Studies on prolactin 48: Isolation and properties of the hormone from horse pituitary glands

Isolation of prolactin from equine pituitary glands has been described. It has a potency of 42 IU/mg in the pigeon crop-sac test and consists of 199 amino acids. The hormone has only four half-cystine residues in contrast to other mammalian prolactins which have six residues. From NH₂-terminal sequence analysis and amino acid composition of cyanogen bromide fragments, the NH₂-terminal disulfide loop is missing in the equine prolactin molecule. Circular dichroism spectra indicate that the α-helical content of equine prolactin appears to be lower (50%) than that found in the ovine hormone (65%).     

Assay of lactogenic hormones using receptors isolated from rabbit liver.

Using 125-I-labelled ovine prolactin and receptors isolated from the livers of rabbits, a sensitive method has been developed suitable for the assay of ovine, bovine, porcine, human and rat prolactins. These hormones showed competitive displacement of 125-I-ovine prolactin which was in general agreement with their respective activities in the pigeon crop sac bioassay. Human and monkey growth hormones and human placental lactogen, which have marked prolactin-like actions on mammary tissue were also effective competitors. Non-primate growth hormones (ovine, bovine, equine and canine) which do not have prolactin-like activity gave little if any displacement as did human FSH, LH, TSH, ACTH and bovine insulin. Preparations of equine and canine prolactin of varying purity gave dose-response curves although their activity as competitors relative to ovine prolactin was poor and not related to their pigeon crop stimulating activity. This indicates species differences between prolactins in hormone-receptor interaction. Experiments with antiserum to human growth hormone have suggested an effective method of making the assay specific in species such as man in which prolactin is not the sole hormone with lactogenic activity.     

Development and characterization of a homologous radioimmunoassay for equine prolactin

A specific and sensitive homologous radioimmunoassay has been developed for equine prolactin, suitable for measuring prolactin concentrations in serum of horses. The sensitivity of the assay ranged from 0.4 to 0.6 ng/ml and the intra- and inter-assay coefficients of variation averaged 6.9 and 15.4%, respectively, for five doses of hormone. Cross-reactivity with other mammalian and nonmammalian prolactins and growth hormones was less than 20 and 0.3%, respectively. Cross-reactivity with equine growth hormone was less than 0.07%. Equine serum and pituitary extracts showed parallel dilution-response curves with equine prolactin. The percentage recovery of exogenous equine prolactin in serum was 89%. Preliminary analysis of several physiological samples (stallions, pregnant, and nonpregnant mares) yielded values from 0.6 to 12.0 ng/ml.     

Elephant prolactin: isolation and characterization

Prolactin was isolated from anterior lobes of elephant pituitary glands. It consisted of 199 amino acids with three disulfide bridges and two tryptophan residues as found in prolactin from other species. The sequence of the NH2-terminal 28 amino acids was determined and shown homologous with the ovine hormone. In comparison with ovine prolactin, a marked difference was seen in the methionine content; the elephant hormone possessed only 18-34% lactogenic potency. The conformation of elephant prolactin was examined by zero order, second order and circular dichroism spectroscopy. The alpha helical content was estimated to be about 60%. In comparison with prolactins from other species, the second order spectra of elephant prolactin suggest that the local microenvironment for one or both tryptophan residues is somewhat different.     

Reaction of the histidines of prolactin with ethoxyformic anhydride. A binding site modification.

