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PubMed1989

NADH-sensitive propionyl-CoA hydrolase in brown-adipose-tissue mitochondria of the rat.

Acyl-CoA hydrolase activity was studied in brown adipose tissue (BAT) mitochondria of rats. The substrate specificity was investigated: total hydrolase activity showed two activity peaks, one sharp peak for propionyl-CoA and a broad peak at medium- to long-chain acyl-CoAs. The propionyl-CoA activity fully comigrated with a mitochondrial matrix marker enzyme in fractionation studies of tissue and mitochondria. The hydrolytic activity against short-chain acyl-CoAs was inhibited by NADH, and analyses of the substrate specificity of the hydrolases in the presence and absence of NADH allowed for the delineation of two distinct acyl-CoA hydrolases. These hydrolases could also be separated by gel filtration. It was concluded that rat BAT mitochondria possess at least two matrix acyl-CoA hydrolases: one broad-spectrum acyl-CoA hydrolase with an apparent native molecular weight of less than 100,000, and a specific propionyl-CoA hydrolase with an apparent native molecular weight at least 240,000; this hydrolase is regulated by NADH. It is suggested that the function of the propionyl-CoA hydrolase is to ensure that the level of propionyl-CoA in the mitochondria is not detrimentally increased.

Adipose Tissue, Brown↗
PubMed1995

Cloning of the Schizosaccharomyces pombe gene encoding diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) asymmetrical hydrolase: sequence similarity with the histidine triad (HIT) protein family.

Diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) asymmetric hydrolase (EC 3.6.1.17) is a specific catabolic enzyme of Ap4A found in Schizosaccharomyces pombe. We have previously described the partial purification of Ap4A hydrolase from S. pombe [Robinson, de la Peña and Barnes (1993) Biochim. Biophys. Acta 1161, 139-148]. We determined the sequence of the N-terminal 20 amino acids of Ap4A hydrolase and designed two degenerate PCR primers based on the sequence. The 60 bp DNA fragment obtained by PCR, which is specific to Ap4A hydrolase, was used to isolate the Ap4A hydrolase gene, aph1, from S. pombe by screening a genomic DNA library in a multicopy plasmid. Ap4A hydrolase activity from the crude supernatant of a positive S. pombe transformant was about 25-fold higher than the control. There was no detectable stimulation of enzymic activity by phosphate. The aph1 gene from S. pombe contains three introns. The intron boundaries were confirmed by sequencing the cDNA of the aph1 gene from a S. pombe cDNA library. The deduced open reading frame of the aph1 gene codes for 182 amino acids. Two regions of significant local similarity were identified between the Ap4A hydrolase and the histidine triad (HIT) protein family [Séraphin (1992) DNA Sequence 3, 177-179]. HIT proteins are present in prokaryotes, yeast, plants and mammals. Their functions are unknown, except that the bovine protein inhibits protein kinase C in vitro. All four histidine residues which are conserved among the HIT proteins, including the HxHxH putative Zn(2+)-binding motif, are conserved in the Ap4A hydrolase. In addition, there are two regions of similarity between the Ap4A phosphorylases I and II from Saccharomyces cerevisiae and Ap4A hydrolase from S. pombe. These regions overlap with the HIT protein similarity regions. The aph1 gene from S. pombe is the first asymmetrical Ap4A hydrolase gene to be cloned and sequenced.

Acid Anhydride Hydrolases↗
PubMed1988

A novel type of short- and medium-chain acyl-CoA hydrolases in brown adipose tissue mitochondria.

