Site‐specific fluorescein labeling of human insulin

Three fluorescein derivatives of human insulin (HI, 1 ) labeled at positions N^αA1, N^αB1 and N^εB29 respectively, were synthesized using an N‐trifluoroacetyl‐based protecting group scheme. The Tfa protecting group introduced by reaction with ethyl trifluoroacetate was found to be stable in aqueous and organic media and efficiently removed under mild basic conditions. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Induction of specific human growth hormone binding sites in rat liver by human growth hormone.

125I-Labeled hGH was bound to liver plasma membranes which were obtained from female rats. The binding was displaced by hGH, hPRL, bPRL, rPRL and bGH but not by rGH. This result indicated that hGH was bound to lactogenic binding sites in rat livers. After hypophysectomy, the binding was markedly decreased. Treatment of hypophysectomized rats with hGH (80 micrograms/day) for 10 days increased the binding sites for hGH. These binding sites were different from those found in normal female rat livers because of their high affinity and specificity for hGH. These results indicate that hGH induces specific binding sites for hGH in rat livers.     

Biological activity of human alpha-interferons studied using specific antisera

Preparation of highly active rabbit antisera (AS) to human recombinant alpha 2-interferon and their use for studying biological properties of natural and plasmid alpha-interferons are described. By exhaustion of AS by alpha 3-interferon there were prepared practically monospecific AS not reacting with antigenic determinants of alpha 3-interferon. It was found that alpha 3-interferon represented a significant portion of human lymphoblastoid interferons and was included in PH-labile alpha-interferon from serum of patients with Kaposi carcinoma. AS to alpha 2-interferon completely neutralized antiviral and antiproliferative activity of the homologous subtype alpha-interferon and stimulation of cytotoxicity of human natural killer cells induced by it. It neutralized also the same effects of the heterologous subtypes (alpha 3 and alpha F/D) and leukocytic interferon, but the neutralization level was lower. The results of the study confirmed the polyfunctional nature of the interferon molecule.     

Isolation and characterization of a sulfoxide and a desamido derivative of biosynthetic human growth hormone

Two derivatives of biosynthetic human growth hormone, a sulfoxide and a mixture of two monodesamido isomers, have been isolated and characterized. The sulfoxide derivative arises from an oxidation of Met-14. The major site of deamidation is at Asn-149 with a minor site at Asn-152. In addition, a fraction has been isolated from a sample of human growth hormone that was maintained at 40 degrees C for 2 weeks. This fraction, the isolated impurities fraction, contains the sulfoxide and the desamido forms, thereby demonstrating that these derivatives are the primary degradation products of biosynthetic human growth hormone. The sulfoxide, the desamido, and the isolated impurities fraction exhibit full biological activity.     

Pepsin inhibition by a high specific activity radioiodinated derivative of pepstatin.

Stop-flow kinetic studies are reported for the reaction of glutathione (GSH) with 2-chloromercuri-4-nitrophenol. The second-order rate constant was found to vary by less than twofold in going from pH 5.9 to 7.3, suggesting that the reaction involves attack of the un-ionized glutathione thiol on the mercurial around pH 7. The rate constant was 1.5 × 10⁷m^?1 s^?1 at pH 7.0 and 8 °C in dilute buffer of ionic strength 0.2. The sensitivity of the kinetics to the nature of the leaving group in the mercurial i.e., to the labile ligand, was investigated in complexes of the mercurial with OH^?, N₃^?, Br^?, Cl^?, CN^?, SCN^?, pyrophosphate, ADP, ATP, EDTA, and adenosine. The addition of most of these substances was observed to lead to rate enhancements or rate retardations, the kinetic effects showing typical binding isotherm behavior when plotted as a function of the added ligand concentration. The kinetic midpoints of such plots showed very good correlation with the independently determined affinities they had toward the mercurial. The results demonstrate that many commonly added compounds of biochemical interest can significantly influence mercurial reactivity toward thiols through rapid exchange at the labile ligand position of the mercurial.     

Enhancement of hormonal activity with a monoclonal antibody specific to porcine growth hormone

Abstract

The potentiation of the biological effects of recombinant porcine growth hormone (pGH) by immunologic manipulation was investigated. A monoclonal antibody (mAb), designated PS‐7.6, was raised against pGH and repeatedly shown to enhance the responsiveness of hypophysectomized (hypox) rats to pGH. As a result, animals receiving a combination treatment of pGH and mAb PS‐7.6 together gained significantly more weight than those receiving the same doses of pGH alone. The enhancing action of the mAb was a rapid process and its effective doses ranged from 0.1 to 2 mg/injection. The ability of the antibody to augment the hormonal activity persisted beyond the 5‐day treatment period and the differences in net weight gain between treated and control animals remained significant for 28 days. Results from treatment frequency studies further suggested that pGH when complexed with mAb PS‐7.6 required fewer injections and produced a greater efficacy than being administered alone. Therefore, present findings suggest that mAb PS‐7.6 may prove useful for not only improving the efficiency of pGH, but also developing a novel formulation for sustained pGH release.     