The seven histidines of bovine prolactin were modified with ethoxyformic anhydride and two classes of reactivity were apparent: 5 histidines were in the more reactive class (k = 0.097 min-1) and 2 histidines were less reactive (k = 0.011 min-1). The activity of the modified prolactins was determined by measuring their ability to bind to prolactin receptors from rabbit mammary glands. This assay showed that prolactin was fully active when 0 to 5 histidines were modified. If all 7 residues were modified, the hormone was unable to bind to its receptor. Circular dichroism studies indicated no significant differences in conformation for prolactins which had 2 to 7 histidines modified. Modification of human growth hormone and human placental lactogen with ethoxyformic anhydride resulted in a loss of the ability of these lactogenic hormones to bind to the prolactin receptor. For all three hormones, essentially full activity was recovered when the modifying group was removed by treatment with hydroxylamine. Sequence comparisons indicate that only 2 of the 3 growth hormone histidines and 2 of 7 placental lactogen histidines were homologous with histidines in bovine prolactin and that, in each case, they correspond to His-27 and His-30 in bovine prolactin. It is postulated that these residues serve to identify a portion of the binding domain of bovine prolactin.     

Molecular cloning of giant panda pituitary prolactin cDNA and its expression in Escherichia coli

cDNA encoding pituitary (PRL) of giant panda was obtained using RT-PCR and expressed in E. coli. The results revealed that panda PRL cDNA encodes a precursor protein of 229 amino acids including a putative signal peptide of 30 amino acids and a mature protein of 199 residues with one potential N-glycosylation site. Sequence comparison indicated that panda PRL shares a high degree of identity to other known PRL sequences ranging from 98% with mink PRL to about 50% with rodent PRL. Six cysteine residues and 29 conserved residues distributed in four domains (PD1, PD2, PD3, and PD4) of PRL were observed. through multiple sequence alignment. Fourteen key residues of binding sites 1 and 2 involved in receptor binding are conserved in panda PRL. GST fused recombinant panda PRL protein was efficiently expressed with the form of insoluble inclusion bodies in E. coli BL21 transformed with a pGEX-4T-1 expression vector containing the DNA sequence encoding mature panda PRL. Western blot analysis indicated that GST-panda PRL recombinant protein could be recognized by antibody against human PRL. Our results would contribute to further elucidating the structural and functional characteristics of pituitary PRL and provide a basis for the production of recombinant panda prolactin for future use in the breeding of giant panda.     

Molecular Cloning of Giant Panda Pituitary Prolactin cDNA and Its Expression in Escherichia coli

cDNA encoding pituitary (PRL) of giant panda was obtained using RT-PCR and expressed in E. coli. The results revealed that panda PRL cDNA encodes a precursor protein of 229 amino acids including a putative signal peptide of 30 amino acids and a mature protein of 199 residues with one potential N-glycosylation site. Sequence comparison indicated that panda PRL shares a high degree of identity to other known PRL sequences ranging from 98% with mink PRL to about 50% with rodent PRL. Six cysteine residues and 29 conserved residues distributed in four domains (PD1, PD2, PD3, and PD4) of PRL were observed through multiple sequence alignment. Fourteen key residues of binding sites 1 and 2 involved in receptor binding are conserved in panda PRL. GST fused recombinant panda PRL protein was efficiently expressed with the form of insoluble inclusion bodies in E. coli BL21 transformed with a pGEX-4T-1 expression vector containing the DNA sequence encoding mature panda PRL. Western blot analysis indicated that GST-panda PRL recombinant protein could be recognized by antibody against human PRL. Our results would contribute to further elucidating the structural and functional characteristics of pituitary PRL and provide a basis for the production of recombinant panda prolactin for future use in the breeding of giant panda.     

The purification and characterization of rabbit placental lactogen.

Rabbit placental lactogen, a polypeptide hormone functionally related to the growth hormone/prolactin family, was isolated from placenta by (NH4)2SO4 precipitation, gel filtration and ion-exchange chromatography on DEAE-and CM-cellulose. The hormone was purified to more than 90% homogeneity, as determined by end-group analysis. On disc gel electrophoresis at pH9.0 it migrates as a pair of closely spaced bands with mobilities of 0.489 (minor band) and 0.511 (major band), and its isoelectric point is 6.1. Its mol.wt. is 20600, as determined by sedimentation--equilibrium centrifugation, and 24200, as estimated by gel electrophoresis in sodium dodecyl sulphate. Its amino acid composition resembles that of rabbit growth hormone and rat prolactin, except for a lower glutamic acid and leucine content. Like the prolactins, rabbit placental lactogen has two tryptophan and six cysteine residues, and its N-terminus, valine, is identical with that for human placental lactogen. By radioimmunoassay, it does not cross-react with antisera to either rat growth hormone or rat prolactin; in addition, it does not cross-react with antisera to bovine placental lactogen by double immunodiffusion. The similarity of the biochemical characteristics of rabbit placental lactogen to the other non-primate placental lactogens lends further support to the hypothesis that these molecules occupy a more central position in the growth hormone/prolactin "tree" than do their primate counterparts.     

