Katherine A. Sukalski

Protein and lipid oxidation of banked human erythrocytes:: Role of glutathione

In banked human erythrocytes (RBCs), biochemical and functional changes are accompanied with vesiculation and reduced in vivo survival. We hypothesized that some of these changes might have resulted from oxidative modification of membrane lipids, proteins, or both as a result of atrophy of the antioxidant defense system(s). In banked RBCs, we observed a time-dependent increase in protein clustering, especially band 3; carbonyl modification of band 4.1; and malondialdehyde, a lipid peroxidation product. Examination of the antioxidative defense system showed a time-dependent decline in glutathione (GSH) concentration and glutathione-peroxidase (GSH-PX) activity, with a concomitant increase in extracellular GSH, cysteine, and homocysteine, and unchanged catalase activity. When subjected to acute oxidant stress by exposure to ferric/ascorbic acid or tert-butylhydroperoxide (tert-BHT), catalase activity showed a steeper decline compared with GSH-PX. The results demonstrate that GSH and GSH-PX appear to provide the primary antioxidant defense in stored RBCs, and their decline, concurrent with an increase in oxidative modifications of membrane lipids and proteins, may destabilize the membrane skeleton, thereby compromising RBC survival.

Multifunctional Glucose-6-Phosphatase: A Critical Review

Glucose-6-phosphatase (D-glucose-6-phosphate phosphohydrolase; EC 3.1.3.9) was reviewed in some detail in the first edition of this work (Nordlie and Jorgenson, 1976). Our intention here is to focus on recent developments since the literature search for that earlier chapter was completed. Accordingly, we attempt to consider here the literature concerning glucose-6-phosphatase which has appeared since 1975. The authors were pleased to note a renewed interest in this complex enzyme, worldwide, more than 150 papers on the subject having appeared in the past seven years. A detailed consideration of the contents of all these papers is impossible in this limited space; we have therefore chosen to allude to many of them through the use of tables. Most of these are straightforward, descriptive studies, the essence of which is included in tables along with the literature references.

In Vivo Oxidative Modification of Erythrocyte Membrane Proteins in Copper Deficiency

Oxidative stress has been postulated to contribute to the pathology associated with dietary copper deficiency. In vivo, erythrocytes are probable targets of oxidative damage because they are exposed to high concentrations of oxygen and contain heme iron that can autoxidize, which results in the formation of superoxide anions. Activity of the important antioxidant enzyme, copper, zinc superoxide dismutase, decreases markedly in erythrocytes during copper deficiency. The effect of dietary copper deficiency on indicators of oxidative stress was examined in erythrocyte membranes of rats maintained on a purified copper-deficient diet for 35 days after weaning. Erythrocytes were separated into young and old populations on a Percoll gradient prior to membrane isolation and quantification of lipid peroxides and protein carbonyls. Protein carbonyls, determined by Western blot immunoassay, were detected predominantly in both the alpha and beta chains of spectrin. Alpha and beta subunits of spectrin in erythrocyte membranes from copper-deficient rats contained higher amounts of carbonyls than controls, regardless of the population of erythrocytes studied. This study suggests that spectrin may be a specific target for oxidative damage when erythrocyte copper, zinc superoxide dismutase activity is reduced by copper deficiency.

Hysteretic behavior of the hepatic microsomal glucose-6-phosphatase system

Carbamyl-P:glucose and PPi:glucose phosphotransferase, but not inorganic pyrophosphatase, activities of the hepatic microsomal glucose-6-phosphatase system demonstrate a time-dependent lag in product production with 1 mM phosphate substrate. Glucose-6-P phosphohydrolase shows a similar behavior with [glucose-6-P] less than or equal to 0.10 mM, but inorganic pyrophosphatase activity does not even at the 0.05 or 0.02 mM level. The hysteretic behavior is abolished when the structural integrity of the microsomes is destroyed by detergent treatment. Calculations indicate that an intramicrosomal glucose-6-P concentration of between 20 and 40 microM must be achieved, whether in response to exogenously added glucose-6-P or via intramicrosomal synthesis by carbamyl-P:glucose or PPi:glucose phosphotransferase activity, before the maximally active form of the enzyme system is achieved. It is suggested that translocase T1, the transport component of the glucose-6-phosphatase system specific for glucose-6-P, is the target for activation by these critical intramicrosomal concentrations of glucose-6-P.

