Biomedical subjects

M D Maines

Publications and source records attributed to M D Maines.

At least 55 records · Page 3Linked to original sources

PubMed1994

Interaction of Fe-protoporphyrin IX and heme analogues with purified recombinant heme oxygenase-2, the constitutive isozyme of the brain and testes.

Heme oxygenase-2 (HO-2) is the predominant form of heme oxygenase in the brain and testes. The enzyme is not readily amenable to isolation from mammalian tissues and has not been characterized for its kinetic properties and interaction with metalloporphyrins. Presently a rat HO-2 cDNA (Rotenberg, M.O., and Maines, M. D. (1990) J. Biol. Chem. 265, 7501-7506) was used to generate a construct with a neutral hydrophobicity profile at its COOH terminus for expression of nearly full-length HO-2 protein in Escherichia coli. The procedures used for HO-1 were of no utility in purification of HO-2. A multistep protocol developed for isolation of HO-2 resulted in a homogeneous protein with a specific activity up to 6,500 nmol of bilirubin/mg/h. Based on SDS-polyacrylamide gel electrophoresis and Western blot analyses, the protein had an apparent molecular mass of approximately 34 kDa. HO-2 binds Fe-protoporphyrin (heme) at near molar unity to give a complex with the absorption maximum at 403 nm. The Soret band has a blue shift to 430 nm when heme iron is reduced, with distinct alpha and beta bands at 485 and 550 nm, respectively. The Soret band of the CO complex of ferrous heme.HO-2 is at 420 nm, and alpha and beta bands are at 540 and 572 nm, respectively. The apparent Km for Fe-protoporphyrin is 0.33 microM, with a Vmax of 0.45 nmol of bilirubin/mg/h. Zn-protoporphyrin is a strong mixed inhibitor of enzyme activity, whereas Co-protoporphyrin is a poor competitive inhibitor of activity. When HO-2 was preincubated (10 min at 4 degrees C) with Fe-protoporphyrin, the cobalt complex did not inhibit enzyme activity, whereas the Zn-protoporphyrin effectively inhibited activity. Calorimetric measurements suggest that HO-2/heme interaction involves one type of association producing a single heat absorption peak upon melting of the complex and that the unfolding is not reversible. The association increases the enthalpy of HO-2 (130 kcal/mol versus 184 kcal/mol) and increases the stability to heat denaturation by 9 degrees C. Heat duration of zinc complex involves at least two stages of unfolding.

Amino Acid Sequence↗

PubMed1994

Site-directed mutagenesis of cysteine residues in biliverdin reductase. Roles in substrate and cofactor binding.

Biliverdin reductase is unique among all enzymes described to date in having two pH optima, 6.75 and 8.7, at which NADH or NADPH, respectively, are required for activity. The enzyme converts biliverdin to bilirubin in mammals. The mature enzyme, which is 293 amino acids long, has 3 cysteine residues, and is sulfhydryl dependent. To understand the role of the cysteine residues in enzyme activity, we examined the effects of the neutral substitution with alanine of each of three residues, individually and in combination, by site-directed mutagenesis. These residues in the predicted amino acid sequence of rat biliverdin reductase correspond to amino acids 73, 280 and 291. The modification of the amino-proximal cysteine (Cys73), which is flanked by a tyrosine residue, completely inactivated the enzyme with NADH at pH 6.75 and NADPH at pH 8.7. The loss of reductase activity was not due to changes in three-dimensional characteristics of the protein as suggested by its mobility in a non-denaturing gel. Although modification of either of the two cysteines located near the C-terminus (Cys280 and Cys291) significantly reduced activity with both cofactors, these mutations did not inactivate the enzyme. Comparison of Km values for the Cys280-->Ala and Cys291-->Ala mutants with the wild type protein, at pH 8.7, suggests that Cys280 principally functions in substrate binding while Cys291 is predominantly involved in cofactor binding. This assignment probably also applies at pH 6.75. Comparison of kcat of the mutants with wild type shows that mutation of Cys280 decreases Vmax of the enzyme. Mutation of both C-terminal cysteines caused inactivation of the enzyme, comparable to that produced by mutation of Cys73. Analysis by circular dichroism at far-ultraviolet wavelengths suggests that the alterations in activity are not the result of changes in the secondary structure of these mutants. These results are consistent with Cys73 having a central role in substrate/cofactor binding while biliverdin reductase can function, albeit at a reduced rate, with only one of the near C-terminus cysteines. The results are further consistent with the suggestion that although the two C-terminal cysteines have preferential affinities, they can serve similar functions in the interaction with substrate/cofactor.

