Isolation and partial characterization of six somatomedin-like peptides from human plasma Cohn fraction IV

Abstract

Six somatomedin-like peptides were purified from human plasma Cohn fraction IV by a six-step procedure which included ethanol precipitation, reversed-phase extraction, gel filtration, chromatofocusing and reversed-phase high pressure liquid chromatography (HPLC). Purification was monitored with a competitive protein binding assay using a crude preparations of somatomedin carrier protein. The peptides isolated were homogeneous by reversed-phase HPLC and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Their apparent isoelectric points determined by chromatofocusing were 9.2 (Sm I), (Sm II), 8.2 (Sm III), 6.7 (Sm IV), 6.3 (Sm V), and 6.15 (Sm VI). SDS-PAGE under reducing conditions revealed that they are composed of a single peptide chain with apparent molecular weights of 6800 for Sm I, II and IV and 6400 for Sm III, V, and VI.They were equally potent in the porcine costal cartilage in vitro bioassay. The basic peptides (Sm I\p=n-\III) were significantly more active in radioimmunoassays for somatomedin C (SmC) and insulin-like growth factor I C-peptide (IGF-I (30\p=n-\41)),while only the slightly acidic peptides were active in a radioimmunoassay for insulin\x=req-\ like growth factor II C-peptide (IGF-II (33\p=n-\40)). When receptor binding was tested with human placental cell membranes and Sm III as tracer, the basic peptides were significantly more potent than Sm IV\p=n-\VI.With rat liver cell membranes and Sm V as tracer the slightly acidic peptides were more potent. These findings suggest 1) that human plasma may contain other somatomedin-like peptides besides the major components IGF-I/SmC and IGF-II, and 2) that the basic peptides are structurally related to IGF-I/SmC and the slightly acidic peptides are related to IGF-II.

Purification, amino acid sequence and characterisation of kangaroo IGF-I

“…A number of variant forms of IGFs have been reported in other studies, including larger forms of IGF-II identified in human plasma (Zumstein et al 1985, Gowan et al 1987. Minor forms of IGF-I which may be due to alternative splicing of mRNA and have different isoelectric points from the major form have also been shown to exist in human sera (Blum et al 1986(Blum et al , 1987. The site of post-translational modification and the possible physiological role for these variants is unclear at this stage.…”

“…However, determination of the complete IGF-II sequence may also help to explain the differences in chromatographic properties between kIGFs and those of other species. Fortuitously, this separation procedure also minimises cross-contamination of IGF-I preparations with IGF-II, which has been reported to be a major problem with other methods for purifying IGFs from biological sources (Blum et al 1986, Bayne et al 1991. Yields of 10·4 and 36·0 µg/l IGF-I and 8·4 and 9·0 µg/l IGF-II were obtained from 2·5 and 4·4 litres serum respectively.…”

Vitreous levels of the insulin-like growth factors I and II, and the insulin-like growth factor binding proteins 2 and 3, increase in neovascular eye disease. Studies in nondiabetic and diabetic subjects.

“…This antibody was of high affinity (half-maximal displacement at 1.1 ng/ml; detection limit, 10 pg/ml) and high specificity (cross reactivity with IGF-II < 0.05%). The standards and the tracers were prepared from recombinant human (h)IGF-I (Kabi-Pharmacia, Stockholm, Sweden) using the chloramine T method for the latter (14). The interference of IGFBPs in the RIA could be completely blocked by performing the assay in the presence of a large excess of IGF-II.…”

Metabolic clearance of recombinant human growth hormone in health and chronic renal failure.

“…To evaluate nonspecific binding to peak II-BP, 5 Ag GH was added to the incubation. IGF I and IGF II were determined using highly specific RIAs after extraction procedures as previously described (25,26). IGF-BP3 was determined by a specific RIA using an antibody against the pure acidstabile 53-kD binding subunit extracted from plasma Cohn fraction IV (27).…”