Production, purification, and crystallization of human interleukin-1 beta converting enzyme derived from an Escherichia coli expression system.

Interleukin-1 beta converting enzyme (ICE) is a cysteine protease that catalyzes the conversion of the inactive precursor form of IL-1 beta to an active mature form. The mature form of IL-1 beta is involved in mediating inflammatory responses and in the progression of autoimmune diseases. We recently reported on the production of active human ICE in insect cells using the baculovirus expression system (Wang XM et al., 1994, Gene 145:273-277). Because the levels of expression achieved with this system were limiting for the purpose of performing detailed biochemical and biophysical studies, we examined the production of ICE in Escherichia coli. By using a tac promoter-based expression system and fusion to thioredoxin we were able to recover high levels of active ICE protein. The expressed protein, which was distributed between the soluble and insoluble fractions, was purified to homogeneity from both fractions using a combination of classical and affinity chromatography. Comparisons of ICE derived from both fractions indicated that they were comparable in their specific activities, subunit composition, and sensitivities to specific ICE inhibitors. The combined yields of ICE obtained from the soluble and insoluble fractions was close to 1 mg/L of induced culture. Recombinant human ICE was crystallized in the presence of a specific ICE inhibitor in a form suitable for X-ray crystallographic analysis. This readily available source of ICE will facilitate the further characterization of this novel and important protease.

Purification and characterization of human biliverdin reductase.

Conversion of biliverdin to bilirubin is catalyzed by the cytosolic enzyme biliverdin reductase. We have purified and characterized the human liver reductase and find it to differ extensively from the previously described rat enzyme (H. Fakhrai and M. D. Maines, 1992, J. Biol. Chem. 267, 4023-4029) in its primary structure/composition, yet share kinetic properties. The human enzyme is substantially larger than the rat enzyme (approximately 41,000-42,000 versus 33,000-34,000), is dual cofactor and dual pH dependent, and requires free-SH groups. At pH 6.0-7.0 the NADH was the more effective cofactor, whereas at pH 8.5-8.75 NADPH was the preferred cofactor. The activity was inhibited by-SH reagents, 5'-dithiobis(2-nitrobenzoic acid) and p-chloromercuribenzoic acid, and protected from these reagents by cofactors and substrate. On two-dimensional electrophoresis, the purified protein resolved into four distinct isoelectric zones (pI 6.03, 5.83, 5.68, and 5.55) and two molecular weight forms (approximately 40,700 and approximately 39,600). Variants with similar pI values were detected in the purified human kidney reductase, although their relative tissue abundance varied. The tryptic map, amino acid composition, and sequence of NH2 terminus and four tryptic peptides of human reductase were compared with those of the rat. The HPLC profile and amino acid composition of the human and the rat enzymes differed vastly, and two tryptic peptides were present in the human that could not be detected in the predicted amino acid sequence of the rat enzyme. At the same time, the first 21 amino acids of the NH2 terminus of rat and human, except for the substitution of glutamic acid in human for lysine (amino acid 4) in the rat, were found identical and two peptides with 78-87% similarity to the rat reductase were found in the human reductase. Of the seven cysteine residues present in the human, four or five were titratable with 5'-dithiobis(2-nitrobenzoic acid).

Differential regulation of heme oxygenase isozymes by Sn- and Zn-protoporphyrins: possible relevance to suppression of hyperbilirubinemia.

