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1.
Genetic basis of inosine triphosphate pyrophosphohydrolase deficiency 总被引:12,自引:0,他引:12
Sumi S Marinaki AM Arenas M Fairbanks L Shobowale-Bakre M Rees DC Thein SL Ansari A Sanderson J De Abreu RA Simmonds HA Duley JA 《Human genetics》2002,111(4-5):360-367
Inosine triphosphate pyrophosphohydrolase (ITPase) deficiency is a common inherited condition characterized by the abnormal accumulation of inosine triphosphate (ITP) in erythrocytes. The genetic basis and pathological consequences of ITPase deficiency are unknown. We have characterized the genomic structure of the ITPA gene, showing that it has eight exons. Five single nucleotide polymorphisms were identified, three silent (138G-->A, 561G-->A, 708G-->A) and two associated with ITPase deficiency (94C-->A, IVS2+21A-->C). Homozygotes for the 94C-->A missense mutation (Pro32 to Thr) had zero erythrocyte ITPase activity, whereas 94C-->A heterozygotes averaged 22.5% of the control mean, a level of activity consistent with impaired subunit association of a dimeric enzyme. ITPase activity of IVS2+21A-->C homozygotes averaged 60% of the control mean. In order to explore further the relationship between mutations and enzyme activity, we examined the association between genotype and ITPase activity in 100 healthy controls. Ten subjects were heterozygous for 94C-->A (allele frequency: 0.06), 24 were heterozygotes for IVS2+21A-->C (allele frequency: 0.13) and two were compound heterozygous for these mutations. The activities of IVS2+21A-->C heterozygotes and 94C-->A/IVS2+21A-->C compound heterozygotes were 60% and 10%, respectively, of the normal control mean, suggesting that the intron mutation affects enzyme activity. In all cases when ITPase activity was below the normal range, one or both mutations were found. The ITPA genotype did not correspond to any identifiable red cell phenotype. A possible relationship between ITPase deficiency and increased drug toxicity of purine analogue drugs is proposed. 相似文献
2.
Analysis of the tetrahydrobiopterin synthesizing system during maturation of murine reticulocytes 总被引:1,自引:0,他引:1
F Kerler L Hültner I Ziegler G Katzenmaier A Bacher 《Journal of cellular physiology》1990,142(2):268-271
The enzymes of tetrahydrobiopterin synthesis have been studied in murine bone marrow, in spleen, in erythrocytes, and in reticulocytes. Mice with chemically induced and with genetically conditioned reticulocytosis as found in the lactate dehydrogenase deficient strain (Ldh-1c/Ldh-1c) were used for analysis of reticulocytic enzyme activities. The activity of the biopterin synthesizing system is highest in bone marrow even though it amounts to only about 10% as compared with liver. The first enzyme of the biosynthetic pathway, GTP-cyclohydrolase, virtually disappears during the final maturation step of reticulocytes. In contrast, the activities of 6-pyruvoyltetrahydropterin synthase and of sepiapterin reductase of erythrocytes are only reduced to about one half of the reticulocyte level. The absence of biopterin in erythrocytes is therefore caused by the loss of the enzyme that initiates the pterin biosynthetic pathway. 相似文献
3.
Summary A case of hereditary nonspherocytic hemolytic anemia associated with partial erythrocyte PFK deficiency without muscular symptoms is reported: erythrocyte enzyme activity in the propositus was 60% of normal. Kinetic studies of erythrocyte PFK revealed increased sensitivity to ATP inhibition and decreased sensitivity to citrate inhibition.Muscle PFK from the patient had a normal enzymatic activity, but was highly unstable to heat, dilution without stabilizer and urea; furthermore its starch gel electrophoretic mobility was markedly faster than the one of a normal control. The results suggested that a muscle type's subunit was deficient in the erythrocyte PFK.The authors hypothesize that there was no PFK deficiency in the patient's muscle because of the active synthesis of proteins by this tissue. In contrast, the deficiency of PFK would be easily detected in erythrocytes, because of the absence of protein synthesis.Attachée de recherche à l'INSERMChargé de recherche à l'INSERM 相似文献
4.
