The UPS locus encoding uroporphyrinogen I synthase is located on human chromosome 11

The expression of the UPS locus encoding uroporphyrinogen I synthase has been investigated in human/mouse somatic cell hybrids. Human and mouse uroporphyrinogen I synthase can be readily distinguished by their isoelectric points. In hybrid cells, both human and mouse isozymes are detected. The multiple human uroporphyrinogen I synthase isozymes segregate as a single unit, as expected if they are the products of a single locus. The absence of new heteropolymers in hybrid cells supports the biochemical evidence that the active enzyme is a monomer. The presence of human uroporphyrinogen I synthase in hybrid clones was correlated with the presence of human chromosome 11, or its enzymatic marker, without exception in 44 independent hybrid lines. All other chromosomes could be eliminated as possible locations for this locus, due to their independent segregation. This report represents the first gene assignment for an enzyme in the heme biosynthesis pathway.     

Purification and characterization of human uroporphyrinogen III synthase expressed in Escherichia coli

The side-chain asymmetry of physiological porphyrins is produced by the cooperative action of hydroxymethylbilane synthase and uroporphyrinogen (uro'gen) III synthase. Although the role of uro'gen III synthase is essential for the chemistry of porphyrin biosynthesis, many aspects, structural as well as mechanical, of uro'gen III synthase have yet to be studied. We report here an expression system in Escherichia coli and a purification procedure for human uro'gen III synthase. The enzyme in the lysate was unstable, but we found that glycerol prevents the activity loss in the lysate. The purified enzyme showed remarkable thermostability, particularly when kept in phosphate buffer containing DTT or EDTA, indicating that the enzyme activity may depend on its oxidation state. Examination of the relationship between the number of Cys residues that are accessible to 5,5'-dithiobis(2-nitrobenzoic acid) and the remaining activity during heat inactivation showed that a particular Cys residue is involved in activity loss. From the crystal structure of human uro'gen III synthase [Mathews et al. (2001) EMBO J. 20, 5832-5839], this Cys residue was considered to be Cys73, which is buried deep inside the enzyme, suggesting that Cys73 of human uro'gen III synthase plays an important role in enzyme activity.     

Localization of the ornithine aminotransferase gene and related sequences on two human chromosomes

Summary We have used a full length cDNA clone to determine the chromosomal location ofthegene encoding human ornithine aminotransferase (OAT), a mitochondrial matrix enzyme. Southern blot analysis of ScaI-digested DNA from 34 human-mouse somatic cell hybrids revealed 11 human fragments. Three fragments mapped to chromosome 10q23-10qter, confirming the previous provisional assignment of the functional gene to this autosome by analysis of OAT expression in somatic cell hybrids (O'Donnell et al. 1985). The remaining eight fragments were assigned to the X chromosome, and regionally assigned to Xp21-Xp11 by use of an X-chromosome mapping panel. These X chromosome sequences could represent pseudogenes, or related members of a multigene family. Two of the X chromosome fragments are alternate alleles of a restriction fragment length polymorphism (RFLP) making this OAT-related locus an excellent genetic marker. The RFLP may now be used to determine any possible relationship between this locus and several X-linked eye defects.     

Genetic and biochemical characterization of mutants of Saccharomyces cerevisiae blocked in six different steps of heme biosynthesis

Summary Heme-deficient mutants of Saccharomyces cerevisiae have been isolated from two isogenic strains with the use of an enrichment method based on photodynamic properties of Zn-protoporphyrin. They defined seven non-overlapping complementation groups. A mutant representative of each group was further analysed. Genetic analysis showed that each mutant carried a single nuclear recessive mutation. Biochemical studies showed that the observed accumulation and/or excretion of the different heme synthesis precursors by the mutant cells correlated well with the enzymatic deficiencies measured in acellular extracts. Six of the seven mutants were blocked in a different enzyme activity: 5-aminolevulinate synthase, porphobilinogen synthase, uroporphyrinogen I synthase, uroporphyrinogen decarboxylase, coproporphyrinogen III oxidase and ferrochelatase. The other mutant had the same phenotype as the mutant deficient in ferrochelatase activity. However, it possessed a normal ferrochelatase activity when measured in vitro, so this mutant was assumed to be deficient in protoporphyrinogen oxidase activity or in the transport and/or reduction of iron.The absence of PBG synthesis led to a total lack of uroporphyrinogen I synthase activity. The absence of heme, the end product, led to an important increase of coproporphyrinogen III oxidase activity, while the activity of 5-aminolevulinate synthase, the first enzyme of the pathway, was not changed. These results are discussed in terms of possible modes of regulation of heme synthesis pathway in yeast.     