Synthetic metalloporphyrins decrease heme oxygenase (HO)-dependent bilirubin formation. Presently, the effects in vivo and in vitro of Sn- and Zn-protoporphyrins on HO-1 (HSP-32) and HO-2 at the protein and transcript levels were examined. Western blot analysis of HO-2 in testes microsomes of Sn-protoporphyrin-treated rats revealed a dramatic disruption of the integrity of the HO-2 protein. Similar observations were made with the liver and adrenal HO-2 and the NADPH-cytochrome P-450 reductase of treated rats. Northern blot analysis, however, suggested unaltered tissue levels of HO-2 transcripts (approximately 1.9 and approximately 1.3 kb). The HO-1 protein integrity in organs of treated rats was less dramatically affected by the metalloporphyrin and an increase in its 1.8 kb mRNA level in the testes was detected. Zn-protoporphyrin also increased HO-1 mRNA level in the testes, but did not affect HO-2 protein integrity. In in vitro studies with purified HO-1 and HO-2, both Sn- and Zn-protoporphyrins were equally inhibitory to HO-1 activity; Sn-protoporphyrin, however, was by far more inhibitory to HO-2-dependent activity than to that of HO-1. Together, these findings and the fact that HO-2 under normal conditions is the predominant form of the enzyme in most organs suggest that loss of HO-2 protein integrity may to a significant degree account for suppression of bilirubin formation by Sn-protoporphyrin. These in turn may reflect differences between HO-1 and HO-2, both at the transcriptional level with HO-2 being noninducible, and in structure/composition of the isozymes, with HO-2 being more labile.

Comparative effects of tin- and zinc-protoporphyrin on steroidogenesis: tin-protoporphyrin is a potent inhibitor of cytochrome P-450-dependent activities in the rat adrenals.

Synthetic metalloporphyins inhibit formation of bilirubin by the heme oxygenase system, an ability that is of considerable experimental and clinical interest for suppression of jaundice in the newborn. The present investigation compares the consequences of treatment with Sn- and Zn-protoporphyrin on hemoprotein-dependent enzymes of the rat adrenals and corticosterone production and defines Sn-protoporphyrin as a potent toxin to adrenal functions. Treatment of rats with Sn-protoporphyrin (two doses of 50 mumols/kg, in 7 d) resulted in a marked reduction of 30-40% in cytochrome P-450-dependent adrenal microsomal 21 alpha-hydroxylase and mitochondrial 11 beta-hydroxylase activities. In the serum, the levels of corticosterone were reduced to about 70% of the control value. In addition, the mitochondrial cytochrome P-450SCC activity was decreased by about 50%. This decrease, however, could not be attributed to a reduced total heme level or an accelerated heme degradatory activity. Disruption by Sn-protoporphyrin of adrenal hemoprotein-dependent functions was not restricted to steroidogenic activities and encompassed drug metabolism activity of the organ; benzo(a)pyrene hydroxylase activity of both the microsomal and the mitochondrial fractions, as well as the microsomal NADPH-cytochrome P-450 reductase activity, were significantly reduced. Zn-protoporphyrin did not cause significant alterations in the above measured parameters although it too was effective in inhibiting the hepatic microsomal heme oxygenase activity. In light of the presently defined adverse effects of Sn-protoporphyrin on adrenal steroidogenesis, we suggest Zn-protoporphyrin is the agent of choice for potential use in treatment of hyperbilirubinemia in humans.

Tin-protoporphyrin: a potent inhibitor of hemoprotein-dependent steroidogenesis in rat adrenals and testes.

The present investigation provides evidence of the ability of Sn-protoporphyrin to cause striking alterations in adrenal and testicular cytochrome P-450-dependent steroidogenesis and defines the potential of this metalloporphyrin to serve as a cellular toxin. Sn-protoporphyrin is currently used on an experimental basis for treatment of hyperbilirubinemias in humans, including newborn infants. Specifically, in the adrenals of rats treated with a moderate regimen of Sn-protoporphyrin (two doses of 50 mumol/kg, s.c.), marked decreases of 60 to 70% in the microsomal 21 alpha-hydroxylase and the mitochondrial 11 beta-hydroxylase activities were observed after 7 days. Concomitant with these decreases was a significant depression in the adrenal mitochondrial cytochrome P-450 content and a notable reduction (approximately 30%) in serum corticosterone levels. Similarly, in the testes, significant decreases in the microsomal and mitochondrial cytochrome P-450 contents and the microsomal 17 alpha-hydroxylase activity were observed. Serum testosterone level, however, was not decreased, reflecting an absence of decrease in side chain cleavage activity. Metalloporphyrin caused a striking decrease of 65 to 80% in the microsomal heme oxygenase activity in the testes and the adrenals, as well as significant reductions in NADPH-cytochrome P-450 reductase activity of the organs. The decrease in heme oxygenase activity, however, as suggested by Western immunoblotting, apparently resulted, to a large extent, from the loss of enzyme protein integrity rather than a competitive inhibition of activity. At the transcript level, Northern blot analysis using a full length rat testis cDNA probe for heme oxygenase-2 mRNA indicated that Sn-protoporphyrin treatment did not decrease the amount of message for either of the heme oxygenase-2 transcripts (1.3 and 1.9 Kb).(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclosporin-mediated depression of luteinizing hormone receptors and heme biosynthesis in rat testes: a possible mechanism for decrease in serum testosterone.