Primary structure of chum salmon prolactins: occurrence of highly conserved regions

The complete amino acid sequence of prolactin from the pituitaries of salmon (Oncorhynchus keta) has been determined. Salmon prolactin comprised two variants, I and II, which were separated by reverse-phase high-performance liquid chromatography. Each variant was reduced, S-carboxymethylated, and then cleaved with cyanogen bromide and enzymes. The resulting fragments were separated by reverse-phase high-performance liquid chromatography, as well as gel filtration, and subjected to sequence analysis by the dansyl-Edman method. Both variants contain 187 amino acid residues with two disulfide linkages at residues 46-160 and 177-187, lack a linkage in the N-terminal portion of mammalian prolactins, and differ from each other by the replacement of only four amino acid residues. Salmon prolactin (sPRL) shows 31% sequence identify with ovine prolactin. Moreover, four restricted regions, i.e., sPRL (3-21), (46-60), (68-83), and (160-178), encompass this significant conservatism between the teleost and the mammalian hormone, with identities of 47, 87, 62, and 68%, respectively. Such considerable identity between these distant phylogenic species strongly suggests that these regions may be responsible for the biological activity of prolactin.     

Expression of new members of the prolactin growth hormone gene family in bovine placenta. Isolation and characterization of two prolactin-like cDNA clones

Two prolactin-like proteins (bPLP-I and bPLP-II) were deduced from the nucleotide sequence analyses of the cDNA clones derived from a bovine (Bos taurus) term placenta. These proteins resembled bovine prolactin but were different from the reported bovine placental lactogens or prolactin-related proteins. The predicted amino acid sequences of these clones showed 45-51% identity with bovine prolactin and 23-24% with bovine growth hormone. The two new clones show 62 and 39% overall homology with each other at the levels of nucleotide and amino acid sequences, respectively. bPLP-I, bPLP-II, placental lactogens, prolactins (PRLs), and other prolactin-like proteins isolated from cow, mouse, and rat share 7 common amino acid residues. Five of the 7 residues are conserved by other members of the family such as growth hormones, suggesting that they may be essential for the common structural features of the gene family. The other 2 residues are uniquely conserved in bovine, mouse, and rat placental lactogens, PRLs, and PRL-like proteins, predicting their indispensable roles in binding to the specific receptors. bPLP-I and bPLP-II, as well as bPLP-III, are shown to be expressed stage specifically and predominantly in full-term bovine placentas.     

The amino acid composition of secreted mouse prolactin,growth hormone,and hamster prolactin: The presence of one tryptophan in mouse prolactin

The amino acid compositions and the electrophoretic properties of secreted mouse prolactin (mPRL), mouse growth hormone (mGH), and hamster prolactin (haPRL) were determined. The amino acid compositions of secreted mPRL and haPRL were similar to the compositions of other rodent prolactins, except that secreted mPRL contained only one tryptophan residue rather than the usual two. The composition of secreted mGH was similar to that of rat growth hormone. On 10% alkaline polyacrylamide gels, mPRL, mGH, and haPRL migrated with R_f's of 0.54, 0.21, and 0.69, respectively. The molecular weights of mPRL, mGH, and haPRL, determined by SDS-gel electrophoresis, were 23,000, 21,000, and 22,000, respectively.     

