Interactions of antisense peptides with ovine prolactin

Two antisense peptides were synthesized to a sense peptide corresponding to amino acid residues 23-35 of ovine prolactin. Both of the antisense peptides formed a saturable complex with the sense peptide and ovine prolactin. The sense peptide inhibited the interaction of ovine prolactin with the antisense peptides. Both of the antisense peptides have a common core sequence VMNV which can bind to ovine prolactin. The lactogenic hormones, rat prolactin and human growth hormone, compete with the binding of ovine prolactin to an antisense peptide whereas a nonlactogen, ovine growth hormone, does not compete indicating a degree of specificity in the interaction.     

Primary structure of whale prolactin

Two CNBr fragments of sea whale prolactin containing 69 and 41 amino acid residues, respectively, were hydrolyzed by trypsin, and the hydrolytic products were separated by the paper peptide mapping technique. The amino acid sequence of 17 homogeneous peptides was studied by the Edman method as well as by hydrolysis with carboxypeptidases A and B. Based on the experimental data and the previously published results the primary structure of sea whale prolactin made up of 199 amino acid residues was proposed.     

Cell-free synthesis of a large translation product of prolactin messenger RNA.

RNA prepared from rat anterior pituitaries or from prolactin-secreting pituitary tumors has been shown to direct the synthesis of a large form of prolactin in a cell-free system derived from wheat germ. Immunoprecipitation of cell-free reactions demonstrated the synthesis of a product which was recognized by a specific antiprolactin antisera. Analysis of the immunoprecipitate on sodium dodecyl sulfate containing polyacrylamide gels suggested that the cell-free product has a molecular weight of approximately 28,000 compared to 22,500 for prolactin. RNA prepared by completely different techniques from rat pituitary and a pituitary tumor resulted in identical large translation products. Translation of tumor RNA in a cell-free system from Krebs ascites cells also resulted in a similar large product. The identity of the cell-free product as prolactin was confirmed by comparing peptides derived from the cell-free product and prolactin. The results of these studies suggest that prolactin messenger RNA directs the cell-free synthesis of a product which contains the amino acid sequence of prolactin but which has an addition at one or both ends of the molecule.     

Peptide products of the cleavage of bovine preprolactin by signal peptidase

Cleavage of preprolactin (pPL) by detergent-solubilized signal peptidase produced mature prolactin and two small peptides derived from the signal peptide region of the pPL molecule. The production of both peptides was dependent on functional signal peptidase; the peptides were not generated at detergent concentrations that abolished signal peptidase activity. The amount of both peptides was proportional to the concentration of signal peptidase in the assay. The appearance of both peptides was insensitive to protease inhibitors, as was signal peptidase activity. The size, labeling characteristics, and amino acid sequence of the larger peptide, peptide 1, corresponded to those of the intact signal peptide of pPL. The smaller peptide, peptide 2, lacked the carboxy terminus of the signal peptide, and was, therefore, a fragment of intact signal peptide. These results demonstrate the endoproteolytic nature of signal peptidase.     

Molecular cloning and functional characterization of a prolactin-releasing peptide homolog from Xenopus laevis

Amino acid sequences for identified prolactin (PRL)-releasing peptides (PrRPs) were conserved in mammals (>90%) or teleost fishes (100%), but there were considerable differences between these classes in the sequence (<65%) as well as in the role of PrRP. In species other than fishes and mammals, we have identified frog PrRP. The cDNA encoding Xenopus laevis prepro-PrRP, which can generate putative PrRPs, was cloned and sequenced. Sequences for the coding region showed higher identity with teleost PrRPs than mammalian homologues, but suggested the occurrence of putative PrRPs of 20 and 31 residues as in mammals. The amino acid sequence of PrRP20 was only one residue different from teleost PrRP20, but shared 70% identity with mammalian PrRP20s. In primary cultures of bullfrog (Rana catesbeiana) pituitary cells, Xenopus PrRPs increased prolactin concentrations in culture medium to 130–160% of the control, but PrRPs was much less potent than thyrotropin-releasing hormone (TRH) causing a three- to four-fold increase in prolactin concentrations. PrRP mRNA levels in the developing Xenopus brain peak in early prometamorphosis, different from prolactin levels. PrRP may not be a major prolactin-releasing factor (PRF), at least in adult frogs, as in mammals.     