The toxic side-effects of the immunosuppressive drug cyclosporin (CsA) include testicular dysfunction and a decline in circulating testosterone. However, mechanisms for the consistently observed CsA-mediated depression of serum testosterone levels are unclear because of conflicting reports concerning circulating gonadotropin levels and incomplete studies of intratesticular steroidogenesis. To elucidate these mechanisms, endocrine-regulated testicular steroidogenesis and heme metabolic parameters were studied in male rats given sc injections of either 25 or 40 mg/kg.day CsA for 6 days and then killed on the seventh day. Consistent with earlier reports, CsA treatment dramatically suppressed serum testosterone levels (less than 20% of control at both CsA doses). Additionally, the intratesticular testosterone content declined with the higher CsA dose. Serum LH and FSH levels were elevated up to 2- to 4-fold after the higher CsA treatment regimen. Measurement of decreases in testicular receptors for LH revealed for the first time that CsA treatment significantly reduced the ability of the testes to respond to normal or elevated circulating levels of LH. In animals receiving higher dose of the drug, cytochrome P-450-dependent mitochondrial cholesterol side-chain cleavage activity, which is the rate-limiting step in steroidogenesis, was markedly reduced to a mere 30% of the control value. Additionally, the activity of the microsomal cytochrome P-450-dependent 17 alpha-hydroxylase was decreased to less than half of the control value. Biotransformation of the prototype drug, benzo(a)pyrene, as well as microsomal cytochrome P450 levels declined significantly after the higher CsA dose, suggesting that CsA has an adverse affect on testicular cytochromes P-450 in general. In addition, CsA treatment altered heme metabolic parameters; significant increases in the activity of uroporphyrinogen-I synthetase and total porphyrin content were noted. Conversely, the activity of ferrochelatase, the enzyme that incorporates iron into porphyrin to form heme molecule, decreased significantly, as did the total heme levels. The latter was reduced to only 61% of control values. The findings suggest the likelihood that the observed inhibition of heme formation may contribute substantially to the reduced levels of microsomal cytochromes P-450 and steroidogenic activities that depend on them. Taken collectively, these data suggest a plausible mechanism by which CsA may induce testicular dysfunction; as the result of a combination of reduction in the number of LH receptors and a suppression of heme formation, the hemoprotein-dependent steroidogenic enzymes activities are compromised, leading to an impairment of normal testicular function.

Heterogeneity of haem oxygenase 1 and 2 isoenzymes. Rat and primate transcripts for isoenzyme 2 differ in number and size.

In Cebus apella monkey, as with other mammalian species tested to date, two different forms of haem oxygenase, HO-1 and HO-2, are detected. With the use of cDNA fragment corresponding to HO-1 nucleotides +71 to +833, blot hybridization of RNA revealed the presence of only one HO-1 mRNA of approx. 1.8 kb in both rat and monkey liver, kidney and brain. With the use of a full-length HO-2 DNA probe, blot hybridization of RNA isolated from the same rat organs revealed the presence of two HO-2 homologous transcripts of approx. 1.3 kb and approx. 1.9 kb. The same probe detected only one message of approx. 1.7 kb in monkey organs. The rat 1.3 kb mRNA has been previously shown [Rotenberg & Maines (1990) J. Biol. Chem. 265, 7501-7506] to encode HO-2 (36 kDa). The monkey 1.7 kb mRNA and the rat 1.3 kb mRNA encode proteins with similar molecular masses and immunochemical properties as indicated by Western-immunoblotting analysis. In rat organs the relative abundance of the two mRNAs differed as follows: in the liver the 1.3 kb mRNA was by far the most abundant form; in the brain equal amounts of the two mRNAs were detected, whereas in the kidney the 1.3 kb mRNA was somewhat more abundant. The protein encoded by the 1.8 kb HO-1 mRNA in the monkey did not exhibit immunochemical reactivity with antibody to rat HO-1 in Western blotting and direct e.l.i.s.a. analysis. The data suggest that, at the primary structural level, both HO-1 and HO-2 share extensive base sequence similarity in the rat and the Cebus apella monkey. The HO-1 protein, however, appears to undergo differential post-translational and/or conformational modifications in the two species, whereas the secondary structure of HO-2 protein and antigenic epitopes are conserved among the two mammalian species.