EMS-induced sh mutants and their heterozygotes were examined for the enzyme, sucrose synthetase, which has previously been shown to be coded by the Sh locus. Complementing heterozygotes have a wild-type phenotype, but show no hybrid protein band after starch gel electrophoresis. The existence of a heteromeric complex, however, is inferred from the two-fold elevation in sucrose cleavage activity in the complementing heterozygotes as compared to the mutant homozygotes. The observations on complementation described here are unique, as the elevation in the activity of this reversible enzyme is noticed only in one direction (viz, sucrose cleavage) of the reaction and not the other (sucrose synthesis). 相似文献
5.
Prolidase (E.C.3.4.13.9) is a homodimeric enzyme encoded at a locus on chromosome 19. Prolidase deficiency is an autosomal recessive disorder with a highly variable clinical phenotype. We purified prolidase to homogeneity from normal human fibroblasts, raised a monospecific rabbit antiserum, and studied biosynthesis of the subunit in normal and prolidase--deficient fibroblasts. Pulse-chase immunoprecipitation experiments showed that the subunit is synthesized and retained in cytosol as a 58-KDa polypeptide. Three types of mutations were identified in six prolidase-deficient cell strains; half conferred a CRM-negative phenotype, while the CRM-positive mutations were of two types, one mutation encoding an enlarged subunit (60 KDa) and the others associated with subunits of normal size. Complementation analysis indicated that these mutations map to one locus. Normal subjects and obligate heterozygotes expressing CRM-negative mutations had thermostable prolidase activity at 50 degrees C in cell extracts, whereas heterozygotes expressing CRM-positive mutations had thermolabile activity under the same condition, implying negative allelic complementation in the putative heterodimer. The occurrence of prolidase-like activity about 5% of normal in amount but with a preference for substrate different from normal, in cells homozygous (or compound) for CRM-negative mutations, identified an alternative cleavage activity not encoded at the prolidase locus. Allelic heterogeneity at the major locus and the amount of alternative peptidase activity encoded elsewhere appear to be determinants of the associated and heterogeneous clinical phenotype. 相似文献
6.
Parche S Burkovski A Sprenger GA Weil B Krämer R Titgemeyer F 《Journal of molecular microbiology and biotechnology》2001,3(3):423-428
The high-GC Gram-positive actinomycete Corynebacterium glutamicum is commercially exploited as a producer of amino acids that are used as animal feed additives and flavor enhancers. Despite its beneficial role, carbon metabolism and its possible influence on amino acid metabolism is poorly understood. We have addressed this issue by analyzing the phosphotransferase system (PTS), which in many bacteria controls the flux of nutrients and therefore regulates carbon metabolism. The general PTS phosphotransferases enzyme I (EI) and HPr were characterized by demonstration of PEP-dependent phosphotransferase activity. An EI mutant exhibited a pleiotropic negative phenotype in carbon utilization. The role of the PTS as a major sugar uptake system was further demonstrated by the finding that glucose and fructose negative mutants were deficient in the respective enzyme II PTS permease activities. These carbon sources also caused repression of glutamate uptake, which suggests an involvement of the PTS in carbon regulation. The observation that no HPr kinase/phosphatase could be detected suggests that the mechanism of carbon regulation in C. glutamicum is different to the one found in low-GC Gram-positive bacteria. 相似文献
7.
