Comparative analytical studies on recombinant and native human somatotropin

In polyacrylamide gel electrophoresis and isoelectric focusing, somatotropin produced by recombinant DNA technology is as heterogeneous as highly purified native pituitary somatotropin. This heterogeneity is not attributable to different degrees of deamination of a single molecular species. In addition to the main protein of 22 kDa, the cloned products contain traces of interchain disulphide dimers of somatotropin. The quantitative amino acid analyses of the three cloned somatotropins investigated are neither identical nor do they correspond to the analysis of the native pituitary hormone. Moreover, there are discrepancies between the amino acid compositions of the hormones studied and the generally recognized amino acid analysis for human somatotropin.     

猪生长激素的提纯及其生物学活性和某些理化性质的研究

通过调pH抽提、硫酸铵分级分离和DEAE-纤维素柱层析等方法从猪垂体中提取了生长激素。平均产率约为0.5g生长激素/1000g鲜垂体。用去垂体大白鼠胫骨测定法测得共促生长效应为1.84。提取的生长激素在高pH不连续缓冲体系聚丙烯酰胺凝胶电泳时呈现两条带;Sephadex G75柱层析得到一个峰;SDS聚丙烯酰胺凝胶电泳呈现一条带,其分子量约为21,900道尔顿;聚丙烯酰胺凝胶等电聚焦电泳呈现五条带,主带在pH7.8处;DNS法测定其N-末端氨基酸为苯丙氨酸一种。此外,还测定了它的氨基酸组成。     

Studies on the pituitary "fettstoffwechselhormon". VI. Preparation of two highly purified lipolytic active peptides from hog pituitary glands.

The preparation of two highly purified lipolytically active hog pituitary peptides, called P-LF II C and P-LF II D is described. The two peptides are free of other pituitary hormone activities. In isolated rat and porcine adipose tissue, both fractions are lipolytically much more active than every other lipolytic active pituitary peptide described to date. By fraction P-LF II D, the first pituitary peptide was isolated which has lipolytic activity in isolated rat adipose tissue than corticotropin, the lipolytically most active pituitary hormone known so far. On isolated porcine adipose tissue, fraction P-LF II D as well as P-LF II C showed without doubt higher activity than corticotropin.     

Preparation and characteristics of porcine growth hormone.

A recently published method for preparation of porcine growth hormone resulted in a highly purified protein with good biological activity. However, after storing for some months the originally homogeneous hormone separated again into several fractions when rechromatographed on ion exchange columns. The biological activity, found in one of these fractions, was clearly diminished compared with the activity of a freshly prepared hormone. In the present paper a modified procedure is described for the isolation of a more stable porcine growth hormone. The influence of ions, involved in buffers of the same molarity and the same pH, upon ion exchange chromatography of porcine growth hormone is discussed. The purified hormone shows high biological activity in the tibia test and is free of activities of other pituitary hormones. The molecular weight is about 20 000; only phenylalanine is found as N-terminal as well as C-terminal amino acid; the amino acid composition resembles neither that of porcine growth hormone described in literature nor that of human growth hormone.     

Isolation and characterization of growth hormone from blue fox pituitary glands

Growth hormone has been purified to homogeneity from blue fox pituitary glands. It has 191 amino acids with two disulfide bridges and a single tryptophan residue. The somatotropin activity is only 8% when compared with the bovine hormone in the receptor-binding assay. From radioimmunoassay data using baboon antisera to porcine or bovine growth hormone, the fox hormone has 14-17% immunoreactivity of bovine or porcine hormone.     

Elephant growth hormone. Isolation and characterization

Growth hormone has been purified to homogeneity from elephant pituitary glands. It has 191 amino acids with two disulfide bridges and a single tryptophan residue. The somatotropin activity is only 15% when compared with the bovine hormone in the radioreceptor binding assay. From circular dichroism spectra alpha-helical content of elephant growth hormone is estimated to be 50%. Difference absorption spectra of the hormone suggest the presence of a hydrogen bond between the single Trp and a carboxylate ion.     