Acyl-CoA hydrolase activities were studied in brown adipose tissue from hamsters. A latent activity was observed in isolated mitochondria. Two peaks of activity were clearly visible in mitochondria, one with an optimum at propionyl-CoA ("short-chain hydrolase") and one with an optimum at nonanoyl-CoA ("medium-chain hydrolase"); there was only low activity toward palmitoyl-CoA and longer-chain acyl-CoAs. In subcellular fractionation experiments, the activity of the short-chain and the medium-chain hydrolase fully followed that of the mitochondrial matrix marker enzyme 2-oxoglutarate dehydrogenase. The specific activity of the hydrolases in the mitochondrial fraction was doubled after cold acclimation. beta-NADH inhibited the short- and medium-chain hydrolases; alpha-NADH, NADPH, and NAD+ were without effect. ADP stimulated the short- and medium-chain hydrolases; ATP and AMP were practically without effect. Evidence is presented to indicate that NADH and ADP interact on the enzyme at the same site and that ADP is essential for the maintenance of the short- and medium-chain enzyme activities. A positive effect of KCl was found on the short- and medium-chain hydrolase activities. Also, the divalent ions Ca2+ and Mg2+ were stimulatory, but only Ca2+ was able to overcome NADH inhibition, possibly due to interaction directly with NADH. It is concluded that brown adipose tissue mitochondria, besides a conventional type of acyl-CoA hydrolase, contain two species of a novel type of acyl-CoA hydrolases which are characterized by being regulated by ADP and NADH (interacting at a common site) and by having an obligatory requirement for ADP.

Acclimatization↗
PubMed1986

Rat cytosolic epoxide hydrolase.

Rat liver microsomal and cytosolic epoxide hydrolase may be distinguished through differences in substrate specificity: styrene 7,8-oxide is preferentially hydrolyzed by the microsomal form, while trans-stilbene oxide is the preferred substrate for cytosolic epoxide hydrolase. Large interindividual differences in the specific activity of Sprague-Dawley (outbred strain) liver cytosolic epoxide hydrolase were observed, varying from 2 to 77 pmol/min X mg protein. Interindividual variations were much lower for microsomal epoxide hydrolase. The specific activity of Fischer F-344 (inbred strain) liver cytosolic epoxide hydrolase varied only by a factor of 2. The specific activity of cytosolic epoxide hydrolase using trans-stilbene oxide as the substrate was highest in kidney and heart, followed by liver, brain, lung, testis, and spleen. For microsomal epoxide hydrolase, the specific activity was much lower in extrahepatic tissues than in liver. None of the commonly used inducers of xenobiotic metabolizing enzymes caused significant changes in rat liver cytosolic epoxide hydrolase. However, peroxisome proliferating drugs were found to drastically increase cytosolic epoxide hydrolase activity. Treatment for one week with a diet containing clofibrate (0.25%), tiadenol (0.5%) or acetylsalicylic acid (1%) caused a 8, 13 and 5 fold increase in cytosolic epoxide hydrolase activity respectively in the liver which parallelled the induction of peroxisomal beta-oxidation activity (13, 19 and 5 fold, respectively).

Animals↗
PubMed1986

Complementary DNA and amino acid sequence of rat liver microsomal, xenobiotic epoxide hydrolase.

The coding nucleotide sequence for rat liver microsomal, xenobiotic epoxide hydrolase was determined from two overlapping cDNA clones, which together contain 1750 nucleotides complementary to epoxide hydrolase mRNA. The single open reading frame of 1365 nucleotides codes for a 455 amino acid polypeptide with a molecular weight of 52,581. The deduced amino acid composition agrees well with those determined by direct amino acid analysis of the rat protein, and the amino acid sequence is 81% identical to that of rabbit epoxide hydrolase. Analysis of codon usage for epoxide hydrolase, and that of rabbit epoxide hydrolase. Analysis of codon usage for epoxide hydrolase, and comparison to codon usage for NADPH-cytochrome P-450 oxidoreductase and cytochromes P-450b, P-450d, and P-450PCN, suggest that epoxide hydrolase is more conserved than cytochromes P-450b and P-450PCN; comparison of the extent of sequence conservation for 12 homologous proteins between the rat and rabbit, including cytochrome P-450b, supports this hypothesis, and indicates that much of epoxide hydrolase is constrained to maintain its hydrophobic character, consistent with its intramembranous location. The predicted membrane topology of epoxide hydrolase delineates 6 membrane-spanning segments, less than the 8 or 10 predicted for two cytochrome P-450 isozymes; the lower number of membrane-spanning segments predicted for epoxide hydrolase correlates with its lesser dependence on the membrane for maintenance of its tertiary structure and catalytic activity.

Amino Acid Sequence↗
PubMed1985

Induction of epoxide hydrolase in cultured rat hepatocytes and hepatoma cell lines.