Metabolic cooperation in cell culture: studies of the mechanisms of cell interaction 总被引:7,自引:0,他引:7
Metabolic cooperation is a form of cell communication in which the mutant phenotype of enzyme deficient cells, as determined by incorporation of labeled substrates, is corrected in culture by contact with normal cells. Previous studies showed that metabolic cooperation between normal and hypoxanthine phosphoribosyl transferase deficient cells (HPRT?) was the result of transfer of product of the enzyme, nucleotide or nucleotide derivative, from normal to mutant cells rather than transfer of enzyme or informational macromolecules leading to the synthesis of the enzyme. In the present study the nature and mechanisms involved in these cell interactions were investigated. Effective communication is observed within one hour of cell contact. Modifications of the extracellular environment including changes in osmolarity, concentration of sodium and divalent ions failed to interfere significantly with transfer. Changes of cell shape induced by cyclic nucleotides, hormones and urea also did not affect communication. Cytochalasin B which dissociates microfilaments and binds to cell membranes reduced metabolic cooperation while colcemide which dissociates microtubules had little effect. Enzymatic oxidation and iodination of cell surface structures abolished metabolic cooperation. The subcellular localization of label in donor cells is important in determining efficiency of transfer. Metabolic cooperation is efficient when radioactive label is primarily located in the nucleus and inefficient if the label is cytoplasmic. Cell lines previously classified as “non-communicating” because they lack gap junctions, ionic coupling and metabolic cooperation were shown in the present study to communicate when incubated with labeled substrates for 20 hours rather than 3. Cell communication is a more generalized phenomenon among cells in contact than previously appreciated. 相似文献
8.
A number of conditional lethal mutants of CHO cells that are defective in protein synthesis have been characterized with respect to their biochemical lesions. A defective aminoacyl-tRNA synthetase appears to be the basis of each mutant phenotype. In each strain, the specific activity in vitro of the synthetase cognate for one of the following amino acids was substantially reduced: arginine, asparagine, glutamine, histidine or methionine. One mutant, Arg-1, gave no detectable arginyl-tRNA synthetase activity, suggesting that it contains an altered enzyme that is unstable in vitro. Most of the mutants correspondingly exhibited impaired aminoacylation in vivo under nonpermissive conditions. However, two mutants, Arg-1 and His-1, appeared to have normal levels of acylated tRNA under the nonpermissive conditions which inhibited protein synthesis to approximately 50% and 10%, respectively. The expression of each mutant's phenotype, measured by rates of protein synthesis or growth, was a function of temperature and/or the concentration of amino acid cognate for the synthetase found to be deficient in vitro. The properties of these mutants make them applicable to diverse problems related to translation in mammalian cells. 相似文献
9.
Mutations in the MGAT2 gene controlling complex N-glycan synthesis cause carbohydrate-deficient glycoprotein syndrome type II, an autosomal recessive disease with defective brain development. 总被引:4,自引:0,他引:4
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Carbohydrate-deficient glycoprotein syndrome (CDGS) type II is a multisystemic congenital disease with severe involvement of the nervous system. Two unrelated CDGS type II patients are shown to have point mutations (one patient having Ser-->Phe and the other having His-->Arg) in the catalytic domain of the gene MGAT2, encoding UDP-GlcNAc:alpha-6-D-mannoside beta-1,2-N- acetylglucosaminyltransferase II (GnT II), an enzyme essential for biosynthesis of complex Asn-linked glycans. Both mutations caused both decreased expression of enzyme protein in a baculovirus/insect cell system and inactivation of enzyme activity. Restriction-endonuclease analysis of DNA from 23 blood relatives of one of these patients showed that 13 donors were heterozygotes; the other relatives and 21 unrelated donors were normal homozygotes. All heterozygotes showed a significant reduction (33%-68%) in mononuclear-cell GnT II activity. The data indicate that CDGS type II is an autosomal recessive disease and that complex Asn-linked glycans are essential for normal neurological development. 相似文献
10.