Inherited pericentric inversion of chromosome no. 2 with Robertsonian translocation (13q 14q) resulting in trisomy for chromosome 13q

This report includes a patient with an inherited pericentric inversion of chromosome No. 2 in addition to a Robertsonian translocation resulting in trisomy for chromosome 13q. The chromosomal constitution of the proband was 46,XX,inv(2) (pter leads to p11 : : q14 leads to p11 : : q14 leads to qter); t(13,14) (13qter leads to 13p11 : : 14q11 leads to 14qter). Sequential QFQ, RFA and GTG banding techniques were employed on the chromosomes of all family members. The chromosomal constitutions of the father and his first child were normal while the mother had an inversion of chromosome No. 2 [46,XX,inv(2) (pter leads to p11 : : q14 leads to p11 : : q14 leads to qter)]. The proband inherited this abnormal chromosome. In addition, she had a de novo Robertsonian translocation involving chromosomes 13q and 14q resulting in trisomy of chromosome 13q.     

Partial trisomy 11q syndrome (11q23.1-->11qter) due to de novo t (11q; 13q) detected by multicolor fluorescence in situ hybridisation

In this report we describe the identification of a de novo 46, XX, 13q + by multicolour fluorescence in situ hybridisation (M-FISH), as a partial distal 11q trisomy (11q23.1-->11qter). The clinical phenotype association with this distal 11q trisomy is briefly reviewed.     

On uroporphyrinogen formation: Studies with 1-aminomethyl-3, 8, 13, 18-tetra(2-carboxyethyl)-2, 7, 12, 17-tetracarboxymethylbilinogen (author's transl)]

The preparation of the aminomethyl-bilinogen which results from formal "head to tail" condensation of porphobilinogen is described. The chemical cyclocondensation of this compound at pH 7.4 yields uroporphyrinogen I. Enzymatic studies with enzyme preparations from Propionibacterium shermanii, which synthesize uroporphyrinogens from porphobilinogen, show that the rate of cyclisation is increased by these enzymes and indicate that the bilinogen also might be used for uroporphyrinogen III formation. This is also suggested by studies on the formation of cobyrinic acid from [4-14C]5-aminolevulinate via uroporphyrinogen III in the presence of the aminomethylbilinogen by cell-free extracts from Clostridium tetanomorphum.     

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.     

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.     

Mapping of the human gene for epidermal growth factor receptor (EGFR) on the p13 leads to q22 region of chromosome 7

We previously reported that the structural gene for epidermal growth factor receptor (EGFR) can be mapped to the p22 leads to qter region of human chromosome 7 (Shimizu et al., 1979, 1980). In the present study, we produced two series of human-mouse cell hybrids by fusing mouse A9 cells that are deficient in EGFR with the human diploid fibroblast lines GM1356, 46,XX,t(1;7)(p34;p13), and GM2068, 46,XX,t(6;7)(q27;q22), both of which possess EGF receptors. Expression of EGF binding ability in the former series of cell hybrids was correlated with the retention of the human translocation chromosome containing the 7p13 leads to qter region, and in the latter series of cell hybrid it was correlated with the retention of the human translocation chromosome containing the 7pter leads to q22 region. Therefore, the EGFR gene can be localized in the p13 leads to q22 region of chromosome 7.     

Direct gene dosage determination in patients with unbalanced chromosomal aberrations using cloned DNA sequences. Application to the regional assignment of the gene for alpha 2(I) procollagen (COLIA2).