Amino Acid Sequence↗

PubMed1994

Coordinated expression and mechanism of induction of HSP32 (heme oxygenase-1) mRNA by hyperthermia in rat organs.

Heme oxygenase isozymes, HO-1 and HO-2, catalyze the cleavage of heme b (Fe-protoporphyrin-IX) at the alpha-meso carbon bridge to form the antioxidant, biliverdin IX alpha, and the putative cellular messenger, carbon monoxide. HO-1 is a heat shock (HSP32) or stress protein, while HO-2 is a noninducible enzyme. Presently, we have examined the time course of expression of HSP32 in liver, kidney, and heart of rats exposed to hyperthermia and investigated the mechanism of induction of HO-1 by hyperthermia. We report a coordinated induction response of all organs to elevated ambient temperature (42 degrees C, 20 min). Specifically, the maximum induction of the 1.8 kb HO-1 mRNA was observed 1 h after hyperthermia and reached a value 20-40-fold that of the control; the transcript level approximated the control value by 6 h after heat stress. In contrast, the levels and the ratio of the 1.3 and 1.9 kb HO-2 transcripts were not affected by hyperthermia. As judged by in vitro nuclear transcription run-on assays, thermal stress caused the stimulation of HO-1 gene transcription. The increase in HO-1 mRNA transcription was accompanied by an increase in binding of nuclear factor(s) to the heat shock element in the promoter region of the gene. The increase of the HO-1 mRNA was reflected in increases in both heme oxygenase activity and in immunoreactive HO-1 protein. We suggest that the induction of heme oxygenase by heat stress is a physiologically relevant defense mechanism whereby both the degradation of heme of denatured hemoproteins and the generation of biologically active products of heme catabolism are enhanced.

Animals↗

PubMed1994

The structure, organization and differential expression of the gene encoding rat heme oxygenase-2.

Overlapping phage lambda clones were utilized to determine the complete nucleotide (nt) sequence of the rat gene encoding HO-2, the major heme oxygenase isozyme in the brain. This isozyme is the constitutive cognate of HSP32 (HO-1). The 12,563-bp gene consists of five exons and four introns, the first two exons are separated by a large intron of 8429 nt. The minus strand of intron 1 contains a nested sequence of 1046 nt with 87% identity to the cDNAs encoding the mouse and human non-histone chromosomal protein, HMG-17. In addition to the coding region, the similarity includes 40 bp upstream from the putative start codon and 800 bp of 3' untranslated sequence. The HO-2 gene lacks a conventional TATA box, but a TATA-like sequence (TAACTA) is found 26 nt upstream from the major transcription start point (tsp), as determined by primer extension. Upstream of the tsp, only a glucocorticoid-response element is found. The structure of the regulatory region is consistent with the previously demonstrated refractory nature of this isozyme to common inducers of gene expression and its apparent response to developmental changes in the adrenal steroid hormone profile. HO-2 is encoded by two transcripts (approx. 1.3 and approx. 1.9 kb), the larger of which is translated less efficiently than the smaller. Presently, we show that the transcripts are the products of a single gene and differ in the use of the polyadenylation signal.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Brain heme oxygenase isoenzymes and nitric oxide synthase are co-localized in select neurons.

