Search PubMed⌕ Search

Biomedical subjects

E Beutler

Publications and source records attributed to E Beutler.

At least 433 records · Page 24Linked to original sources
PubMed1976

Acid hydrolases in leukocytes and platelets of normal subjects and in patients with Gaucher's and Fabry's disease.

Lymphocytes, monocytes, neutrophilic granulocytes and platelets were each separated to greater than 95% purity from six normal subjects, three patients with Gaucher's disease, two heterozygotes for Gaucher's disease, and one patient with Fabry's disease. Activities of the following acid hydrolases were determined: "acid" (pH 4.0) beta-glucosidase, pH 5.0 beta-glucosidase, alpha-galactosidase, alpha-arabinosidase, alpha-mannosidase, alpha-glucosidase, beta-glucuronidase, beta-galactosidase, beta-hexosaminidase, and acid phosphatase. Enzymatic activity varied greatly with cell type and the enzyme being measured; the importance of assaying pure preparations especially for heterozygote detection is emphasized. Gaucher's disease patients' cells were found to be deficient in the pH 4.0 acid beta-glucosidase, variable in the pH 5.0 beta-glucosidase, and normal in all other acid hydrolases tested, including acid phosphatase, the activity of which is known to be elevated in plasma. Blood cells of a patient with Fabry's disease were deficient in alpha-galactosidase and normal in all other acid hydrolases tested.

Acid Phosphatase↗
PubMed1976

Characterization of heteropolymeric hexosaminidase A in human X mouse hybrid cells.

Expression of heteropolymeric hexosaminidase A activity is reported in a human X mouse hybrid cell line that contains an X/15 translocation chromosome but lacks human chromosome 5 and has no detectable human hexosaminidase B activity. (Hexosaminidase is beta-N-acetylglucosaminidase; EC 3.2.1.30; 2-acetamido-2-deoxy-beta-D-glucoside acetamidodeoxyglucohydrolase.) That the "hexosaminidase A" enzyme of these cells contains the human hexosaminidase-alpha subunit and not the human hexosaminidase-beta subunit is indicated by the cells' reactions to specific antisera prepared against the alpha subunit and against the beta subunit. Our results indicate that the "hexosaminidase A" activity in this hybrid cell line is the expression of a hybrid molecule composed of human hexosaminidase-alpha subunit and a mouse hexosaminidase subunit. These results are consistent with the hypothesis that the human hexosaminidase A enzyme is formed from alpha and beta subunits coded for by genes on chromosomes 15 and 5, respectively.

Animals↗
PubMed1976

Enzyme replacement therapy in Gaucher's disease: a rapid, high-yield method for purification of glucocerebrosidase.

Gaucher's disease is caused by a deficiency of the lysosomal enzyme glucocerebrosidase (glucosylceramidase; D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45); this disorder has been a leading candidate for enzyme replacement trials. A rapid, high-yield method for purification of glucocerebrosidase has been developed. Detergent extraction of human placenta was followed by salt fractionation, concanavalin A-Sepharose chromatography, organic solvent precipitation, and affinity chromatography on phosphatidylserine-agarose; the total yield is 60% with 6000-fold purification. Purified glucocerebrosidase has been administered intravenously to a volunteer Gaucher's patient on two separate occasions. For the first injection, the enzyme was entrapped in resealed erythrocytes; for the second injection, the enzyme was given without any carrier. The enzyme infusions caused no untoward effects.

Erythrocyte Membrane↗
PubMed1976

Explanation for the apparent lack of ouabain inhibition of pyruvate production in hemolysates: the "backward" PGK reaction.

The concept that ouabain-sensitive membrane (Na+ + K+)-ATPase-generated adenosine diphosphate (ADP) preferentially serves as the substrate for the phosphoglycerate kinase (PGK) step of erythrocyte glycolysis has been reexamined. Membrane ATPase readily provides ADP for and utilizes ATP generated in the pyruvate kinase (PK) step and is ouabain sensitive. Earlier reports in the literature, which have suggested that in hemolysates the ATPase reaction facilitating the PK reaction is ouabain-insensitive, are reinterpreted: in crude hemolysates ADP generated in the "backward" PGK reaction can account for these data. We conclude that there is no convincing evidence of selective linkage of (Na+ + K+)-ATPase with the PGK reaction.

Adenosine Triphosphatases↗
PubMed1976

Human red cells protein kinase in normal subjects and patients with hereditary spherocytosis, sickle cell disease, and autoimmune hemolytic anemia.