Microheterogeneity of biliverdin reductase in rat liver and spleen: selective suppression of enzyme variants in liver by bromobenzene.

Recently we have reported the detection of multiple net-charge and molecular mass variants of biliverdin reductase in the rat liver. We now report an apparent selective change in the electrophoretic profile of the reductase variants in the liver by in vivo bromobenzene treatment (2 mmol/kg, sc, 24 h). Using two-dimensional electrophoresis and isoelectric focusing, one molecular mass species of the reductase (Mr 30,400) appeared to be selectively suppressed by bromobenzene treatment. This molecular mass species was the main component of two isoelectric focusing bands with pI6.23 and 5.91. The effect in vivo of bromobenzene could not be duplicated by in vitro experiments involving treatment of purified enzyme with bromobenzene in the presence of a NADPH-dependent microsomal drug metabolizing system. The phenomenon of multiplicity of the reductase was not limited to the liver. Multiplicity of the enzyme was detected also in the spleen; however, the pattern of composition of the reductase variants vastly differed from that of the liver. In the spleen, variants with pI 5.76, 5.61, and 5.48 were the prevalent forms; the variant with pI 6.23 was absent, and pI 5.91 was present in a minute amount. Further, bromobenzene did not affect the composition pattern of net-charge variants in this organ. Also, the splenic biliverdin reductase activity was refractory to in vivo bromobenzene treatment, whereas the liver reductase activity with both NADH and NADPH was altered by the treatment. The possible significance of the presence of multiple variants of biliverdin reductase and the change in their composition caused by bromobenzene is discussed.

Multiple forms of biliverdin reductase: modification of the pattern of expression in rat liver by bromobenzene.

Rat liver biliverdin reductase was purified from control and bromobenzene-treated rats and was designated as C-BVR-T and B-BVR-T, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the existence of two molecular weight variants (30,100 and 29,800) in C-BVR-T but only one form (30,100) in B-BVR-T. Western immunoblotting confirmed that both molecular weight variants were biliverdin reductase. Nondenaturing electrophoresis separated C-BVR-T and B-BVR-T preparations into groups of four variants, designated as BVR ND1 to ND4. However, the C-BVR-T preparation contained three major forms (BVR ND1, ND2, and ND3) while the B-BVR-T preparation contained two major forms (BVR ND2 and ND3). In vitro treatment of biliverdin reductase preparations with either bromobenzene or dithiothreitol did not interconvert the variants of the enzyme. QAE-Sepharose anion-exchange chromatography was used to isolate the ND2 and ND3 variants for physiochemical analysis. The amino acid composition of the variants was rather similar except for their Tyr content. Also, the peptide maps were similar except for a series of moderately early chromatographic peaks. These findings implied secondary modifications to the protein rather than substantial differences in primary structure. The pH-dependent cofactor requirements for enzyme activity were examined. Both variants exhibited 2 pH optima that were cofactor dependent; maximum activity with NADPH and NADH was observed at pH 8.5 and 6.7, respectively. However, both variants exhibited a higher catalytic rate with NADH than with NADPH at their pH optima. Furthermore, BVR ND3 exhibited a higher catalytic rate than BVR ND2 with either cofactor throughout the pH range 6.5-9.

Detection of 10 variants of biliverdin reductase in rat liver by two-dimensional gel electrophoresis.

