ePrl was (- 4.09) and QMEANbrane for ePrlr was (- 8.45), which proves the structures' reliability. This study will implement another tool in equine genomics in order to give light to interactions of these molecules, structural and functional alterations and therefore help diagnosing fertility problems, contributing in the selection of a high genetic herd.

N-glycoprofiling analysis in a simple glycoprotein model: a comparison between recombinant and pituitary glycosylated human prolactin.

Human prolactin (hPRL) is a polypeptide hormone occurring in the non-glycosylated (NG-hPRL) and glycosylated (G-hPRL) forms, with MM of approximately 23 and 25kDa, respectively. It has a single, partially occupied N-glycosylation site located at Asn-31, which makes it a particularly simple and interesting model for glycosylation studies. The bioactivity of G-hPRL is lower than that of NG-hPRL (by ca. 4-fold) and its physiological function is not clear. However, carbohydrate moieties generally play important roles in the biosynthesis, secretion, biological activity, and plasma survival of glycohormones and can vary depending on the host cell. The main objective of this study was to determine the N-glycan structures present in native, pituitary G-hPRL and compare them with those present in the recombinant hormone. To obtain recombinant G-hPRL, genetically modified Chinese hamster ovary cells (CHO), adapted to growth in suspension, were treated with cycloheximide, thus increasing the glycosylation site occupancy from 5.5% to 38.3%, thereby facilitating G-hPRL purification. CHO cell-derived G-hPRL (CHO-G-hPRL) was compared to pituitary G-hPRL (pit-G-hPRL) especially with regard to N-glycoprofiling. Among the main differences found in the pituitary sample were an extremely low presence of sialylated (1.7%) and a high percentage of sulfated (74.0%) and of fucosylated (90.5%) glycans. A ∼6-fold lower in vitro bioactivity and a higher clearance rate in mice were also found for pit-G-hPRL versus CHO-G-hPRL. N-Glycan profiling proved to be a useful and accurate methodology also for MM and carbohydrate content determination for the two G-hPRL preparations, in good agreement with the values obtained directly via MALDI-TOF-MS.

Novel fusion protein derived from vasostatin 30 and vasoinhibin II-14.1 potently inhibits coronary endothelial cell proliferation.

Angiogenesis has been considered an important target for cancer therapy. The inhibition of angiogenesis represents a promising strategy for anti-cancer treatment, tumor growth inhibition, and metastasis. Vasostatin 30 (Vs30), and the 14.1 kDa vasoinhibin (Vi-II-14.1) are two peptides with remarkable anti-tumor and anti-angiogenic effect. The aim of this study was to produce a novel fusion protein between Vs30 and Vi-II-14.1, denominated VS_VI, to obtain a new protein with higher biological activity. The protein fusion genes were cloned into a T7 promoter-based vector, expressed in Escherichia coli BL21-SI and purified by affinity column chromatography. In vitro assays showed that the recombinant fusion protein inhibited rat coronary endothelial cell proliferation at 65.5 % at 10 nM, whereas recombinant Vs30 and Vi-II-14.1 inhibited at 33 and 50.5 % respectively, at the same concentration. The results showed that VS_VI is significantly more active than the Vs30 and Vi-II-14.1 separately. In addition, a practical classification of the vasoinhibins based on the peptide origin and theoretical molecular weight is proposed. This is the first study to produce a new fusion protein derived from Vs30 and Vi-II-14.1, both of them proposed as promising therapeutic agents.

N-terminal prolactin-derived fragments, vasoinhibins, are proapoptoptic and antiproliferative in the anterior pituitary.

