Thomas A. Bewley

Purification and properties of teleost growth hormone

Highly purified growth hormone (GH) has been prepared from the pituitary glands of a euryhaline teleost, Tilapia mosambica. Tilapia GH was obtained in a yield of 1400 mg/kg wet weight tissue. It was found to have a molecular weight (gel filtration) of 22,200 daltons, a sedimentation coefficient (s20,w) of 2.19, and an α-helix content (circular dichroism) of 50%. Isoleucine was found to be the major amino-terminal residue; leucine was found to be COOH terminal. The amino acid composition, disc gel electrophoresis pattern, and circular dichroism spectra were similar to those of mammalian GHs. Tilapia GH was found to have a low but significant activity in the rat tibia assay and showed immunological relatedness to mammalian GH in a rat GH radioimmunoassay. Antiserum was prepared against the Tilapia GH and characterized in agar diffusion experiments and radioimmunoassay. Results from these investigations demonstrated a significant degree of cross-reaction between Tilapia GH and pituitary extract from another teleost (perch), but purified tetrapod GHs were essentially nonreactive. The data indicate a significant resemblance between Tilapia GH and mammalian GHs and suggest that the GH structure has been strongly conserved during evolution.

Isolation and properties of teleost prolactin.

Highly purified prolactin (PRL) has been isolated from the pituitary tissue of a euryhaline teleost, Tilapia mossambica. It has a molecular weight of 19,400 daltons, with a single NH2-terminal residue, valine, and a single COOH-terminal residue, half-cystine. Amino acid composition data revealed the presence of one tryptophan and four half-cystine residues, which is characteristic of all known vertebrate growth hormones (GH) but not of mammalian PRLs. The circular dichroism spectrum of Tilapia PRL was similar to that of porcine PRL and showed an α-helix content of 65%. Tilapia PRL was considerably more potent than ovine PRL in two teleost PRL bioassays: the sodium-retaining assay in Tilapia and the reduction of water permeability in the urinary bladder of Gillichthys mirabilis, but it did not stimulate mammary tissue or the pigeon crop sac. Tilapia PRL had equivocal but suggestive activity in the rat tibia assay, and showed cross reaction in two GH (rat and snapping turtle) radioimmunoassays. A specific Tilapia PRL antiserum was prepared in a rabbit which gave a precipitin reaction against Tilapia PRL in agar diffusion but showed no cross reaction with purified mammalian PRLs or pituitary extracts from other teleosts. The data show that Tilapia PRL has features common to both mammalian PRLs and GHs as well as to Tilapia GH, lending support to the hypothesis that PRL and GH originated from a common ancestral molecule.

Primary Structures of Human Pituitary Growth Hormone and Sheep Pituitary Lactogenic Hormone Compared

The primary structures of human pituitary growth hormone and sheep pituitary lactogenic hormone have been compared with regard to similarities in their sequence. Three homologous segments in each molecule, comprising approximately 45 percent of either peptide chain, have been found. These two proteins have apparently evolved from a common ancestor molecule.

A novel procedure for determining protein concentrations from absorption spectra of enzyme digests

Abstract A novel procedure for determining molar extinction coefficients (EM), and hence protein concentrations, has been tested on ribonuclease A, bovine serum albumin, β-lactoglobulins A and B, α-lactalbumin, chymotrypsinogen A, and lysozyme. EM values were obtained from a combination of the absorption spectrum of the native protein and a difference spectrum generated between two aliquots of a thermolysin digest of the protein titrated to pH 1.5 and pH ≥ 13. Enzymatic digestion was shown to minimize conformational contributions to the difference spectrum. The EM of the native protein is then calculated from the tyrosine molarity of the digest and the previously determined tyrosine content of the protein. These EM values displayed a maximum error of −2.2% and an average absolute error of 1.4%. Samples as small as 100 μg give satisfactory results. Accurate sample weight and assumptions with regard to moisture and ash content are not required. In addition, the use of second-derivative (second-order) absorption spectroscopy to access the degree of spectral normalization produced by enzymatic digestion is described.

The kinetics of dissociation and reassociation of ovine pituitary interstitial cell stimulating hormone

Abstract The dissociation of ovine interstitial cell stimulating hormone and the reassociation of its subunits have been followed by circular dichroism and sedimentation velocity measurements. The dissociation was found to be a rapid, first-order reaction accompanied by the exposure of previously buried tyrosyl groups. The reassociation reaction begins as a rapid, probably second-order formation of a complex between the α and β subunits. This is followed by a much slower, first-order rearrangement of the conformation resulting in the reburying of at least two tyrosyl groups, one contributed by each subunit.