The molecular evolution of pituitary growth hormone prolactin and placental lactogen: A protein family showing variable rates of evolution

Summary Pituitary growth hormone and prolactin, together with the homologous placental hormones, comprise a family of related protein hormones. Complete or partial amino acid sequences of seven mammalian growth hormones, six mammalian prolactins and one placental lactogen are available, and have been compared. A phylogenetic tree has been constructed which describes the relationships within the family. At least two gene duplications have occurred during the evolution of these proteins. Rates of evolution in the family have been quite variable, the overall rate of evolution having been apparently fairly slow, but having increased markedly on several occasions, most notably in the evolution of human (and, on the basis of immunological relationships, probably other primate) growth hormones and rat (and possibly other rodent) prolactins.     

The triose phosphate-3-phosphoglycerate-phosphate translocator from spinach chloroplasts: nucleotide sequence of a full-length cDNA clone and import of the in vitro synthesized precursor protein into chloroplasts.

The nucleotide sequence of several cDNA clones coding for the phosphate translocator from spinach chloroplasts has been determined. The cDNA clones were selected from a lambda gt10 library prepared from poly(A)+ mRNA of spinach leaves using oligonucleotide probes modeled from amino acid sequences of tryptic peptides prepared from the isolated translocator protein. A 1439 bp insert of one of the clones codes for the entire 404 amino acid residues of the precursor protein corresponding to a mol. wt of 44,234. The full-length clone includes 21 bp at the transcribed non-coding 5' region with the ribosome initiation sequence ACAATGG, a 1212 bp coding region and 199 bp at the non-coding 3' region excluding the poly(A) tail which starts 17 bp downstream from a putative polyadenylation signal, AATAAT. According to secondary structure predictions the mature part of the chloroplast phosphate translocator exhibits high hydrophobicity and consists of at least seven membrane-spanning segments. Using plasmid-programmed wheat germ lysate the precursor protein was synthesized in vitro and could be imported into spinach chloroplasts where it is inserted into the inner envelope membrane.     

Development of an homologous radioimmunoassay for secreted prolactin from the California ground squirrel (Spermophilus beecheyi)

A sensitive homologous radioimmunoassay was developed for secreted prolactin from the California ground squirrel (Spermophilus beecheyi). S. beecheyi plasma and pituitary extracts displaced 125I-labeled S. beecheyi prolactin in a parallel manner with S. beecheyi prolactin (sbPRL). Mean minimum sensitivity of the assay was 0.21 ng/ml, and mean intra- and interassay coefficients of variation were 4.1% and 14.5%, respectively. The assay was used to measure basal prolactin concentrations in male and female ground squirrels at various stages of their annual reproductive cycles. Mean concentrations in nonpregnant, nonlactating young females, pregnant females, and lactating females were 1.63, 11.35, and 10.86 ng/ml, respectively. Mean concentrations in nonreproductive and breeding males were 1.50 and 9.81 ng/ml, respectively. Finally, the assay was used to evaluate cross-reactivity between sbPRL and prolactins and growth hormones from other rodent species. Of the tested hormones, only hamster prolactin showed any cross-reactivity with sbPRL (about 0.03%).     

A giant prolactinoma and the effect of chronic bromocriptine therapy on basal and TRH-stimulated serum prolactin levels.

The role of bromocriptine as primary therapy for prolactin-producing tumors is currently well accepted in the literature. Bromocriptine decreases the concentration of serum prolactin and this decrease precludes tumor shrinkage, despite the lack of correlation between amount of decrease in tumor size and baseline serum prolactin. We submit the case of a patient on chronic bromocriptine therapy followed by measuring baseline and thyrotropin-releasing hormone (TRH)-stimulated serum prolactins. Bromocriptine affects both release and storage of prolactin. The literature has suggested that the effects of bromocriptine on storage and synthesis may be responsible for its effects on tumor size. It was felt that TRH stimulation would more accurately reflect storage and synthesis, and thus correlate better with tumor size. The pituitary was initially debulked via a right frontal approach; then the patient was placed on bromocriptine therapy and postoperatively followed with baseline and TRH-stimulated serum prolactins. The size of the pituitary was measured by computed tomography. Baseline serum prolactin levels rapidly decreased, but despite the slow decrease in TRH-stimulated prolactins no change was noted in tumor size. Because of the time difference between the baseline and TRH-stimulated prolactin levels, we conclude that clinically bromocriptine affects primarily secretion of prolactin and secondarily storage and synthesis. We also show that TRH-stimulated prolactin does not correlate with size of prolactin-secreting pituitary tumors and therefore tumor size should be independently measured. The literature has shown that prolactinomas do not respond well to TRH stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of prolactin receptors in hepatic nuclei.

