Human red cell glucose-6-phosphate dehydrogenase: all active enzyme has sequence predicted by the X chromosome-encoded cDNA.
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Amino Acid Sequence↗
Biomedical subjects
W Kuhl
Publications and source records attributed to W Kuhl.
Linkage between a PvuII restriction fragment length polymorphism and G6PD A-202A/376G: evidence for a single origin of the common G6PD A- mutation.
DNA samples from 29 males with the G6PD A- phenotype and 14 males with a G6PD B phenotype were studied for the presence of each of four polymorphic restriction sites in the glucose-6-phosphate dehydrogenase locus. All G6PD A- subjects with the G6PD A-202A/376G genotype, regardless of population origin, shared identical haplotypes. In view of the fact that at least one of the restriction sites, the PvuII site in the intron between exon 5 and 6, has thus far been uncommon in the populations studied, it seems likely that the G6PD A- mutation at nucleotide 202 arose relatively recently and in a single individual.
Base Sequence↗
The NT 1311 polymorphism of G6PD: G6PD Mediterranean mutation may have originated independently in Europe and Asia.
A polymorphic site exists in exon 11 of G6PD: in the wild-type enzyme, nucleotide (NT) 1311 is a C, but is some individuals from diverse populations a T is present instead. Nine of 54 X chromosomes from Europeans of mixed origins, nine of 41 X chromosomes of Ashkenazi Jewish subjects, three of 18 X chromosomes of Sicilians, five of 20 African X chromosomes, and nine of 20 Asian Indian X chromosomes had the mutant genotype. In contrast, the mutation was found in only three of 59 Oriental X chromosomes and in three of 30 Central/South American X chromosomes. The mutation was absent from four samples of chimpanzee DNA. Twenty-one of 22 male subjects from Mediterranean countries who had the G6PD Mediterranean 563T genotype investigated in the present study or reported previously had a T at NT 1311. Only one had the normal C at NT 1311. In contrast, both G6PD Mediterranean563T males from the Indian subcontinent had the normal C at NT 1311. These findings suggest that the same mutation at nucleotide 563 giving rise to G6PD Mediterranean may have arisen independently in Europe and in Asia.
Animals↗
Molecular biology of G 6 PD variants.
Glucose-6-phosphate dehydrogenase (G6PD, E.C. 1.1.1.49) deficiency is probably the most common disease-producing enzyme deficiency of man. Originally described in the 1950's in Black Americans and regarded a single disorder, it soon became apparent that this enzyme defect occurred in many populations and that it was biochemically heterogeneous. By 1965 a considerable number of distinct variants had been described and a WHO Scientific Group was convened to standardize methods of characterization of variants, so as to allow meaningful interlaboratory comparisons to be made. In the succeeding quarter of a century nearly 400 variants believed to be unique have been characterized, most of them by the standard methods that had been adopted in 1967. Helpful as standardization proved to be, however, comparison of variants with one another proved to be difficult. Electrophoretic mobilities and kinetic constants vary with changes in reagents over which investigators have no control, and the lack of stability of enzymes and their kinetic characteristics makes side-by-side comparison a goal that can be achieved only rarely. It is not surprising, then, that in at least one instance variants that appeared to be quite different proved to have been obtained from two members of the same family, and were undoubtedly identical. It has thus been clear for many years that a true appreciation of the extent of G6PD mutations would require the acquisition of incontrovertible structural data.(ABSTRACT TRUNCATED AT 250 WORDS)
Africa↗
Molecular genetics of the glucose-6-phosphate dehydrogenase (G6PD) Mediterranean variant and description of a new G6PD mutant, G6PD Andalus1361A.
Glucose-6-phosphate dehydrogenase (G6PD; E.C.1.1.1.49) deficiency is the most common human enzymopathy; more than 300 different biochemical variants of the enzyme have been described. In many parts of the world the Mediterranean type of G6PD deficiency is prevalent. However, G6PD Mediterranean has come to be regarded as a generic term applied to similar G6PD mutations thought, however, to represent a somewhat heterogeneous group. A C----T mutation at nucleotide 563 of G6PD Mediterranean has been identified by Vulliamy et al., and the same mutation has been found by De Vita et al. in G6PD Mediterranean, G6PD Sassari, and G6PD Cagliari. The latter subjects had an additional mutation, at nucleotide 1311, that did not produce a coding change. We have examined genomic DNA of five patients--four of Spanish origin and one of Jewish origin--having enzymatically documented G6PD Mediterranean. All had both the mutation at nucleotide 563 and that at nucleotide 1311. A sixth sample, resembling G6PD Mediterranean kinetically but with a slightly rapid electrophoretic mobility, was designated G6PD Andalus and was found to have a different mutation, a G----A transition at nucleotide 1361, producing an arginine-to-histidine substitution. These studies suggest that G6PD Mediterranean is, after all, relatively homogeneous.