The expression of epoxide hydrolases was studied in cultured rat hepatocytes and hepatoma cell lines. Styrene 7,8-oxide and benzo[a]pyrene 4,5-oxide were used as substrates for microsomal epoxide hydrolase and trans-stilbene oxide for the cytosolic form of this enzyme. In freshly isolated hepatocytes from control rats, microsomal epoxide hydrolase activity was 7.7 and 10.8 nmoles/mg cellular protein/min with benzo[a]pyrene 4,5-oxide and styrene 7,8-oxide as substrates respectively. This enzyme activity increased by more than 2-fold in hepatocytes after 24 hr in culture and remained elevated throughout 96 hr using both substrates. In cultured hepatocytes from rats pretreated in vivo with phenobarbital, trans-stilbene oxide, 2-acetylaminofluorene and N-hydroxy-2-acetylaminofluorene, both benzo[a]pyrene 4,5-oxide and styrene 7,8-oxide hydrolase activities were increased greater than 1.8 relative to controls. Hepatocytes from 2-acetylaminofluorene-pretreated animals at 24 hr in culture had approximately 9-fold higher activities than control hepatocytes. In marked contrast to microsomal epoxide hydrolase activity, the cytosolic enzyme showed an initial activity of 191 pmoles/mg cellular protein/min in freshly isolated hepatocytes, decreased by 75% after 24 hr in culture, and was barely detectable at 96 hr. A similar trend was apparent in hepatocytes from the pretreated animals. In vitro treatment of hepatocytes with trans-stilbene oxide and phenobarbital increased microsomal epoxide hydrolase, while this activity was refractory to 2-acetylaminofluorene treatment. Styrene 7,8-oxide hydrolase activity was increased in the McA-RH-7777 rat hepatoma cell line by phenobarbital, trans-stilbene oxide and 2-acetylaminofluorene treatment. Similarly, benzo[a]pyrene 4,5-oxide hydrolase activity was also increased in this cell line by treatment with phenobarbital and trans-stilbene oxide but not by 2-acetylaminofluorene. Microsomal epoxide hydrolase activity in rat H4-II-E hepatoma cells was refractory to induction, except by trans-stilbene oxide treatment, which caused a 70% increase in benzo[a]pyrene 4,5-oxide hydrolase activity.

2-Acetylaminofluorene↗
PubMed1988

Organ distribution of epoxide hydrolases in cytosolic and microsomal fractions of normal and nafenopin-treated male DBA/2 mice.

Using trans-stilbene oxide and styrene oxide as substrates, epoxide hydrolase activities were measured in cytosolic and microsomal fractions from liver, kidney, heart, lung and testis of male DBA/2 mice. The activities towards these two substrates are remarkably organ specific: trans-stilbene oxide was most effectively hydrolyzed in subcellular fractions from liver, kidney and heart, whereas styrene oxide was predominantly hydrolyzed in those from liver, lung and testis. Immunoblotting experiments were performed with two polyclonal antibodies isolated from goat antisera. Using an anti-mouse liver cytosolic epoxide hydrolase antibody, the corresponding antigen protein was predominantly detected in both cytosolic and microsomal fractions from liver, kidney and heart. An anti-rat liver microsomal epoxide hydrolase antibody proved to be cross-reactive with the mouse enzyme and stained SDS-gels run with microsomal fractions from liver, lung and testis. The anti-mouse liver cytosolic epoxide hydrolase antibody precipitated cytosolic epoxide hydrolase activities from liver, kidney and heart cytosolic fractions. Dietary exposure to the hypolipidemic agent nafenopin (2000 ppm/10 days) caused an induction of trans-stilbene oxide hydrolase and styrene oxide hydrolase activities in cytosolic and microsomal liver fractions whereas, in the other organs, the same activities were unaffected by this treatment. This finding was in accordance with the increased amounts of antigen protein as detected with the antibodies in liver fractions from treated animals. The anti-mouse liver cytosolic epoxide hydrolase antibody was found to precipitate the whole trans-stilbene oxide hydrolase activity also from liver cytosol of nafenopin-treated mice, which indicates the presence of a single cytosolic epoxide hydrolase following induction.

