Kirsten J. van der Merwe

Mechanism for the stabilization in vivo of the aziridine precursor 2-(4-acetoxyphenyl)-2-chloro-N-methyl-ethylammonium chloride by serum proteins

Oral and intraperitoneal administration of 2-(4-acetoxyphenyl)-2-chloro-N-methyl-ethylammonium chloride (Compound A), an analogue of phenyl aziridine precursors that occur in the shrub Salsola tuberculatiformis Botsch, had a contraceptive effect on female Wistar rats with a concomitant decrease in total body, uterus, and every mass and an increase in abronal mass. Compound A elicited a Type II difference spectrum and inhibited the Type I deoxycorticosterone (DOC) induced difference spectrum of sheep adrenal cytochrome P450c11 in a manner similar to that of S2, a biologically active fraction isolated from S. tuberculatiformis. The effects of Compound A on the spectral properties of P450c11 were diminished with time in PBS. Electrospray mass spectrometry (ES-MS) indicated that the rate of cyclization of Compound A to the corresponding aziridine followed a time course similar to the attenuation of cytochrome P450c11 inhibition. It was concluded that the aziridine precursor. Compound A, rather than aziridine itself, was the inhibiting agent of sheep adrenal P450c11. Addition of sheep and rat plasma prevented the attenuation of the effect of Compound A on the spectral properties of cytochrome P450c11. Subsequent ES-MS analysis indicated that Compound A was stabilized in plasma by sex hormone binding globulin and corticosteroid binding globulin. These results suggest a mechanism whereby natural plant products, which are highly reactive and unstable in vitro, can be stabilized by binding to plasma proteins, and so remain biologically active in vivo.

Structure elucidation of fusarin C, a mutagen produced by Fusarium moniliforme

The assignment of structure (1) to fusarin C, a mutagen isolated from cultures of Fusarium moniliforme is based on a detailed study of its high-field 1H and 13C n.m.r. spectra and X-ray crystallography of the 8Z isomer of (1) which defined the substitution pattern and relative configuration of the 2-pyrrolidone moiety; nuclear Overhauser enhancement experiments indicate that the 2E,4E,6E,8E,10E polyene chromophore of (1) exists in solution as an equilibrium between two conformers with s-cis and s-trans topology of the C-5–C-6 single bond.

Metabolic activation and deactivation of fusarin C, a mutagen produced by Fusarium moniliforme

The metabolic activation and deactivation of fusarin C, a mutagen produced by Fusarium moniliforme strain MRC 826, was studied by the Salmonella typhimurium mutagenicity assay using tester strain TA 100. A microsomal monooxygenase, preferably induced by phenobarbitone (PB) activities the mutagen to its active mutagenic form. Deactivation of the mutagenic metabolite seems to occur through chemical binding to thiol groups and by enzymatic conjugation mediated by a cytosolic glutathione -S-transferase.

The chemical and enzymatic interaction of glutathione with the fungal metabolite, fusarin C

Glutathione (GSH) interacts both chemically and enzymatically with fusarin C, a mutagenic metabolite produced by Fusarium moniliforme. The chemical reaction, which is pH-dependent, results in the formation of both fusarin A and a compound that lacks the 2-pyrrolidone moiety thereby suggesting an interaction at the C-13-C-14 epoxide. Enzymatic interaction of fusarin C with GSH also appears to occur at this site as fusarins A and D, which lack the epoxide, do not serve as substrates for GSH-S-transferases. The interaction of GSH with fusarin C appears to be an important deactivation step which could explain the lack of carcinogenicity observed for fusarin C in rats.

Expression of human P450C17 as an export protein in saccharomyces cerevisiae

Cytochrome P450c17 (P450c17), together with cytochrome P450c21 (P450c21), plays an important role in progesterone metabolism in the mammalian adrenal cortex. Low levels of expression and the presence of other steroidogenic enzymes in adrenal cortex endoplasmic reticulum (ER) impedes purification and characterisation of wild type as well as mutant forms of the hemoprotein. Heterologous gene expression systems have previously been used successfully to express active P450c17. Heterologous expression can also be used for the preparation of anti-P450c17-IgG. For antibody production larger amounts of pure P450c17 peptide, rather than the active protein, is, however, desirable. If the expressed protein can be affinity tagged and secreted into the medium, isolation and purification will be facilitated. Saccharomyces cerevisiae, YPH259, was transformed with a modified YCplac111 yeast expression-secretion vector (pPRL2). The gene coding for a truncated human P450c17 (signal anchor sequence 1-18 was removed) was inserted, in reading frame, downstream from the leader sequence MF alpha. A histidine tag was incorporated at the C-terminus. The modified yeast expression vector was expressed in yeast, the secreted P450c17-peptide purified by affinity chromatography and identified by immunoblot analysis.

An Apparatus for the Concentration of Large Volumes of Dilute Protein Solutions to A Predetermined Volume

An apparatus was designed and manufactured that can be used for the concentration of large quantities of dilute protein solutions to a predetermined volume. The method is based on the osmotic transfer of water to a concentrated hydrophilic polymer (poly ethylene glycol, PEG) through a protein-stopping dialysis membrane and is a refinement of a method previously reported by van Oss. The apparatus is made of perspex. Concentration takes place through a commercially available dialysis tube and is aided by a 40% polyethylene glycol solution. Large volumes (5l) of dilute protein solution could be reduced to 50 ml at a rate of 30 ml per hour with no significant loss in biological activity.

A Novel Method for the Preparation of Substrate-Free Cytochrome P-45011β

Abstract A new method for the removal of the stabilizing substrate, deoxycorticosterone, from adrenal cytochrome P-45011β, has been developed. Dextran coated charcoal is used for the adsorption of the steroid and the adsorbed steroid is separated from the cytochrome P-450-preparation by low speed centrifugation. The substrate-free enzyme, obtained in this manner, has all the characteristic spectral properties of low-spin cytochrome P-45011β, and may be converted to the high-spin form by the addition of deoxycorticosterone. The dextran coated charcoal method has the following advantages over the previously used method of substrate removal. It does not require the addition of the cofactors for cytochrome P-450-dependant hydroxyla-tion of deoxycorticosterone, small amounts of enzyme may be prepared in a short time and the enzyme preparation is not diluted to any great extent during the process.