Isolation and characterization of fin whale prolactin.

A highly purified prolactin preparation has been obtained from fin whale pituitaries by extraction with acid acetone. salt precipitation, isoelectric fractionation, and exclusion chromatography on Sephadex G-100 and ion-exchange chromatography on DEAE-CELLULOSE. Fin whale prolactin was isolated in a yield of 250 mg/kg wet weight tissue. It was found to have a molecular weight (SDS disc gel electrophoresis) of 23,600 daltons and an alpha-helix content (circular dichroism) of 50%. The amino acid composition and circular dichroism spectra were very similar to those of porcine prolactin. The partial amino acid sequence has been determined by the method of fluorescein-isothiocyanate. Fin whale prolactin was found to be 80% as potent as ovine prolactin with regard to pigeon crop-sac assay.     

In silico identification of new secretory peptide genes in Drosophila melanogaster

Bioactive peptides play critical roles in regulating most biological processes in animals. The elucidation of the amino acid sequence of these regulatory peptides is crucial for our understanding of animal physiology. Most of the (neuro)peptides currently known were identified by purification and subsequent amino acid sequencing. With the entire genome sequence of some animals now available, it has become possible to predict novel putative peptides. In this way, BLAST (Basic Local Alignment Searching Tool) analysis of the Drosophila melanogaster genome has allowed annotation of 36 secretory peptide genes so far. Peptide precursor genes are, however, poorly predicted by this algorithm, thus prompting an alternative approach described here. With the described searching program we scanned the Drosophila genome for predicted proteins with the structural hallmarks of neuropeptide precursors. As a result, 76 additional putative secretory peptide genes were predicted in addition to the 43 annotated ones. These putative (neuro)peptide genes contain conserved motifs reminiscent of known neuropeptides from other animal species. Peptides that display sequence similarities to the mammalian vasopressin, atrial natriuretic peptide, and prolactin precursors and the invertebrate peptides orcokinin, prothoracicotropic hormones, trypsin modulating oostatic factor, and Drosophila immune induced peptides (DIMs) among others were discovered. Our data hence provide further evidence that many neuropeptide genes were already present in the ancestor of Protostomia and Deuterostomia prior to their divergence. This bioinformatic study opens perspectives for the genome-wide analysis of peptide genes in other eukaryotic model organisms.     

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.     

Cloning and sequence analysis of cDNA for mouse prolactin

The present study was undertaken to find out whether or not sexual dimorphism in biological activities and amino acid compositions of mouse prolactin might be due to heterogeneity in mRNA for mouse prolactin Cloned cDNAs for mouse prolactin were first isolated from a mouse pituitary cDNA library by hybridization with a rat prolactin cDNA. Then, one clone of about 140 positive clones obtained from 2000 transformants was subjected to nucleotide sequence analysis and verified to contain a nearly full length of cDNA sequence coding for mouse prolactin precursor. The deduced complete amino acid sequence indicates that the precursor molecule consists of 31 amino acids as the signal peptide and 197 amino acids of prolactin, in which two amino acids were found to be different from the amino acid sequence previously published elsewhere. S1 nuclease mapping analysis using male and female pituitary RNAs indicates that mouse preprolactin is encoded by two mRNAs in both sexes. The two mRNAs differ from each other based upon the deletion of three nucleotides in the coding region for the signal peptide determined by the nucleotide sequence analysis in other cDNA clones. In the present study, no sexual difference was revealed in murine prolactin mRNA.     

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.     

Regulation of enzyme turnover during tissue differentiation. Interactions of insulin, prolactin and cortisol in controlling the turnover of fatty acid synthetase in rabbit mammary gland in organ culture