Isolation of Chinese hamster ovary cells with reduced poly(ADP-ribose) polymerase activity 总被引:1,自引:0,他引:1
The biological function of poly(ADP-ribose) polymerase in DNA repair, cell-cycle regulation and cellular differentiation has yet to be defined. Isolation of cells which are deficient in poly(ADP-ribose) synthesis would greatly facilitate the determination of the biological role of this enzyme. A method is described for isolating Chinese hamster ovary (CHO) cells deficient in the poly(ADP-ribose) polymerase activity by direct screening of colonies for enzyme activity. Colonies with decreased production of poly(ADP-ribose) are recovered from nylon replicas for further analysis. Using this method we have isolated a series of CHO cells which have 50% or less poly(ADP-ribose) polymerase activity. These mutants have normal generation times and are 20% more sensitive to the effects of DNA (m)ethylating agents than the parental cell. However, these mutants display normal sensitivity to gamma-rays. 相似文献
11.
Characterization and genetic mapping of fructose phosphotransferase mutations in Pseudomonas aeruginosa 总被引:12,自引:8,他引:4
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Pseudomonas aeruginosa transports and phosphorylates fructose via a phosphoenolpyruvate-dependent fructose phosphotransferase system (PTS). Mutant strains deficient in both PTS activity and glucose-6-phosphate dehydrogenase activity were isolated and were used to select mannitol-utilizing revertant strains singly deficient in PTS activity. These mutants were unable to utilize fructose as a carbon source and failed to accumulate exogenously provided [14C]fructose, and crude cell extracts lacked phosphoenolpyruvate-dependent fructose PTS activity. Thus, the PTS was essential for the uptake and utilization of exogenously provided fructose by P. aeruginosa. Mutations at a locus designated pts, which resulted in a loss of PTS activity, exhibited 57% linkage to argF at 55 min on the chromosome in plasmid R68.45-mediated conjugational crosses. The pts mutations in four independently isolated mutant strains exhibited from 11 to 20% linkage to argF, and one of these mutations exhibited 3% linkage to lys-9015 in phage F116L-mediated transductional crosses. 相似文献
12.
Hair root versus red cell individual phenotype in Sardinian heterozygotes for G6PD deficiency (Mediterranean type). 总被引:1,自引:1,他引:0
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G6PD activity was assayed in 20 Sardinian heterozygotes for G6PD deficiency and related to that of LDH and MDH. One of these heterozygotes showed a deficient phenotype in all her follicles, while the remaining 19 had different proportions of deficient, intermediate, and normal follicles. This is in accordance with a previous estimate. Because of the broad fiducial limits at the 5% level and because of some developmental considerations, this value cannot be interpreted as indicative of the number of primordial cells for scalp epidermis at the time of X-chromosome inactivation, as previously stated. The assay of single hair follicles is, however, a very valuable tool for establishing the role of cell selection in the same or in a different tissue, like peripheral blood. 相似文献
13.
Mizoguchi T Arakawa Y Kobayashi M Fujishima M 《Biochemical and biophysical research communications》2011,(1):121-126
Glutathione (GSH) is present in all mammalian tissues and plays a crucial role in many cellular processes. The second and final step in the synthesis involves the formation of GSH from gamma-glutamylcysteine (γ-GC) and glycine and is catalyzed by glutathione synthetase (GS). GS deficiency is a rare autosomal recessive disorder, and is present in patients with a range of phenotypes, from mild hemolytic anemia and metabolic acidosis to severe neurologic disorders or even death in infancy. The substrate for GS, γ-GC, has been suggested as playing a protective role, by substituting for GSH as an antioxidant in GS deficient patients. To examine the role of GS and GSH metabolites in development, we generated mice deficient in GSH by targeted disruption of the GS gene (Gss). Homozygous mice died before embryonic day (E) 7.5, but heterozygous mice survived with no distinct phenotype. GS protein levels and enzyme activity, as well as GSH metabolites, were investigated in multiple tissues. Protein levels and enzyme activity of GS in heterozygous mice were diminished by 50%, while GSH levels remained intact. γ-GC could not be detected in any investigated tissue. These data demonstrate that GSH is essential for mammalian development, and GSH synthesis via GS is an indispensable pathway for survival. 相似文献
14.