We have identified and characterized multiple forms of biliverdin reductase (BVR) in control rat liver cytosol. Two-dimensional electrophoresis of the purified BVR resolved a minimum of 10 discrete protein zones. All 10 proteins were BVR as judged by immunological cross-reactivity toward rabbit anti-rat BVR. Based on the isoelectric focusing pattern of separation, the BVR variants could be organized into five net-charge groups designated as BVR-IEF1 to BVR-IEF5 and three molecular mass groups designated as BVR-MW1-BVR-MW3, respectively. The pI values of the net-charge groups were: BVR-IEF1, 6.23; IEF2, 5.91; IEF3, 5.76; IEF4, 5.61; IEF5, 5.48. The Mr values of the molecular mass groups were: BVR-MW1, 30,400; MW2, 30,700; MW3, 31,400. Single dimension slab gel isoelectric focusing offered greater resolution of the net charge variants, and BVR-IEF3 was further resolved into two variants, IEF3a and IEF3b, with pIs of 5.77 and 5.75, respectively. The six net-charge variants also resolved on a preparative chromatofocusing column and were designated as BVR-CF1-BVR-CF6. The pH values of the peak fractions were: BVR-CF1, 6.91; CF2, 6.33; CF3, 6.03; CF4, 5.82; CF5, 5.45; CF6, 5.27. Correspondence between the isoelectric focusing net-charge variants and the chromatofocusing net-charge variants was established. The Mr and net-charge variants did not represent partially degraded forms of biliverdin reductase produced during purification since the pattern of resolution of variants on slab gel isoelectric focusing or two-dimensional electrophoresis did not change by purifying the proteins in the presence of protease inhibitors and 5 mM EDTA. BVR-CF2 and BVR-CF4 were purified and examined for pH-dependent cofactor requirements for activity. Both net-charge variants and two pH optima that were cofactor-dependent; maximum activity with NADPH, however, was at pH 8.5 and with NADH at pH 6.7. With both variants, however, a higher catalytic rate was observed with NADH than with NADPH at their respective pH optima. Furthermore, BVR-CF2 exhibited a higher catalytic rate than did BVR-CF4 with either cofactor throughout the pH range of 5-9.

Multiplicity of heme oxygenase isozymes. HO-1 and HO-2 are different molecular species in rat and rabbit.

We report on the detection and characterization of two forms of heme oxygenase in rabbit tissues and provide data suggesting that heme oxygenases in rat and rabbit are not identical and constitute a group of heterogenous proteins. Certain molecular properties, however, are shared by the isozymes in rat and rabbit; the predominant form of the enzyme in control liver and testis is HO-2, in the liver HO-1 is the inducible form, and in the brain HO-1 is not detectable. HO-1 was purified from liver of rabbits treated with bromobenzene to near homogeneity with a specific activity of 8,270 nmol of bilirubin/mg/h and compared with a homogenous preparation of rat HO-1 with a specific activity of 6,220, also obtained from bromobenzene-treated animals. Rat and rabbit HO-1, on sodium dodecyl sulfate-polyacrylamide gel, had molecular weights of 30,000 and 30,700, respectively. Rabbit HO-2 was partially purified from testis to a specific activity of 386 nmol of bilirubin/mg/h and compared with a purified preparation of rat testis HO-2 with a specific activity of 5,700. Using Western immunoblotting, rabbit HO-2 displayed intense cross-reactivity with antibody raised in rabbit to sodium dodecyl sulfate-denatured rat HO-2, and had a substantially larger molecular weight than the rat HO-2 (42,000 versus 36,000). Rabbit HO-1 did not cross-react with antibody to rat HO-1 which was also raised in rabbit. Unlike the rat enzymes, rabbit HO-1 and HO-2 did not differ in thermolability. It is speculated that HO-1 in rat and rabbit, and possibly HO-2, have evolved from divergent evolution of a common ancestral gene(s).

Detection of two heme oxygenase isoforms in the human testis.

This study shows heme oxygenase multiplicity is common to rat and human tissues. The isozymes in man and rat, however, are heterogenous proteins that share certain characteristics. Two forms of heme oxygenase, HO-1 and HO-2, were identified in human testis. HO-2 form was the prevalent form. Human and rat HO-1 differed in chromatographic behavior and molecular weight; human HO-1 was a larger molecule (35,400 vs 30,000). The two forms, however, were similar in that immunochemically human HO-1 exhibited reactivity toward antibody to rat HO-1. Human and rat HO-2 also were dissimilar in chromatographic behavior and showed only a weak immunological cross-reactivity. Human and rat HO-1 were essentially the same size. As in rat organs, the microsomal cytochrome P-450 content in human testis was reciprocal to heme oxygenase activity.