Two isoforms of the enzyme heme oxygenase are expressed in distinct populations of neurons in the brain. These enzymes catalyse the oxidative cleavage of heme to the cellular antioxidant biliverdin resulting in the release of carbon monoxide in the process. Both heme and carbon monoxide may play important roles in regulating the nitric oxide-cyclic guanosine monophosphate signal transduction system. Thus we have examined the distributions of both isoforms of heme oxygenase in the rat brain, and compared their localizations with that of nitric oxide synthase determined with the NADPH-diaphorase histochemical technique. Heme oxygenase-1 is highly expressed in a few select populations of neurons including cells in the hilus of the dentate gyrus, in the hypothalamus, cerebellum and brainstem. This enzyme appears to be coexpressed with nitric oxide synthase only in a few cells in the dentate gyrus. Heme oxygenase-2 is much more widely expressed. It is present in mitral cells in the olfactory bulb, pyramidal cells in the cortex and hippocampus, granule cells in the dentate gyrus, many neurons in the thalamus, hypothalamus, cerebellum and caudal brainstem. However, only some of these labelled neurons also displayed nitric oxide synthase. Instead, many neurons expressing heme oxygenase-2 correspond to those known to express high levels of the hemoprotein soluble guanylyl cyclase. These results suggest that heme oxygenase may play a role in modulating guanylyl cyclase independent of nitric oxide synthase. This may result from regulation of intracellular heme and carbon monoxide levels by the heme oxygenase system.

Amino Acid Oxidoreductases↗

PubMed1994

Corticosterone regulates heme oxygenase-2 and NO synthase transcription and protein expression in rat brain.

Heme oxygenase (HO)-1 and -2 produce carbon monoxide, which is suspected, as is nitric oxide (NO), to function as a neuronal messenger. We report on glucocorticoid-mediated modulation of HO-2 and NO synthase expression in brain and the differential response of the two proteins to corticosterone in different brain regions. Corticosterone treatment (40 mg/kg, 20 days) had opposing effects on HO-2 and NO synthase transcript levels: increasing the 1.3- and 1.9-kb HO-2 mRNAs and decreasing that of the brain-specific 10.5-kb NO synthase. Corticosterone did not uniformly affect HO-2 protein expression in all regions, but appeared to cause a universal reduction in NO synthase, e.g., HO-2 was decreased in hippocampus (CA1 and dentate gyrus), but not in cerebellum. In contrast, NADPH diaphorase staining was reduced in hippocampus and in molecular and granule layers of cerebellum (not detected in Purkinje cells). Striking deficits in neuronal morphology and number of diaphorase-staining neurons were observed in the lateral tegmental area, paraventricular nucleus, and frontal cortex; HO-2 expression was only selectively affected. In cerebellum, activity of NO synthase, but not that of HO, was reduced. Consistent with the possibility that carbon monoxide can generate cyclic GMP, the change in cyclic GMP level did not mirror the decrease in NO synthase. We suggest that glucocorticoid-mediated deficits in hippocampal functions may reflect their negative effect on messenger-generating systems.

Amino Acid Oxidoreductases↗

PubMed1994

Induction of heart heme oxygenase-1 (HSP32) by hyperthermia: possible role in stress-mediated elevation of cyclic 3':5'-guanosine monophosphate.

Presently we have investigated the carbon monoxide generating capacity of the cardiovascular system under normal and stress conditions by examining the microsomal heme oxygenase system at the transcript, protein and activity levels; and have assessed response of heart nitric oxide (NO) synthase activity and cyclic GMP levels to stress. Heme oxygenase (HO) isozymes, HO-1 (HSP32) and HO-2, catalyze the rate limiting step in the only known pathway in eukaryotes for the generation of the potential cellular message, carbon monoxide, and the antioxidant, bilirubin. We show expression of HO-1 and HO-2 at both the transcription and protein levels under normal conditions in the heart and descending aorta, and demonstrate the sensitivity of only the HO-1 isozyme to heat stress in these tissues. The ratio of the two HO-2 homologous transcripts (approximately 1.9 and 1.3 Kb) present in the atrium, ventricles and descending aorta and their levels were not altered by hyperthermia (42 degrees C, 20 min) when measured 1 or 6 hr after treatment. In contrast, hyperthermia caused a rapid, robust and coordinate increase of approximately 10- to 32-fold in the approximately 1.8-Kb HO-1 mRNA in these tissues when measured 1-hr post-treatment. Hyperthermia also caused a significant increase in both HO-1 protein and heme degradation capacity in the heart. Furthermore, the induction of HO-1 protein in the heart was accompanied by a significant elevation in tissue cyclic GMP level first detected 1-hr post-treatment and was sustained 6 hr after heat shock.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗

PubMed1993

Inactivation of phytochrome- and phycobiliprotein-chromophore precursors by rat liver biliverdin reductase.

