Midkine (MK), a retinoic acid (RA)-inducible gene product, produced in E. coli acts on neuronal and HL60 leukemia cells.

We have shown previously that (i) retinoic acid (RA), an anti-neoplastic agent, activates the midkine (MK) gene in mammalian embryonic carcinoma cells, and that (ii) the MK of 118 amino acids, purified from L cells, induces neurite outgrowth of mammalian embryonic brain cells. In this paper, we describe an unconventional strategy for the purification of a fully active MK from E. coli with a high yield. The MK was overproduced in E. coli as a glutathione S-transferase (GST) fusion protein. The MK fusion protein extracted from the bacterial inclusion bodies with guanidine-HCl was renatured, refolded slowly and cleaved by thrombin at the site where the GST links to the MK. The purified free MK, like RA, induced neurite outgrowth from central neurons of the mouse spinal cord, and suppressed the growth of human HL60 leukemia cells in vitro. Unlike RA, however, the MK did not induce granulocytic differentiation of HL60 cells. Furthermore, the MK supported the survival of an NGF-insensitive sensory neuron subpopulation(s) from chicken embryo dorsal root ganglion. Thus, the actions of the MK and leukemia inhibitory factor (LIF) are surprisingly similar. There is no sequence similarity between MK and LIF, however, and unlike MK, LIF production does not appear to be RA-inducible.

Differences in the Elution profiles of corticotropin-releasing activity in rat plasma and hypothalamic extracts following high performance liquid chromatography.

Extracts of rat plasma and hypothalami were fractionated by reversed-phase high performance liquid chromatography (HPLC) and the fractions were assayed in an in vitro bioassay for corticotropin-releasing activity. Corticotropin releasing factor (CRF) bioactivity was found in many fractions with retention times of 20-50 min. In contrast, material derived from the plasma of rats stressed by ether anaesthesia yielded only one peak of CRF bioactivity, with a retention time of 47-48 min. These results suggest that ACTH can be released from isolated pituitary cells by a number of components derived from HPLC of extracts of rat hypothalami. It is possible, however, that some of these are artefacts of the extraction process from post-mortem material. Because the CRF activity found in plasma was homogeneous by HPLC, plasma can be used to obtain physiologically active CRF.

Use of hydrophobic interaction methods in the isolation of proteins from endocrine and paraendocrine tissues and cells by high-performance liquid chromatography.

We have recently described the separation of a large number of polypeptide hormones, related peptides and some protein standards by hydrophobic interaction high-performance liquid chromatography (HPLC). This paper reports the practical application of these methods to the reproducible isolation and separation of components of a mixture of immunoreactive calcitonin-like proteins (less than 25 kD) synthesised and secreted by human tumour cells in vitro. Using hydrophobic interaction HPLC on ODS-silica for both preliminary bulk fractionation and subsequent analytical separation greater than 80% recoveries of small (ng) quantities of immunoreactive proteins were obtained from samples containing less than 100 mg total protein, and characteristic profiles of synthesised and secreted materials were established. Using a partially purified hypothalamic extract, containing a number of small proteins (12--25 kD), we have also examined the effects of varying chromatographic conditions in an attempt to modify the separations obtained with ODS-silica using an acid-saline-acetonitrile gradient elution system at ambient temperature, and achieve further resolution of its components. No useful selective effects were observed when temperature, organic modifier, gradient profile or hydrophobic stationary phase were altered. These techniques may not therefore be inherently capable of completely resolving all components of natural protein mixtures. They do, however, offer an adjunct to and in certain cases a substitute for conventional methods of protein separation.