Animals↗
PubMed1999

Differential subcellular localization of endogenous and transfected soluble epoxide hydrolase in mammalian cells: evidence for isozyme variants.

Endogenous, constitutive soluble epoxide hydrolase in mice 3T3 cells was localized via immunofluorescence microscopy exclusively in peroxisomes, whereas transiently expressed mouse soluble epoxide hydrolase (from clofibrate-treated liver) accumulated only in the cytosol of 3T3 and HeLa cells. When the C-terminal lie of mouse soluble epoxide hydrolase was mutated to generate a prototypic putative type 1 PTS (-SKI to -SKL), the enzyme targeted to peroxisomes. The possibility that soluble epoxide hydrolase-SKI was sorted slowly to peroxiosmes from the cytosol was examined by stably expressing rat soluble epoxide hydrolase-SKI appended to the green fluorescent protein. Green fluorescent protein soluble epoxide hydrolase-SKI was strictly cytosolic, indicating that -SKI was not a temporally inefficient putative type 1 PTS. Import of soluble epoxide hydrolase-SKI into peroxisomes in plant cells revealed that the context of -SKI on soluble epoxide hydrolase was targeting permissible. These results show that the C-terminal -SKI is a non-functional putative type 1 PTS on soluble epoxide hydrolase and suggest the existence of distinct cytosolic and peroxisomal targeting variants of soluble epoxide hydrolase in mouse and rat.

3T3 Cells↗
PubMed1989

Bleomycin hydrolase: molecular cloning, sequencing, and biochemical studies reveal membership in the cysteine proteinase family.

Bleomycin (BLM) hydrolase catalyzes the inactivation of the antitumor drug BLM and is believed to protect normal and malignant cells from BLM toxicity. The normal physiological function of BLM hydrolase is not known. We now provide evidence for its membership in the cysteine proteinase family. BLM hydrolase was purified to homogeneity from rabbit lungs, and a partial amino acid sequence was determined from a tryptic digest peptide. On the basis of this sequence a 36-mer oligonucleotide was synthesized. The 36-mer oligonucleotide probe hybridized to a single mRNA species of 2.5 kb from several species and was used to isolate an 832-bp cDNA insert from a lambda gt11 rabbit liver cDNA library. This insert encoded the tryptic digest peptide previously identified in rabbit lung BLM hydrolase by amino acid sequencing. Analysis of the predicted amino acid sequence coded by the 832-bp BLM hydrolase cDNA fragment indicated no significant homology with any currently known proteins except for a 15 amino acid portion, which displayed remarkable homology with the active site of cysteine proteinases. Within this active-site region, 10 of the amino acid residues of papain and 9 of aleurain, cathepsin H, and cathepsin L were identical with those of rabbit liver BLM hydrolase. The catalytic cysteine of thiol proteinases was also conserved in BLM hydrolase, and cysteine proteinase specific inhibitors, such as E-64, were found to be potent inhibitors of BLM hydrolase activity. Furthermore, bleomycin hydrolase exhibited cathepsin H like enzymatic activity. Bleomycin hydrolase had, however, no significant cathepsin B or L activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2001

Saccharomyces cerevisiae leukotriene A4 hydrolase: formation of leukotriene B4 and identification of catalytic residues.

