Co-purification of mitochondrial HSP70 and mature protein disulfide isomerase with a functional rat kidney high-M(r) cysteine S-conjugate beta-lyase.

S-(1,1,2,2-Tetrafluoroethyl)-L-cysteine (TFEC, the cysteine S-conjugate of tetrafluoroethylene) is an example of a nephrotoxic, halogenated cysteine S-conjugate. Toxicity results in part from the cysteine S-conjugate beta-lyase(s)-catalyzed conversion of TFEC to a thioacylating fragment with the associated production of pyruvate and ammonia. In the present study, we have demonstrated that rat kidney homogenates contain at least three enzyme fractions that are capable of catalyzing a cysteine S-conjugate beta-lyase reaction with TFEC. One of these fractions contains a high-M(r) lyase. At least two proteins co-purify with this high-M(r) complex. N-Terminal analysis (15 cycles) revealed that the smaller species was mature protein disulfide isomerase (M(r) approximately 54,200) from which the 24 amino acid endoplasmic reticulum signal peptide had been removed. Internal amino acid sequencing (15 cycles) revealed that the larger species was mitochondrial HSP70 (mtHSP70; M(r) approximately 75,000). The present findings offer an explanation for the previous observation that mtHSP70 in kidney mitochondria is heavily thioacylated when rats are injected with TFEC (Bruschi et al., J Biol Chem 1993;268:23157-61).

Human aromatase in high yield and purity by perfusion chromatography and its characterization by difference spectroscopy and mass spectrometry.

Expression of human cytochrome P450 aromatase (CYP19A1, aromatase) was accomplished at a high level using a baculovirus expression system in an insect cell suspension culture. Using the relatively new chromatographic technique of perfusion chromatography, a very rapid procedure for purification of the protein from solubilized cells was developed. At extraordinary flow rates of between 3 and 9 column volumes per minute, all chromatographic procedures could be performed, including setup, equilibration, and column regeneration steps, in less than 2 h, not including brief dialysis periods. Total yields were 40-52% and resulted in preparations with specific content values of 17.1 nmol aromatase/mg protein. Final purified preparations showed virtually no typical P450 spectra under standard conditions, but displayed full activity with typical enzyme kinetic parameters. These unusual results suggest that standard methods of P450 measurement are inappropriate when applied to aromatase. The findings are fully consistent with those encountered previously for purified preparations from a human placental source and led us to a new aromatase quantification method based on ligand-induced difference spectroscopy. A new HPLC assay is described which rapidly separates heme and apoprotein while measuring total heme content. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry was employed with both glycosylated and deglycosylated forms of the final purified product to confirm its identity as a glycosylated cytochrome P450.

Subnanomolar quantification of caffeine's in vitro metabolites by stable isotope dilution gas chromatography-mass spectrometry.

A method for the quantification of subnanomolar levels of in vitro metabolites of caffeine by an isotope dilution gas chromatographic-mass spectrometric (GC-MS) assay has been developed and applied. Trideuteromethylated analogs of each primary metabolite were synthesized and added after incubations of caffeine with human liver microsomes high in cytochrome P4501A2. HPLC separation of the metabolites prior to GC-MS quantification allowed the isolation of theobromine and paraxanthine which coeluted by GC and enabled quantification over a larger dynamic range. Quantitative analysis was performed on the n-propylated derivatives by selected-ion monitoring of either the M+. ions for the dimethylxanthines or [M-C3H6]+. ions for 1,3,7-trimethyluric acid. For the least abundant metabolite (1,3,7-trimethyluric acid), the detection level on column was 200 pg. Replicate analyses exhibited intra- and inter-day variability of 4.2 and 7.9%, respectively. This assay has been successfully used in the quantification of caffeine's primary metabolites in more than 180 incubations, at varying substrate concentrations and with multiple enzyme sources.