Knowledge of the G6PD genotype and its associated enzyme activity is significant for population genetics, diagnosis of disease, and management of patients. We tested 2,872 unrelated subjects from a Hakka population in China for G6PD activity by the WHO standard method and for genotype by DHPLC and DNA sequencing. Among female heterozygotes, 78.5% had relatively normal enzyme activity. The phenotype frequency of G6PD deficiency is 0.028, and the causal allele frequency is 0.060 in females. The accuracy, sensitivity, and specificity of DHPLC are more than 98% for detecting G6PD-deficient hemizygotes, heterozygotes, and homozygotes. Measuring enzyme activity alone is not sufficient for the diagnosis of heterozygotes. A combination of enzyme activity and DNA analysis should be used. 相似文献
15.
Model involving gene inactivation in the generation of autosomal recessive mutants in mammalian cells in culture. 总被引:8,自引:7,他引:1
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We present evidence for a two-step model for expression of the recessive phenotype at the diploid adenine phosphoribosyl transferase (aprt) locus in Chinese hamster ovary cells. This model proposes a high-frequency event leading to allelic inactivation and a low-frequency event leading to a structural alteration of the APRT protein. Either event can occur first, resulting in two types of heterozygous cells. The proposed model is based on analysis of Chinese hamster ovary presumptive aprt heterozygotes and APRT- mutants, derived by two different laboratories. The major class of heterozygotes (class 1) had approximately 50% parental APRT activity, 50% immunologically precipitable APRT protein, and only wild-type enzyme as based on two-dimensional gel electrophoresis and thermal inactivation studies. We propose that one allele at the aprt locus has been inactivated in these heterozygotes. APRT- mutants derived from any single class 1 heterozygote arose at a low frequency and contained either no immunologically detectable APRT protein or an APRT enzyme which was, in most cases, demonstrably altered. The second class of heterozygotes, consisting of two independent isolates, gave rise to APRT- cells at a high frequency (10(-3) to 10(-5). These heterozygous cell lines had 50% of parental APRT activity and only wild-type spot, or wild-type and an electrophoretic variant spot, on two-dimensional gels. These aprt heterozygotes appear to have arisen by mutation at one allele. APRT- mutants derived from either heterozygote of this class had all lost the wild-type activity, consistent with the proposed model. 相似文献
16.
Subjects heterozygous for the Lesch-Nyhan syndrome with a deficiency of the X-linked gene for the enzyme hypoxanthine-guanine phosphoribosyl transferase (PRT) would be expected to have two populations of erythrocytes in roughly equal proportions—one type with the normal enzyme and the other type exhibiting the mutant form of the enzyme. In contrast to this prediction, previous studies utilizing an X-linked gene for another enzyme as a marker for the PRT locus have suggested that erythrocytes from heterozygotes consist largely of cells with the normal form of the enzyme. We have recently described a mutant form of hypoxanthine-guanine phosphoribosyl tranferase with altered kinetic properties which allow it to be measured in artificial mixtures with the normal enzyme. The mutant enzyme could not be detected in erythrocyte lysates from a proven heterozygote for both the normal and this mutant form of the enzyme. This provides additional evidence that either inactivation of the X-chromosome in erythropoietic tissue from the heterozygote for PRT deficiency is not random or that random X-chromosome inactivation is followed by selection against erythrocyte precursors with the mutant enzyme.This study was supported in part by USPHS Research Grant No. AM14362, USPHS Training Grant No. AM05620, and a grant (RR-30) from the General Clinical Research Centers Program of the Division of Research Resources, National Institutes of Health. 相似文献
17.