Prolactin is a trophic hormone which may act directly at the hepatocyte nucleus. In this study, specific prolactin binding sites were sought in purified rat liver nuclei. Saturable and specific, high affinity 125I-prolactin binding sites were demonstrated to be on or within the nucleus. Prolactin binding was competitively inhibited by rat and ovine prolactins but not by rat growth hormone. Using immunogold electron microscopy, we detected prolactin receptors throughout the nucleus, in association with heterochromatin. Furthermore, endogenous immunoreactive prolactin was demonstrated to be within hepatic nuclei. We conclude that rat liver nuclei possess prolactin binding sites which likely participate in hormone-directed growth processes.     

High homology is present in the primary structures between murine senile amyloid protein (ASSAM) and human apolipoprotein A-II

The primary structure of a murine senile amyloid protein (ASSAM) was determined. The protein consists of a single polypeptide chain of 78 amino acid residues. The amino-terminus is blocked with pyrrolidone-carboxylic acid. The sequence differs from that of the known murine amyloid A protein and is highly homologous to human apolipoprotein (apo) A-II. The result indicates that the putative precursor of the senile amyloid protein is apo A-II in mice.     

Functional epitopes for site 1 of human prolactin

Human prolactin (hPRL) binds two human prolactin receptor molecules, creating active heterotrimeric complexes. Receptors bind dissimilar hormone surfaces termed site 1 and site 2 in an obligate ordered process. We sought to map the functional epitopes in site 1 of hPRL. Extensive alanine mutagenesis (102 of the 199 residues) showed approximately 40% of these mutant hPRLs changed the ΔG for site 1 receptor binding. Six of these residues are within 3.5 ? of the receptor and form the site 1 functional epitopes. We identified a set of noncovalent interactions between these six residues and the receptor. We identified a second group of site 1 residues that are between 3.5 and 5 ? from the receptor where alanine mutations reduced the affinity. This second group has noncovalent interactions with other hormone residues and stabilized the topology of the functional epitopes by linking these to the body of the protein. Finally, we identified a third group of residues that are outside site 1 (>5 ?) and extend to site 2 and whose mutation to alanine significantly weakened receptor binding at site 1 of prolactin. These three groups of residues form a contiguous structural motif between sites 1 and 2 of human prolactin and may constitute structural features that functionally couple sites 1 and 2. This work identifies the residues that form the functional epitopes for site 1 of human prolactin and also identifies a set of residues that support the concept that sites 1 and 2 are functionally coupled by an allosteric mechanism.     

Isoforms of turkey prolactin: evidence for differences in glycosylation and in tryptic peptide mapping.

1. Three isoforms of turkey pituitary prolactin have been isolated, including a nonglycosylated isoform of 22,500 mol. wt and two glycosylated isoforms of 24,500 mol. wt. 2. The glycosylated turkey prolactins differed in carbohydrate composition, with one isoform apparently containing only O-linked carbohydrate. 3. Tryptic peptide maps showed a few peptides distinctly different among the three prolactin isoforms. 4. Amino acid sequencing of the first 40 residues of the three prolactin isoforms showed arginine at position 24 and histidine at position 27, for the nonglycosylated form, but no identifiable amino acids were detected at this position for the glycosylated isoforms.