Uroporphyrinogen synthase in human blood: developmental studies.

Uroporphyrinogen synthase (URO-S), the enzyme that catalyzes the conversion of porphobilinogen to uroporphyrinogen I, has been measured in whole blood lysates by a fluorometric microassay. Cord and fetal bloods have 3 and 6 times the specific activity, respectively, of adult control subjects. The three groups seem to present a similar genetic heterogenity with ratios of highest to lowest URO-S specific activity close to 2. These results establish normal ranges for URO-S activity in human blood, which may be useful for the early detection of carriers of a gene for acute intermittent porphyria.     

Simultaneous determination of hydroxymethylbilane synthase and uroporphyrinogen III synthase in erythrocytes by high-performance liquid chromatography.

A high-performance-liquid-chromatographic method is developed for the simultaneous determination of hydroxymethylbilane synthase and uroporphyrinogen III synthase activity in erythrocytes. Effective separation of uroporphyrin I and III isomers allows the accurate quantification of individual isomers and the total uroporphyrin concentration. Total uroporphyrin production is used to calculate hydroxymethylbilane synthase activity, and the amount of uroporphyrin III formed represents the activity of uroporphyrinogen III synthase. Normal ranges are established for the two enzymes.     

Purification and properties of uroporphyrinogen III synthase from human erythrocytes

Uroporphyrinogen III synthase (hydroxymethylbilane hydro-lyase (cyclizing); EC 4.2.1.75), the fourth enzyme in the heme biosynthetic pathway, was purified to homogeneity from human erythrocytes. For enzyme purification and characterization, a sensitive coupled enzyme assay was used which generated the substrate, hydroxymethylbilane; the oxidized product, uroporphyrin III, was quantitated by high pressure liquid chromatography. Uroporphyrinogen III synthase was initially separated from delta-aminolevulinate dehydratase and hydroxymethylbilane synthase by a preparative anion exchange chromatographic step. Subsequent chromatography on hydroxyapatite, phenyl-Sepharose, and Sephadex G-100 purified the enzyme about 70,000-fold with an 8% yield. Homogeneous enzyme was obtained following a final C4-reversed phase high pressure liquid chromatographic step which removed a single major and several minor protein contaminants from the enzyme. The purified enzyme had a specific activity of over 300,000 units/mg, an isoelectric point of 5.5, and was thermolabile (t1/2 at 60 degrees C approximately 1 min). Molecular weight studies by gel filtration (Mr approximately equal to 30,000) and analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr approximately equal to 29,500) were consistent with the enzyme being a monomer. Using hydroxymethylbilane as substrate, the purified enzyme formed uroporphyrinogen III in the absence of hydroxymethylbilane synthase or other cofactors. The pH optimum was 7.4 and the Km for hydroxymethylbilane was 5-20 microM. The enzyme was activated by Na+, K+, Mg+, and Ca2+ and was inhibited by Cd2+, Cu2+, Hg2+, and Zn2+. Amino acid composition analysis was performed, and the N-terminal sequence, Met-Lys-Val-Leu-Leu-Leu, was determined by microsequencing. The availability of the purified enzyme should permit investigation of its reaction mechanism as well as facilitate biochemical and molecular studies of the genetic defect in congenital erythropoietic porphyria.     

A simple rapid purification scheme for hydroxymethylbilane synthase from human erythrocytes.

Hydroxymethylbilane synthase from human erythrocytes was purified 47,000-fold to greater than 95% homogeneity and 7.5% yield by a simple and rapid procedure using heat treatment (80 degrees C, in the presence of proteinase inhibitors, to convert one of two chromatographically separable forms into the other), DEAE-cellulose and Cibacron Blue F3G-A-Sepharose chromatographies and Sephadex G-75 gel filtration. The purified enzyme was similar to the enzyme purified from other species in showing hyperbolic dependence of velocity on substrate concentration, a non-linear progress curve for uroporphyrinogen appearance, and was monomeric, having an Mr of 44,000 by gel filtration on Sephadex G-100 and h.p.l.c. and an Mr of 45,000 on SDS/polyacrylamide-gel electrophoresis. The enzyme showed a sharp pH profile for Vmax, and various folates were shown to accelerate neither the enzymic formation of hydroxymethylbilane nor ring-closure of hydroxymethylbilane.     