1. Explants of mammary gland from mid-pregnant rabbits were cultured in Medium 199 containing combinations of insulin, prolactin and cortisol. With hormone combinations which included prolactin, a sustained increase in the apparent rate of synthesis and in the amount of fatty acid synthetase was measurable immunologically. Maximum increase was produced with insulin, prolactin and cortisol present together. 2. With prolactin present alone, synthetase activity in the explants decreased to undetectable values after 1 day in culture, whereas the incorporation of l-[U-(14)C]leucine into immunodetectable material increased. Prolactin may therefore direct the synthesis of immunologically cross-reactive precursors of fatty acid synthetase which are enzymically inactive. 3. Culture with dibutyryl cyclic AMP plus theophylline in the presence of insulin, prolactin and cortisol delayed the increase in the rate of synthesis and accumulation of the synthetase. These compounds may also prevent the apparent decrease in the rate of degradation of the synthetase which occurs on day 2 of culture. 4. A large decrease in the apparent rate of degradation of the synthetase on day 2 of culture occurs during culture with hormone combinations which include prolactin. The protein obtained by centrifugation of explant homogenates for 6min at 14000g(av.) is degraded continuously throughout the culture period. 5. This decrease in the apparent rate of degradation of the synthetase was measured by radio-immunological precipitation. It is probably part of a regulated programme of enzyme degradation and not a reflexion of the reutilization of radioactive amino acids for the following reasons. (a) The calculated increase in the amount of the synthetase in explants on day 2 of culture with insulin, prolactin and cortisol was approximately equal to the measured increase of the enzyme complex which accumulates in the explants. This suggests little or no enzyme degradation has occurred. (b) Explants were cultured for 24h with insulin, prolactin and cortisol. They were then incubated with l-[U-(14)C]leucine, washed and incubated again for up to 4(1/2)h. l-[U-(14)C]Leucine rapidly equilibrated with the intracellular amino acid pool. Within 10min of incubation after washing explants to remove endogenous l-[U-(14)C]leucine the previously linear incorporation of l-[U-(14)C]-leucine into total explant protein ceased. This suggests that protein is synthesized from an amino acid pool which rapidly equilibrates with amino acids in the culture medium. (c) Explants were cultured for 24h as described in (b) but after washing they were cultured with insulin, prolactin and cortisol for 24h. Approx. 90% of the radioactivity lost from the ;free' intracellular amino acid pool and from amino acids derived from the degradation of explant protein in this period was detected in the culture medium. This suggests that the ;free' intracellular amino acids and amino acids derived from protein degradation can equilibrate with amino acids in the medium. A residual ;free' radioactive amino acid pool was present in the tissue. (d) Casein represents approx. 20% of the protein synthesized after 1 day in culture with insulin, prolactin and cortisol. Histological evidence suggests that on day 2 of culture, casein is unlikely to be degraded in the tissue. No increase in the radioactivity incorporated into casein can be measured in the 23h after incubation of explants with l-[U-(14)C]leucine as described in (b). This suggests that the incorporation of radioactivity into proteins during culture after incubation with l-[U-(14)C]leucine is minimal. (e) Inhibition of protein synthesis in explants by cycloheximide after incubation with l-[U-(14)C]leucine does not reveal a latent continuous degradation of fatty acid synthetase on day 2 of culture which might have been masked by the high rates of protein synthesis and therefore the accumulation of the enzyme. 6. The conclusion is discussed that there is a real decrease (or even cessation) in the rate of degradation of fatty acid synthetase during the period when the enzyme accumulates in explants cultured with hormone combinations which contain prolactin.     

Synthesis, cloning and determination of the primary structure of DNA complementary to mRNA of prolactin from the human pituitary gland

The poly(A)-containing mRNA from human pituitary and prolactinoma have been purified and translated in the cell-free system from rabbit reticulocytes. mRNA from prolactinoma was shown to be enriched with specific prolactin mRNA. DNA complementary to the prolactin mRNA from human pituitary was obtained and cloned. Sequencing of the 900 bp insert by the Maxam-Gilbert technique suggested the cDNA cloned to cole for the previously published amino acid sequence, mismatches with mRNA from prolactinoma occurring at the third positions of codons and thus not causing amino acid substitutions.     

Purification, cloning, and expression of the prolactin receptor

The rat liver prolactin receptor has been purified to homogeneity, and partial amino acid sequences have been obtained. The structure of the receptor has been deduced from a single complementary DNA clone. The mature protein of 291 amino acids has a relatively long extracellular region, a single transmembrane segment, and a short (57 amino acids) cytoplasmic domain. With the rat cDNA used as a probe, the prolactin receptor in rabbit mammary gland and human hepatoma cells has also been isolated. These tissues contain a second, longer form of the receptor (592 and 598 amino acids, respectively). Both the short and long forms of the prolactin receptor show regions of strong sequence identity with the human and rabbit growth hormone receptors, suggesting that the prolactin and growth hormone receptors originate from a common ancestor.     

