Following the recent identification and characterization of a highly specific binding protein(s) for human growth hormone (hGH) in human sera, we now report our initial studies on its purification. Outdated blood blank serum was first fractionated by ammonium sulphate precipitation. The majority of the binding activity appeared in the 30-45% saturation fraction, which was then applied in sequence to an hGH-affinity column, to strong anion exchange (SAX) chromatography (a Mono Q column 0.01-0.2 M phosphate gradient), to a TSK phenyl hydrophobic interaction column (HIC) (0.75-0 M sulphate gradient in 0.1 M phosphate) and finally to a second SAX step. By Scatchard analysis the final product was purified approximately 16,500-fold compared to serum with an overall recovery of 5%. The binding affinity (Ka) of the purified material was approximately 0.4 X 10(9) M-1, very similar to that of whole serum (approximately 0.5 X 10(9) M-1). On silver-stained, sodium dodecyl sulphate-polyacrylamide gels, the final SAX product yielded a major doublet, under reduced conditions, of molecular weights (MW) 55,000 and 59,000. Although it is not known whether one or both of these bands might have hGH binding activity, the MW 59,000 band is very similar in size to one (MW 60,000) of two specific binding proteins observed by covalent crosslinking techniques. These data indicate that the specific hGH binding activity of human serum can be substantially purified and that it appears to exist in at least two forms. Further studies are required to determine the biochemical and physiological relationship, if any, between the two forms.

Chromatography, Affinity↗

PubMed1987

A comparison of cyclosporine A and Nva2-cyclosporine (cyclosporine G) in a rat renal allograft model.

We compared CsG and CsA in the DA-to-Lewis rat renal allograft model. At equivalent oral doses, plasma radioimmunoassay (RIA) CsG levels were higher than CsA (P less than 0.02). Neither drug prevented rejection at doses of 5 mg/kg/day. CsG-treated rats had a higher rejection rate at doses of 7.5 mg/kg/day (P less than 0.05). Both drugs were equally effective in preventing rejection at doses of 10 mg/kg/day. Neither drug was nephrotoxic at the doses used in this study. CsG is a potent immunosuppressant, and thus a potential clinical successor to CsA. Since CsG and CsA provide equivalent immunosuppression at therapeutic doses, CsG's clinical significance will ultimately depend on its nephrotoxicity in man.

Animals↗

PubMed1987

Posttranslational processing of corticotropin-releasing factor in the ovine tuberoinfundibular system and pituitary.

The present studies were undertaken to characterize the immunoreactive-corticotropin-releasing factor (ir-CRF) in two areas of the ovine tuberoinfundibular system, hypophysial portal blood, and pituitary. With an antiserum raised against synthetic ovine (o)CRF(1-41) and 125I-Tyro-oCRF(1-41) as the tracer, concentrations of ir-CRF (pg/mg wet weight, n = 5) were: paraventricular hypothalamus (PVN), 11.7 +/- 2.5; median eminence (ME), 2276 +/- 296; anterior pituitary (AP), less than 0.5; posterior pituitary (PP), 10.0 +/- 2.2. Analysis of the ir-CRF in these areas on G-75 Sephadex chromatography revealed two main peaks--a 'major' peak which coeluted with synthetic oCRF(1-41) and a 'minor' peak which eluted eight fractions later. These two immunoreactive species of CRF were also found in hypophysial portal blood. When ME extract was analyzed by reverse-phase high performance liquid chromatography (HPLC), the 'minor' peak of ir-CRF eluted before that of CRF(1-41). Since CRF contains Arg35-Lys36 within its sequence, we tested the hypothesis that the 'minor' peak of ir-CRF represented a fragment, or fragments, of the molecule derived by proteolytic cleavage at this site. Tyro-oCRF(34-41) was digested with trypsin and the reaction products were identified by amino acid analysis. Two of these products were CRF(36-41) and CRF(37-41), and both migrated in the 'minor' peak area on G-75 Sephadex chromatography and HPLC. In the CRF(1-41) RIA, serial dilution of both fragments yielded nonparallel displacement curves. However, with 125I-Tyro-oCRF(34-41) as the radiolabeled ligand and Tyro-oCRF(34-41) as the standard, serial dilutions of CRF(1-41), CRF(36-41), and CRF(37-41) generated parallel displacement curves, and the molar cross-reactivities were 90%, 45% and 10% respectively. When the ir-CRF in HPLC fractions of ovine ME was measured in the Tyro-oCRF(34-41) RIA, the molar abundance of the hexapeptide and pentapeptide could be obtained. Calculations based on the premise that the 'minor' peak was solely composed of either the hexapeptide or pentapeptide indicated that CRF(36-41) could account for up to 37% of the total ir-CRF, or that CRF(37-41) could account for up to 73% of the total immunoreactivity. On more discriminating HPLC systems, immunoreactive (ir-) oCRF in the sheep median eminence (ME) could be resolved into five different molecular forms. On three distinct chromatographic systems, four of these immunoreactive species shared retention characteristics identical with synthetic oCRF(37-41), oCRF(36-41), oCRF(16-41) and oCRF(1-41), with the fifth immunoreactive peak as yet unidentified.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