Porphobilinogen deaminase and uroporphyrinogen III synthase: Structure,molecular biology,and mechanism

Porphobilinogen deaminase (hydroxymethylbilane synthase) and uroporphyrinogen III synthase (uroporphyrinogen III cosynthase) catalyze the transformation of four molecules of porphobilinogen, via the 1-hydroxymethylbilane, preuroporphyrinogen, into uroporphyrinogen III. A combination of studies involving protein chemistry, molecular biology, site-directed mutagenesis, and the use of chemically synthesized substrate analogs and inhibitors is helping to unravel the complex mechanisms by which the two enzymes function. The determination of the X-ray structure ofE. coli porphobilinogen deaminase at 1.76 Å resolution has provided the springboard for the design of further experiments to elucidate the precise mechanism for the assembly of both the dipyrromethane cofactor and the tetrapyrrole chain. The human deaminase structure has been modeled from theE. coli structure and has led to a molecular explanation for the disease acute intermittent porphyria. Molecular modeling has also been employed to simulate the spiro-mechanism of uroporphyrinogen III synthase.     

Inhibition of uroporphyrinogen I synthase activity and depression of microsomal heme and cytochrome P-450 in rat liver by bilirubin

Purified rat hepatic uroporphyrinogen (UROgen) I synthase (URO-S) was inhibited by bilirubin or the ditaurine derivative. Inhibition was reversible and non-competitive to the substrate porphobilinogen (PBG). The inhibition constants (Ki values) for bilirubin and the conjugate were 1.5 microM and 0.26 microM respectively. Rats afflicted with hyperbilirubinemia caused by biliary obstruction had decreased levels of hepatic microsomal heme (58% of control) and cytochrome P-450 (60% of control) at day 3. Hepatic delta-aminolevulinic acid synthetase (ALAS) activity was increased (39% of control) at day 3.     

Cell-based bioluminescent assays for all three proteasome activities in a homogeneous format

A luminescent method to individually measure the chymotrypsin-like, trypsin-like, or caspase-like activities of the proteasome in cultured cells was developed. Each assay uses a specific luminogenic peptide substrate in a buffer optimized for cell permeabilization, proteasome activity, and luciferase activity. Luminescence is generated in a coupled-enzyme format in which proteasome cleavage of the peptide conjugated substrate generates aminoluciferin, which is a substrate for luciferase. The homogeneous method eliminates the need to prepare individual cell extracts as samples. Luminogenic proteasome substrates and buffer formulations enabled development of a single reagent addition method with adequate sensitivity for 96- and 384-well plate formats. Proteasome trypsin-like specificity was enhanced by incorporating a mixture of protease inhibitors that significantly reduce nonspecific serum and cellular backgrounds. The assays were used to determine EC₅₀ values for the specific proteasome inhibitors epoxomicin and bortezomib for each of the catalytic sites using a variety of cancer lines. These cell-based proteasome assays are direct, simple, and sensitive, making them ideal for high-throughput screening.     

Determination of δ-aminolevulinate dehydratase activity by a specific fluorometric coupled-enzyme assay

A coupled-enzyme assay for the specific and sensitive determination of δ-aminolevulinate dehydratase activity has been developed. The assay specifically measured picomole quantities of the product, porphobilinogen, by its enzymatic conversion to uroporphyrinogen I and the fluorometric detection of oxidized uroporphyrin I. The coupled-enzyme assay was linear with time and protein concentration and required less than 3 h for 20 individual determinations. Under the standard assay conditions, 10 to 100 pmol of uroporphyrin I was reliably measured, representing 0.085 to 0.850 nmol/h of δ-aminolevulinate dehydratase activity per assay. In addition, the fluorometric assay was more sensitive than either the standard or the semimicro colorimetric methods. The specificity, rapidity, and sensitivity of this new fluorometric method facilitates the reliable determination of low levels of aminolevulinate dehydratase activity in small amounts of crude tissue homogenates or in cultured cells.     