We describe a new method of direct gene dosage determination in patients with unbalanced chromosomal aberrations using cloned DNA sequences: the intensity of the signal obtained by hybridization of the radioactive probe to the corresponding DNA fragments can be compared with the intensity of the DNA fragments that hybridize with a nonsyntenic probe used as an internal control. This has been demonstrated by densitometer tracing of the autoradiogram, using an X-specific DNA sequence, beta globin and alpha 2(I) collagen probes, in normal men and women, in one patient trisomic for 11p, and in one patient trisomic for segment 7q21 leads to 7qter. The ratio men/women for the X-specific sequence (DXS) was close to the expected value 0.5, while the ratio trisomy 11/normal control and trisomy 7/normal control were close to 1.5 for beta globin (HBB) and alpha 2(I) collagen (COLIA2), respectively. The gene coding for COLIA2 can therefore be assigned to 7q21 leads to 7qter. This method should also apply to noncoding sequences: the increasing number of cloned DNA segments that have already been assigned to a specific chromosome represent a new tool for prenatal and premorbid diagnosis of unbalanced chromosomal aberrations.     

Reduced substrate affinity for human erythrocyte uroporphyrinogen decarboxylase constitutes the inherent biochemical defect in porphyria cutanea tarda

The pathogenesis of human porphyria cutanea tarda (PCT) is associated with an intrinsic abnormality of the uroporphyrinogen decarboxylase enzyme. To characterize this, we studied the kinetic properties of the red cell enzyme procured from patients with various forms of PCT and non-porphyric controls. The enzyme activity (units/mg hemoglobin) in the red cell hemolysate was close to normal in sporadic PCT but about 75% diminished in the familial PCT. The Michaelis constants (Km) of 200-fold purified red cell enzyme preparations, determined by using pentacarboxylic porphyrinogen I and uroporphyrinogen I as substrates, were more than 3.8-4.0 times higher, and the maximum velocity (Vmax) was about 70% diminished in familial PCT, whereas the Km was about 1.7-1.9 times higher and the Vmax was more or less normal for sporadic PCT. These observations suggest for the first time that the primary lesion in familial PCT is a genetically determined kinetic abnormality of uroporphyrinogen decarboxylase which appears to be different from the sporadic form of the disease.     

Uroporphyrinogen-I-synthetase in erythrocytes in acute intermittent porphyria

The primary genetic defect in acute intermittent porphyria is a decreased uroporphyrinogen I-synthetase [EC.4.3.1.8] activity. As a beginning of a genealogical study of the known families with members suffering from this disease in the People's Republic of Bulgaria, the red cell uroporphyrinogen I-synthetase was determined in 3 families by the method of Mandel et al [8]. Except for the three propositi, an enzyme deficiency was established in 3 latent carriers of the pathological gene, two of whom had normal values of the urinary epsilon-aminolevulinic acid and porphobilinogen. The determination of red cell uroporphyrinogen I-synthetase proved to be a valuable parameter for revealing the latent AIP.     

Unique properties of Plasmodium falciparum porphobilinogen deaminase

The hybrid pathway for heme biosynthesis in the malarial parasite proposes the involvement of parasite genome-coded enzymes of the pathway localized in different compartments such as apicoplast, mitochondria, and cytosol. However, knowledge on the functionality and localization of many of these enzymes is not available. In this study, we demonstrate that porphobilinogen deaminase encoded by the Plasmodium falciparum genome (PfPBGD) has several unique biochemical properties. Studies carried out with PfPBGD partially purified from parasite membrane fraction, as well as recombinant PfPBGD lacking N-terminal 64 amino acids expressed and purified from Escherichia coli cells (DeltaPfPBGD), indicate that both the proteins are catalytically active. Surprisingly, PfPBGD catalyzes the conversion of porphobilinogen to uroporphyrinogen III (UROGEN III), indicating that it also possesses uroporphyrinogen III synthase (UROS) activity, catalyzing the next step. This obviates the necessity to have a separate gene for UROS that has not been so far annotated in the parasite genome. Interestingly, DeltaPfP-BGD gives rise to UROGEN III even after heat treatment, although UROS from other sources is known to be heat-sensitive. Based on the analysis of active site residues, a DeltaPfPBGDL116K mutant enzyme was created and the specific activity of this recombinant mutant enzyme is 5-fold higher than DeltaPfPBGD. More interestingly, DeltaPfPBGDL116K catalyzes the formation of uroporphyrinogen I (UROGEN I) in addition to UROGEN III, indicating that with increased PBGD activity the UROS activity of PBGD may perhaps become rate-limiting, thus leading to non-enzymatic cyclization of preuroporphyrinogen to UROGEN I. PfPBGD is localized to the apicoplast and is catalytically very inefficient compared with the host red cell enzyme.     