The phytochrome chromophore precursor, 3E-phytochromobilin, and the phycobiliprotein chromophore precursors, 3E-phycocyanobilin and 3E-phycoerythrobilin, are enzymatically converted to novel rubinoid products by purified rat liver biliverdin reductase. Phytochromobilin and phycocyanobilin are particularly good substrates for biliverdin reductase with Km and Vmax values very similar to those of the natural substrate, biliverdin IX alpha. Phycoerythrobilin is the least preferred of the three bilin substrates. 1H NMR spectroscopy of phycocyanorubin, the product of phycocyanobilin catalysis by biliverdin reductase, and comparison of absorption spectra of all three rubinoid products reveal that the C10 methine bridge is selectively reduced by biliverdin reductase without altering the A-ring ethylidene substituent. In vitro phytochrome assembly experiments demonstrate that the phytorubin products do not form photoactive adducts with recombinant apophytochrome. These results suggest that ectopic expression of biliverdin reductase in plants will prevent assembly of the functional photoreceptor and thus will potentially alter light-mediated plant growth and development.

Animals↗

PubMed1993

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).

Amino Acid Sequence↗

PubMed1993

Domains of rat heme oxygenase-2: the amino terminus and histidine 151 are required for heme oxidation.

Cleavage of heme b (Fe-protoporphyrin IX) at the alpha-meso carbon bridge is catalyzed by heme oxygenase isozymes, HO-1 and HO-2, to form biliverdin IX alpha. Currently, we have examined the requirement for the amino terminus and the hydrophobic carboxy terminus of rat HO-2 expressed in Escherichia coli for heme degradation activity and have assessed the importance of His 151 for this activity. His 151 is in the longest span of amino acids (24 residues) which are present, with only a single conservative substitution, in seven cloned heme oxygenases including the apparent single isozyme in chicken. We show His 151 is essential for cleavage of heme, as substitution of alanine for this residue by site-directed mutagenesis resulted in expression of an inactive protein with immunoreactivity toward antibody to rat HO-2. A cDNA construct in which nucleotides encoding the 33 N-terminal amino acid residues were deleted, when expressed, produced a protein of predicted size and immunoreactivity with antibody to HO-2 but also devoid of heme degrading activity. The presence of additional residues at this terminus, for the most part, accounts for the larger size of HO-2 compared to HO-1. Conversely, the hydrophobic region at the carboxy terminus did not appear to be essential for heme degradation. A construct in which the sequence encoding the primarily hydrophobic amino acids of the carboxy terminus was replaced by a sequence encoding predominantly hydrophilic residues expressed a protein which retained full capability to convert heme to biliverdin. Further, the construct with a hydrophilic carboxy terminus was not appreciably associated with bacterial membranes, suggesting that the carboxy terminus in the wild-type protein serves as a membrane anchor for this enzyme.

Amino Acid Sequence↗

PubMed1993

Glutathione depletion induces heme oxygenase-1 (HSP32) mRNA and protein in rat brain.

In mammalian systems, the heme oxygenase (HO) isozymes HO-1 (HSP32) and HO-2 oxidatively cleave the heme molecule to produce bile pigments and carbon monoxide. Although HO-1 is inducible by various chemicals in systemic organs and cell culture systems, this communication reports for the first time the induction of this stress protein and its transcript by a chemical in the brain. In addition, this study demonstrates expression of HO-1 in select populations of cells in the brain in response to GSH depletion. Specifically, treatment of adult rats with diethyl maleate (DEM; 4.7 mmol/kg) caused a pronounced decrease in brain GSH content within 1 h. GSH levels remained significantly depressed for at least 24 h postinjection. Northern blot analysis of brain poly(A)+ mRNA following DEM treatment revealed on the average a sixfold increase in the 1.8-kb HO-1 mRNA level compared with that of controls; concomitant with this change was a decrease in GSH levels. Total brain HO activity was not significantly altered along with the increase in HO-1 mRNA level. The increase in transcription of HO-1 was a direct response to GSH depletion, as judged by the observation that treatment of neonatal rats with L-buthionine-(S,R)-sulfoximine (BSO) (3 mmol/kg, twice daily, for 2 days), a selective inhibitor of GSH synthesis, caused a marked depression in total brain GSH level and a concomitant increase in brain 1.8-kb HO-1 mRNA content. The magnitude of the increase was up to approximately 11.5-fold that of the control level, as evidenced by northern blot analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Biliverdin reductase is heat resistant and coexpressed with constitutive and heat shock forms of heme oxygenase in brain.