Rapid determination of δ-aminolevulinate synthase activity by a specific fluorometric coupled enzyme assay

The rapid and specific determination of picomole quantities of δ-aminolevulinate has been accomplished by its specific enzymatic conversion to uroporphyrinogen I and fluorometric detection of the oxidized uroporphyrin I. The coupled enzyme assay was linear with time and protein concentration and required less than 3 h for 25 individual determinations. Under the standard assay conditions, 1 to 100 pmol of uroporphyrin I was reliably quantitated; these values corresponded to a range of ALA synthase activities from 0.15 to 15 nmol/h/ml of enzyme. The sensitivity of this method was comparable to the more time-consuming radiochemical determinations of ALA synthase. In addition, this method was at least 10 times more sensitive than the colorimetric assays for ALA synthase activity. The rapidity, specificity, and sensitivity of this new method make it useful for monitoring the purification of ALA synthase and for reliable determinations of low levels of ALA synthase activity in crude tissue or cultured cell homogenates.     

先天性红细胞生成性卟啉症患者酶活性的研究

先天性红细胞生成性卟啉症（ｃｏｎｇｅｎｉｔａｌｅｒｙ－ｔｈｒｏｐｏｉｅｔｉｃｐｏｒｐｈｙｒｉａ,ＣＥＰ）是Ｇｕｎｔｈｅｒ于１９１１年首先提出并加以描述,有时亦称Ｇｕｎｔｈｅｒ病．该病是因遗传性缺陷所致卟啉代谢中有关酶的异常造成的卟啉代谢紊乱而发生的一...     

Immobilized uroporphyrinogen I synthetase from Rhodopseudomonas palustris

Rhodopseudomonas palustris uroporphyrinogen I synthetase (URO-S) has been chemically attached to Sepharose 4B and some of its properties have been studied. When 7-8 mg protein/ml activated Sepharose was used, immobilized URO-S retained 45% of the activity of the original soluble preparation, with a coupling yield of 66% after a period of 15 h. Optimal incubation conditions for the activity of gel-enzyme were determined. Unlike the soluble enzyme, the Sepharose-bound URO-S showed a biphasic substrate saturation curve, indicating that a protein conformational change had occurred during the process of immobilization. Immobilized URO-S stored at 4 degrees C for 35 days retained 90% of activity and when repeatedly used, up to 5 times, retained 48% of the original activity. Attachment of URO-S to Sepharose led to an enhanced thermal stability.     

Inhibition of the biosynthesis of uroporphyrinogen and heme in rat liver during obstructive jaundice produced by bile duct ligation

Altered hepatic microsomal drug metabolism has been reported to occur in afflicted with hyperbilirubinemia. Similarities of the chemical structures of hydroxymethylbilane, an intermediate in the biosynthesis of uroporphyrinogen, to bilirubin prompted investigations of the effect of bilirubin on the activity of uroporphyrinogen I synthase (porphobilinogen deaminase, EC 4.3.1.8) and the biosynthesis of heme. Bilirubin was found to be a reversible, noncompetitive inhibitor of uroporphyrinogen I synthase. The inhibition constant (Ki) for bilirubin was 1.5 microM. Bile acids had no effect on rat hepatic uroporphyrinogen I synthase activity. Hyperbilirubinemia was achieved in rats by biliary ligation in order to investigate whether elevated levels of bilirubin impair the biosynthesis of hepatic heme in vivo. The relative rate of heme biosynthesis, as measured by the rate of incorporation of delta-[4-14C]aminolevulinic acid into heme, was decreased 59% 24 h after biliary obstruction. The levels of hepatic microsomal heme and cytochrome P-450 were decreased by 43 and 40%, respectively, 72 h after biliary obstruction. The activities of hepatic delta-aminolevulinic acid synthase and uroporphyrinogen I synthase were increased by 39 and 46%, respectively, 72 h after biliary obstruction. During the 48- to 72-h period following biliary obstruction, the urinary excretion of porphobilinogen and uroporphyrin was increased 3.0- and 3.5-fold, respectively, whereas, the urinary excretion of delta-aminolevulinic acid was not altered. During this 48-to 72-h time interval following biliary obstruction, 100% of the uroporphyrin was excreted as isomer I. These results indicate that bilirubin is capable of depressing the biosynthesis of rat hepatic heme and thus cytochrome P-450-mediated drug metabolism by inhibition of the formation of uroporphyrinogen. These findings are a plausible mechanism for reports of impaired clearance of various drugs in patients afflicted with hyperbilirubinemic disease states.     