Primary structure of cyanogen bromide fragments of sei whale (Balaenoptera borealis) pituitary somatotropin]

5 fragments are isolated after the degradation of somatotropin from sei whale pituitary glands with cyanogen bromide: N-terminal 4-segmented; C-terminal 12-segmented with the internal disulfide bond; middle 25- and 30-segmented and a high molecular weight fragment following N-terminal tetrapeptide and bound with disulfide bond to 30-segmented fragment. Complete amino acid sequence of three shortest cyanogen bromide fragments is deciphered and N- and C-terminal sequence is investigated in two large fragments after their uncoupling under performic acid oxidation. Amino acid sequence is deciphered of a peptide obtained after trypsine hydrolysis of 30-segmented cyanogen bromide fragment. Comparison of amino acid sequence of whale somatotropin fragments with that of sheep, beef and human somatotropin has revealed that 57 out of 61 identified amino acid residues of whale somatotropin repeat amino acid residues in similar regions of beef somatotropin, 56--of sheep and only 42--of human somatotropins. Besdies, 4 of 5 revealed amino acid substitutions in whale hormone, as compared with sheep somatotropin, are amino acids which are present at the same positions in human hormone.     

Biosynthesis and degradation of prolactin in the rat anterior pituitary gland. Time course of incorporation of label in vitro and evidence for rapid degradation.

Biosynthesis of prolactin was studied in anterior pituitary glands from female rats, incubated in vitro. In this system [3H]leucine was incorporated into pituitary proteins, including somatotropin (growth hormone) and prolactin. The rate of uptake of label into prolactin (and to a lesser extent into total protein) slowed considerably during the first 2 h of incubation, although the rate of uptake into somatotropin was constant for 8 h. The most probable explanation for this apparent decrease in the rate of prolactin synthesis is degradation of prolactin in the gland. Degradation of this hormone was also demonstrated by incubating prelabelled pituitaries in unlabelled medium and following the content of labelled prolactin, and by studying the hormonal content of pituitary glands (by radioimmunoassay) before and after incubation. Degradation of prolactin appears to be much more rapid than that of somatotropin, and may represent a physiological mechanism whereby over-accumulation of prolactin is prevented when secretion of the hormone has been rapidly switched off.     

Purification and characterization of pituitary bovine somatotropin

Bovine somatotropin (bST) has been isolated from pituitary glands and compared in a variety of chemical analyses and bioassays with somatotropin derived from recombinant Escherichia coli. Comparison of pituitary extracts and purified bST by Western blot analysis of two-dimensional gels suggested that the immunoreactive somatotropin species present in the extract were also present in the purified material, with no significant losses or degradation as a result of the purification method. NH2-terminal sequence analysis indicated the presence of equal quantities of Ala-Phe-Pro-Ala-Met-Ser-Leu-Ser- and Phe-Pro-Ala-Met-Ser-Leu-Ser- sequences. The Met-Ser-Leu-Ser-NH2-terminal sequence, a degradation product observed in NIH standard lots, was not detected. Assay of bioactivity in a bovine liver receptor-binding assay and in a female rat growth assay showed pituitary bST and recombinant methionyl-bovine somatotropin to be equipotent. Tryptic maps and sequence analysis of pituitary-derived somatotropin suggest the presence of isoaspartate derivatization at Asp128.     

Preparation of internally labelled rat pituitary somatotropin (growth hormone).

Rat somatotropin (growth hormone) was labelled biosynthetically by incubating anterior pituitary lobes with radioactive amino acids for 24 h in a simple buffered salts medium containing glucose. The labelled hormone was isolated by preparative polyacrylamide-gel electrophoresis or by chromatography on Sephadex G-100 and then DEAE-cellulose. The labelled material was pure by several criteria and cross-reacted immunologically with unlabelled rat somatotropin. When a mixture of 14C-labelled amino acids was used for labelling the protein, label could be introduced into these same amino acids of somatotropin, though relative specific radioactivities varied considerably. Somatotropin labelled by the procedures described in the present paper was suitable for structural studies and could be used for a variety of other biochemical experiments.     

Follitropin receptors in rat testis. Characterization with enzymatically 125I-labeled human follitropin.