We have developed a procedure for testing iduronate sulfatase, the enzyme deficient in Hunter syndrome, in single hair roots. Beta-Hexosaminidase was used as the reference enzyme. The ratio of iduronate sulfatase to beta--hexosaminidase, expressed in arbitrary units of activity, is near zero for Hunter patients and greater than 0.6 in almost all roots of normal individuals. Hair roots of Hunter heterozygotes show a characteristic continuum of activity ratios, ranging from totally deficient up to and including the normal range. The results are consistent with the origin of hair roots from a small number of progenitor cells which obey the Lyon hypothesis. The proportion of roots with low activity can be used to discriminate between normal and heterozygous individuals. 相似文献
18.
A family with congenital non-spherocytic hemolytic anemia associated with glucose-6-phosphate dehydrogenase (G6PD) deficiency was studied. Two females, heterozygous for the enzyme deficency, had evidence of a hemolytic anemia. The results of chromium-51 erythrocyte life span studies prior to, during, and after periods of primaquine administration suggested that the hemolytic anemia in these women was due to the presence of two populations of red blood cells in their circulation. One population had normal G6PD levels and a normal life span, whereas the other had diminished enzyme activity and a shortened life span.In vitro metabolic studies of the erythrocytes of a heterozygous female and a hemizygous male suggested that, in spite of G6PD deficiency, the synthesis and breakdown of adenosine triphosphate and 2,3-diphosphoglyceric acid was similar to that in normal erythrocytes. 相似文献
19.
S H Chen K S Lin C L Chen 《Proceedings of the National Science Council, Republic of China. Part B, Life sciences》1985,9(4):251-254
The activity of glucose-6-phosphate dehydrogenase (G-6-PD) in leucocytes was studied for erythrocyte G-6-PD deficiency using 49 hemizygous males, 16 heterozygous females, and 19 normal controls. The mean G-6-PD activity in leucocytes of the affected neonates (9.2 +/- 5.4 units) and the children (11.2 +/- 5.3 units) were significantly lower than those of normal newborns (22.9 +/- 5.1 units, P less than 0.01). Seventy percent of the effected newborns and 58% of the children with G-6-PD deficiency had the leucocyte enzyme activity of less than 13 IU/10(9)WBC. The leucocyte enzyme activity (14.6 +/- 8.6 units) of 16 heterozygous G-6-PD deficient mothers was also lower than that of normal controls (23.1 +/- 7.0 units). The present study thus concludes that, in G-6-PD deficient Chinese, the enzyme defect is demonstrable not only in erythrocytes but also in leucocytes. 相似文献
20.
Orvisky E Stubblefield B Long RT Martin BM Sidransky E Krasnewich D 《Analytical biochemistry》2003,317(1):12-18
Congenital disorders of glycosylation (CDG) are a group of multisystemic disorders resulting from defects in the synthesis and processing of N-linked oligosaccharides. The most common form, CDG type Ia (CDG-Ia), results from a deficiency of the enzyme phosphomannomutase (PMM). PMM converts mannose 6-phosphate (man-6-P) to mannose-1-phosphate (man-1-P), which is required for the synthesis of GDP-mannose, a substrate for dolichol-linked oligosaccharide synthesis. The traditional assay for PMM, a coupled enzyme system based on the reduction of NADP(+) to NADPH using man-1-P as a substrate, has limitations in accuracy and reproducibility. Therefore, a more sensitive, direct test for PMM activity, based on the detection of the conversion of man-1-P to man-6-P by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), was developed. Using this assay, the activity of PMM was markedly deficient in fibroblasts and lymphoblasts from 23 patients with CDG-Ia (range 0-15.3% of control, average 4.9+/-4.7%) and also decreased in seven obligate heterozygotes (range 33.0-72.0% of control, average 52.2+/-14.7%). Unlike the spectrophotometric method, there was no overlap in PMM activity among patients, obligate heterozygotes, or controls. Thus, the PMM assay based on HPAEC-PAD has increased utility in the clinical setting, and can be used, together with transferrin isoelectric focusing, to diagnose patients with CDG-Ia and to identify heterozygotes when clinically indicated. 相似文献