An HPLC-Based Assay of Adenylosuccinate Lyase in Erythrocytes

ADSL deficiency is a disorder of purine metabolism with a broad clinical spectrum. A rapid and simple HPLC-based assay to measure ADSL activity in erythrocytes was developed. The suitability of DBSs was assessed. ADSL activity was measured in erythrocyte lysates and DBS using succinyl-AMP as the substrate. Detection and quantification were performed using isocratic ion-pairing reversed-phase HPLC with UV-detection. Reference values in erythrocyte lysates were established. The intra- and interassay variations were 2% and 8%, respectively. ADSL deficiency was easily recognized. ADSL activity in DBS was highly unstable, disqualifying DBS for diagnostic procedures.     

An HPLC-based assay of adenylosuccinate lyase in erythrocytes

ADSL deficiency is a disorder of purine metabolism with a broad clinical spectrum. A rapid and simple HPLC-based assay to measure ADSL activity in erythrocytes was developed. The suitability of DBSs was assessed. ADSL activity was measured in erythrocyte lysates and DBS using succinyl-AMP as the substrate. Detection and quantification were performed using isocratic ion-pairing reversed-phase HPLC with UV-detection. Reference values in erythrocyte lysates were established. The intra- and interassay variations were 2% and 8%, respectively. ADSL deficiency was easily recognized. ADSL activity in DBS was highly unstable, disqualifying DBS for diagnostic procedures.     

Separation of E. coli tRNA-Tyr precursor RNA from E. coli tRNA by hydroxyapatite chromatography

An assay procedure is described for triosephosphate isomerase based on measurement of the ellipticity of l-glyceraldehyde 3-phosphate remaining when d,l-glyceraldehyde 3-phosphate is the source of substrate and d-glyceraldehyde 3-phosphate is converted by triosephosphate isomerase to dihydroxyacetone phosphate. The assay method has advantages over the conventional coupled-enzyme assays in that it circumvents the difficulties posed by instability of the coupling enzymes and their cofactors, as well as by inhibitors of triosephosphate isomerase which may be present in preparations of the coupling enzymes. Although the method is not suited for routine assays during purification or in most clinical applications, it has advantages for detailed kinetic studies where pH, temperature, or other factors cause the coupled-enzyme assay procedures to be unreliable.     

Quantitative detection of blood-brain barrier-associated enzymes in cultured endothelial cells of procine brain microvessels

Summary The present study deals with a rapid and convenient assay for blood-brain barrier (BBB)-associated enzymes, γ-glutamyl transpeptidase (γ-GTP) and alkaline phosphatase (ALP), in cultured endothelial cells and other cells. These enzyme activities in cultured cells could be efficiently measured by direct incubation of each substrate in the culture plates without pretreatment of the cells. This new direct in situ-in plate assay was more rapid and convenient than conventional ex-plate assays, and these assays gave similar values for specific enzyme activities. γ-GTP and ALP activities could be detected by this in situ method in primary-cultured endothelial cells of porcine brain microvessels, but their levels were lower than those before culture. The degree of loss due to culture differed, between γ-GTP and ALP; a relatively large amount of ALP remained but the γ-GTP level decreased greatly In this direct in situ-in plate assay, cultured porcine aortic endothelial cells exhibited negligibly small activities for both enzymes, whereas cultured astroglial cells of neonatal porcine brain showed moderate γ-GTP activity and a trace of ALP activity. This direct in situ-in plate assay can be used for microculture and automatic measurement and offers a convenient means for studying the possible regulatory mechanisms of the expression of the BBB-associated enzymes.     

