Partial purification of cytochrome P-450 from human normal granulocytes.

Cytochrome P-450 was partially purified from human normal granulocytes, using four different purification procedures. PEG-6000 and ammonium sulfate fractionation of human granulocyte post-mitochondrial supernatant (S1) fraction resulted in 3.9 and 2.25 fold purification of cytochrome P-450 respectively. On the other hand, granulocyte S1 fractions subjected to noctylamino sepharose 4B and DEAE cellulose column chromatography separately revealed about 11 fold purification of cytochrome P-450. When these fractions were submitted to SDS-polyacrylamide gel electrophoresis, PEG-6000 and ammonium sulfate precipitated fractions showed multiple protein bands whereas n-octylamino sepharose 4B and DEAE eluates were relatively pure showing one or few bands. There was no indication of the existence of multiple P-450 species in the partially purified human granulocyte cytochrome P-450.

Depression of mouse liver microsomal mixed function oxidase enzymes by adriamycin.

Levels of cytochrome P-450 and cytochrome b5, and activities of NADPH cytochrome c reductase and 7-ethoxycoumarin O-deethylase, were found to be significantly decreased in hepatic microsomes prepared from mice killed 24 h after administration of a single intraperitoneal (i.p.) dose of adriamycin (ADR, 5 mg/kg). In contrast, both ascorbate-induced lipid peroxidation and conjugated dienes were increased in the same preparations. In vitro addition of ADR (5 micrograms/ml) to hepatic microsomal preparations (1 mg/ml protein) from the control mice also led to a substantial decrease in the mixed function oxidase (MFO) enzymes. A characteristic spectral change with an absorption peak at 408 nm and trough at 422 nm was associated with this in vitro interaction. It is suggested that the loss of cytochrome P-450 and related MFO enzymes due to ADR treatment is related to the generation of free radicals and subsequent lipid peroxidation in the liver.

Substrate-induced spectral changes in human normal and chronic myeloid leukemic granulocytes.

Several drugs/chemicals were allowed to interact with the cytochrome P-450 dependent mixed function oxidase system in the postmitochrondrial supernatant fractions of Ficoll-Hypaque-separated granulocytes from human normal subjects and patients with chronic myeloid leukemia. The substrate-induced spectral changes were followed by recording the difference spectra. Compounds conventionally classified as type I and type II substrates, on addition to S1 fractions of both normal and leukemic granulocytes, caused spectral changes that were reverse to those reported for the rat liver microsomes. Aminopyrine, phenobarbital, and Tween 80 evoked a reverse type I spectral change with a peak at 420-430 nm and a trough at 380-400 nm, whereas aniline and pyridine induced a modified type I (a reverse type II) spectral change characterized by a peak at 408 nm and a trough at 421 nm. These changes were found to be quantitatively proportional to the amounts of substrate added. However, the magnitude of the peaks and troughs was considerably less in the S1 fraction of the leukemic granulocytes. Correspondingly, total heme content was significantly decreased in S1 fractions of CML granulocytes as compared to similar fractions of normal granulocytes.

Plasma membranes from normal and chronic myeloid leukemic granulocytes: isolation and two-dimensional polyacrylamide gel electrophoretic analysis.

Normal human granulocytes obtained by Ficoll-Hypaque sedimentation were subjected to mild hypotonic shock and disruption by shear. The homogenate was fractionated by differential centrifugation and equilibrium ultracentrifugation to yield a plasma membrane preparation constituting 1% of the total cellular protein and enriched fifteen- and six-fold in alkaline phosphatase and Mg2+-adenosine triphosphatase activities, respectively. Granulocytes obtained from patients with chronic myeloid leukemia (CML) were identically processed. The protein constituents of both the normal and CML granulocyte plasma membranes were resolved by two-dimensional polyacrylamide gel electrophoresis. Comparison of the stained gels revealed CML-associated quantitative changes in four out of the fifteen protein spots examined. Thus, this analysis has permitted identification of those protein moieties that deserve attention for further isolation and purification.

Mixed function oxidase enzymes in human normal granulocytes and chronic myeloid leukemia cells. I. Detection and estimation.

Subcellular fractions prepared from human normal granulocytes and leukemic cells from patients with chronic myeloid leukemia (CML), were examined for 7-ethoxycoumarin O-deethylase activity as well as cytochrome P-450 and cytochrome b5 contents. The average 7-ethoxycoumarin O-deethylase activity was found to be significantly increased in all the fractions of CML cells when compared to the corresponding fractions of the normal granulocytes. The CML cells also differed from the normal cells by exhibiting decreased levels of both cytochrome P-450 and cytochrome b5. Examination of the microsomal and cytosolic fractions of both normal granulocytes and CML cells showed distribution of 7-ethoxycoumarin O-deethylase, cytochrome P-450, and cytochrome b5 in both the fractions. Such distribution seems to be unique for human leukocytes. Solubilization of protein in the postmitochondrial (S1) fractions of both the cell types with sodium cholate in the presence of 20% glycerol and further fractionation with 10% polyethylene glycol 6000 (PEG-6000) yielded a partially purified preparation with enriched 7-ethoxycoumarin O-deethylase activity and cytochrome P-450 as well as cytochrome b5 contents.

Ethanol-inducible liver cytochrome P-450 in the rat: relative specificity for ethanol oxidation activity in vitro.

Ethanol induces in rat liver microsomes a form of cytochrome P-450 differing in substrate specificity from cytochrome P-450 species present in controls or in rats treated with phenobarbital or 3-methylcholanthrene. Its relative capacity to oxidize ethanol remained to be determined and was studied using highly purified cytochrome P-450 preparations and comparing their activity in a reconstituted microsomal ethanol oxidizing system (MEOS). On a molar basis, reconstituted MEOS activity was found to be highest when assayed with ethanol-inducible cytochrome P-450, intermediate with cytochrome P-450 from phenobarbital-treated rats, and lowest with cytochrome P-450 from controls or 3-methylcholanthrene-treated rats. A decrease in apparent Km for ethanol was also found to be associated with higher MEOS activity of ethanol-inducible cytochrome P-450. The induction by ethanol of a catalytically distinct form of cytochrome P-450, and the comparatively high ethanol oxidizing activity of this cytochrome P-450 species, are in accordance with the MEOS hypothesis.