Gordon C. Mills

Glutathione peroxidase and the destruction of hydrogen peroxide in animal tissues.

Abstract Rat liver contains a very active glutathione peroxidase, and lung, heart, and kidney of the rat also show a significant activity. Experiments have been carried out to show that reduction of hydrogen peroxide in the presence of glutathione and glutathione peroxidase may be coupled to oxidation of glucose 6-phosphate. The significance of this mechanism in the physiological destruction of hydrogen peroxide is discussed.

Increased purine nucleotides in adenosinedeaminase-deficient lymphocytes

It was found that ATP and cyclic AMP were greatly increased in human blood lymphocytes which were deficient in ADA. Certain other purine and pyrimidine nucleotides were elevated but to a lesser degree. Energy production in these cells may be inhibited by the increase in nucleotides since the ATP:ADP ratio was significantly below normal. Thus it appears that the immunologic deficiency in human ADA deficiency is related to increased nucleotide concentrations in the lymphocytes.

Limited effect of erythrocyte and plasma infusions in adenosine deaminase deficiency

A 10-month-old child with a profound deficiency of adenosine deaminase and severe combined immunodeficiency was treated for a period of 17 months with red cell and plasma transfusions containing normal amounts of the deficient enzyme. Following each transfusion, the plasma adenosine, red cell and lymphocyte ATP, urinary adenine, and urinary deoxyadenosine decreased transiently. During this period, the absolute blood lymphocyte count rose and a limited increased in the response of the lymphocytes to PHA-P was observed. Delayed hypersensitivity skin tests remained negative during the transfusion periods. A quantitative elevation of serum immunoglobulins occurred, but specific antibody formation was not elicited. In contrast to a previous report of successful therapy of ADA deficiency with red cell and plasma infusions, this patient responded poorly to enzyme replacement therapy. The difference may be related to a more profound enzyme deficiency in our patient.

The metabolism of nucleotides and other phosphate esters in erythrocytes during in vitro incubation at 37

Abstract Anion exchange chromatography of organic phosphates has been utilized to study the changes in human red cells during in vitro incubation at 37 °C. The changes in the three adenine nucleotides, inosine monophosphate, and 2,3-diphosphoglycerate were particularly noteworthy. The significance of these changes in relation to erythrocyte metabolism has been discussed.

Urinary excretion of purines, purine nucleosides, and pseudouridine in adenosine deaminase deficiency

Abstract Previous studies on the urine composition of an adenosine deaminase deficient child have been extended. New analytical procedures have been developed for determination of adenine, deoxyadenosine and adenosine in urine. Of these compounds, deoxyadenosine was the major urinary component. After initiation of a red cell transfusion regimen as treatment for the child, a statistically significant reduction was noted in the excretion of deoxyadenosine and adenosine. Catabolic products (hypoxanthine, xanthine, uric acid) of the major purine bases, as well as excretory products (7-methylguanine, pseudouridine) of some of the minor bases found in nucleic acids, have also been determined by ion exchange analysis. A decrease in the excretion level of 7-methylguanine following initiation of red cell infusions suggested an effect of adenosine deaminase deficiency on nucleic acid methylation. We have discussed the role of adenosine deaminase in adenosylmethionine metabolic pathways and have reviewed the evidence suggesting that increased levels of adenosine might play a major role by altering nucleic acid methylation reactions, particularly those involving methylation of the guanine cap of messenger RNA. We have also contrasted purine excretory patterns of our adenosine deaminase-deficient child with excretory patterns noted by others in a child with purine nucleoside phosphorylase deficiency. Despite similarities in the clinical syndrome, marked differences in purine excretory patterns have been noted.

Metabolic control mechanisms in human erythrocytes. The role of glyceraldehyde phosphate dehydrogenase.