Base Sequence↗
Interference of heparin with the polymerase chain reaction.
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Centrifugation↗
Molecular heterogeneity of glucose-6-phosphate dehydrogenase A-.
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is probably the most common disease-producing genetic polymorphism of humans. Virtually all G6PD-deficient Africans show the G6PD A- phenotype, an electrophoretically rapid, deficient enzyme. The recently acquired ability to identify the point mutations producing the different variants has given us new insights into the population genetics of G6PD variants. Twenty-nine males with the G6PD A- phenotype were studied. They were of African, Mexican, Spanish, and US white ethnic origin. All had the A---G transition at nucleotide 376 characteristic of G6PD A. In each case, one of three additional mutations was present, at nucleotides 202, 680, or 968. That in this population second mutations producing G6PD deficiency occurred only on the genetic background of G6PD A suggests that G6PD A was at one time the most common type of G6PD in Africa. However, the nucleotide sequence of the chimpanzee (Pan troglodytes) G6PD indicates that the primordial human type of G6PD was G6PD B.
Base Sequence↗
Identification of the binding domain for NADP+ of human glucose-6-phosphate dehydrogenase by sequence analysis of mutants.
Human erythrocyte glucose-6-phosphate dehydrogenase is normally quite stable in the presence of 10 microM NADP+. Certain glucose-6-phosphate dehydrogenase variants lose virtually all their activity at this concentration of NADP+ but are reactivated by 200 microM NADP+. Such variants presumably have a defect in their NADP+-binding site. We analyzed the sequence of cDNA or genomic DNA from seven unrelated patients with hemolytic anemia due to the inheritance of variants that are reactivated by NADP+. Six patients had substitutions of one of three adjacent amino acids, and the seventh patient had another amino acid substitution 23 residues downstream. These amino acids are highly conserved, all being present in rat and all but one being found also in Drosophila. The anomalous electrophoretic behavior of some of the variants can be explained by their loss of ability to bind NADP+. We conclude that the region in which these mutations occur defines the binding domain for NADP+ and that binding NADP+ that has been designated as "structural" and as "catalytic" probably occurs at the same site.
Amino Acid Sequence↗
A long term study of the health status and performance of sows on different feed allowances during late pregnancy. I. Clinical observations, with special reference to agalactia post partum.
Thirty nine pairs of full sibs were investigated over 6 parities in a long term study on the effects of late pregnancy feed allowance on the occurrence of agalactia post partum and on the performance of sows and piglets. A careful examination of all sows with a rectal temperature exceeding 39.5 degrees C was performed by a veterinarian within the first 48 h after farrowing. Milk-samples were taken from sows with elevated rectal temperatures and showing clinical symptoms of agalactia. During the last 15 days of gestation the sows in the control group were fed 3.4 kg daily and the sows in the experimental group 1.0 kg daily of a commercial type of diet. In 26.6% of the farrowings in the control group the sows were agalactic whereas the corresponding figure in the experimental group was 14.4%. On clinical examination udder changes were observed in a majority of the diseased sows in both groups. However, the agalactic sows in the control group were generally more affected, with lower water and feed consumption than in the experimental group. No effects of age of the sow (parity number) or length of the gestation period on the incidence of agalactia were demonstrated. The rectal temperature of agalactic sows was significantly higher than in the healthy sows already 1 day before farrowing. The agalactic sows farrowed a larger number of stillborn piglets, which indicates an early establishment of the disease. The number of weaned piglets at 6 weeks did not differ between agalactic and healthy animals. The interval from weaning to first oestrus was not influenced by agalactia in the preceding lactation.
Animal Feed↗
Complete correction of the enzymatic defect of type I Gaucher disease fibroblasts by retroviral-mediated gene transfer.