The anterior pituitary is under a constant cell turnover modulated by gonadal steroids. In the rat, an increase in the rate of apoptosis occurs at proestrus whereas a peak of proliferation takes place at estrus. At proestrus, concomitant with the maximum rate of apoptosis, a peak in circulating levels of prolactin is observed. Prolactin can be cleaved to different N-terminal fragments, vasoinhibins, which are proapoptotic and antiproliferative factors for endothelial cells. It was reported that a 16 kDa vasoinhibin is produced in the rat anterior pituitary by cathepsin D. In the present study we investigated the anterior pituitary production of N-terminal prolactin-derived fragments along the estrous cycle and the involvement of estrogens in this process. In addition, we studied the effects of a recombinant vasoinhibin, 16 kDa prolactin, on anterior pituitary apoptosis and proliferation. We observed by Western Blot that N-terminal prolactin-derived fragments production in the anterior pituitary was higher at proestrus with respect to diestrus and that the content and release of these prolactin forms from anterior pituitary cells in culture were increased by estradiol. A recombinant preparation of 16 kDa prolactin induced apoptosis (determined by TUNEL assay and flow cytometry) of cultured anterior pituitary cells and lactotropes from ovariectomized rats only in the presence of estradiol, as previously reported for other proapoptotic factors in the anterior pituitary. In addition, 16 kDa prolactin decreased forskolin-induced proliferation (evaluated by BrdU incorporation) of rat total anterior pituitary cells and lactotropes in culture and decreased the proportion of cells in S-phase of the cell cycle (determined by flow cytometry). In conclusion, our study indicates that the anterior pituitary production of 16 kDa prolactin is variable along the estrous cycle and increased by estrogens. The antiproliferative and estradiol-dependent proapoptotic actions of this vasoinhibin may be involved in the control of anterior pituitary cell renewal.

Synthesis, purification and characterization of recombinant glycosylated human prolactin (G-hPRL) secreted by cycloheximide-treated CHO cells.

Human prolactin (hPRL) is a 199 aminoacid protein hormone with a wide spectrum of biological activities which is best known for its stimulation of lactation and development of mammary gland. This protein contains only one potential asparagine-linked glycosylation site, which is partially (10-30%) occupied when the protein is synthesized in eukaryotic cells. Although the biological activity of glycosylated hPRL (G-hPRL) has been found to be approximately 4-fold lower than that of hPRL, its physiological function is not yet well defined. In order to better characterize and study this hormone variant, we carried out its laboratory scale purification from conditioned medium of genetically modified CHO cells that had been supplemented with cycloheximide. Addition of cycloheximide increased the absolute concentration of G-hPRL approximately 4-fold and the glycosylated versus non-glycosylated hPRL concentration ratio by approximately 7-fold. G-hPRL purification was carried out via a two-step process based on a cationic exchanger and a size-exclusion HPLC (HPSEC) column. Characterization was carried out by HPSEC, Western blotting, MALDI-TOF-MS and in vitro bioassay based on Nb2 and Ba/F3-LLP cells, the biological activity being of the same order (11-15 IU mg(-1)) in the two assays. Our results show that addition of cycloheximide can be an important strategy for increasing glycosylated protein production, facilitating the purification and characterization of these isoforms.

A molecular mimic of phosphorylated prolactin (S179D PRL) secreted by eukaryotic cells has a conformation with an increased positive surface charge compared to that of unmodified prolactin.

S179D prolactin (S179D PRL) is a pseudophosphorylated form of human PRL which has potent antitumor and anti-angiogenic activities in vivo. This molecule binds to the same forms of the PRL receptor (PRLR) as unmodified PRL, yet this binding results in different intracellular signaling and biological end points. Since it is now clear that PRLRs are predimerized and therefore that ligand binding must initiate signaling by inducing a conformational change in the receptor dimer, we hypothesized that S179D PRL had an altered conformation compared to unmodified PRL. The conformation of the ligand-receptor ternary complex would therefore also have an altered conformation, and thus, different signaling molecules would be activated. Here we present evidence in support of this hypothesis by demonstrating, in contrast to unmodified PRL, that S179D PRL has reduced nickel and zinc binding capacity and a higher affinity for heparin and DEAE. Conformational changes have occurred since these features are counterintuitive on the basis of the simple substitution of a serine with a negatively charged aspartate residue. To demonstrate that these particular properties of S179D PRL were not due to misfolding of the molecule during production, S179D PRL was expressed in two different mammalian cell lines. Also investigated was the potential for production of S179D PRL as a soluble cytoplasmic, or secreted periplasmic, protein in Escherichia coli.

Prolactin-like hormone in the nematode Trichinella spiralis larvae.

Expression of prolactin (PRL) or prolactin-like hormone has been reported in invertebrates. We investigated the larval phase of Trichinella spiralis: (a) to express 23 kDa PRL, (b) to define its localization and (c) to test its possible biological activity. Immunostaining in isolated larvae demonstrated positive material to 23 kDa PRL by all along the stichosome, specifically in the stichocytes. Homogenized immunoblot larvae showed a 23 kDa protein band. To assess PRL release and its biological activity, larvae were incubated in culture medium and the excretory/secretory products were analyzed by the Nb2 cells bioassay. A cellular growth equivalent until 10 nM PRL and using antibody against 23 kDa PRL, the growth was blocked. In conclusion our result provides evidence that PRL-like hormone is expressed and secreted by the larvae of T. spiralis.

