R.G. Slee

Lipid peroxidation of rat liver microsomes

1. The NADPH-dependent lipid peroxidation process was studied with microsomes and also the effects of addition of superoxide dismutase, catalase and thiourea. Only catalase and thiourea were able to inhibit lipid peroxidation. It seems that the initiating radical is the OH. radical formed by the Fenton reaction. 2. During lipid peroxidation glucose-6-phosphatase is inactivated, whilst the microsomal enzyme palmitoyl-CoA hydrolase is practically not affected. Because glucose-6-phosphatase activity decreases during ageing and palmitoyl-CoA hydrolase does not, a possible relationship with the ageing process is thought to exist. 3. Chromolipids are formed by the NADPH-dependent lipid peroxidation. These chromolipids have the same excitation-emission spectra as described for lipofuscin. The formation of these chromolipids is blocked by the addition of catalase and thiourea. 4. High-molecular weight proteins are formed during the NADPH-dependent lipid peroxidation. This process can be associated with the inactivation of enzymes. Also polymerisation is prevented by catalase and thiourea.

Lipid peroxidation of human erythrocyte ghosts induced by organic hydroperoxides

Isolated human erythrocyte ghosts perform lipid peroxidation, measured as malondialdehyde, induced by cumene hydroperoxide and t-butyl hydroperoxide but not by H2O2. In contrast to Ames et al. (Ames, B.N., Cathcart, R., Schwiers, E. and Hochstein, P. (1981) Proc. Natl. Acad. Sci. 78, 6858-6862), no inhibition is found by uric acid, only an increase in lag-time of the malondialdehyde production. In parallel with the malondialdehyde production, fluorescent chromolipids are also formed. Both processes are blocked by the addition of desferal, a potent iron chelator. The malondialdehyde production is also inhibited by the OH radical scavenger, thiourea, and by the anti-oxidant, butylated hydroxytoluene. Treatment of erythrocyte ghosts with cumene hydroperoxide or t-butyl hydroperoxide leads to the genesis of high-molecular-weight protein, but not with H2O2. The appearance of high-molecular-weight proteins is accompanied by disappearance of protein bands, e.g., the alpha- and beta-spectrin band, the anion-exchanger and some other smaller bands. Furthermore, a protein band is formed in the lower-molecular-weight region. 4. The addition of desferal does not reveal any blockade of the high-molecular-weight protein genesis. In contrast, a marked diminution of high-molecular-weight proteins is observed by the addition of thiourea, accompanied by a protection of the protein bands which would otherwise disappear. Similar results are obtained with butylated hydroxytoluene. 5. It is concluded that under oxidative stress the process of high-molecular-weight protein genesis can occur independently of the lipid peroxidation process, measured as the revealing of malondialdehyde.

COMPARISON OF THE INACTIVATION OF MICROSOMAL GLUCOSE-6-PHOSPHATASE BY IN SITU LIPID PEROXIDATION-DERIVED 4-HYDROXYNONENAL AND EXOGENOUS 4-HYDROXYNONENAL

1) The effect of 4-hydroxynonenal and lipid peroxidation on the activities of glucose-6-phosphatase and palmitoyl CoA hydrolase were studied. 2) 4-Hydroxynonenal inactivates glucose-6-phosphatase but has no effect on palmitoyl-CoA hydrolase. These effects are similar with those observed during lipid peroxidation of microsomes. 3) The inhibition of glucose-6-phosphatase by 4-hydroxynonenal can be prevented by glutathione but not by vitamin E. The inactivation of glucose-6-phosphatase during lipid peroxidation is prevented by glutathione and delayed by vitamin E. 4) The formation of 4-hydroxynonenal during lipid peroxidation was followed in relation to the inactivation of glucose-6-phosphatase. At 50% inactivation of glucose-6-phosphatase the 4-hydroxynonenal concentration was 1.5 microM. To obtain 50% inactivation of glucose-6-phosphatase by added 4-hydroxynonenal a concentration of 150 microM or 300 microM was needed with a preincubation time of 30 and 60 min, respectively. 5) It is concluded that the glucose-6-phosphatase inactivation during lipid peroxidation can be due to the formation of 4-hydroxynonenal. The formed 4-hydroxynonenal which inactivates glucose-6-phosphatase is located in the membrane. If this mechanism is valid it implies that a functional SH group of glucose-6-phosphatase is layered in the membrane. However, an inactivation of glucose-6-phosphatase by desintegration of the membrane by lipid peroxidation cannot be ruled out.

On the lipid peroxidation of rat liver hepatocytes, the formation of fluorescent chromolipids and high molecular weight protein

1. The formation of malondialdehyde by intact hepatocytes, induced by ADP/Fe3+ or cumene hydroperoxide, can be inhibited by the addition of thiourea. This may indicate that hydroxyl radicals are involved in this process. 2. Lipid peroxidation of intact hepatocytes leads to the formation of fluorescent chromolipids. When similar amounts of malondialdehyde are formed by either ADP/Fe3+ or cumene hydroperoxide, the lipid peroxidation induced by cumene hydroperoxide generates more fluorescent chromolipids than does the lipid peroxidation induced by ADP/Fe3+. 3. The formation of chromolipids is accompanied by the genesis of high molecular weight protein. With cumene hydroperoxide more high molecular weight protein is formed than with ADP/Fe3+. 4. It can be concluded that the defense system against lipid peroxidation of intact hepatocytes does not prevent the formation of lipofuscin-like chromolipids and high molecular weight protein as found earlier in microsomes. Cumene hydroperoxide, at least in this system, can be considered as an effective inducer of chromolipids.

