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Relationships between activities of delta-aminolevulinate synthase and heme oxygenase, respectively the rate-limiting enzymes of heme biosynthesis and degradation, have been studied in chick embryo liver cell cultures following exposure of the cultures to glutethimide and iron, a combination known to produce a synergistic induction of both enzymes. In time-course experiments, synergistic induction of heme oxygenase activity by glutethimide and iron preceded that of delta-aminolevulinate synthase by 4 h. Effects of selective inhibitors of both heme synthesis and degradation have also been studied with respect to effects on delta-aminolevulinate synthase and heme oxygenase activities. The synergistic induction of heme oxygenase by glutethimide and iron appears to be dependent upon cellular heme synthesis because addition of inhibitors of heme biosynthesis, 4,6-dioxoheptanoic acid or N-methyl-mesoporphyrin abolishes this synergistic induction. Exposure of cultures to tin-mesoporphyrin, a potent inhibitor of heme oxygenase, prevented the synergistic induction of delta-aminolevulinate synthase produced by glutethimide and iron, or, when added after induction was already established, promptly halted any further induction. These results suggest that the level of activity of heme oxygenase can reciprocally modulate intracellular heme levels and thus activity of delta-aminolevulinate synthase.

Monospecific polyclonal rabbit antibodies to a purified form of haem oxygenase of chick liver, showing sequence similarity to mammalian haem oxygenase-1, were raised and used to study characteristics of the oxygenase. The antibodies inhibited activity of the purified oxygenase, but not other enzyme components (NADPH:cytochrome reductase and biliverdin reductase) of the standard assay mixture of haem oxygenase. In addition, the antibodies inhibited activity of haem oxygenase in microsomes (microsomal fractions) from Cd(2+)-treated chick liver, spleen, testis and brain. Western (immuno-) blots of microsomal proteins of selected organs from chick, rat and man, and homogenates of chick-embryo liver-cell cultures, probed with the antibodies, showed a major protein with a molecular mass of 33-34 kDa and a lower-molecular-mass protein (28-29 kDa) of variable intensity. Studies with trypsin and selected proteinase inhibitors established that the smaller peptide was a proteolytic product of the larger. Treatment of chick-embryo liver-cell cultures with CdCl2, a potent inducer of haem oxygenase, increased the degree of proteinase-mediated cleavage of the 33 kDa protein to the lower-molecular-mass form. These results indicate that, under at least some conditions, such cultures should be homogenized in the presence of trypsin inhibitor to prevent proteolytic degradation of the enzyme and allow maximal expression of haem oxygenase activity. The antibodies also reacted with haem oxygenase from spleen, testis and brain of both chicks and rats, and the spleen of humans. A method for quantifying the amount of haem oxygenase protein was developed with use of slot-blots and laser densitometry; linearity was observed from 0 to 5 ng of haem oxygenase protein per slot, and the method was applied to sonicated cultured chick-embryo liver cells treated with Cd2+ (0.3 mM) or iron plus glutethimide. In both cases, increases in enzyme activity were of similar magnitude to increases in amounts of enzyme protein. Approximate amounts of haem oxygenase protein in microsomes of several organs from intact animals could also be estimated by the use of slot-blot-laser densitometry, and the amounts measured were increased by the addition of purified haem oxygenase to the microsomal preparations. Results of these studies indicated that haem oxygenase-1 could be detected in microsomes from all chick or rat organs studied, including testis and brain.(ABSTRACT TRUNCATED AT 400 WORDS)

A cDNA from a chick liver library that encodes for haem oxygenase has been cloned and sequenced. Positive clones were identified with monospecific antibodies to the purified enzyme from chick liver and a cDNA of rat haem oxygenase-1. The length of the cDNA is 1258 bases. An open reading frame of 888 bases was identified by comparison of nucleotide and amino acid sequences with those previously identified for haem oxygenase of mammalian or avian origin. The protein corresponding to this fragment of DNA is composed of 296 amino acid residues and has a molecular mass of 33,509 Da, which is similar to that previously estimated for haem oxygenase purified from chick liver. Unequivocal identification of this clone as that complementary to haem oxygenase was provided by (a) comparison of amino acid compositions and partial sequences with those previously established for the purified enzyme, (b) comparison with nucleotide and amino acid sequences for haem oxygenase from rat and human sources and (c) expression in Escherichia coli with production of high levels of mRNA, protein and haem oxygenase activity after exposure of the transfected bacteria to isopropyl beta-D-thiogalactopyranoside (IPTG). Overall, the similarity of chick haem oxygenase to rat and human haem oxygenase (nucleotides 66% and amino acids 62%) is moderately high. The region between proline-129 and alanine-157 is identical in all three enzymes, including histidine-135, which is proposed to play a key role in binding the substrate haem at the active centre of the enzyme. Northern blots also show that treatment of chicks with CdCl2, a potent inducer of haem oxygenase, results in increases in 1.65-1.70 kb mRNA, which hybridizes selectively to the full-length cDNA or to a synthetic 24-base oligonucleotide with sequence identical to that of a portion of the haem oxygenase cDNA. These results suggest that Cd-dependent induction of haem oxygenase is due to increased transcription of the gene or stabilization of its message.

Heme oxygenase, the rate controlling enzyme for heme catabolism, is inducible by a variety of treatments, some of which induce by a heme-dependent mechanism and others by a heme-independent mechanism. This work shows that, in cultured chick embryo liver cells, synergistic induction of heme oxygenase by iron, added with the phenobarbital-like drug, glutethimide was heme-dependent. Addition of an inhibitor of heme biosynthesis abolished the synergistic induction of heme oxygenase providing evidence for the heme-dependent mechanism of induction. Glutethimide and iron appeared to induce at the transcriptional level since both heme oxygenase mRNA and protein levels correlate with changes in heme oxygenase activity.