Characterization of glutathione S-transferases in rat kidney. Alteration of composition by cis-platinum.

We have developed chromatographic and mathematical protocols that allowed the high resolution of glutathione S-transferase (GST) subunits, and the identification of a previously unresolved GST monomer in rat kidney cytosol; the monomer was identified tentatively as subunit 6. Also, an aberrant form of GST 7-7 dimer appeared to be present in the kidney. This development was utilized to illustrate the response of rat kidney GST following cis-platinum treatment in vivo. Rat kidney cytosol was separated into three 'affinity families' of GST activity after elution from a GSH-agarose matrix. The affinity peaks were characterized by quantitative differences in their subunit and dimeric compositions as determined by subsequent chromatography on a cation-exchange matrix and specific activity towards substrates. By use of these criteria, the major GST dimers of affinity peaks were tentatively identified. The major GST dimers in peak I were GST 1-1 and 1-2, in affinity peak II it was GST 2-2, and in peak III they were GST 3-3 and 7-7. GST 3-6 and/or 4-6, which have not been previously resolved in kidney cytosol, were also present in peak II. Alterations in the kidney cytosolic GST composition of male rats were detected subsequent to the administration of cis-platinum (7.0 mg/kg subcutaneously, 6 days). This treatment caused a pronounced alteration in the GST profile, and the pattern of alteration was markedly different from that reported for other chemicals in the kidney or in the liver. In general, the cellular contents of the GSTs of the Alpha and the Mu classes decreased and increased respectively. It is postulated that the decrease in the Alpha class of GSTs by cis-platinum treatment may be related to renal cortical damage and the loss of GSTs in the urine. The increase in the Mu class of GSTs could potentially stem from a lowered serum concentration of testosterone; the latter is a known effect of cis-platinum treatment.

Resolution of the rat brain heme oxygenase activity: absence of a detectable amount of the inducible form (HO-1).

In the present study we report on the detection of a distinct pattern of heme oxygenase isoform composition in the rat brain. In this organ only the noninducible form of heme oxygenase, HO-2, could be clearly detected. This pattern of composition distinguishes the brain from other organs tested to date, namely the liver, testis, and spleen. The rat brain microsomal fraction displayed a rather impressive rate of heme oxygenase activity. This fraction also exhibited a rate of NADPH-cytochrome P-450 reductase activity that was sufficient to fully support the oxygenase activity. The brain microsomal fraction was solubilized and subjected to ion-exchange chromatography on DEAE-Sephacel. The chromatographic elution pattern of heme oxygenase activity was compared with those of the liver and testis. In the brain only one peak of heme oxygenase activity was detected. The peak exhibited an elution profile similar to that of HO-2 of the liver and the testis. The presence of an activity peak was not detected in the elution profile at the region where the inducible isoform of heme oxygenase, HO-1, was expected. Cross-reactivity was observed between the solubilized brain microsomal fraction and antiserum to the testis HO-2 when subjected to Ouchterlony double diffusion immunoanalysis. A reaction was not observed when antiserum to liver HO-1 was employed. The presence of HO-2 in the brain microsomal preparation was also established by Western immunoblotting analysis. A protein having a mobility that was identical to the purified testicular HO-2 (Mr 36,000) was present in the brain microsomal preparation when probed with antiserum to HO-2. However, our attempts to demonstrate the presence of HO-1 in the brain microsomal preparation by a similar technique, but using antiserum to HO-1, were not successful. It is proposed that HO-2 is responsible for the bulk, if not all, of the brain microsomal heme oxygenase activity. It is further proposed that tissue-specific regulatory mechanisms are responsible for both the refractory response of the brain heme oxygenase to known metallic inducers and the absence of a detectable amount of the HO-1 isoform.

Regulation of the activity of heme degradative enzymes in K562 erythroleukemic cells: induction by thymidine.