The interaction between enzymatically radioiodinated human follitropin and the follitropin receptors in testis homogenate was investigated in immature and adult rats. The 125I-labeled human follitropin exhibited high binding activity with specific binding of up to 17% in the presence of an excess of testis homogenate. Approx. 50% of the bound hormone could be eluted at pH 5, and the receptor purified tracer exhibited a 3.6-fold increase in binding activity when compared with the original tracer preparation. Quantitative analysis of equilibrium binding data was performed with corrections for the measured specific activity and maximum binding activity of the tracer hormone. The equilibrium association constants (Ka) determined 24 degrees C were not significantly different in immature and adult rat testis, and the mean value for Ka was 3.9 . 10(9) M-1. At 37 degrees C, the Ka value obtained using immature rat testis was 1.3 . 10(10) M-1. The association of 125I-labeled human follitropin with immature rat testis homogenate was time and temperature dependent. In the presence of an excess of unlabeled hormone, 30--60% of the preformed hormone . receptor complex was dissociated after 24 h incubation. A specific and sensitive radioligand-receptor assay for follitropin was developed using immature rat testis homogenate. The minimum detectable dose of purified human follitropin was 0.6 ng, and human urinary and pituitary follitropin, ovine follitropin and pregnant mare serum gonadotropin reacted in the assay with equivalent slopes. The potencies of highly purified pregnent mare serum gonadotropin and highly purified human follitropin were similar in the radioligand-receptor assay, consistent with the follitropin bioactivity of the equine gonadotropin.     

An enzyme-linked immunosorbent assay (ELISA) to measure growth hormone level in serum and milk of buffaloes (Bubalus bubalis)

An indirect Sandwich ELISA to measure growth hormone level in serum and milk of buffaloes was developed. The assay was based on purified anti rbST IgG raised in rabbits and chicken and rabbit anti chicken IgG horseradish peroxidase. The assay was validated in terms of sensitivity, specificity, precision and recovery. Parallelism was demonstrated between the standard curve and serially diluted serum, milk and pituitary derived growth hormone. Sensitivity of the assay was 0.1 ng/ml. Recovery of exogenous bovine somatotropin from serum and milk ranged from 90 to 102% and 96 to 108% respectively. The intra and inter assay variations to measure growth hormone in serum and milk were 3.36 to 8.81% and 6.01 to 12.31% respectively. Statistical analysis for parallelism and cross-reactivity of rbST with serum of other species confirmed the reproducibility of the assay.     

Conformation of equine pituitary somatotropin

Equine pituitary somatotropin (growth hormone) has been studied by zero-order and second-order absorption spectroscopy, and by circular dichroism. Difference absorption spectra have also been generated during proteolytic digestion of the hormone. The molar extinction coefficient of the native protein was found to be 16,050 +/- 330 M-1 cm-1 at 278.1 nm. Comparison of the conformations of equine somatotropin and somatotropins isolated from several other mammalian species indicates a close structural relationship between these molecules. With the increasing number of species which have been studied, it is becoming evident that with regard to conformation, the somatotropins can be subdivided into at least three major groups.     

The conformation of monkey pituitary somatotropin

Monkey pituitary somatotropin has been studied by zero-order, second-order, and circular dichroism spectroscopy. Difference absorption spectra have also been generated during proteolytic digestion of the hormone. The molar extinction coefficient of the native protein was found to be 23,800 +/- 550 (M-1 cm-1) at 276.6 nm. A comparison of the conformations of monkey and human pituitary somatotropins indicates a close relationship between the two molecules, including alpha-helix contents of 55 +/- 5%.     

An investigation of sites that bind human somatotropin (growth hormone) in the liver of the pregnant rabbit.