Physico-chemical and biological characterizations of two human prolactin analogs exhibiting controversial bioactivity, synthesized in Chinese hamster ovary (CHO) cells.

The synthesis, purification and characterization of G129R-hPRL and S179D-hPRL, the two better-studied antagonists of human prolactin (hPRL), is described. Both of these have been expressed for the first time, in their authentic form, by a stable CHO cell line, at secretion levels of 7.7 and 4.3 microg/10(6) cells/day, respectively. Previous studies had shown that these hPRL analogs, when produced in bacterial cytoplasm, consistently contained misfolded forms and multimers according to the specific denaturation, refolding and purification conditions. These versions also have an N-terminal extra methionine. An extensive physico-chemical characterization was carried out after a practical two-step purification process and included SDS-PAGE and Western blotting analysis, matrix-assisted laser-desorption ionization time-of-flight mass spectral (MALDI-TOF-MS) analysis, high-performance size-exclusion chromatography (HPSEC) and reversed-phase high-performance liquid chromatography (RP-HPLC). This last technique revealed a considerable difference in hydrophobicity due to a single amino acid substitution, with S179D-hPRL less (t(RR) = 0.85 +/- 0.010) and G129R-hPRL more (t(RR) = 1.10 +/- 0.013) hydrophobic than hPRL, where t(RR) is the relative retention time. The biological characterization was based on further refinement of a sensitive proliferation assay using the pro-B murine cell line (Ba/F3) transfected with the long form hPRL receptor cDNA such that the minimal detectable dose was 0.04 ng of hPRL/mL, the Ba/F3-LLP assay. On the basis of this assay, the relative residual agonistic activity of these two products, determined against a hPRL international standard in four independent assays, was 53 x 10(-3) for S179D-hPRL and 70 x 10(-5) for G129R-hPRL. We believe that the present synthesis and characterization could be extremely helpful for studies of these two proteins, which have been reported to antagonize tumor growth-promoting effects of hPRL in vivo in animal models of breast and prostate cancer.

The application of NIR Raman spectroscopy in the assessment of serum thyroid-stimulating hormone in rats.

Serum blood samples of euthyroid and thyroidectomized rats treated with thyrotropin-releasing hormone (TRH) were analyzed on aluminum substrates using the near-infrared Raman spectroscopy (830 nm). Spectra of thyroid-stimulating hormone (TSH), TRH and prolactin standards were obtained. Differences between Raman spectra profiles of control and Tx + TRH samples groups were found. These differences were confirmed by the linear discriminant analysis (LDA), which presents a good classification between groups. It is supposed that these differences are produced by the increment of TSH in the thyroidectomized rats.

Analysis of molecular heterogeneity of prolactin in human systemic lupus erythematosus.

Hyperprolactinemia without clinical manifestations has been reported in some patients with systemic lupus erythematosus (SLE) because an increase of prolactin (PRL) is produced due to the BIG/BIG molecular variant (molecular variant < 150 kD). This research project aimed to determine levels of PRL: its bioactive form, the little nonglycosylated form (NGPRL) and variants with decreased bioactivity such as the BIG/BIG and the little glycosylated (GPRL), in 29 women and five men with SLE. PRL was assayed by IRMA with a kit from Immunotech Laboratory, the BIG/BIG form by precipitation with polyethyleneglycol 6000, and the NGPRL and GPRL by chromatography on Concanavalin-A- Sepharose. Increased PRL was detected in seven patients (20.6%) of whom three had increased BIG/BIG, six had increased GPRL and only four had increased NGPRL. The three cases with increased BIG/BIG were contrasted by chromatography on Sephadex G-100. No increased PRL or any of the other variants assayed were found in men. Results were similar when PRL was evaluated in the same blood samples by a different IRMA (DPC Laboratory). The etiology of the hyperprolactinemia in some of these patients is unknown, but their lack of symptoms (galactorrhea or amenorrhea) could be due to the BIG/BIG forms and basically to the glycosylation of the hormone. As for the relation between PRL and SLE activity, we found that hyperprolactinemic patients were younger, had a shorter history of illness, although it was not statistically significant, and a higher SLEDAI score. This would indicate a relation between hyperprolactinemia and lupus activity. The patients with increased BIG/BIG form also had a very active illness at the time of the study.