Leukotriene A(4) hydrolase in mammals is a bifunctional zinc metalloenzyme that catalyzes the hydrolysis of leukotriene A(4) into the proinflammatory mediator leukotriene B(4), and also possesses an aminopeptidase activity. Recently we cloned and characterized an leukotriene A(4) hydrolase from Saccharomyces cerevisiae as a leucyl aminopeptidase with an epoxide hydrolase activity. Here we show that S. cerevisiae leukotriene A(4) hydrolase is a metalloenzyme containing one zinc atom complexed to His-340, His-344, and Glu-363. Mutagenetic analysis indicates that the aminopeptidase activity follows a general base mechanism with Glu-341 and Tyr-429 as the base and proton donor, respectively. Furthermore, the yeast enzyme hydrolyzes leukotriene A(4) into three compounds, viz., 5S,6S-dihydroxy-7,9-trans-11,14-cis-eicosatetraenoic acid, leukotriene B(4), and Delta(6)-trans-Delta(8)-cis-leukotriene B(4), with a relative formation of 1:0.2:0.1. In addition, exposure of S. cerevisiae leukotriene A(4) hydrolase to leukotriene A(4) selectively inactivates the epoxide hydrolase activity with a simultaneous stimulation of the aminopeptidase activity. Moreover, kinetic analyses of wild-type and mutated S. cerevisiae leukotriene A(4) hydrolase suggest that leukotriene A(4) binds in one catalytic mode and one tight-binding, regulatory mode. Exchange of a Phe-424 in S. cerevisiae leukotriene A(4) hydrolase for a Tyr, the corresponding residue in human leukotriene A(4) hydrolase, results in a protein that converts leukotriene A(4) into leukotriene B(4) with an improved efficiency and specificity. Hence, by a single point mutation, we could make the active site better suited to bind and turn over the substrate leukotriene A(4), thus mimicking a distinct step in the molecular evolution of S. cerevisiae leukotriene A(4) hydrolase toward its mammalian counterparts.

Amino Acid Sequence↗
PubMed2001

Co-localization of leukotriene a4 hydrolase with 5-lipoxygenase in nuclei of alveolar macrophages and rat basophilic leukemia cells but not neutrophils.

The synthesis of leukotriene B(4) from arachidonic acid requires the sequential action of two enzymes: 5-lipoxygenase and leukotriene A(4) hydrolase. 5-Lipoxygenase is known to be present in the cytoplasm of some leukocytes and able to accumulate in the nucleoplasm of others. In this study, we asked if leukotriene A(4) hydrolase co-localizes with 5-lipoxygenase in different types of leukocytes. Examination of rat basophilic leukemia cells by both immunocytochemistry and immunofluorescence revealed that leukotriene A(4) hydrolase, like 5-lipoxygenase, was most abundant in the nucleus, with only minor occurrences in the cytoplasm. The finding of abundant leukotriene A(4) hydrolase in the soluble nuclear fraction was substantiated by two different cell fractionation techniques. Leukotriene A(4) hydrolase was also found to accumulate together with 5-lipoxygenase in the nucleus of alveolar macrophages. This result was obtained using both in situ and ex vivo techniques. In contrast to these results, peripheral blood neutrophils contained both leukotriene A(4) hydrolase and 5-lipoxygenase exclusively in the cytoplasm. After adherence of neutrophils, 5-lipoxygenase was rapidly imported into the nucleus, whereas leukotriene A(4) hydrolase remained cytosolic. Similarly, 5-lipoxygenase was localized in the nucleus of neutrophils recruited into inflamed appendix tissue, whereas leukotriene A(4) hydrolase remained cytosolic. These results demonstrate for the first time that leukotriene A(4) hydrolase can be accumulated in the nucleus, where it co-localizes with 5-lipoxygenase. As with 5-lipoxygenase, the subcellular distribution of leukotriene A(4) hydrolase is cell-specific and dynamic, but differences in the mechanisms regulating nuclear import must exist. The degree to which these two enzymes are co-localized may influence their metabolic coupling in the conversion of arachidonic acid to leukotriene B(4).

Animals↗
PubMed2001

Paraoxon sensitive phenylvalerate hydrolase in assessing the severity of acute paraoxon poisoning.