Heme oxygenase is rate-limiting in the heme degradative pathway, and its activity is induced by a host of chemicals. In K562 human erythroleukemic cells, heme oxygenase activity was not increased by exposure to potent inducers, such as cobalt chloride, bromobenzene, and heme. Indeed heme treatment severely suppressed the enzyme activity, and at 18 h the activity measured less than 5% of the control. Heme and cobalt chloride did not inhibit activities of NADPH-cytochrome c (P-450) reductase and biliverdin reductase to a marked degree. In contrast, treatment of cells with thymidine/hypoxanthine alone, or in combination with cobalt chloride, caused an increase in the activity of three enzymes of heme degradation. It is suggested that with thymidine, which is a committing inducer of hemoglobin synthesis, the induction of activity of the three enzymes of the heme degradation pathway is coupled with cell differentiation. On the other hand, in the case of heme, a noncommitting inducer of hemoglobin synthesis, induction of hemoglobin synthesis and increase in heme degradation activity may be independent.

Characterization of two heme oxygenase isoforms in rat spleen: comparison with the hematin-induced and constitutive isoforms of the liver.

Two isoforms of heme oxygenase, designated as HO-1 and HO-2, were identified in rat spleen. The most abundant form was HO-1, wherein a relative ratio of about 5:1 of HO-1 to HO-2 was detected. The splenic HO-1 and HO-2 were immunochemically similar to the purified isoforms obtained from the liver and the testis. Moreover, the elution properties of splenic HO-1 as well as those of the constitutive liver HO-1 and the hematin-induced liver HO-1 on a DEAE-sephacel column were similar. However, the splenic HO-1 activity could not be induced by hematin. It is suggested that in the spleen heme oxygenase activity is maintained in the induced state as the result of constant exposure to hemoglobin released in the course of disruption of senescent erythrocytes.

Cadmium-mediated inhibition of testicular heme oxygenase activity: the role of NADPH-cytochrome c (P-450) reductase.

The concerted activity of two microsomal enzymes, heme oxygenase and NADPH-cytochrome c (P-450) reductase, is required for isomer-specific oxidation of heme molecule; heme oxygenase is commonly believed to be rate limiting in this activity. In this report, we provide evidence strongly suggesting the rate-limiting role of the reductase in oxidation of heme molecule in rat testis. In the testis and the liver of rats treated with Cd (20 mumol/kg, sc, 24 h) heme oxygenase activity, assessed by the formation of bilirubin, was decreased by 50% and increased by 7-fold, respectively. In these animals, the reductase activity was decreased by nearly 75% in the testis, but remained unchanged in the liver. Similarly, the reductase activity in the liver was not altered when heme oxygenase activity was increased by 20-fold in response to bromobenzene treatment. Addition of purified testicular reductase preparation (purified over 4000-fold), or hepatic reductase, to the testicular microsomes of Cd-treated rats obliterated the Cd-mediated inhibition of heme oxygenase activity. The chromatographic separation of heme oxygenase and the reductase of the testicular microsomal fractions revealed that the reductase activity was markedly decreased (75%) while the heme oxygenase activity, when assessed in the presence of exogenous reductase, was not affected by in vivo Cd treatment. In vitro, the membrane-bound reductase preparation obtained from the testis was more sensitive to the inhibitory effect of Cd than the liver preparation. However, the purified reductase preparations from the testis and the liver exhibited a similar degree of sensitivity to Cd. Based on the molar ratio of heme oxygenase to the reductase in the microsomal membranes of the liver and the testis it appeared that the testicular heme oxygenase, which is predominantly HO-2 isoform, interacts with the reductase less effectively than HO-1; in the induced liver, heme oxygenase is predominantly the HO-1 isoform. It is suggested that due to the low abundance of NADPH-cytochrome c (P-450) reductase and the apparently lower affinity of the enzyme for HO-2, the reductase exerts a regulatory action on heme oxygenase activity in the testis.

Purification and characterization of the major constitutive form of testicular heme oxygenase. The noninducible isoform.