The influence of glucose 1,6-diphosphate on the enzymatic activity of pyruvate kinase

Abstract 1. 1. The influence of Glc-1,6-P2 on hepatic and red blood cell pyruvate kinase (ATP: pyruvate phosphotransferase, EC 2.7.1.40) is quite similar to that of Fru-1,6-P2. The hexose diphosphates can replace each other in stimulating pyruvate kinase; after maximal stimulation by one of the compounds, the other is not capable of further stimulation. 2. 2. The regulatory role of Fru-1,6-P2 on the activity of pyruvate kinase is discussed in view of the results obtained.

The use of leucocytes as an aid in the diagnosis of glycogen storage disease type II (Pompe's disease)

Abstract The acid maltase activities of human lymphocytes and polymorphonuclear cells from controls and patients suffering from Pompes disease, have been investigated. The effect of antibody against human liver acid maltase on leucocyte acid maltase was studied. Furthermore, by using glycogen as substrate, it was possible to detect heterozygotes by calculating the ratio of acid and neutral maltase activities in dextran-isolated leucocyte preparations.

Identity and activities of superoxide dismutase in parenchymal and nonparenchymal cells from rat liver.

Abstract Intact and pure parenchymal and nonparenchymal cells were isolated from rat liver. The activities of Superoxide dismutase in these cell types were determined by two different methods. With both methods the specific activity of this enzyme is 1.5 times higher in parenchymal than in nonparenchymal liver cells. It can be calculated that about 7% of the total rat liver Superoxide dismutase activity is localized in the nonparenchymal liver cells. Electrophoresis on polyacrylamide gels indicates that the isolated parenchymal cells contain both cytosolic and mitochondrial isoenzymes, whereas with nonparenchymal cells only the cytosolic enzyme could be detected. The mitochondrial band observed in isolated parenchymal cells is absent in the original total liver homogenate. This isoenzyme seems to be activated during the parenchymal cell isolation procedure. Isoelectrofocusing indicates that the cytosolic Superoxide dismutase consists in four different isoelectric forms in both parenchymal and nonparenchymal cells. With the mitochondrial isoenzyme two bands are obtained. The possibility that O 2 − is an important intermediate in H 2 O 2 formation in nonparenchymal liver cells is discussed. In this respect, Superoxide dismutase might not only protect the cell against a toxic reagent as O 2 t - , but might also help to regulate the level of the important antimicrobial agent, H 2 O 2 .

Some properties of human liver acid α-glucosidase

Abstract 1. 1. Albumin activates human liver acid α-glucosidase (α- d -glucoside hydrolase, EC 3.2.1.20). From the Arrhenius plot, pH-dependence and LineweaverBurk plots it can be concluded that this activation is not only due to stabilisation of the enzyme, but also influences the enzymatic activity. It is proposed that for optimal functioning human liver acid α-glucosidase needs a protein environment. 2. 2. Glycogen has a competitive inhibitory effect on the hydrolysis of 4methylumbelliferyl-α- d -glucopyranoside, in contrast to maltose which exhibits a non-competitive type of inhibition. It is concluded that two catalytic sites exist, one for glycogen and one for maltose, while both sites influence each other. With glycogen as substrate a break in the Arrhenius plot is found. This is not the case when maltose is used as substrate. 3. 3. The effect of antibody raised against human liver acid α-glucosidase on the activity of human liver acid α-glucosidase is studied. No cross-reacting material could be demonstrated in the liver of a patient with glycogen storage disease Type II (M. Pompe, acid α-glucosidase deficiency).

Canine glycogen storage disease type II a biochemical study of an acid α-glucosidase-deficient Lapland dog

A biochemical study was performed in a Lapland dog suspected of glycogen storage disease type II (acid alpha-glucosidase deficiency, Pompes disease). Glycogen content was substantially elevated in heart and skeletal muscle but not in the liver. Severely reduced activities of acid alpha-glucosidase (EC 3.2.1.20) were found in heart, skeletal muscle, liver and cultured tongue fibroblasts. The deficiency was located in the glycoprotein fraction, which supported its lysosomal origin. The electrophorogram showed after acid incubation that the affected dog was missing the activity band, while after neutral incubation the pattern was similar to control. The obtained biochemical data are compared with the known data of the human pathology.

Physico-chemical and immunological properties of acid α-glucosidase from various human tissues in relation to glycogenosis type II (pompe's disease)

Abstract The physico-chemical and immunological properties of acid α-glucosidase from various human tissues have been studied. Heat stability of acid α-glucosidase from heart, liver and skeletal muscle is identical, but for kidney some different results are obtained. Identical isoelectrofocussing patterns are found for heart, liver and skeletal muscle. Furthermore, the effect of antiserum against human liver acid α-glucosidase on the activity of acid α-glucosidase from various tissues is studied. The results are discussed in relation to glycogenosis type II (Pompes disease).