Heterogeneity of circulating prolactin in the bitch.

Different molecular forms of circulating prolactin (PRL) are known to occur in several species. As no such information was available in dogs, we assessed the molecular profile of circulating PRL in bitches. Pooled sera from covertly (CTRL) and overtly pseudopregnant (PSPT) diestrous bitches with high or low (> 10 or < 10 ng x mL(-1), respectively) serum PRL (measured by ELISA) were analyzed by Sephadex G-100 and Concanavalin A-Sepharose column chromatography. Four serum PRL fractions were identified and termed big-big, big (> 67 kDa), native (23 kDa) and fragmented (< 20) kDa) PRL. The percentages of these fractions were roughly similar in CTRL and PSPT animals, irrespective of their serum PRL levels (higher in PSPT than in CTRL bitches). A large proportion of glycosylated PRL (between 69 and 100%) was also detected in these sera. We conclude that in dogs, circulating PRL occurs in multiple molecular forms, whose relative abundance is comparable in covertly and overtly pseudopregnant bitches.

Macroprolactinemia in childhood and adolescence: a cause of asymptomatic hyperprolactinemia.

Asymptomatic hyperprolactinemias associated with altered proportions of molecular forms of circulating prolactin (PRL) have been reported in adults. The scarce references available in children and adolescents prompted us to report our experience in the evaluation and follow-up of patients with macroprolactinemia. We studied 5 patients (1 male and 4 females) aged 11.6-18 years with incidentally discovered asymptomatic hyperprolactinemia. Patients underwent repeated evaluations for a period of 3 months to 8 years, and their PRL levels remained elevated (34.4-516 ng/ml). Structural variants of PRL >/=45 kD ranged between 58.9 and 78.6%. Chromatographic profiles showed increases in Big Big PRL in the 5 cases, ranging between 40 and 72% (normal: 9-21%), and in Big PRL in 3 cases, ranging between 30.0 and 32.6% (normal: 5-25%). Little PRL was decreased in all cases, ranging between 20.6 and 41.1% (normal: 50-90%). In conclusion, upon detection of hyperprolactinemia with no clinical manifestations and no alteration of the remaining endocrine functions, macroprolactinemia should be considered as a possible diagnosis. The confirmed absence of functional alterations during the follow-up would favor a no-treatment approach and at the same time avoid repeating imaging studies.

Caracterización bioquímica de prolactina de diferentes especies / Biochemical characterization of prolactin of different species

Se utilizaron diferentes técnicas para determinar las características bioquímicas de las preparaciones de prolactina (Prl) de origen hipofisario con preparados que se utilizan en centros de investigaciones para el desarrollo de sistemas in vivo e in vitro, bioanálisis, radioinmunoanálisis y análisis inmunoenzimáticos. Los preparados se analizaron por electroforesis bajo condiciones reductoras (SDS-PAGE), electrotransferencia e inmunodetección, enfoque isoeléctrico, cromatografía líquida de alta presión (HPLC), y capacidad de unión a receptores microsomales (RRA). Se encontró que en las Prl (s) porcina (pPrl), ovina (oPrl) y humana (hPrl) existen proporciones significativas de la forma glucosilada (30-40 porciento), no así en la Prl bovina (bPrl) y en la de rata (rPrl). Se comprobó que es posible detectar impurezas o mezclas de las diferentes formas moleculares de la Prl presentes en estos preparados hipofisarios utilizando HPLC. El análisis por enfoque isoeléctrico de las Prl (s) hipofisarias de diferentes especies reveló que en la bPrl existen 4 isoformas de carga eléctrica, la oPrl y la hPrl están compuestas por 3 isoformas mientras que la rPrl y la pPrl presentan 1 y 2 isoformas, respectivamente. Aún se desconoce cuál podría ser el significado fisiológico de las isoformas de carga eléctrica en la Prl hipofisaria de diferentes especies. Según los resultados obtenidos a partir de las curvas de desplazamiento y de las gráficas de Scatchard, se encontró que existen diferencias entre las constantes de disociación (Kd) y la capacidad de unión de las prolactinas a los receptores microsomales. Finalmente es necesario conocer con mayor exactitud las propiedades bioquímicas, inmunológicas y biológicas de los preparados de las prolactinas que son utilizadas en diferentes estudios tanto in vivo como in vitro, porque en esas residen las funciones biológicas de la hormona. Se confirmó y se amplió la heterogeneidad estructural de la Prl en diferentes especies de mamíferos, que dan lugar a la existencia de diferentes variantes moleculares de la hormona y a la diversidad de funciones que ejerce. Se comprobó la necesidad de desarrollar nuevos métodos para el estudio del significado biológico de la heterogeneidad estructural de la Prl y de obtener las diferentes variantes moleculares de la Prl con un alto grado de pureza, que permitan establecer nuevos métodos cuantitativos, útiles para ser aplicados en estudios clínicos y bioquímicos(AU)