Multiple forms of type I glycogen storage disease: underlying mechanisms

Abstract Three variants of Type I glycogen storage disease have been identified and their underlying biochemistry determined. All are characterized by a much diminished or absent function of glucose-6-phosphatase. The current concept is that Type Ia glycogenosis results from the congenital absence of the catalytic unit of glucose-6-phosphatase. With Types Ib and Ic, the catalytic unit remains intact, but the microsomal membrane transport function specific for glucose 6-phosphate is absent in Type Ib, and aberrant function of a second transporter specific for orthophosphate, inorganic pyrophosphate and carbamyl-phosphate is involved in the Type Ic condition.

Stimulation by 3-mercaptopicolinate of net glusose uptake by perfused livers from diabetic rats

Glucose uptake/production was studied as a function of varied glucose loads in isolated perfused livers from glucagon-treated alloxan-diabetic rats. Uptake of D-[U-14C]glucose was seen at all levels studied - 9.5-71 mM. In studies with unlabelled D-glucose carried out in the absence of 3-mercaptopicolinate, livers of diabetic rats showed a net production of glucose with perfusate glucose levels less than 22 mM. Above this level, these livers exhibited a time- and concentration-dependent net uptake of glucose for a period of 20-30 min. When 4 mM 3-mercaptopicolinate, which inhibited gluconeogenesis from endogenous substrates, was included in perfusates, a continuous net uptake of unlabelled glucose was observed at all levels above 4 mM. This lowering of the null-point, cross-over glucose concentration was shown to relate mechanistically to the observed reduction in steady-state hepatic glucose 6-phosphate level produced by mercaptopicolinate. The need for supplemental mechanisms of glucose utilization by high Km hepatic enzyme(s) operative in the virtual absence of insulin-dependent glucokinase also is indicated by these observations and by kinetic analysis.

Hysteresis at near-physiologic substrate concentrations underlies apparent sigmoid kinetics of the glucose-6-phosphatase system

Although Canfield and Arion (J. Biol. Chem. 263, 7458-7460 (1990)) have described the kinetics as hyperbolic, Traxinger and Nordlie (J. Biol. Chem. 262, 10015-10019 (1987)) reported sigmoid kinetics in the glucose-6-phosphatase system of intact microsomes at near-physiologic glucose-6-P concentrations. We show here that apparent sigmoidal kinetics, most clearly seen as sharp upward inflections in Hanes plots as substrate concentration approaches zero, are a consequence of the hysteretic lag in product formation during the first minutes of incubation of the enzyme with low concentrations of substrate. The appearance of sigmoidicity, observed when reaction velocities are calculated from changes in Pi concentration between 0 and 6 min of incubation, is not present when velocity is determined from slopes of [product]-time plots after linearity is achieved. The Km,glucose-6-P value, 0.86 mM, based on these hysteresis-corrected velocity values determined with intact microsomes from normal, control rats at low substrate concentrations, approached the upper limit of physiologic hepatic glucose-6-P concentrations. This suggests that glucose-6-phosphatase activity may be regulated by factors other than substrate concentrations alone. We propose that the hysteretic behavior, not sigmoid kinetics of the glucose-6-phosphatase enzyme system, may be a prime regulatory feature.

N-Acetylglucosamine-insensitive glucose phosphorylation in isolated hepatocytes from rats fasted for 48 h

Glucose phosphorylation in isolated hepatocytes was studied by the release of 3H from D-[2-3H]glucose. Glucokinase activity was decreased by fasting rats for 48 h and was further reduced in cells by adding 30 mM GlcNAc, a potent competitive inhibitor. Although this treatment resulted in the loss of more than 97% of glucokinase activity in hepatocytes, glucose phosphorylation proceeded at an appreciable rate. These observations demonstrate the involvement of a high -K0.5 enzyme system in addition to glucokinase in hepatocyte glucose phosphorylation.