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Biomedical subjects

G L Dale

Publications and source records attributed to G L Dale.

66 records · Page 4Linked to original sources
PubMed1980

Simple and rapid purification of inside-out vesicles from human erythrocytes.

The preparation of inside-out vesicles from human erythrocytes requires their separation from contaminating right-side-out vesicles. We have taken advantage of the fact that there are no glycoproteins on the internal side of the erythrocyte membrane; therefore, inside-out vesicles do not interact with the lectin, concanavalin A, while right-side-out vesicles do interact. A concanavalin A-cellulose affinity matrix has been utilized to separate easily inside-out vesicles with a purity comparable to those prepared by prolonged centrifugation.

Cell Fractionation↗
PubMed1980

Glutathione transport by inside-out vesicles from human erythrocytes.

Purified inside-out vesicles from human erythrocytes were used to investigate the active transport of oxidized glutathione (GSSG). Incubation of vesicles and GSSG in the presence of ATP resulted in the transport of GSSG into the vesicles. When vesicles were incubated with reduced glutathione (GSH), no transport was observed. At GSSG concentrations of less than 5 mM, transport was linear up to 4 hr at 37 degrees C. A Lineweaver-Burk plot of the transport rate as a function of GSSG concentration was biphasic and gave apparent Km values of 0.1 and 7.1 mM. The Km for ATP . Mg in this transport process was 0.63 mM at a GSSG concentration of 20 microM and 1.25 mM at a GSSG concentration of 5 mM. The transport rate at low GSSG concentrations was inhibited by CTP or UTP, which acted as competitive inhibitors of ATP; Ki=0.51 mM. This inhibition may account for the high erythrocyte GSH levels observed in pyrimidine-5'-nucleotidase deficiency, a disorder in which erythrocytic levels of CTP and UTP are elevated.

Biological Transport, Active↗
PubMed1979

Incorporation of glucocerebrosidase into Gaucher's disease monocytes in vitro.

Several carriers were evaluated for use in the delivery of exogenous glucocerebrosidase to monocytes from Gaucher's disease patients. Only gamma globulin-coated, resealed erythrocytes proved to be an effective vehicle for enzyme delivery. Glucocerebrosidase added in this manner normalized intracellular enzyme levels for at least 18 hr. In this model system for the study of enzyme replacement therapy, soluble enzyme, enzyme in uncoated resealed erythrocytes, and enzyme incorporated into liposomes were ineffective.

Cells, Cultured↗
PubMed1979

Transport accounts for glutathione turnover in human erythrocytes.

Human erythrocytes were incubated with 3H-glycine to label the glutathione pool. These cells were then used to determine the rate of oxidized glutathione (GSSG) transport out of erythrocytes. For 6 normal individuals, the mean transport rate was 6.7 nmole GSSG/hr/ml red cells. This transport rate would suggest a half-life of 4.7 days for the erythrocytic glutathione, which is in close agreemwnt with the observed in vivo half-life of 4 days. These data demonstrate that GSSG transport can account for the observed turnover of erythrocytic glutathione.

Biological Transport↗
PubMed1979

Electrophoresis of glucocerebrosidase from normal and Gaucher disease fibroblasts.

A polyacrylamide gel electrophoresis system for glucocerebrosidase has been developed. This method was used to characterize the glucocerebrosidase activity of normal and Gaucher disease fibroblasts; the residual glucocerebrosidase activity in adult Gaucher disease fibroblasts co-migrates with the activity from normal fibroblasts.

Cells, Cultured↗
PubMed1977

Enzyme replacement therapy in Gaucher's disease: preliminary clinical trial of a new enzyme preparation.

A patient with far-advanced adult type Gaucher's disease was treated with solubilized, highly purified placental glucocerebrosidase administered after entrapment in human erythrocytes or by direct intravenous injection. In some instances the enzyme-containing erythrocytes were coated with gamma globulin. No toxic side effects were observed after enzyme infusion. There were suggestive, but not conclusive, findings that enzyme infusion may have been beneficial. After therapy, there was a decrease in transfusion requirement, some improvement of liver function, possible decrease in liver size, and relief of subjective symptoms. Erythrocyte and plasma glucocerebroside levels were unchanged during therapy, but there was a possibly significant decrease in leukocyte and platelet levels of the glycolipid. No changes occurred in serum acid phosphatase or angiotensin-converting enzyme activity.

Acid Phosphatase↗
PubMed1976

Purification of normal and inactive galactosemic galactose-1-phosphate uridylyltransferase from human red cells.

A rapid method is described for the purification of galactose-1-phosphate uridylyltransferase (EC 2.7.7.12) from human red blood cells by the use of DEAE-cellulose and two steps of affinity chromatography on a "uridine-aminohexyl" agarose column. The enzyme consists of two identical subunits of 31,000 molecular weight as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Chromatography on Sephadex G-200 chromatography gave a molecular weight of 69,000 for the native enzyme, it being eluted from the column with bovine serum albumin. Cross-linking of the enzyme with dimethylsuberimidate followed by analysis of the products by sodium dodecyl sulfate polyacrylamide gel electrophoresis caused the near-disappearance of the 31,000 molecular weight subunit and the appearance of a protein with a molecular weight of about 65,000 without the appearance of higher polymers.

Amino Acids↗
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↗