The common origins of the pigments of life—early steps of chlorophyll biosynthesis

The complex pathway of tetrapyrrole biosynthesis can be dissected into five sections: the pathways that produce 5-aminolevulinate (the C-4 and the C-5 pathways), the steps that transform ALA to uroporphyrinogen III, which are ubiquitous in the biosynthesis of all tetrapyrroles, and the three branches producing specialized end products. These end products include corrins and siroheme, chlorophylls and hemes and linear tetrapyrroles. These branches have been subjects of recent reviews. This review concentrates on the early steps leading up to uroporphyrinogen III formation which have been investigated intensively in recent years in animals, in plants, and in a wide range of bacteria.Abbreviations ALA 5-aminolevulinic acid - ALAS 5-aminolevulinic acid synthase - GR glutamyl-tRNA reductase - GSA glutamate-1-semialdehyde - GSAT glutamate-1-semialdehyde aminotransferase - HMB hydroxymethylbilane - PBG porphobilinogen - PBGD porphobilinogen deaminase - PBGS porphobilinogen synthase - URO uroporphyrin - URO'gen uroporphyrinogen - US uroporphyrinogen III synthase     

Effects of polychlorinated biphenyl compounds, 2,3,7,8-tetrachlorodibenzo-p-dioxin, phenobarbital and iron on hepatic uroporphyrinogen decarboxylase. Implications for the pathogenesis of porphyria.

Treatment of cultured chick embryo hepatocytes with phenobarbital, polychlorinated biphenyl compounds and 2,3,7,8-tetrachlorodibenzo-p-dioxin resulted in increased delta-aminolaevulinate synthase and decreased uroporphyrinogen decarboxylase activities and porphyrin accumulation; uroporphyrin and heptacarboxyporphyrin predominated. Iron had no effect on these changes. Simultaneous treatment of cultures with dioxin and phenobarbital produced a synergistic response in delta-aminolaevulinate synthase induction, uroporphyrinogen decarboxylase inhibition and porphyrin accumulation. These data suggest that an inhibitor of uroporphyrinogen decarboxylase may be generated in the liver from polychlorinated biphenyl compounds or dioxin by metabolic activation. Additionally these findings bear on the postulated role of these and related chemicals in determining the low levels of uroporphyrinogen decarboxylase activity in porphyria cutanea tarda patients.     

Structural diversity in metal ion chelation and the structure of uroporphyrinogen III synthase

All tetrapyrroles are synthesized through a branched pathway, and although each tetrapyrrole receives unique modifications around the ring periphery, they all share the unifying feature of a central metal ion. Each pathway maintains a unique metal ion chelatase, and several tertiary structures have been determined, including those of the protoporphyrin ferrochelatase from both human and Bacillus subtilus, and the cobalt chelatase CbiK. These enzymes exhibit strong structural similarity and appear to function by a similar mechanism. Met8p, from Saccharomyces cerevisiae, catalyses ferrochelation during the synthesis of sirohaem, and the structure reveals a novel chelatase architecture whereby both ferrochelation and NAD(+)-dependent dehydrogenation take place in a single bifunctional active site. Asp-141 appears to participate in both catalytic reactions. The final common biosynthetic step in tetrapyrrole biosynthesis is the generation of uroporphyrinogen by uroporphyrinogen III synthase, whereby the D ring of hydroxymethylbilane is flipped during ring closure to generate the asymmetrical structure of uroporphyrinogen III. The recently derived structure of uroporphyrinogen III synthase reveals a bi-lobed structure in which the active site lies between the domains.     

Molecular and tissue-specific heterogeneity in HPRT deficiency

In several patients with different degrees of HPRT deficiencies, residual activities have been determined in both lysed and intact erythrocytes. No close correlation could be found between the degree of HPRT deficiency and the severity of the clinical expression. Unless HPRT activity in both intact and lysed erythrocytes was below detection level, the residual activity in intact red blood cells was higher than in lysates. Tissue-specific heterogeneity was illustrated with a patient suffering from X-linked gout. Lysates from erythrocytes, leukocytes, and cultured fibroblasts showed 1%, 8%, and 100% of normal HPRT activity, respectively. Characterization of the erythrocyte and fibroblast HPRT from this patient showed no kinetic abnormalities. However, there was a decreased heat stability. It is concluded that for a better understanding of the pathophysiology in HPRT deficiency studies on nucleated cells from the different tissues are needed.