The binding of 125I-labelled human somatotropin (growth hormone) to a crude membrane preparation from the liver of pregnant rabbit, and to receptors solubilized from this fraction by Triton X-100, was dependent on time, temperature and receptor concentration. At 4 degrees C a steady state was reached after 20 h, and maximum specific binding (as a percentage of total tracer added) was approx. 50% for both membrane-bound and solubilized receptors. Solubilization did not significantly affect the binding properties of the receptor at low concentrations of Triton X-100 (less than 0.05%, v/v, in the assay tube). However, at higher concentrations (approx. 0.1%, v/v), the detergent lowered the ability of some hormones, for example ovine prolactin, to displace 125I-labelled human somatotropin, but did not affect other hormones such as bovine somatotropin. Some somatogenic hormones, such as bovine somatotropin, and some lactogenic hormones, such as ovine prolactin, displaced 125I-labelled human somatotropin from membrane-bound and solubilized receptor preparations. Furthermore, 85% of 125I-labelled bovine somatotropin was displaced from membrane-bound receptors by ovine prolactin, and 125I-labelled ovine prolactin was almost completely displaced by bovine somatotropin. Scatchard analysis of the binding data for human somatotropin suggested a single class of binding sites in the membrane-bound receptor preparation, with an affinity (Ka) of 1.9 X 10(9) M-1 and a capacity of 1726 fmol/mg of protein; these values were slightly increased by solubilization (Ka = 3.2 X 10(9) M-1, capacity = 2103 fmol/mg of protein). Scatchard analysis of binding to membrane-bound receptors also indicated a single class of high-affinity binding sites for bovine somatotropin (Ka = 4.8 X 10(9) M-1, capacity = 769 fmol/mg) and for ovine prolactin (Ka = 6.1 X 10(9) M-1, capacity = 187 fmol/mg).     

Use of electrophoresis and gel filtration to characterize human pituitary somatotropin preparations after storage.

Fresh and stored preparations of human pituitary somatotropin examined by different polyacrylamide gel electrophoresis techniques and by gel exclusion chromatography (Ultrogel) clearly demonstrated that the hormone undergoes a structural alteration during storage for 3 years. The formed aggregates were readily quantitated from the gel filtration profile and from polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate (SDS-PAGE). According to gel filtration the amount of aggregates in fresh and stored hormone was determined to be 2 and 10%, respectively, while SDS-PAGE indicated 6 and 14%.     

Isolation and properties of the electrophoretic components of human growth hormone by Sephadex-gel filtration and preparative polyacrylamide-gel electrophoresis

1. Human growth hormone was prepared from acetone-dried residues after extraction of gonadotrophins from pituitary glands. 2. Crude growth hormone was purified by gel filtration on Sephadex, resulting in a product that is soluble in water or 0.5% sodium chloride. It is painless on injection and shows a twofold increase in biological potency. Aggregation of growth hormone on Sephadex columns can be avoided by the addition of urea (6m) and EDTA (1mm) to the buffer. 3. Growth hormone appeared as a single component from Sephadex and ion-exchange columns and sedimented as a single boundary in the ultracentrifuge. In the circular disk electrophoresis, however, the growth hormone showed one faster and two slower-moving anionic components. 4. These components were isolated by preparative electrophoresis on polyacrylamide columns. The purified growth hormone and its three components sedimented as single boundaries with coefficients 2.62, 2.66, 2.66 and 2.83s respectively. 5. Amino acid analyses of the purified growth hormone and its components were closely related. End-group analysis of purified growth hormone and its components showed only phenylalanine at both N- and C-terminals. 6. The purified growth hormone and its components were essentially free of other pituitary hormones, but contained significant prolactin activity. The biochemical similarities among the electrophoretic components of human growth hormone and the presence of the same three components in the growth hormone prepared from a single human pituitary gland suggest polymorphism of a biologically active protein molecule.     

Biological activity and metabolic clearance of recombinant human follicle stimulating hormone produced in Sp2/0 myeloma cells