Rat hepatic uroporphyrinogen III co-synthase. Purification and evidence for a bound folate coenzyme participating in the biosynthesis of uroporphyrinogen III.

Rat hepatic uroporphyrinogen III co-synthase was isolated and purified 73-fold with a 13% yield by (NH4)2SO4 fractionation and sequential chromatography on DEAE-Sephacel, Sephadex G-100 (superfine grade) and folate-AH-Sepharose 4B. The purified co-synthase has an Mr of approx. 42 000, and is resolved into two bands, each possessing co-synthase activity, by polyacrylamide-gel electrophoresis. A factor was dissociated from the purified co-synthase. Results of both microbiological and competitive protein-binding assays suggest that it is a pteroylpolyglutamate. The isolated pteroylpolyglutamate factor was co-eluted with authentic N5-methyltetrahydropteroylheptaglutamate on DEAE-Sephacel. Uroporphyrinogen III is formed by cosynthase-free preparations of uroporphyrinogen I synthase in the presence of tetrahydropteroylglutamate. Tetrahydropeteroylheptaglutamate is also able to direct the formation of equivalent amounts of uroporphyrinogen III at a concentration approximately one-hundredth that of tetrahydropteroylmonoglutamate. These results suggest that a reduced pteroylpolyglutamate factor is associated with rat hepatic uroporphyrinogen III co-synthase, and that this may function as a coenzyme for the biosynthesis of uroporphyrinogen III.     

Rat hepatic uroporphyrinogen III cosynthase: Purification,properties, and inhibition by metal ions

Rat hepatic uroporphyrinogen III cosynthase has been isolated and purified 50-fold with a 36% yield by ammonium sulfate fractionation and sequential chromatography on DEAE-Sephacel and Sephadex G-100SF. Inhibition of uroporphyrinogen III formation with increasing porphobilinogen concentration was observed. Cosynthase was shown to be thermolabile, and a time-dependent loss of enzyme activity during reaction with uroporphyrinogen I synthase and porphobilinogen was observed. The pH optimum for the complete system (synthase and cosynthase) was pH 7.8 in 50 mm Tris-HCl or 50 mm sodium phosphate buffer. Various metals (KCl, NaCl, MgCl₂, CaCl₂) increased formation of Uroporphyrinogen III. Heavy metals including ZnCl₂, CdCl₂, and CuCl₂ were shown to selectively inhibit cosynthase activity, whereas other metals (HgCl₂, PbCl₂) were less selective and inhibited both synthase and cosynthase at similar concentrations.     

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...     

Identification and characterization of the Arabidopsis gene encoding the tetrapyrrole biosynthesis enzyme uroporphyrinogen III synthase

UROS (uroporphyrinogen III synthase; EC 4.2.1.75) is the enzyme responsible for the formation of uroporphyrinogen III, the precursor of all cellular tetrapyrroles including haem, chlorophyll and bilins. Although UROS genes have been cloned from many organisms, the level of sequence conservation between them is low, making sequence similarity searches difficult. As an alternative approach to identify the UROS gene from plants, we used functional complementation, since this does not require conservation of primary sequence. A mutant of Saccharomyces cerevisiae was constructed in which the HEM4 gene encoding UROS was deleted. This mutant was transformed with an Arabidopsis thaliana cDNA library in a yeast expression vector and two colonies were obtained that could grow in the absence of haem. The rescuing plasmids encoded an ORF (open reading frame) of 321 amino acids which, when subcloned into an Escherichia coli expression vector, was able to complement an E. coli hemD mutant defective in UROS. Final proof that the ORF encoded UROS came from the fact that the recombinant protein expressed with an N-terminal histidine-tag was found to have UROS activity. Comparison of the sequence of AtUROS (A. thaliana UROS) with the human enzyme found that the seven invariant residues previously identified were conserved, including three shown to be important for enzyme activity. Furthermore, a structure-based homology search of the protein database with AtUROS identified the human crystal structure. AtUROS has an N-terminal extension compared with orthologues from other organisms, suggesting that this might act as a targeting sequence. The precursor protein of 34 kDa translated in vitro was imported into isolated chloroplasts and processed to the mature size of 29 kDa. Confocal microscopy of plant cells transiently expressing a fusion protein of AtUROS with GFP (green fluorescent protein) confirmed that AtUROS was targeted exclusively to chloroplasts in vivo.