Biological characterization of purified native 20-kDa human growth hormone

Because of the propensity of the 20-kDa variant of human growth hormone (GH) to aggregate with itself and with 22-kDa human GH, it has been difficult to prepare monomeric 20-kDa GH in highly purified form. This has been a major complicating factor in determining whether 20-kDa GH has a biological activity profile distinct from that of 22-kDa GH. In the present study, native 20-kDa GH was isolated from a human GH dimer concentrate and purified by a procedure that included column electrophoresis in agarose suspension as a final separation step. This procedure yielded highly purified monomeric 20-kDa GH, which was contaminated to an extent of less than 1% with 22-kDa GH, and which exhibited only a small degree of dimerization upon storage. The native 20-kDa GH was quite active in stimulating growth in hypophysectomized rats, when growth was assessed by body weight gain, longitudinal bone growth, the stimulation of sulfation of cartilage, and the elevation of serum IGF-1 level. However, in all of these growth assays, the 20-kDa GH was somewhat less active than the native 22-kDa GH to which it was compared; e.g., in the body weight gain and longitudinal bone growth assays, it had an estimated potency of 0.6 relative to the 22-kDa GH. The 20-kDa GH exhibited substantial diabetogenic activity when tested for the ability to raise fasting blood glucose concentration and to impair glucose tolerance in ob/ob mice. Also, the native 20-kDa GH had significant in vitro insulin-like activity, although its potency was approximately 20% that of the native 22-kDa GH to which it was compared. Thus, the biological activity profile of native 20-kDa GH differs from that of 22-kDa GH primarily in that insulin-like activity is markedly attenuated.     

Sequences required for cell-type specific thyroid hormone regulation of rat growth hormone promoter activity

We have located sequences within the rat growth hormone (rGH) promoter region which are required for pituitary cell-type specific responsiveness to T3 (thyroid hormone, 3,5,3'-L-triiodothyronine). Transient transfections with a series of plasmids containing as few as 202 nucleotides upstream of the start site of the rat growth hormone mRNA showed specific induction by T3 in rat pituitary cell lines. Both the magnitude and the kinetics of this response were similar to those of the endogenous rGH gene, showing a strong early induction followed by a decline in T3 effect. Deletion of an additional 19 base pairs (to -183 relative to the start site) eliminated this induction. Plasmids containing sequences up to -237 or -202 showed significant promoter activity but no T3 responsiveness in transfections of mouse fibroblasts or monkey kidney cells. The presence of high affinity nuclear T3 binding proteins was demonstrated in both cell types. These results show that sequences between -183 and -202 are required for pituitary cell specific T3 regulation of the rGH promoter. The lack of T3 responsiveness in non-pituitary cells suggests that such regulation may be mediated by factors present in pituitary cells and absent in other cells.     

Chimeric bovine-human growth hormone prepared by recombinant DNA technology: binding properties and biological activity

A chimeric bovine GH (amino acids Met-Asp-Gln-greater than 1-23) and human GH (hGH) (amino acids 24-191) plasmid was constructed and expressed in Escherichia coli. The purified protein (chimeric GH) exhibited a 2-3 order of magnitude lower affinity toward lactogenic receptors in Nb2 lymphoma cells, microsomal fractions from bovine mammary gland and male rat liver. The affinity towards somatogenic receptors in IM-9 human lymphocytes and male rat liver was decreased to a much lesser degree. This diminished affinity towards lactogenic receptors was accompanied by a parallel decrease in the ability of the chimeric GH to stimulate proliferation of Nb2-11C lymphoma cells and the lipogenesis in bovine mammary gland. This implies that occupation of the respective receptors by either chimeric GH or hGH leads to identical postreceptoral effects. The chimeric GH was also capable of down-regulating the lactogenic receptors in Nb2 lymphoma cells and was recognized by three anti-hGH monoclonal antibodies. These and previously published results indicate that the N-terminal part of hGH is essential for the high affinity binding to lactogenic receptors and subsequent biological effect. Removal or replacement by a corresponding part of bovine GH converts the hormone, respectively to weak antagonist or agonists. Analysis of our data, based on hydropathy index leads us to suggest that the high affinity binding site of the hGH towards lactogenic receptors is mainly confined to amino acids nos. 8-18.     

Inactivation of human S-adenosylhomocysteine hydrolase by covalent labeling of cysteine 195 with thionucleoside derivatives

A new series of 5'-thioadenosine derivatives 1-4 were synthesized for selectively targeting (195)Cys of human AdoHcy hydrolase. Their incubation with the enzyme resulted in time- and concentration-dependent inactivation, without major modifications of the NAD(+)/NADH ratio. The electrospray mass analysis of the inactivated enzyme with 1, 2, 3, and 4b showed that inhibition was accompanied by the formation of a specific and covalent labeling of each AdoHcy hydrolase subunit. Proteolytic cleavage (endo-Lys-C) and subsequent peptide characterization of the labeled enzyme revealed that (195)Cys was the residue modified during the inactivation process.     