INTRODUCTION: Intoxications with organophosphorous compounds, especially paraoxon, are frequent. Organophosphorous compounds inhibit serine hydrolases such as acetylcholine, butyrilcholine, and carboxyl esterases although acetylcholine and butyrylcholine are too sensitive to paraoxon to be useful markers of severity. They cannot show a dose-dependent inhibition during an acute organophosphorous compounds exposure because maximal enzyme inhibition is reached at very low organophosphorous compounds concentrations. PURPOSE: To determine in vitro the dose-effect relationship between the activity of the paraoxon-sensitive phenylvalerate hydrolase, a member of the carboxvl esterases family, and the paraoxon dose, and to assess its utility as a putatively less sensitive enzyme marker to monitor the severity of an acute paraoxon intoxication. MATERIALS AND METHODS: Phenylvalerate hydrolase and butyrylcholine activities were determined in serum of nine healthy human volunteers before and after addition of different concentrations of paraoxon. The determination of phenyl-valerate hydrolase activity was carried out using a modification of the method described by Johnson. A commercially available kit was used to measure butyrylcholine activity. RESULTS: Paraoxon inhibits phenyl-valerate hydrolase activity at concentrations above 10 M. Maximal inhibition (approximately 50% of baseline) is achieved at concentrations above 2.5 x 10(-7) M. The IC50 value of paraoxon for phenyl-valerate hydrolase is 34+/-2 nM. The uninhibited phenyl-valerate hydrolase activity is due to paraoxon-resistent isoforms. Paraoxon begins inhibiting butyrylcholine activity at concentrations above 10(-9) M. At concentrations above 5 x 10(-5) M, no butyrylcholine activity is measulrable. The IC50 value of paraoxon for butyrylcholine is 150+/-23 nM. CONCLUSION: The paraoxon-sensitive subunit of phenyl-valerate hydrolase shows dose-dependent inhibition when exposed to paraoxon in vitro, but it is even more sensitive than butyrylcholine to paraoxon inhibition. Determinations of phenyl-valerate hydrolase activity to assess the severity of an acute organophosphorous compounds poisoning cannot be recommended, but phenyl-valerate hydrolase may have utility in worker surveillance.

Biomarkers↗
PubMed1998

Human large luteal cells in the menstrual cycle and early pregnancy express leukotriene A4 hydrolase.

Leukotriene (LT) A4 hydrolase (EC3.3.2.6) converts LTA4 to LTB4 which shows a chemotactic activity to leukocytes. To investigate the involvement of LTB4 in human corpus luteum (CL) function, the localization of LTA4 hydrolase in human ovarian tissues was examined by immunohistochemistry with a rabbit polyclonal antibody against LTA4 hydrolase. The enzyme was weakly expressed on granulosa cells of the follicles. After ovulation, the intensity of LTA4 hydrolase on large luteal cells increased and was highest in the midluteal phase. High expression was also observed in the CL of early pregnancy. In theca interna and small luteal cells, LTA4 hydrolase was weakly detected in all developmental stages. Immunoblot analysis demonstrated that the molecular mass of LTA4 hydrolase expressed in CL was 62 kDa, and confirmed that LTA4 hydrolase expression increased during CL formation and remained high in early pregnancy. The sequence encoding mRNA of LTA4 hydrolase, which was isolated from CL and amplified by polymerase chain reaction, was shown to be identical to the previously reported one. Immunocytochemistry showed that LTA4 hydrolase expression in cultured granulosa cells increased over 4 days in vitro and was enhanced by human chorionic gonadotrophin treatment. These expression profiles of LTA4 hydrolase suggest the involvement of LTB4 in luteal cell function during CL formation and early pregnancy.

Adolescent↗
PubMed1996

Colocalization of lysosomal hydrolase and beta-amyloid in diffuse plaques of the cerebellum and striatum in Alzheimer's disease and Down's syndrome.

The lysosomal hydrolases, cathepsin D (Cat D) and beta-hexosaminidase A (HEX), which are normally intracellular enzymes, colocalize with beta-amyloid in a subgroup of diffuse plaques in the cerebellum and striatum of individuals with Alzheimer's disease or Down's syndrome. Using specific antisera in combination with single- and double-label immunocytochemical techniques, extracellular hydrolase was detected in 30 to 40% of the diffuse plaques in the cerebellar molecular layer and nearly all of the diffuse plaques in the striatum. In both Alzheimer's disease and Down's syndrome, about 5 to 10% of the cerebellar Purkinje cells contained abnormally increased numbers of hydrolase-positive lysosomes despite their normal appearance by conventional histologic stains. Occasional atrophic Purkinje cells identified by Nissl stain were intensely immunostained. By confocal imaging analysis, abnormal hydrolase-laden Purkinje cell dendrites were seen coursing through some hydrolase-positive plaques and were continuous with dendritic branches that terminated within deposits of extracellular hydrolase and beta-amyloid. In the striatum, intensely immunostained abnormal-appearing neurons were commonly associated with extracellular deposits of hydrolase immunoreactivity and beta-amyloid within diffuse plaques and in the less commonly seen classical plaques. In both brain regions, other hydrolase-negative beta-amyloid deposits were seen, these being associated with blood vessels. The presence of HEX immunoreactivity in neurons, but not in glia, and its abundance in plaques support earlier studies, suggesting that neurons are the principal source of plaque hydrolase. An endosomal-lysosomal system upregulation, with increased hydrolase expression and extracellular enzyme deposition in plaques, is, like beta-amyloid deposition, an early marker of metabolic dysfunction potentially related to primary etiologic events in Alzheimer's disease and Down's syndrome.