Amino acid sequence of chitinase from Streptomyces erythraeus

The amino acid sequence of chitinase from Streptomyces erythraeus was determined by the conventional method. The amino acid sequences of tryptic peptides of the reduced and S-carboxymethylated protein were determined. The tryptic peptides were aligned by overlapping the amino acid sequences of chymotryptic peptides, lysyl endopeptidase peptides and cyanogen bromide fragments. S. erythraeus chitinase consists of 290 amino acid residues with the molecular weight of 30,400 and has two disulfide bridges at Cys(45)-Cys(89) and Cys(265)-Cys(272). The enzyme has no significant homology with other chitinases, lysozymes, and other proteins.     

Primary structure of otter (Lutra lutra L.) myoglobin. II. Pepsin peptides of trypsin hydrolysate. Reconstruction of the polypeptide chain of the otter myoglobin globin component

13 peptic peptides have been isolated from the insoluble (at pH 5.0) fraction of the tryptic hydrolysate of main chromatographic component of otter myoglobin and their amino acid composition and N-terminal amino acid sequences have been determined. The isolated peptides contain in total 40 amino acid residues. The results obtained, along with those on tryptic peptides and the comparison with homologous portions of myoglobins of the known primary structure, allowed reconstructing the complete amino acid sequence of otter myoglobin.     

Cloning and expression of the rat prolactin receptor, a member of the growth hormone/prolactin receptor gene family

The primary structure of the rat liver prolactin receptor has been deduced from a single complementary DNA clone. The sequence begins with a putative 19 amino acid signal peptide followed by the 291 amino acid receptor that includes a single 24 amino acid transmembrane segment. In spite of the fact that the prolactin receptor has a much shorter cytoplasmic region than the growth hormone receptor, there is strong localized sequence identity between these two receptors in both the extracellular and cytoplasmic domains, suggesting that the two receptors originated from a common ancestor.     

Somatocrinin,growth hormone releasing factor,stimulates secretion of growth hormone in anesthetized rats

The synthetic replicate of a 44 amino acid peptide isolated from a human pancreatic tumor which had caused acromegaly possesses high specific activity to release growth hormone (GH) in anesthetized male rats. The GH secretion induced by this peptide is dose-dependent from 50 ng to 1 μg, with plasma GH concentrations increasing more than 10-fold within 5 min of iv administration at the higher doses. Two enzymatic degradation products of the 44 residue peptide were also isolated and consist of the first 37 and 40 amino acids. All three peptides appear to possess similar potency, on a molar basis, $\text{in}\text{vivo}$, contrary to $\text{in}\text{vitro}$ results. The specificity of these peptides on GH release was shown by their failure to alter plasma concentrations of prolactin (PRL), thyroid-stimulating hormone (TSH), luteinizing hormone (LH), follicle-stimulating hormone (FSH) and corticosterone. Based on these $\text{in}\text{vivo}$ results, the three peptides with serve as powerful tools with which to investigate the mechanisms of GH secretion.     

Peptide and protein pheromones in amphibians

Purification, characterization and biological activity of urodele and anuran sex-pheromones were reviewed. Female-attracting pheromones obtained from the abdominal gland of Cynops pyrrhogaster and C. ensicauda males are peptides consisting of 10 amino acid residues being designated sodefrin and silefrin, respectively. Each pheromone attracted only conspecific females. Molecular cloning of cDNAs encoding sodefrin and silefrin revealed that both are generated from precursor proteins. Synthesis of these pheromones is regulated by prolactin (PRL) and androgen. Responsiveness of the female vomeronasal epithelium to sodefrin is enhanced by PRL and estrogen. The submandibular gland of the male terrestrial salamander, Plethodon jardani secretes a 22-kD proteinaceous pheromone that enhances female receptivity. It was revealed that every salamander synthesizes multiple isoforms of this pheromone, Plethodontid receptivity factor. The magnificent tree frog, Litoria splendida breed in an aquatic environment. The skin glands of the male secrete a female-attracting peptide pheromone, splendipherin, comprising 25 amino acid residues. The significance of the structure of the amphibian sex-pheromone as peptide and protein is discussed in terms of their species specificity.