No abstract

I n the sunflower (Helianthus annuus L.) cotyledons, two distinguishable thiolase activities have been identified: acetoacetyl CoA thiolase (AACT), specifically active during oxidation of short chain acetoacetyl CoA, and 3oxoacyl CoA thiolase (OACT), active towards short, medium, and long-chain acyl CoAs. The purpose of this research was to optimize the purification of the AACT expressed in bacteria. Escherischia coli (E. coli) with the full-length sunflower AACT cDNA cloned into the expression vector pBAD-His B was induced for production of the AACT using 0.2% arabinose. Optimizing the conditions for protein purification is often an extremely empirical process requiring several iterations of a basic protocol each with specific changes. This iterative process was simplified using the programmable Äkta chromatography system from Amersham Pharmacia (now GE Healthcare). Programming this system for automatic sample injection and variation of elution conditions allows for optimization of the protein purification protocol to be achieved overnight as opposed to taking a day for each iteration done manually. Once optimized, soluble AACT was purified from E. coli bacteria in one step using a His HiTrap HP column containing Ni-Sepharose resin in conjunction with the Äkta chromatography system. AACT activity was detected in the induced bacteria, whereas no activity was observed with the uninduced control. ( JALA 2006;11:85-7)

The number and type of glucuronic acid derivatives known to be formed in the body includes compounds which are hormones, metabolic intermediates or administered drugs containing hydroxy (Isselbacher and Axelrod, 1955), carboxyl (Billing and Lathe, 1956), amine (Axelrod, Inscoe and Tomkins, 1958) and sulphydryl groups (Clapp, 1956). All of these groups act as electron donating nucleophiles and the reaction leading to the formation of glucuronides by glucuronyl transferase with UDPGA is now well understood. Less is known about the hydrolysis of glucuronides by the~ glucuronidases present throughout the body especially in the gastro-intestinal tract, the urogenital system and in the liver. Abnormal~ glucuronidase activity has been associated with the neoplastic diseases (Goldberg et al, 1959), a decreased glucose tolerance (Hagenfeldt and Wahlberg, 1965), atherosclerotic disease (Kayahan, 1960) and may indicate the extent of lysosomal damage (Pagliaro et al, 1964). Correct evaluation of~-glucuronidase activity in mammalian tissues has been obscured by the presence of a number of components showing different substrate specificity. These fractions probably contain different enzymes each of which is associated with a particular sub-cellular structure.Those methods which are currently applied to the assay of~-glucuronidase activity in homogenised tissues or purified preparations have been re-examined. Media containing phenolphthalein glucuronide in an acetate buffer (Fishman, Springer and Brunetti, 1948)were shown to cause considerable changes in activity when the buffer ions were altered. The results from~-glucuroni dases prepared from patella vulgata, bovine liver and E. coli were shown to be unrelated and substrate dependant. For example, the mammalian enzyme was more active in buffers containing phosphate if phenolphthalein glucuronide was the substrate than when androsterone glucuronide was hydrolysed. Citrate was a better medium for the latter substrate except in the presence of the molluscan enzyme which then displayed only 50 % of the activity in acetate.The reported multiple pH optima for some glucuronidases (Wakabayashi and Fishman, 1961) may be due to changes in buffer ions over the pH range studied. Substrate inhibition of the mammalian and bacterial enzymes occurred at concentrations equivalent to the total glucuronides present in some biological fluids as well as those recommended for the assay.Although~-glucuronidases may be usefully employed as hydrolases when more drastic methods would destroy labile compounds, prolonged incubation for two or three days is often necessary. With all three types of enzyme, the hydrolysis equilibrium was reached when 72-78 % of steroid glucuronides had been split. This equilibrium was displaced by including compounds acting as acceptors for the glucuronyl radical. Selection of the appropriate enzyme, buffer and acceptor made it possible to increase the yields of freed aglycone, with much shorter incubation times, for example, to 94 % recovery of etiocholanolone from etioc...