Two heme oxygenase (HO) isozymes--HO-1, which is a heat shock protein (HSP32), and HO-2--catalyze the isomer-specific production of biliverdin IX alpha and carbon monoxide. The latter has the potential of functioning as a neurotransmitter, whereas the reduced form of biliverdin, bilirubin, has potent antioxidant activity. Formation of bilirubin is catalyzed by biliverdin reductase (BVR). The reductase is a unique enzyme in being dual pyridine nucleotide and dual pH dependent. Here, we show that the reductase is resistant to thermal stress at both the protein and message level. We further demonstrate that the reductase is coexpressed in cells that display HO-1 and/or HO-2 under normal conditions, as well as in regions and cell types that have the potential to express heat shock-inducible HO-1 protein. Exposure of male rats to 42 degrees C for 20 min did not decrease brain BVR activity, but caused a slight increase in NADPH- and NADH-dependent activities at 1 and 6 h following hyperthermia. High levels of the approximately 1.5-kb BVR mRNA were detected in control brain; it too displayed thermal tolerance. Similarly, the pattern of multiplicity of net charge variants of the enzyme purified from brain of heat-shocked rats did not differ from the control pattern. Immunochemical localization of BVR protein in normal brain correlated well with the presence of HO-1 and/or HO-2 throughout the forebrain, diencephalon, cerebellum, and brainstem regions. There were select neuronal and nonneuronal cells in the substantia nigra and cerebellum that did express the reductase under normal conditions, wherein no HO isozymes could be detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Induction of kidney heme oxygenase-1 (HSP32) mRNA and protein by ischemia/reperfusion: possible role of heme as both promotor of tissue damage and regulator of HSP32.

Presently we describe, for the first time, induction of microsomal heme oxygenase-1 (HO-1) mRNA and protein in response to ischemia/reperfusion and therefore define HO-1 as stress protein in the kidney. Specifically, Northern blot analysis of kidneys of rats subjected to bilateral ischemia for 30 min revealed an increase of 8- to 10-fold in the level of 1.8 Kb HO-1 mRNA 6 hr after reperfusion. The increase in transcript level was maintained when assessed after 24 hr. The levels of 1.3 and 1.9 Kb transcripts for the second isozyme of HO, HO-2, were decreased at both time points. The increase in HO-1 mRNA was reflected in HO-1 protein level, as judged by Western blot analysis and at the level of activity as judged by the rate of bilirubin formation. An absence of change in adrenal HO-1 mRNA level subsequent to renal ischemia/reperfusion suggested that the induction of kidney HO-1 did not reflect a generalized response of the rat organs to stress; rather, it was a target organ specific response. Moreover, in kidneys subjected to ischemia 6 and 24 hr after reperfusion, significant increases in the cellular content of heme were observed; heme is a known inducer of HO-1 synthesis. Ischemia/reperfusion also adversely affected concentration of cytochrome P-450 in both mitochondrial and the microsomal fractions of the kidney. We suggest that increase in tissue heme levels may be a significant factor in damage caused by ischemia/reperfusion to renal tissue, whereby the metalloporphyrin promotes oxygen-free radical formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

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.

Adrenal Glands↗

PubMed1992

Human heme oxygenase-2: characterization and expression of a full-length cDNA and evidence suggesting that the two HO-2 transcripts may differ by choice of polyadenylation signal.