Different techniques were used to determine the biochemical characteristics of the Prolactin (Prl) preparations of hypophyseal origin with preparations that are used in research centers for the development of in vitro and in vivo systems, bioanalyses, radioimmunoanalyses and immunoenzimatic analyses. The preparations were analyzed by electrophoresis under reducing conditions (SDS-PAGE), electrotrasnference and immunodetection, isoelectric focussing, high pressure liquid chromatography (HPLC) and binding capacity to microsomal receptors (RRA). It was observed that in the porcine Prl (pPrl), ovine Prl (oPrl) and human Prl (hPrl) there are significant proportions of the glycosilated form (30-40%) that are not found in the bovine Prl (bPrl) and in the rat Prl (rPrl). It was proved that it is possible to detect impurities or mixtures of the different molecular forms of Prl that appear in these hypophyseal preparations by using HPLC. The analysis by isoelectric focussing of the hypophyseal prolactins of different species revealed that in the bPrl there are 4 isoforms of electric charge, that the oPrl and the hPrl are composed of 3 isoforms, whereas the rPrl and the pPrl have 1 and 2 isoforms, respectively. The physiological meaning of the isoforms of electric charge in the hypophyseal Prl of different species is still unknown. According to the results obtained from the displacement curves and from the Scatchard graphs, it was found that there are differences among the dissociation constants (Kd) and the binding capacity of prolactins to microsomal receptors. Finally, it is necessary to know with more precision the biochemical, immunological and biological properties of the preparations of prolactins that are used in different studies, both in vivo and in vitro, since that's where the biological functions of the hormone are. The structural heterogeneity of the Prl was confirmed and widened in different species of mammals giving rise to the existence of different molecular variants of the hormone and to the diversity of functions it performs. The need to develop new methods for studying the biological meaning of the structural heterogeneity of Prl and to obtain different molecular variants of Prl with a degree of purity that allow to establish new quantitative methods useful to be applied in clinical and biochemical studies was proved. The adequate understanding of the structural modifications of prolactins and of their biological consequences will make possible to establish the synthesis and secretion mechanisms of this hormone, including its multifunctional properties(AU)

Ovine placental lactogen and ovine prolactin: partial proteolysis and conformational stability.

The high-resolution structure of ovine placental lactogen (oPL) and ovine prolactin (oPRL), not yet established in detail, was probed by limited proteolysis with the Glu-specific protease from Staphylococcus aureus V8. While in hGH there were no cleavage sites inside of any of the four alpha-helices, the analysis of the fragments obtained after partial proteolysis of oPL showed a site of cleavage at the putative third helix, suggesting that this helix is partially unwound at this point. The partial proteolysis of the rest of the molecule was compatible with a similar folding pattern for oPL, hGH and pGH, on the basis of the crystal structure of these last hormones. In the case of oPRL, proteolytic cleavage occurred at Glu residues which would be located at the end of the first helix and the beginning of the second in the hGH folding model, suggesting that these helices are shorter in oPRL than in hGH. In order to gain further insight on the folding of these molecules, circular dichroism and intrinsic fluorescence measurements were used to examine the effect of denaturing conditions on oPL and oPRL. After exposure to 6 M guanidine the unfolding of both proteins was completely reversed upon elimination of the denaturing agent. In contrast, exposure to pH 3.0 caused an irreversible decrease in the alpha-helical content in both hormones, most striking for oPL, indicating that this hormone is less stable than oPRL or hGH.

Synthesis and characterization of recombinant, authentic human prolactin secreted into the periplasmic space of Escherichia coli.