Recently we reported on the presence of two isoforms of heme oxygenase in rat liver microsomes, referred to as HO-1 and HO-2, and that only HO-1 is inducible (Maines, M. D., Trakshel, G. M., and Kutty, R. K. (1986) J. Biol. Chem. 261, 411-419). Presently we report on the detection of two isoforms of the enzyme in rat testis and purification to near homogeneity of the noninducible isoform, HO-2. A comparative characterization of the liver HO-1 and the testicular HO-2 is also provided. The relative abundance of the isoforms in the two organs was dissimilar. In the testis, the predominant form was HO-2, and only minute amounts of HO-1 were detected. In the liver, however, a 1:2 ratio of HO-1 to HO-2 was noted. The activity of HO-2 in both organs was refractory to cadmium, an inducer of the hepatic HO-1. Under nondenaturing electrophoresis conditions, HO-2 showed a higher mobility than HO-1; on a sodium dodecyl sulfate-polyacrylamide gel, HO-2 displayed a higher monomeric Mr. The apparent Mr values for HO-2 and HO-1 were 36,000 and 30,000, respectively. The isoforms differed in immunochemical properties. Antiserum to the liver HO-1 did not recognize the testicular HO-2 when examined by double immunodiffusion or by Western immunoblotting. HO-2 was more sensitive to heat inactivation than HO-1. When exposed at 65 degrees C (10 min), 70% of HO-1 activity was retained; however, nearly 80% of HO-2 activity was lost. The apparent Km values for heme for HO-1 and HO-2 were 0.24 and 0.40 microM, respectively. HO-1 and HO-2 had similar requirements for cofactor and flavoprotein reductase and were inhibited by heme-ligands (CO, KCN, NaN3). HO-2 utilized as substrate, Fe-protoporphyrin, Fe-hematoporphyrin, and Fe-hematoporphyrin acetate; it did not degrade intact purified rat liver cytochromes b5 and P-450 LM2, catalase, cytochrome c, hemoglobin, or myoglobin.

Characterization of two constitutive forms of rat liver microsomal heme oxygenase. Only one molecular species of the enzyme is inducible.

The present report describes, for the first time, the identification of two constitutive forms of heme oxygenase, designated as HO-1 and HO-2, in rat liver microsomal fractions. HO-1 was purified to homogeneity and exhibited a specific activity of up to 4000 nmol of bilirubin/mg of protein/h. HO-2 was partially purified to a specific activity of 250 nmol of bilirubin/mg of protein/h. In the native state, the relative activity of HO-2 surpassed that of HO-1 by 2-3-fold. However, a remarkable difference existed in the regulatory mechanism(s) for the production of the two enzyme forms. Whereas the activity of HO-1 was increased up to 100-fold in response to cobalt, cadmium, hematin, phenylhydrazine, and bromobenzene, that of HO-2 was fully refractory to these agents. The two forms differed in their apparent Km, thermolability, ammonium sulfate precipitation, antigenicity, electrophoretic mobility under nondenaturing conditions, and chromatographic behavior. Specifically, for HO-1 the apparent Km value was 0.24 microM, whereas that for HO-2 was 0.67 microM. HO-2 preparation was more susceptible to heat inactivation; nearly 65% activity was retained by HO-1 preparation after exposure to 60 degrees C for 10 min, whereas under the same conditions only about 25% of HO-2 activity was retained. When subjected to ammonium sulfate precipitation the bulk of HO-1 activity precipitated between 0 and 35% saturation, whereas that of HO-2 was precipitated between 35 and 65% saturation. The two forms appeared as immunologically different entities, in so far as a crossreactivity between antibody raised against HO-1 in rabbit and HO-2 could not be detected. Similarities were observed in respect to cofactor requirements for activity, sensitivity to inhibitors, as well as their reactivity towards the substrates used in this study, i.e. hematin, hematoheme, and cytochrome c. Specifically both forms of the enzyme required NADPH-cytochrome c (P-450) reductase, NADPH or NADH, and O2 for activity, and reactions were inhibited by KCN, NaN3, and CO. Both forms cleaved the tetrapyrrole molecule exclusively at the alpha-meso bridge to form biliverdin IX alpha isomer. HO-1 and HO-2 utilized hematin and hematoheme as substrates but not intact cytochrome c.