A somewhat simplified modification of a previously described method for the measurement of red cell membrane phosphorylation by ATP has been devised. Phosphorylation of membranes was linear with time for only 5-10 min, and linearity with membrane concentration was observed only when assays were limited to short incubation times. Protein kinase activity of hereditary spherocytosis (HS) membranes was found to be normal. However, the average phosphorylation after 60 min incubation was less in HS membranes than in normal membranes. Findings similar to those in HS membranes were observed in sickle cell disease. The Km of red cell protein kinase for ATP is approximately 10(-5) M. Membrane phosphate binding sites are not saturated in either HS or normal membranes after 1 hr incubation with ATP. Approximately 27% of phosphorylating activity is lost after 1 hr incubation at 37 degrees C. GTP is a very inefficient phosphate donor. Under the conditions of measurement employed, the enzyme is slightly stimulated by 1 muM cAMP, but is not stimulated by 1 muM cGMP. Dephosphorylation of red cell membranes after labeling occurs at a similar rate in HS as in normal membranes. Although a mild abnormally in membrane phosphorylation is observed in HS, this could not be demonstrated to be due to a decrease in protein kinase activity or in alterations of its kinetic properties. The abnormally seen is not specific for HS.

Adenosine Diphosphate↗
PubMed1976

Biochemical and electrophoretic studies of erythrocyte pyridoxine kinase in white and black Americans.

The mean PNK activity in red blood cells from black subjects was only about 40% of that in whites. Among 51 whites examined, one was found to have enzyme deficiency. The estimated gene frequencies for PNKH (the common allele in whites which codes for higher enzyme activity) and PNKL (the common allele in blacks which codes for lower enzyme activity) were .35 and .65, respectively, for black donors, and .81 and .19, respectively, for white donors, The variant enzyme in persons with enzyme deficiency was associated with an increased rate of degradation in red cells during aging. No other biochemical or electrophoretic differences were detected.

Alleles↗
PubMed1976

Studies on gamma-glutamyl transpeptidase in human and rabbit erythrocytes.

Gamma-glutamyl transpeptidase transfers the gamma-glutamyl moiety of glutathione to a variety of acceptor amino acids. Through the operation of the gamma-glutamyl-cyclotransferase cycle, this enzyme has been implicated in the transport of amino acids into cells, especially the cells of the proximal tubules of kidney. It has been reported to be present in rabbit erythrocytes. However, using white cell-free preparations, we have not been able to demonstrate the presence of gamma-glutamyl transpeptidase in human or rabbit erythrocytes either by measuring the utilization of GSH or by following the formation of the product. 14C-L-methionine was used as acceptor amino acid, and the formation of gamma-glutamyl-14C-L-methionine was followed. Using similar conditions, we have been able to demonstrate the presence of gamma-glutamyl transpeptidase in human and rabbit leukocytes and in human kidney. In contrast to a previous report, we were unable to find the accumulation of 5-oxoproline, an intermediate of the gamma-glutamyl-cyclotransferase pathway in human red cells incubated in Krebs-Ringer solution. Immunologic studies demonstrated that human red cell membranes contained no protein antigenically similar to kidney gamma-glutamyl transpeptidase. Thus our studies indicated that in human and rabbit erythrocytes, the gamma-glutamyl transpeptidase-cyclotransferase pathway was not operative.

Animals↗
PubMed1976

The removal of leukocytes and platelets from whole blood.

Various methods for the removal of leukocytes from whole blood have been compared and a new technique has been devised. This procedure consists of passing whole blood through a bed composed of microcrystalline cellulose and alpha-cellulose. The method is rapid, reliable, removes over 99.75 per cent of the leukocytes from blood, and does not seem selectively to retain reticulocytes or to release a significant proportion of leukocyte enzymes. Most of the platelets are also removed from anticoagulated blood, and platelet-free red cells can be obtained by passing defibrinated blood over the microcrystalline cellulose-alpha-cellulose bed.

Blood↗
PubMed1976

A simple, rapid, efficient method for the preparation of gamma 32P-labeled guanosine triphosphate (GTP) and adenosine triphosphate (ATP).

A simplified new method for synthesis fo gamma 32P GTP and gamma 32P ATP is described. Radiophosphorus is incorporated into GDP and ADP to form GTP and ATP, respectively, in the phosphoglycerate kinase reaction. The product is separated by ion exchange resin chromatography. The procedure is simple, gives a product a high purity, and the label is confined to the gamma position.

Adenosine Triphosphate↗
PubMed1975

Genetic disorders of human red blood cells.