Hemolyzates of human red cells have been utilized to evaluate the role of glyceraldehyde 3-phosphate dehydrogenase as a control enzyme in the glycolytic pathway. The kinetic data obtained at pH 7.3 indicate Km values of 21 μ m , 143 μ m , and 6.6m m , respectively, for glyceraldehyde 3-phosphate, NAD, and inorganic phosphate. NADH is a competitive inhibitor of NAD with a Ki of 9.1 μ m . At physiologic concentrations of substrates and NADH, glyceraldehyde phosphate dehydrogenase is operating at less than 1% of maximal velocity. Consequently, despite the relatively high activity reported for this enzyme in erythrocytes, it appears likely that it is a site of metabolic control. It is clearly the rate-limiting reaction when the pH of the cells is raised or when the phosphate concentration of the external medium is increased markedly.

Urinary excretion of modified purines and nucleosides in immunodeficient children

Studies have been carried out using an XAD-4 resin and ion-exchange chromatography for determination of urinary purines and nucleosides in seven children with severe combined immunodeficiency and in six normal children. These studies have included analyses for five methylated purines or nucleosides produced by catabolism of nucleic acids. The following compounds have been quantitatively determined: 1-methyladenosine, 1-methylinosine, 1-methylguanosine, 1-methylguanine, 3-methylcytidine, adenosine, methylthioadenosine sulfoxide, cytidine, and deoxycytidine. 1-Methyladenosine and 1-methylinosine were most consistently elevated in the urine of immunodeficient children. Methylthioadenosine sulfoxide was very markedly increased in urine of two of the immunodeficient children while more moderate increases were noted with a number of other nucleosides. The germ-free child with severe combined immunodeficiency showed consistently lower excretion levels of these compounds when compared to normal children.

Removal of salts from purines, pyrimidines and nucleosides using an xad—4 resin

Abstract In the present study, procedures are described for using columns of XAD-4 resin beads, a styrene—divinylbenzene copolymer, as adsorbents for purines, pyrimidines and their nucleosides. Elution of these nitrogenous compounds from the beads is carried out with either water or 19% ethanol, depending upon how tightly the particular compound is bound to the beads. The various salts [i.e., sodium chloride, sodium acetate, sodium phosphates, tris(hydroxymethyl)aminomethane (Tris), etc,] that have been tested are not adsorbed on the columns and appear in the first few fractions of the column effluent. Studies have been carried out with approximately 30 different compounds, including a number of methylated purines and purine nucleosides. Nearly all of the compounds were obtained entirely free of salt with an average recovery from the column of 96%.

Purification and properties of acyl phosphatase from human erythrocytes

Abstract A specific acyl phosphatase was purified 900-fold from human erythrocytes by utilizing ion-exchange cellulose-column chromatography. The retention of the enzyme on a carboxymethyl-cellulose column at pH 7.5, and on a diethylaminoethyl-cellulose column at pH 9.4, indicates a basic protein. The enzyme hydrolyzes acetyl phosphate and 1,3-diphosphoglycerate, but does not act on carbamyl phosphate, nor on a variety of phosphate esters. Maximal activity of the enzyme, with acetyl phosphate as substrate, is noted at pH 5. The K m of erythrocyte acyl phosphatase is 10.3 m m , with acetyl phosphate as substrate, and 117 μ m , with 1,3-diphosphoglycerate as substrate (pH 7.50 at 37 °). Adenosine triphosphate, carbamyl phosphate, and inorganic phosphate all inhibit the enzyme competitively, with K i values of 4.4, 6.9, and 3.4 m m respectively (with acetyl phosphate as substrate). Iodoacetate and p -chloromercuri-benzoate are not inhibitory. In contrast to acyl phosphatases purified from muscle and brain, the erythrocyte acyl phosphatase is thermolabile. The possible role of erythrocyte acyl phosphatase in the regulation of metabolism is discussed.

Isolation and Identification of 5′-Methylthioadenosine Sulfoxide from Human Urine

Abstract An adenine nucleoside isolated from human urine has been identified by mass spectra and other techniques as 5′-deoxy-5′-methyl-thioadenosine sulfoxide. Elevated levels (3–5 nmols/μmol creatinine) were noted in two children with severe combined immunodeficiency.