Human pituitary growth hormone: XXII. The reduction and reoxidation of the hormone

Abstract The two disulfide bridges in human pituitary growth hormone were reduced with dithiothreitol in the absence of denaturant. The reduced protein was allowed to autooxidize at pH 8.4, and a reoxidized monomer was obtained in good yield. The reoxidized hormone retained the full biological potency of the native hormone as a growth-promoting and lactogenic agent. Small differences between the native and reoxidized proteins were revealed by viscosity measurements and the rates of tryptic hydrolysis. However, these differences are much smaller than those found between the native hormone and a biologically active reduced tetra-S-carbamidomethylated derivative reported previously. The circular dichroism spectra of the reoxidized and native proteins at pH 8.2 were found to be identical between 205 and 300 mμ. The significance of these findings is discussed in terms of the role of the disulfide bonds in this protein.

Circular dichroism studies on human chorionic somatomammotropin

Abstract The conformation of human chorionic somatomammotropin has been studied by means of circular dichroism spectra. The protein appears to contain about 45% α-helix in the native state. Circular dichroism bands in the region of side chain absorption have been assigned to phenylalanine and tryptophan residues. Tentative assignments has also been made to bands probably arising mostly from tyrosine residues. The stability of the native structure has been assessed by challenging the protein with four perturbing solvents. With the exception of 0.1 n NaOH which produced permanent denaturation, all conformational changes produced by the perturbants were fully reversible. In addition, the monomer molecular weight has been evaluated by gel filtration and osmotic pressure measurements. A value of 21,600 ± 900 was found by osmotic pressure at pH 8.4. The results have been compared with similar findings on human pituitary growth hormone and ovine pituitary lactogenic hormone.

Physicochemical and biological characterizations of pregnant mare serum gonadotropin and its subunits

Pregnant mare serum gonadotropin and its subunits have been further characterized. Ultracentrifugation of the gonadotropin at pH 1.3 and 11.5 showed little evidence of dissociation compared to pH 8.2. Highly purified subunits are obtained by urea dissociation and ion-exchange chromatography followed by gel-filtration. Circular dichroism spectra of the gonadotropin and its subunits are much like those of ovine lutropin and its subunits in that there is little evidence for secondary structure and one or more tyrosine residues are inaccessible in the intact gonadotropin compared to the subunits. The alpha-subunit possesses almost 3 times as much total carbohydrate as the beta-subunit; the individual sugar composition of each was determined as well as the amino acid composition. The alpha-subunit begins with the sequence NH2-Phe-Pro (Gly or Pro) ... and terminates with isoleucine. The beta-subunit has the sequence NH2-Ser-Pro-Gly ...; no C-terminal residue is detectable by either carboxypeptidase or hydrazinolysis. Biological studies show the gonadotropin to be active in assays specific for both lutropin and follitropin. Precipitin test in agar with rabbit antiserum against the gonadotropin show that the beta subunit cross-reacts whereas the alpha subunit does not.

Comparison of secreted and extracted forms of rat pituitary prolactin

Prolactin secreted by rat anterior pituitary explants into organ culture medium was purified by salt fractionation and gel filtration. A yield of 22 mg/g was obtained, which clearly represented de novo synthesis and secretion of the hormone. Comparative characterization studies were performed on the secreted prolactin and pituitary extracted rat prolactin obtained from the National Institute of Arthritis, Metabolism and Digestive Disease. The biological and immunological activity estimates of both forms were comparable, although the specific activities of the secreted prolactin were somewhat lower than those of the pituitary prolactin. The secreted and extracted forms of prolactin appeared to be identical in primary structure as evidenced by similar amino acid compositions and identical NH2-terminal sequences. Circular dichroism spectra suggested that there may be differences in tertiary structure, since the positive tryptophan band at 292 nm, which eas observed with extracted hormone, was absent in the secreted prolactin.

Conformational Comparison of Human Pituitary Growth Hormone and Human Chorionic Somatomammotropin (Human Placental Lactogen) by Second-Order Absorption Spectroscopy

Second-order absorption spectra strongly suggest the presence of a hydrogen bond between the single Trp of human pituitary growth hormone (hGH) and a carboxylate ion. This hydrogen-bonded complex is buried within the hydrophobic interior of the hGH molecule. Although the homologous Trp in human chorionic somatomammotropin [human placental lactogen, HCS(hPL)] is also buried within the hydrophobic interior of the molecule, there is no evidence that it is hydrogen bonded in the native protein. However, during the early stages of thermolysin digestion of HCS(hPL), both difference and second-order absorption spectra do indicate the transient presence of a similar hydrogen-bonded Trp-carboxylate complex. The molar extinction coefficients of hGH and HCS(hPL) have been refined.