Purification and biochemical characterisation of human and murine stem cell inhibitors (SCI).
We have recently characterised an inhibitor of haemopoietic stem cell proliferation (SCI/MIP-1 alpha) and report here on its purification and initial biological and biochemical characterisation. The activity can be detected by direct addition to the CFU-A stem cell assay and this simple test for inhibitory activity has greatly facilitated the purification of the molecule. The purification involves a combination of Mono Q ion exchange chromatography, heparin-sepharose affinity chromatography and Blue Sepharose affinity chromatography. The purified stem cell inhibitor is an 8 kD peptide which is identical to the previously described peptide macrophage inflammatory protein 1 alpha. The peptide has a natural tendency to form large self-aggregates and appears, in physiological buffers, to have a native molecular weight of around 90 kD. SCI is a heat stable, protease sensitive protein which is half maximally active at between 10 and 25 pM in the CFU-A assay. The self-aggregates can be disrupted by dilute solutions of acetic acid and it appears that disruption increases the specific activity of SCI preparations. We also report the characterisation of the human homologue of the stem cell inhibitor (human SCI/MIP-1 alpha) which is 74% identical to murine MIP-1 alpha and which shares all the above features of the murine inhibitor.
Graham
G J
GJ
CRC Beatson Laboratories, Beatson Institute for Cancer Research, Bearsden, Glasgow.
Freshney
M G
MG
Donaldson
D
D
Pragnell
I B
IB
D000818:Animals; D001483:Base Sequence; D054405:Chemokine CCL3; D054407:Chemokine CCL4; D003001:Cloning, Molecular; D003114:Colony-Forming Units Assay; D016207:Cytokines / Q000302:isolation & purification* / Q000378:metabolism; D004247:DNA / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D006801:Humans; D019402:Macrophage Inflammatory Proteins; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015846:Monokines / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D054405:Chemokine CCL3; D054407:Chemokine CCL4; D016207:Cytokines; D019402:Macrophage Inflammatory Proteins; D015846:Monokines; D011994:Recombinant Proteins; D004247:DNA