Purification and characterization of three molecular forms of insulin-like growth factor II from human Cohn paste IV.
The somatomedins or insulin-like growth factors (IGFs) are a family of peptides present in human serum. They are bound to specific carrier proteins and are thought to mediate growth-promoting actions of human growth hormone. Starting from Cohn fraction IV of human plasma, we describe here a rapid and highly efficient procedure for the purification to homogeneity, in addition to IGF I. of three forms of insulin-like growth factor II: IGF IIA (10-12 kDa), IGF IIB (the "classical" 7.5 kDa IGF II) and IGF IIC, identified as the IGF II variant of Jansen by fast-atom bombardment mass spectrometry. The procedure is based on ion-exchange chromatography and gel permeation chromatography on Biogel P10. As judged by specific radioimmunoassay methods for IGF I and IGF II, one of the most striking advantages of this process at this stage is the yield of IGF I not contaminated by 7.5 kDa IGF II. Isoelectric focusing or chromatofocusing, which require affinity chromatography to separate proteins from the polybuffers, are not necessary in this procedure. Final purification was directly achieved by preparative, followed by analytical high-performance liquid chromatography. The N-terminal sequence of peptide IGF IIB (39 amino acids) and peptide IGF I (29 amino acids) showed total homology with those previously described by Rinderknecht and Humbel [FEBS Lett., 89 (1978) 283]. The final yields of purified human IGF I and IGF IIB were 15 and 25 micrograms, respectively, from 11 of serum. All peptides interact with specific receptors on human lymphocytes and red blood cells, and are biologically active (stimulation of 35S uptake, increasing [3H]thymidine incorporation in human and chick embryo fibroblasts).
Straczek
J
J
Biochemistry Laboratory, Faculty of Medicine, University of Nancy I, Vandoeuvre, France.
Heulin
M H
MH
Chenut
A M
AM
Lasbennes
A
A
Geschier
C
C
Belleville
F
F
Nabet
P
P
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002642:Chick Embryo; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004247:DNA / Q000096:biosynthesis; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000302:isolation & purification / Q000494:pharmacology; D007335:Insulin-Like Growth Factor II / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D016339:Spectrometry, Mass, Fast Atom Bombardment
D000596:Amino Acids; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II; D004247:DNA