Human follicle stimulating hormone is a pituitary glycoprotein that is essential for the maintenance of ovarian follicle development and testicular spermatogenesis. Like other members of the glycoprotein hormone family, it contains a common a subunit and a hormone specific subunit. Each subunit contains two glycosylation sites. The specific structures of the oligosaccharides of human follicle stimulating hormone have been shown to influence both thein vitro andin vivo bioactivity. Since the carbohydrate structure of a protein reflects the glycosylation apparatus of the host cells in which the protein is expressed, we examined the isoform profiles,in vitro bioactivity and metabolic clearance of a preparation of purified recombinant human follicle stimulating hormone derived from a stable, transfected Sp2/0 myeloma cell line, and pituitary human follicle stimulating hormone. Isoelectric focussing and chromatofocussing studies of human follicle stimulating hormone preparations both showed a more basic isoform profile for the recombinant human follicle stimulating hormone compared to that of pituitary human follicle stimulating hormone. The recombinant human follicle stimulating hormone had a significantly higher radioreceptor activity compared to that of pituitary human follicle stimulating hormone, consistent with a greaterin vitro potency. Pharmacokinetic studies in rats indicated a similar terminal half life (124 min) to that of the pituitary human follicle stimulating hormone (119 min). Preliminary carbohydrate analysis showed recombinant human follicle stimulating hormone to contain high mannose and/or hybrid type, in addition to complex type carbohydrate chains, terminating with both2,3 and2,6 linked sialic acids. These results demonstrate that recombinant human follicle stimulating hormone made in the Sp2/0 myeloma cells is sialylated, has a more basic isoform profile, and has a greaterin vitro biological potency compared to those of the pituitary human follicle stimulating hormone.     

Stimulation of anterior pituitary prostaglandin E content and somatotropin (growth hormone) synthesis by phospholipase A.

The prostaglandin E content of dispersed rat anterior pituitary glands was found to increase in the presence of phospholipase A or arachidonic acid. The increases were abolished by the addition of indomethacin. Similarly, the rate of somatotropin (growth hormone) synthesis was increased by these two agents, and the increases were again abolished by indomethacin. Phospholipase A also stimulated somatotropin release. The stimulation of prostaglandin E accumulation was a specific response to those fatty acids that are precursors for prostaglandin synthesis. One such precursor, [3H]arachidonic acid, was incorporated by rat anterior pituitary glands in vitro, and found to be associated mainly with phosphatidylethanolamine-like material. It is concluded that the intracellular concentration of prostaglandin E is limited by the availability of precursor fatty acids and that this can be increased by the addition of exogenous precursors or by the action of exogenous phospholipase A on the cellular phospholipid. Factors that increased prostaglandin E concentrations also increase the rate of synthesis of somatotropin, providing further evidence for the concept that prostaglandin E is involved in modulation of the rate of synthesis of this hormone.     

Conformational restrictions of the sheep testicular receptor discriminates pituitary lutropin and placental gonadotropins

A membrane preparation from the testis of maturing Dorset-Leicester-Suffolk sheep, capable of discriminating pituitary LH (lutropin) from placental gonadotropins human choriogonadotropin (hCG) and equine choriogonadotropin is described. Maximum binding of 125I-oLH (ovine lutropin) to the testicular receptors occurred at 4 degrees C in a rapid manner, attaining equilibrium in 12-16 h. Under such optimal conditions, only unlabeled ovine LH or the structurally identical bovine LH effectively competed for receptor occupation. Other highly purified pituitary LH preparations from rat and human pituitaries were weakly (4-10%) active in displacement assays. Purified hCG or equine choriogonadotropin, which were highly potent in rat testicular LH receptor assays, could not compete with 125I-oLH for binding to the sheep LH receptor at 4 degrees C. Thus, the sheep testicular LH receptor was highly specific in recognizing pituitary LH conformation. The presence of an ovine/bovine LH alpha- or beta-subunit in recombinants with hCG subunit counterparts was required to generate an effective conformation capable of receptor recognition. Chemically deglycosylated hCG, containing 75% less carbohydrate and which showed greater binding to other LH receptors, failed to recognize sheep LH receptor, suggesting that excess carbohydrate in hCG was not a factor in hindering binding of the native placental hormone. Scatchard analysis using 125I-hCG/125I-oLH revealed that there were separate sites with similar affinities but vastly different capacities. The hCG binding sites, which could also be effectively occupied by oLH, were less than 10% of oLH binding sites. Thus, the Dorset-Leicester-Suffolk sheep testicular receptor provides an important and unique in vitro test system to distinguish pituitary LH from placental LH-like hormones. We infer that temperature-dependent conformational restrictions of the sheep testicular LH receptor are involved in recognizing differences in these highly similar and structurally homologous hormones.