Reconstitution of a kidney chloride channel and its identification by covalent labeling

The basolateral membrane of the thick ascending loop of Henle (TALH) of the mammalian kidney is characterized by its high content of Na+/K(+)-ATPase and a Cl- conductance, which function in parallel in salt reabsorption. In order to reconstitute the Cl- channels, TALH membrane vesicles were solubilized in 1% sodium cholate in buffer containing 200 mM KCl, followed by dilution with soybean lipids (final ratio of protein/detergent/lipid of 1:3:15 in mg) and removal of the detergent by gel filtration on Sephadex G-50. Cl- channel activity in the liposomes was determined by a 36Cl- uptake assay where the accumulation of the radioactive tracer against its chemical gradient is driven by the membrane potential (positive inside) generated by an outward Cl- gradient. The 36Cl- uptake by the KCl-loaded liposomes was dependent on the inclusion of membrane protein and was abolished by valinomycin, indicating the involvement of a conductive pathway. It was also inhibited by 36% by 100 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). Solubilization of the Cl- channels in cholate was optimal in the presence of 200 mm KCl, but was found to decrease markedly at low ionic strength. SDS-PAGE analysis of the proteins extracted by cholate at high and low salt concentrations showed that the Cl- channel-containing high KCl extract was enriched in the 96 and 55 kDa alpha- and beta-subunits of the Na+/K(+)-ATPase (the major proteins in the membrane preparation) and several minor protein bands. Treatment of the membrane vesicles with the radioactive analogue of DIDS, [3H]2DIDS, labeled primarily a 65 and a 31 kDa protein. The solubilization of the 31 kDa protein by cholate depended markedly on the ionic strength and thus paralleled the solubilization pattern of Cl- channel activity. Furthermore, the labeling of the 31 kDa protein was prevented by nonradioactive DIDS and by NPPB but not by other compounds, indicating that it may be a Cl- channel component.     

Inhibition of voluntary activity by growth hormone

During food deprivation and subsequent refeeding there are alterations in growth hormone (GH) secretion and wheel-running activity such that wheel running increases when GH secretion decreases and vice versa. To investigate if there might be a causal relationship between GH secretion and wheel running, the effect of GH administration on wheel running was studied. Female Sprague-Dawley rats were placed singly in activity-wheel cages shortly after weaning. Beginning at 70–75 days of age the rats received 14 daily control injections of saline followed by 14 daily injections of GH and a further 14 days of control injections. Wheel-running activity was inhibited during the period of growth hormone injections. Possible implications of this result are discussed.     

Characterisation and covalent labeling of the human placental methyltrienolone binding protein

Human placental cytosol contains an androgen binding protein which binds the synthetic androgen methyltrienolone (R 1881) with high affinity (Kd 8.7 nM) and with an average binding capacity of 518 fmol/mg cytosol protein. This study provides further evidence that this protein is distinguishable from classical androgen receptors on the basis of steroid specificity and sulphydryl group sensitivity. Covalent labeling studies have shown this protein, which we have called "the methyltrienolone binding protein", to have a mol. wt of 67,000 daltons.     

Regulation of glycogen synthase activity in muscle by a C-terminal part sequence of human growth hormone

The synthetic peptide hGH 177–191, corresponding to the last 15 residues at the carboxyl terminus of human pituitary growth hormone, promotes the conversion of glycogen synthase α to glycogen synthase b in muscle. When injected, the peptide was found to produce inactivation of glycogen synthase phosphatase activity in rat skeletal muscle. The time course of phosphatase inactivation was closely correlated with that for glycogen synthase. The peptide had no effect either on muscle 3′,5′-cyclic AMP levels or on synthase kinase activity. These results can be explained in terms of a dynamic cycle of interconversion of synthase between active and inactive forms, by the simultaneous action of synthase kinases and synthase phosphatases. A decrease in the ratio of phosphatase to kinase activity would result in a decrease in the steady-state level of synthase α activity.     

Affinity purification and structural characterization of a specific binding protein for human growth hormone in human serum

A highly specific binding protein for human growth hormone (hGH) has been isolated from human serum by hGH-affinity chromatography. A purification of approximately 1500-fold with a 30-40% recovery was obtained with essentially no alteration in binding characteristics. Covalent cross-linking of 125I-hGH to the binding protein, followed by analysis by SDS-polyacrylamide gel electrophoresis and autoradiography, revealed two specifically labeled complexes. Allowing for a 1:1 binding stoichiometry the binding proteins themselves had mean mol wts of 57,000 and 69,300. These increased slightly to mol wt 60,300 and 72,000 respectively in the presence of 100 mM dithiothreitol, suggesting the presence of intramolecular but not inter-subunit disulfide linkages. These data confirm the presence of the hGH binding protein(s) in human serum and define their gross structural nature.     

Biological activity of follicle-stimulating hormone in the pituitary gland of human fetuses

Biological methods were used to study the changes in the folliculo-stimulating hormone (FSH) level in the hypophysis of human fetuses. FSH was found to be present in the hypophysis of fetuses during the whole period of investigation: from the 8th to the 34th week. Marked sex differences both in the total and in the relative FSH content were revealed in the adenohypophysis from the 17th to the 34th weeks of gestation.