Glucocerebrosidase cDNA and the neomycin-resistance gene (neo) were cloned into a retrovirus vector. Mouse fibroblasts infected with this vector expressed human glucocerebrosidase, which was readily distinguished from the mouse enzyme using mouse monoclonal anti-glucocerebrosidase antibodies. Cultured fibroblasts and transformed lymphoblasts from patients with type I Gaucher disease were infected with the retrovirus rescued from the mouse fibroblasts by a helper virus. Transformed cells were selected with the antibiotic G418. The enzyme activity of cells infected with virus containing glucocerebrosidase cDNA was restored to normal, while uninfected cells or cells infected with virus containing only the neo gene did not produce glucocerebrosidase.
Animals↗
The human glucocerebrosidase gene has two functional ATG initiator codons.
Gaucher disease is due to a deficiency in the activity of the enzyme glucocerebrosidase. Glucocerebrosidase is a lysosomal enzyme that presumably requires a signal peptide for transport across the membrane of the rough endoplasmic reticulum and glycosylation for transport into lysosomes. Human glucocerebrosidase cDNA contains two potential ATG start codons in its long open reading frame. The signal peptides that are initiated from each ATG are quite different in their hydrophobicity. We demonstrate that either ATG can function independently to produce active glucocerebrosidase enzyme in cultured fibroblasts. The glucocerebrosidase activity produced from translation products initiated at either ATG is found predominantly in the lysosomes.
Amino Acid Sequence↗
Glucocerebrosidase processing in normal fibroblasts and in fibroblasts from patients with type I, type II, and type III Gaucher disease.
Fibroblasts from normal subjects and patients with the three types of Gaucher disease were labeled with [3H]leucine. Glucocerebrosidase antigen was immunoprecipitated using affinity-purified Sepharose-bound antibody. Normal cells initially formed a 60-kDa polypeptide antigen that was gradually replaced by a broad band of antigen averaging 63 kDa. This position corresponds with that of mature fibroblast and placental enzyme. Processing of glucocerebrosidase in six unrelated patients with type I Gaucher disease and one patient with type III Gaucher disease was exactly the same as normal. In contrast, three patients with the severe infantile (type II) form of the disease manifested a very unstable enzyme; the 60-kDa band appeared transiently and the mature 63-kDa band was never seen. These results indicate that type II Gaucher disease may well be distinguishable from type I disease by virtue of the very unstable enzyme precursor. Contrary to some earlier reports, processing of glucocerebrosidase in type I disease appears to be entirely normal.
Cells, Cultured↗
Gaucher disease: retrovirus-mediated correction of the enzymatic defect in cultured cells.
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Amino Acid Sequence↗
Blood cell phosphogluconolactonase: assay and properties.
6-Phosphogluconolactonase (6-PGL) catalyses the second reaction of the hexosemonophosphate pathway. Although the delta-lactone of 6-phosphogluconic acid is the natural substrate for this enzyme, the more stable gamma-lactone may also be used. We prepared the gamma-lactone of 6-phosphogluconic acid from 6-phosphogluconate. When stored in dimethylsulfoxide, this material was found to be stable in liquid nitrogen for several months. A method for measuring 6-phosphogluconolactonase (6-PGL) using the gamma-lactone as substrate has been developed, after defining conditions under which spontaneous hydrolysis of the lactone is relatively slow and the enzymatic velocity is relatively rapid. The enzyme had no divalent cation requirement and was not significantly inhibited by a 50-fold excess of gluconolactone. It was distinct, therefore, from gluconolactonase. At 25 degrees C normal human red cells were found to contain approximately 50 IU of 6-phosphogluconolactonase/g Hb. The activity of the enzyme was independent of red cell age. Based on protein content, human lymphocytes, monocytes, granulocytes and platelets, contained approximately 10 times the activity of red blood cells. The activity of 6-PGL was stable for at least 6 d in red cells stored at 22 degrees C and for at least 20 d in red cells stored at 4 degrees C.
Adult↗
Characteristics and significance of the reverse glucose-6-phosphate dehydrogenase reaction.
