Nerve growth factor (b-NGF), a neurotrophin required for the development and survival of specific neuronal populations, is translated as a prepro-protein in vivo. While the presequence mediates translocation into the endoplasmic reticulum, the function of the pro-peptide is so far unknown. As the pro-sequences of several proteins are known to promote folding of the mature part, the renaturation behaviour of recombinant human b-NGF pro-protein was compared to that of the mature form. Expression of rh-pro-NGF in Escherichia coli led to the formation of inclusion bodies (IBs). The presence of the covalently attached pro-sequence significantly increased the yield and rate of refolding with concomitant disulfide bond formation when compared to the in vitro refolding of mature NGF (rh-NGF). Physicochemical characterization revealed that rh-pro-NGF is a dimer. The pro-peptide could be removed by limited proteolysis with trypsin yielding biologically active, mature rh-NGF. Furthermore, rh-pro-NGF exhibited biological activity in the same concentration range as rh-NGF.

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Selenium‐containing glutathione peroxidase (EC 1.11.1.9) was purified 6000‐fold from bovine red blood cells to apparent homogeneity. Lipoxygenase (EC 1.13.11.12) was enriched 20‐fold from soybean acetone powder. Linoleic acid was peroxidized with lipoxygenase and then used as a substrate in the glutathione peroxidase reaction. Analogous experiments were conducted with synthetic 1,2‐dilinoleoyl‐l‐α‐glycerophosphocholine and with natural bovine heart cardiolipin. The peroxidized phospholipids were reactive with glutathione peroxidase only after enzymatic attack by phospholipase A2 (EC 3.1.1.4). This result implies that the membrane‐protective function of glutathione peroxidase includes preceeding phospholipase action and excludes a direct interaction of this enzyme with membrane‐bound lipid hydroperoxides.

Transgenic rabbits carrying gene constructs encoding human nerve growth factor beta (hNGF-L L) cDNA were generated. Expression of hNGF-L L mRNA was restricted to the mammary gland of lactating rabbits. Western Blot analysis revealed a polypeptide of 13.2 kDa in the milk of transgenic animals. hNGF-L L was purified from the milk by a two-step chromatographic procedure. Electrospray mass spectroscopy analysis of purified hNGF-L L depicted a molecular weight of 13 261 Da per subunit. The biological activity of the hNGF-L L was tested using PC12W2 cells and cultures of dorsal root ganglion neurons from chicken embryos. Crude defatted milk from transgenic animals and purified hNGF-L L demonstrated full biological activity when compared to commercial recombinant hNGF-L L.z 1999 Federation of European Biochemical Societies.