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Biomedical subjects

E Beutler

Publications and source records attributed to E Beutler.

At least 271 records · Page 15Linked to original sources
PubMed1987

Effect of oxalate and malonate on red cell metabolism.

The addition of oxalate to blood stored in Citrate-phosphate-dextrose (CPD) produces a marked improvement in 2,3-diphosphoglycerate (2,3-DPG) preservation; an increase in 2,3-DPG levels can also be documented in short-term incubation studies. Oxalate is a potent in vitro inhibitor of red cell lactate dehydrogenase, monophosphoglycerate mutase, and pyruvate kinase (PK). In the presence of fructose 1,6-diphosphate the latter inhibitory effect is competitive with phospho(enol)pyruvate (PEP). Determination of the levels of intermediate compounds in red cells incubated with oxalate suggest the presence of inhibition at the PK step, indicating that this is the site of oxalate action. Apparent inhibition at the glyceraldehyde phosphate dehydrogenase step is apparently due to an increase in the NADH/NAD ratio. Oxalate had no effect on the in vivo viability of rabbit red cells stored in CPD preservatives for 21 days. Greater understanding of the toxicity of oxalate is required before it can be considered suitable as a component of preservative media, but appreciation of the mechanism by which it affects 2,3-DPG levels may be important in design of other blood additives. Malonate, the 3-carbon dicarboxylic acid analogue of oxalate late did not inhibit pyruvate kinase nor affect 2,3-DPG levels.

2,3-Diphosphoglycerate↗
PubMed1987

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↗
PubMed1986

Improved assay of the enzymes of glutathione synthesis: gamma-glutamylcysteine synthetase and glutathione synthetase.

New methods for the estimation of red cell gamma-glutamylcysteine synthetase and glutathione synthetase have been developed. gamma-32P ATP is allowed to equilibrate until the gamma and beta phosphate groups are equally labelled. The amount of 32Pi released in the presence of glutamic acid and cysteine, the substrates for GC-S or in the presence of gamma-glutamylcysteine and glycine, the substrates of GSH-S, is measured. This is accomplished by extraction of the phosphomolybdate complex into isobutanol-benzene. The methods are linear with time and hemolysate concentration. Normal values are presented.

Adenosine Triphosphate↗
PubMed1986

G-6-PD Walter Reed: possible insight into "structural" NADP in G-6-PD.

A new G-6-PD variant, G-6-PD Walter Reed, causing hereditary nonspherocytic hemolytic anemia is characterized. This variant is unusual in that its stability requires the presence of high concentrations of NADP, while its Km for NADP is normal. This finding is consistent with the suggestion that G-6-PD has two separate binding sites, a high affinity "structural" site and a lower affinity catalytic site. The mutation in G-6-PD Walter Reed, like that of the previously described variant, G-6-PD Torrance, may be due to a mutation of the "structural" site for NADP.

Anemia, Hemolytic, Congenital↗
PubMed1986

Microcytosis in Hodgkin disease associated with unbalanced globin chain synthesis.

A review of 162 patients with Hodgkin disease disclosed 36 with microcytic anemia (mean corpuscular hemoglobin values [MCV] less than 80 fl). Three patients had iron deficiency, and one had beta-thalassemia. Of the remaining 32 patients, 24 had microcytic anemia at the time of diagnosis of Hodgkin disease, and ten, including two patients with this finding initially, developed microcytic anemia in association with recurrence of Hodgkin disease. Seven patients with Hodgkin disease and normal MCV had normal alpha-to-beta-globin chain ratios (1.0 +/- 0.14). Seven patients with Hodgkin disease and MCV less than 80 fl had significantly lower alpha-to-beta chain ratios (0.66 +/- 0.05). Twelve normal controls and four with iron-deficiency anemia and MCV less than 80 fl had normal ratios. Anemia was corrected, and MCV returned to normal in all patients who responded to therapy for Hodgkin disease. In the two patients studied sequentially, abnormal alpha-to-beta-chain ratio was corrected along with the anemia.

Anemia↗
PubMed1986

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↗
PubMed1986

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↗
PubMed1986

Erythrocyte glutathione synthetase deficiency leads not only to glutathione but also to glutathione-S-transferase deficiency.

Glutathione synthetase (GSH-S) is one of the two known hereditary causes of glutathione deficiency. We describe a family whose two children have hemolytic anemia. The children's erythrocytes lack GSH and are severely deficient in GSH-S activity. No neurologic findings or 5-oxoprolinuria were present. A concurrent deficiency of glutathione-S-transferase (GST) was also detected in the erythrocytes. Residual glutathione could be detected in the erythrocytes using a sensitive cycling assay. The deficiency was found to be most severe in reticulocyte-depleted preparations. The GSH-S activity of the erythrocytes of the parents was one-half normal, while the glutathione S-transferase activity was normal. We conclude that the primary defect is one of GSH-S. Glutathione stabilizes GST in vitro, and it is assumed that the deficiency of GST in the erythrocytes of the patients is due to the instability of this enzyme in the absence of adequate intracellular GSH levels.

Adolescent↗
PubMed1986

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↗
PubMed1986

The mechanism of removal of leukocytes by cellulose columns.

Cellulose columns efficiently remove leukocytes from whole blood. Interaction of leukocytes with cellulose particles is not affected by glucose, galactose, fructose, mannose, or cellobiose. Although red cells normally pass through cellulose columns, they are retained after fixation in glutaraldehyde. We conclude that the leukocyte-removing activity of cellulose columns is due to mechanical filtration rather than to specific adherence of leukocytes to the cellulose particles.

Carbohydrates↗
PubMed1985

A new glucose-6-phosphate dehydrogenase variant, Gd(-) Tepic, characterized by moderate enzyme deficiency and mild episodes of hemolytic anemia.

A 16-year-old Mexican male of Japanese ancestry was found to have a new glucose-6-phosphate dehydrogenase (G-6-PD) deficient variant, named Gd(-) Tepic after the birthplace of the maternal grandmother. A younger brother was also affected and the two sisters were heterozygous. The mother, an obligatory heterozygote, did not show the abnormal variant and the possible explanation of this phenomenon is discussed. From the clinical standpoint, the propositus has had three mild hemolytic episodes while his siblings are so far asymptomatic.

Adolescent↗
PubMed1985

G-6-PD Jalisco and G-6-PD Morelia: two new Mexican variants.

Two new G-6-PD variants designated G-6-PD Jalisco and G-6-PD Morelia were identified in two unrelated Mexican families. An additional G-6-PD variant was found in each family: G-6-PD Trinacria and G-6-PD A-. In both families compound heterozygotes were identified. G-6-PD Jalisco and G-6-PD Morelia belong to Classes 3 and 4, respectively. G-6-PD Morelia is the first variant from its class with a high Km for NADP and a low Ki for NADPH.

Adult↗
PubMed1985

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↗