Cooperative interactions in neurophysin-neuropeptide hormonecomplexes

The structural interdependence between neurophysin (NP) self-association and ligand binding surfaces has been studied by analytical affinity chromatography of several NP sequence variants and derivatives on Met-Tyr-Phe-aminobutyl-agarose and bovine NP-II Sepharose. Elutions of radiolabeled NP's from both matrices show that hybrid dimers can form between major bovine NP's (I and II, or VLDV- and MSEL-NP's, respectively), as well as between human and bovine NP's, with affinities close to that for homologous dimer formation. Such evidence supports the view that the region of NP involved in NP-NP contact is composed primarily of conserved structural elements of the protein. NP antibodies which recognize surfaces close to or in the NP-NP contact region have been detected by their effects on bovine NP-II elution on NP-II Sepharose. Elutions of [3-nitro-Tyr 49] BNP-II from Met-Tyr-Phe-aminobutyl-agarose showed that nitration has little effect on the chromatographic properties of NP-II. This evidence substantiates previous arguments (Angal, S. & Chaiken, I.M. (1982) Biochemistry 21 , 1574–1580) that the chromatographic behavior of native NP's on the affinity matrices is an expression of the interdependence of NP self-association and ligand binding surfaces and not due to bivalent peptide binding by NP monomer. The affinity chromatographic properties of NP derivatives, including bovine NP-II photolabeled in the ligand binding site and tryptic fragments of bovine     

Conformational dynamics and kinetics of peptide antagonist interactions with interleukin-1 receptor. Fluorescence studies using the NBD-labelled peptide AF12415

Interleukin-1 plays a key role in the inflammatory response provoked by various disease states and inhibition of its action can bring therapeutic benefits. Steady-state and time-resolved studies of the intrinsic tryptophan fluorescence of the free soluble Type I form of interleukin-1 receptor (IL-1R) reveal that the rotational motions of the three major domains are strongly associated. Bound peptide antagonists are buried in hydrophobic regions, but a flexible association permits access to species from the aqueous phase. Ligand binding does not lead to rigidification of the receptor structure. The kinetics and mechanism of complex formation and dissociation, involving IL-1R with receptor antagonist protein (IL-1ra) and with peptides AF11733 (15 aa) and AF10961 (21 aa) were determined with the aid of peptide AF12415 (15 aa) labeled at its N-terminus by the NBD fluorophore, which exhibits a five-fold increase in emission intensity at 540 nm on binding of the peptide to IL-1R. The magnitude of the ON rate constant, typically 1 × 10⁶ M^?1 s^?1, implies the existence of an intermediate ‘encounter complex’ involving interactions of low specificity. Readjustments of the initial encounter complex leads to a final complex where very specific interactions dominate. The first-order rate constant for this latter process is the most sensitive indicator of the true peptide affinity for the receptor binding site, and thus provides a better criterion than the apparent OFF rate (typically 2 × 10^?3 s^?1) for discrimination of peptide antagonists. © Munksgaard 1997.     

Growth hormone isoform testing in capillary dried blood spots: Results from single and multiple dose administration studies and large-scale field collections

A multiphase study was designed to examine the detectability of human growth hormone (GH) use in capillary dried blood spots (DBS). First, 13 subjects self-injected a single, 2-mg dose of somatropin and collected capillary DBS samples for 24 h. Next, nine subjects self-injected 2-mg somatropin, six times over the course of 11 days; DBS were collected intermittently following dosing. Finally, a nondrug, large-scale field study involved DBS collections from an athlete and staff population over 3 years. All DBS samples were self-collected using the Tasso M20 device and were analyzed for the presence of GH using the WADA-approved GH isoforms test. Following the single dose, positive detection within 12 h of dosing was 86% and 56% sensitive on Kits 1 and 2, respectively. In the multidose study, detection within 12 h was 85% and 69% sensitive on Kits 1 and 2, respectively. No positives were detected outside the 12-h window following a single dose, wherein detection was 5.6% sensitive at 24-h in the multidose study. Combining the 12-h windows from both studies, 100% of samples had measurable recombinant (REC) and pituitary (PIT) GH concentrations above the assay LoD, 0.041 ng/ml. Finally, 1213 samples were collected in the large-scale field study: 189 showed REC and PIT concentrations above the LoD; none returned positive results. GH is detectable in capillary DBS using the isoforms method for 12–24 h following use. While detection is short lived, transitioning to a DBS self-collection method can allow more frequent testing and increase deterrence to GH abuse.     

Mapping the receptor binding regions of human chorionic gonadotropin (hCG) using disulfide peptides of its β‐subunit: possible involvement of the disulfide bonds Cys9−Cys57 and Cys23−Cys72 in receptor binding of the hormone

Abstract: Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone essential for the establishment and maintenance of pregnancy. The α‐ and β‐subunits of hCG are highly cross‐linked internally by disulfide bonds which seem to stabilize the tertiary structures required for the noncovalent association of the subunits to generate hormonal activity. The purpose of this study was to delineate the role of the disulfide bonds of hCGβ in receptor binding of the hormone. Six disulfide peptides incorporating each of the six disulfide bonds of hCGβ were synthesized and screened, along with their linear counterparts, for their ability to competitively inhibit the binding of [¹²⁵I] hCG to sheep ovarian corpora luteal LH/CG receptor. Disulfide peptide Cys (9?57) was found to be ≈ 4‐fold more potent than the most active of its linear counterparts in inhibiting radiolabeled hCG from binding to its receptor. Similarly, disulfide peptide Cys (23?72) exhibited receptor binding inhibition activity, whereas the constituent linear peptides were found to be inactive. The results suggest the involvement of the disulfide bonds Cys⁹?Cys⁵⁷ and Cys²³?Cys⁷² of the β‐subunit of hCG in receptor binding of the hormone. This study is the first of its kind to use disulfide peptides rather than linear peptides to map the receptor binding regions of hCG.