Adult↗
PubMed2000

Localization of S-adenosylhomocysteine hydrolase in the rat kidney.

S-adenosylhomocysteine (SAH) hydrolase is a cytosolic enzyme present in the kidney. Enzyme activities of SAH hydrolase were measured in the kidney in isolated glomeruli and tubules. SAH hydrolase activity was 0.62 +/- 0.02 mU/mg in the kidney, 0.32 +/- 0.03 mU/mg in the glomeruli, and 0.50 +/- 0.02 mU/mg in isolated tubules. Using immunohistochemical methods, we describe the localization of the enzyme SAH hydrolase in rat kidney with a highly specific antibody raised in rabbits against purified SAH hydrolase from bovine kidney. This antibody crossreacts to almost the same extent with the SAH hydrolase from different species such as rat, pig, and human. Using light microscopy, SAH hydrolase was visualized by the biotin-streptavidin-alkaline phosphatase immunohistochemical procedure. SAH hydrolase immunostaining was observed in glomeruli and in the epithelium of the proximal and distal tubules. The collecting ducts of the cortex and medulla were homogeneously stained. By using double immunofluorescence staining and two-channel immunofluorescence confocal laser scanning microscopy, we differentiated the glomerular cells (endothelium, mesangium, podocytes) and found intensive staining of podocytes. Our results show that the enzyme SAH hydrolase is found ubiquitously in the rat kidney. The prominent staining of SAH hydrolase in the podocytes may reflect high rates of transmethylation. (J Histochem Cytochem 48:211-218, 2000)

Adenosylhomocysteinase↗
PubMed1996

Cyclosporin A down-regulates the LTA4 hydrolase level in human keratinocyte cultures.

Leukotriene A4 hydrolase is a key enzyme in the biosynthesis of leukotriene B4, a potent pro-inflammatory compound. The purpose of this study was to determine the capacity of antiinflammatory and anti-proliferative compounds to regulate the levels and activity of leukotriene A4 hydrolase in cultured human keratinocytes. The content of leukotriene A4 hydrolase was determined by Western blot analysis, and the activity of leukotriene A4 hydrolase was expressed as the leukotriene B4 formation after incubation of keratinocyte cultures with leukotriene A4. Leukotriene B4 was measured by revered-phase high performance liquid chromatography. Preincubation for 10 min of the cultured keratinocytes with the leukotriene A4 hydrolase inhibitor RP 64699 (0.1-10 microM) caused a significant dose-dependent inhibition of leukotriene B4 formation (IC50 = 0.7 microM). Cyclosporin A (0.1 micrograms/ml and 1.0 micrograms/ml) had no direct effect on leukotriene A4 hydrolase activity, but after incubation for 72 h there was a decrease in the mean leukotriene B4 formation per culture dish (35% and 48%, respectively). The decreased leukotriene B4 formation was caused mainly by a decrease in the mean leukotriene A4 hydrolase content per mg protein (30.1% at 0.1 micrograms/ml cyclosporin A and 40.0% at 1.0 micrograms/ml cyclosporin A), although keratinocyte proliferation was also slightly decreased. Incubations with 1.25-dihydroxyvitamin D3 (10(-7)-10(-10) M), all-trans retinoic acid (10(-6)-10(-10) M), eicosartienoic acid (10(-6)-10(-8) M), dexamethasone (10(-5)-10(-7) M), interferon-gamma (10 and 100 units/ml) or methotrexate (0.1-10 micrograms/ml) had no effect on either the leukotriene B4 formation or the amount of leukotriene A4 hydrolase in keratinocyte cultures. These results show that cyclosporin A, in contrast to other anti-inflammatory and anti-proliferative compounds, inhibits the level of leukotriene A4 hydrolase in keratinocyte cultures. Since similar cyclosporin A concentrations are obtained during treatment of psoriasis with cyclosporin A, the effect on leukotriene A4 hydrolase may play a role in the anti-inflammatory action of cyclosporin A.