We show by Northern blot analysis that human HO-2 is encoded by two transcripts (1.3 and 1.7 kb) and is a single-copy gene as judged by Southern blot analysis. We further provide evidence based on Northern blot and sequence analysis of a cDNA representing the larger transcript that the transcripts differ in the 3' untranslated region. A 274-base-pair DNA fragment from the rat heme oxygenase-2 gene (I. Cruse and M.D. Maines, 1988, J. Biol. Chem. 263, 3348-3353) was used to isolate a human HO-2 cDNA from a fetal kidney library in lambda gt11. The clone, designated hK-1, was sequenced and the cDNA insert was determined to be 1625 base pairs in length, encoding a protein of 313 amino acids. Two consensus polyadenylation signals separated by 440 nucleotides were identified in the 3' untranslated region. The size of the cDNA insert closely approximated the larger of two mRNAs. The nucleotide sequence was 88% identical to the rat HO-2 gene within the predicted coding region and the putative translation product was also estimated to be 88% identical to the rat gene product (M. O. Rotenberg and D. Maines, 1990, J. Biol. Chem. 265, 7501). The predicted size, 36 kDa, corresponded well with HO-2 detected in human testis microsomes by Western blot analysis. Further, the fusion protein expressed in Escherichia coli displayed significant heme oxygenase activity, which was inhibited by Zn- and Sn-protoporphyrins, known inhibitors of eukaryotic heme oxygenase, but not by sulfhydryl reagents.

Amino Acid Sequence↗

PubMed1992

Expression and characterization of a cDNA for rat kidney biliverdin reductase. Evidence suggesting the liver and kidney enzymes are the same transcript product.

Biliverdin reductase is a unique dual cofactor- and pH-dependent enzyme that converts biliverdin to bilirubin and displays extensive inter-organ pI and molecular weight microheterogeneity. Presently we have explored the molecular basis for these properties. The amino acid composition and the sequences of NH2 termini plus five tryptic fragments of purified rat liver and kidney enzymes were obtained. A 62-nucleotide DNA probe was designed and in combination with antibody was used to screen a rat kidney cDNA library. A cDNA sequence of 1108 base pairs (bp) containing an 885-bp open reading frame was generated. The cloned cDNA probe detected a single mRNA of approximately 1500 bp in liver and kidney. The open reading frame encodes a 295 amino acid protein. Methionine and aspartic acid residues at positions 1 and 2 of the deduced protein are removed during processing. The deduced amino acid composition of the mature protein closely matched that of the purified rat liver and kidney enzymes. All liver peptides were found in the deduced amino acid sequence of kidney enzyme and the NH2 termini of both enzymes were identical. The expressed protein co-migrated with purified reductase and was recognized by antiserum to the enzyme. The expressed reductase displayed two distinct pH optima using a different cofactor at each pH: NADH at the lower pH 6.7-6.9 range and NADPH at pH 8.5-8.7. The findings suggest that the liver and kidney enzymes are the products of the same transcript(s) and that their microheterogeneity may reflect tissue-specific post-translational modifications.

Amino Acid Sequence↗

PubMed1992

Normal and heat-induced patterns of expression of heme oxygenase-1 (HSP32) in rat brain: hyperthermia causes rapid induction of mRNA and protein.

Most cells possess a variety of mechanisms, such as high levels of glutathione, that guard against cytotoxic free radicals, which are suspected in the etiology of various neurological deficits. Neurons, however, are deficient in this antioxidant source. The list of other potent antioxidants includes the bile pigments biliverdin and bilirubin. Heme oxygenase (HO) isozymes, HO-1 (HSP32) and HO-2, catalyze the rate-limiting step in the only biological pathway by which bile pigments are produced. In this study, heat shock is identified as the only stimulus reported to date that can alter expression in brain HO-1 of protein and mRNA in vivo. Using a HO-1 cDNA probe, we examined the level of HO-1 mRNA in normal rat brain and in brain 1 and 6 h following heat shock. Exposure of male rats to 42 degrees C for 20 min caused a 20-fold increase in brain HO-1 1.8-kb mRNA within 1 h after treatment. Quantification of brain HO-1 protein by HO-1 radioimmunoassay revealed a fourfold increase at 6 h posttreatment. In normal brain, HO-1 protein was sparsely expressed in few select neuronal and nonneuronal cell populations in forebrain, diencephalon, cerebellum, and brainstem regions. Six hours following heat shock, an intense increase in HO-1 protein in glia throughout the brain, ependyma lining the ventricles of the brain, paraventricular nucleus, Purkinje cell layer of the cerebellum, and cochlear nucleus of brainstem was observed. We suggest that increases in HO-1 transcript and protein reflect a means to elevate levels of antioxidants in cells with compromised defense mechanisms caused by stress.

Animals↗

PubMed1992

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.

Adrenal Glands↗