Recombinant, fully bioactive, authentic human prolactin (aut-hPRL) has been synthesized in transformed Escherichia coli HB2151 bacteria in a soluble, non-glycosylated form, which is secreted into the bacterial periplasm. Use was made of a bacterial expression vector, containing tac promoter-controlled sequences for the translation enhancer from bacteriophage T7 gene 10, and for a cellulase leader peptide from Cellulomonas fimi joined to sequences coding for aut-hPRL. This vector was derived from a previously described vector containing sequences of an hPRL variant, tag-hPRL (containing a 12-amino-acid peptide tag at the N-terminal end), using site-specific mutagenesis to delete the tag sequence. SDS/PAGE, partial N-terminal amino acid sequence analysis, Western blot analysis and Nb2 lymphoma cell in vitro bioassay indicated correct processing of the hormone. Periplasmic secretion of aut-hPRL, as measured by immunoassay, was relatively low (approx. 0.08 microgram/ml per A600 unit), in contrast to that of tag-hPRL which was approximately 8-fold higher, apparently a consequence of the tag sequence. This is the first report describing periplasmic secretion of biologically active, authentic hPRL.

Production and characterization of biologically active Ala-Ser-(His)6-Ile-Glu-Gly-Arg-human prolactin (tag-hPRL) secreted in the periplasmic space of Escherichia coli.

Human prolactin (hPRL) cDNA was obtained by screening of a pituitary cDNA library with a synthetic 21-mer oligonucleotide and with rat PRL cDNA. For its expression, use was made of a vector, p3SN8, containing tac-promoter-controlled sequences for a bacterial cellulase leader joined to sequences coding for Ala-Ser, a chromatographic affinity site consisting of six histidines and a Factor Xa cleavage site. The hPRL cDNA was inserted at the 3' end of the cleavage-site sequences. Expression in Escherichia coli led to secretion in the periplasmic space of a fully bioactive hPRL variant constituting authentic hPRL with a peptide tag, i.e. Ala-Ser-(His)6-Ile-Glu-Gly-Arg, at its N-terminal. This tag-hPRL could be rapidly and efficiently purified by metal-chelate affinity chromatography. The correct processing and quality of tag-hPRL was monitored by SDS/PAGE, Western-blot analysis, immunoassay and Nb2-lymphoma-cell bioassay. Treatment with Factor Xa for tag removal was only partially successful. Periplasmic secretion of tag-hPRL of the order of 0.7 micrograms/ml per A600 unit and one-step purification indicate feasibility for tag-hPRL production for in vitro diagnostic and research applications. This is the first report describing periplasmic secretion of a bioactive form of hPRL.

Modification of arginine residues in ovine prolactin by 1,2-cyclohexanedione. Effect on binding capacity to lactogenic receptors.

The reactivity of arginine residues in ovine prolactin was studied by reaction with 1,2-cyclohexanedione. Kinetic analysis of the data showed a good fit with two simultaneous pseudo-first-order equations with apparent velocity constants of 0.28 and 1.2 x 10(-2) min-1, corresponding to 1.8 'fast' and 8.7 'slow' residues, respectively. Modification led to a decrease in binding capacity to lactogenic rat liver receptors, and apparently the modification of the two 'fast' reacting arginine residues is responsible for the rapid loss of this capacity. The presence of a non-reacting arginine has been described in human and bovine growth hormones, and it is located near the carboxy-terminus. This lack of reactivity is probably due to the formation of a salt bridge, since the arginine residue becomes susceptible to modification once the peptide is separated from the rest of the molecule. This salt bridge is absent in ovine prolactin, since the homologous arginine residue is reactive with cyclohexanedione. This result suggests that there could be a difference between the three-dimensional structure of ovine prolactin and of the growth hormones, at least near the carboxy-terminal region of the molecule.

[Current concepts on prolactin physiology: molecular variants and mechanisms of action]. / Conceptos actuales sobre fisiología de la prolactina: variantes moleculares y mecanismos de acción.

Prolactin is a polymorphic hormone with multiple biological functions. The versatility in the actions of prolactin depends on its structural polymorphism and perhaps on the feasibility of prolactin to be converted into different bioactive forms. In this review we attempt to summarize information concerning the synthesis, structure and mechanisms of action, including the heterogeneous nature of prolactin and its possible physiological significance in humans.