Human red blood cells (RBCs) are subject to an enormous degree of genetic diversity. The variability that occurs may result in anemia, cyanosis, polycythemia, or may cause no hematologic alterations. Genetic abnormalities affecting hemoglobin include the sickling disorders, the unstable hemoglobinopathies, hemoglobinopathies associated with polycythemia or with methemoglobinemia, and the alpha- and beta-thalassemias. The most common enzymatic abnormality of RBCs is glucose-6-phosphate dehydrogenase deficiency, but defects of many other enzymes leading to hemolytic anemia have been identified. Deficiences of RBC enzymes may also be important in the diagnosis of nonhematologic disease and in the evaluation of dietary status.

Anemia, Hemolytic, Congenital↗
PubMed1975

Purification and properties of human erythrocyte glutathione peroxidase.

Glutathione peroxidase has been purified to homogeneity from human erythrocytes. The purification steps involved ammonium sulfate precipitation of hemolysate, CM-cellulose (CM-52), DEAE-cellulose (DE52), Sephadex G-200, and DEAE-Sephadex column chromatography. In the last step, i.e. DEAE-Sephadex A-25 column chromatography, the enzyme was eluted in a major peak and tailing fraction. The major peak was found to be homogeneous on polyacrylamide disc electrophoresis and disignated as glutathione peroxidase A (GSH-Px A). The tail fraction, however, separated into two protein bands on polyacrylamide disc electrophoresis. One of the bands corresponded to GSH-Px A while the other band was slower moving and was designated as GSH-Px B. GSH-Px A and GSH-Px B had specific activity of 103 and 4 enzyme units per mg of protein, respectively. Antibodies raised against the homogeneous GSH-Px A have been found to cross-react with GSH-Px B. Both, GSH-Px A and B are selenoproteins. GSH-Px A has been found to contain 3.5 g atoms of selenium per mol of protein. Selenium content of GSH-Px B, however, could not be determined accurately due to insufficient material. The molecular weight of GSH-Px A as determined by the sedimentation equilibrium method is 95,000 plus or minus 3,000. On urea-sodium dodecyl sulfate-polyacrylamide disc electrophoresis GSH-Px A and B dissociate into single subunits. The molecular weight of the subunits of GSH-Px A is 23,000 and that of GSH-Px B is 47,000. Thus, it appears that GSH-Px A is a tetramer. Our results suggest that GSH-Px B is probably an altered form of the major component, GSH-Px A, or its precursor. The properties of GSH-Px A have been studied. The isoelectric pH was found to be 4.9 and the optimum pH for enzyme activity was 8.5. The energy of activation was 8.2 kcal. The Km of the enzyme for GSH was 4.1 mM while the Km for t-butyl hydroperoxide was 52 mu-M. The effect of sulfhydryl reagents and the metal ions on the enzyme was also studied.

Animals↗
PubMed1975

Purification and characterization of human erythrocyte pyridoxine kinase.

A 6000-fold purification of pyridoxine kinase from human erythrocytes has been achieved by a combination of DEAE-cellulose chromatography, ammonium sulfate fractionation, gel filtration and preparative disc polyacrylamide gel electrophoresis. Analytic disc polyacrylamide gel electrophoresis at pH 8.7 reveals two protein bands with similar mobility in the purified enzyme, but only one of these has catalytic activity. The enzyme is found to have a pI of 5.5 and a molecular weight of 65000 by gel filtration, and a broad pH optimum of 8.5. The enzyme is labile at acidic pH. The order of activation of divalent metal ions on the enzyme is Co-2+ greater than Mn-2+ greater than Mg-2+ greater than Zn-2+ greater than Cu-2+ greater than Ni-2+ greater than Fe-2+. Of the monovalent cations studied, K+ is the most effective activator, NH+4 is slightly less effective while Na+ is inhibitory. ATP is the specific phosphate donor for the enzyme. Sulfhydryl reagents did not significantly inhibit the enzyme. The Km for pyridoxine is 5.7 with 10-minus 6 M. Pyridoxal, pyridoxamine and 4-deoxypyridoxine inhibit pyridoxine kinase. Inhibition by pyridoxal is competitive and pyridoxal is observed to be phosphorylated effectively by the enzyme. Young human red cells have a higher activity of pyridoxine kinase than old red cells.

Cations, Divalent↗
PubMed1975

Pyruvate kinase deficiency anemia with terminal myelofibrosis and osteosclerosis in a beagle.

A 15-month-old male Beagle with chronic hemolytic anemia was found to have erythrocytic pyruvate kinase deficiency and, terminally, myelofibrosis and osteosclerosis. The dog's erythron was studied by procedures that enabled close comparison with congenital hemolytic anemia (pyruvate kinase deficiency) of Basenji dogs. The affected dog's sire, dam, and one littermate--each clinically and hematologically normal--were found to have 50% reduction in erythrocytic pyruvate kinase (PK) activity.

Ancylostomiasis↗