8,11,14-Eicosatrienoic Acid↗
PubMed1981

Cytosolic and microsomal epoxide hydrolases are immunologically distinguishable from each other in the rat and mouse.

Antibodies raised to homogeneous rat liver microsomal epoxide hydrolase were used to distinguish microsomal epoxide hydrolase from epoxide hydrolase of cytosolic origin in mice and rats. Using double diffusion analysis in agarose gels, we show that anti-rat liver microsomal epoxide hydrolase forms a single precipitin line with solubilized microsomes from rat and mouse liver, but no reaction is seen with the corresponding cytosolic fractions. Rat or mouse microsomal epoxide hydrolase activity (using benzo[a]pyrene 4,5-oxide as substrate) can be completely precipitated out of solubilized preparations by the antibody, which is equipotent against rat and mouse microsomal epoxide hydrolase. No precipitation of cytosolic hydrolase activity (using trans-beta-ethyl styrene oxide as substrate) is seen with any concentration of the antibody tested. Thus, in the case of microsomal epoxide hydrolase, extensive immunological cross-reactivity exists between the two species, rat and mouse. In contrast, no cross-reactivity is detectable between cytosolic and microsomal epoxide hydrolase, even when enzymes from the same species are compared. We conclude that microsomal and cytosolic epoxide hydrolase activities represent distinct and immunologically non-cross-reactive protein species.

Animals↗
PubMed1982

Reactivation of S-adenosylhomocysteine hydrolase activity in cells exposed to 9-beta-D-arabinofuranosyladenine.

9-beta-D-Arabinofuranosyladenine (ara-A) inactivates isolated S-adenosyl-L-homocysteine (AdoHcy) hydrolase (EC 3.3.1.1) as well as AdoHcy hydrolase in intact cells. Whereas the inactivation in cell-free systems is an irreversible process, the AdoHcy hydrolase activity in rat hepatocytes exposed to ara-A gradually recovered upon prolonged incubation of the cells in a medium devoid of ara-A. This process, tentatively termed reactivation of the enzyme, was nearly totally dependent on a high level of adenosine deaminase in the extracellular medium, which induced a decrease in intracellular content of adenosine as well as ara-A. Reactivation of intracellular enzyme was inhibited by adenosine deaminase inhibitors [2'-deoxycoformycin and erythro-9-(2-hydroxy-3-nonyl)adenine] and the synthetic substrate for AdoHcy hydrolase, 3-deazaadenosine. An inhibitor of protein synthesis (cycloheximide) was without effect. Homocysteine, which protected the intracellular AdoHcy hydrolase against inactivation by ara-A, induced no reactivation of the enzyme. The half-life of the intracellular ara-A-AdoHcy hydrolase complex was about 90 min and was not affected by adenosine deaminase, 3-deazaadenosine, or homocysteine added to the cell suspension. However, the rate of elimination of the complex in the hepatocytes exceeded the rate of reactivation of AdoHcy hydrolase. Thus, the elimination process accounted for the reactivation, but not correlation between these two processes was observed. Reactivation of intracellular AdoHcy hydrolase caused a pronounced fall in cellular content of AdoHcy. The possibility that reduced cellular level of AdoHcy induced the reactivation of AdoHcy hydrolase seemed unlikely. This statement was based on the observation that reactivation was observed also under conditions of high concentrations of AdoHcy (obtained by the addition of homocysteine to the cell suspension). Reactivation of AdoHcy hydrolase with a concomitant decrease in cellular level of AdoHcy could also be demonstrated with mouse plasmacytoma (MPC-11) cells and mouse fibroblasts (L-929) exposed to ara-A, but the reactivation process was far less pronounced than with hepatocytes.

Adenosine Deaminase↗