Glucose-6-phosphate dehydrogenase (G-6-PD) is the first enzyme of the hexose monophosphate pathway, and this important reaction is often considered to be irreversible. However, its apparent irreversibility is caused by the rapid removal of the immediate product, 6-phosphoglucono-delta-lactone. We have now investigated the reverse G-6-PD reaction, namely, the oxidation of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) by 6-phosphoglucono-delta-lactone to form glucose-6-phosphate and nicotinamide-adenine dinucleotide phosphate (NADP). The substrate of the reaction, 6-phosphoglucono-delta-lactone, was rapidly generated from glucose-6-phosphate and NADP. The lactone was stabilized by addition of perchloric acid. A substrate analogue, 6-phosphoglucono-gamma-lactone, was synthesized by dehydrating 6-phosphogluconic acid. At pH 2.3 the t 1/2 of the delta-lactone was 2.4 hours; that of the gamma-lactone was 57 hours. The following kinetic parameters were established: Km delta-lactone 1027 +/- 183 mumol/L; Km gamma-lactone 266 +/- 71 mumol/L; Km NADPH less than 10 mumol/L; ratio of the Vmax G-6-PD forward/reverse reaction 2.0. Glucose-6-phosphate was found to be a competitive inhibitor with both lactones in the reverse G-6-PD reaction. Genetic mutants of humans in which the Km of G-6-PD for glucose-6-phosphate was diminished also had a diminished Km for 6-phosphoglucono-delta-lactone. Thus, it appears that the same active site on the enzyme binds glucose-6-phosphate in the forward reaction and 6-phosphogluconolactone in the reverse reaction.(ABSTRACT TRUNCATED AT 250 WORDS)
Drug Stability↗
6-Phosphogluconolactonase deficiency, a hereditary erythrocyte enzyme deficiency: possible interaction with glucose-6-phosphate dehydrogenase deficiency.
Partial deficiency of 6-phosphogluconolactonase (EC 3.1.1.31) of the erythrocytes was discovered as an autosomal dominant disorder. Hemolytic anemia occurred in an individual who had inherited both the gene for 6-phosphogluconolactonase deficiency and that for deficiency of a nonhemolytic variant of glucose-6-phosphate dehydrogenase (EC 1.1.1.49). It is proposed that the interaction of this hereditary erythrocyte abnormality with glucose-6-phosphate dehydrogenase deficiency may explain hemolysis in some other patients who have inherited polymorphic variants of glucose-6-phosphate dehydrogenase.
Anemia, Hemolytic↗
Limiting role of 6-phosphogluconolactonase in erythrocyte hexose monophosphate pathway metabolism.
The natural product of the glucose-6-phosphate dehydrogenase reaction is 6-phosphoglucono-delta-lactone, which must be hydrolyzed to 6-phosphogluconic acid before it can be further metabolized by 6-phosphogluconate dehydrogenase. Because this lactone is very unstable, it has been uncertain whether the enzyme that hydrolyzes it, 6-phosphogluconolactonase, is required for functioning of the hexose monophosphate pathway. We have purified glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, and 6-phosphogluconate dehydrogenase from human erythrocytes to the point where each enzyme is essentially free of each of the other activities. We constructed an artificial hexose monophosphate pathway from these enzymes, providing as substrate 14C-labeled glucose-6-phosphate either directly or by continual generation from 14C-glucose by yeast hexokinase and adenosine triphosphate. The oxidation of 6-phosphogluconic acid was estimated by measuring the CO2 formed. In the absence of a reduced nicotinamide-adenine dinucleotide phosphate (NADPH)-oxidizing system, such as oxidized glutathione (GSSG)-glutathione reductase or phenazine methosulfate, little CO2 was formed, and the presence of 6-phosphogluconolactonase did not affect the amount that was produced. When the hexose monophosphate pathway was stimulated by providing an NADPH-oxidizing system, CO2 was produced two and a half to five times as fast in the presence of 6-phosphogluconolactonase as in its absence. These studies suggest that 6-phosphogluconolactonase is required for the functioning of the hexose monophosphate pathway when the rate of oxidation of NADPH is accelerated.
Carbon Dioxide↗
Glucocerebrosidase "processing" and gene expression in various forms of Gaucher disease.
Immunoblots were prepared using extracts of fibroblasts derived from five controls and from four unrelated patients with type I, three with type II, and two with type III Gaucher disease. Five monoclonal antisera and two rabbit sera, crude and affinity purified, were utilized to detect antigen transferred to nitrocellulose paper. Only a band of 63,000 molecular weight (Mr) was consistently detected. We found no 56-K band either in normal or in Gaucher disease fibroblast extracts. Thus, using a variety of antisera, we are unable to verify the claim that the types of Gaucher disease can be differentiated from one another by immunoblotting.
Cell Line↗