Th.J.C. Van Berkel

Distribution of L- and M-type pyruvate kinase between parenchymal and Kupffer cells of rat liver

Abstract 1. 1.|Parenchymal cells were isolated from rat liver by using EDTA + lysozyme or citrate. These cells contained only the L-type pyruvate kinase (ATP:pyruvate phosphotransferase, EC 2.7.1.40). 2. 2.|Kupffer cells were isolated from rat liver by using pronase. The Kupffer cell preparation showed a ratio of M-type to L-type pyruvate kinase 10–20 times higher than the ratio in a total liver homogenate, suggesting that Kupffer cells only (probably) contain the M-type pyruvate kinase. 3. 3.|The results obtained suggest that gluconeogenesis is confined to the parenchymal cells of rat liver.

Two interconvertible forms of L-type pyruvate kinase from rat liver.

Abstract 1. 1. Reduced L-type pyruvate kinase (ATP:pyruvate phosphotransferase, EC 2.7.1.40) from rat liver can be converted into an oxidized form by incubation with oxidized mercaptoethanol and oxidized glutathione. This interconversion can be completely reversed by incubation with reduced mercaptoethanol. 2. 2. The kinetic and allosteric properties of the reduced and oxidized forms are described. 3. 3. The results are discussed in view of a possible regulation of the enzyme.

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.

On the molecular basis pyruvate kinase deficiency: I. Primary defect or consequence of increased glutathione disulfide concentration

Abstract 1. 1. Human erythrocyte pyruvate kinase (ATP:pyruvate phosphotransferase EC 2.7.1.40) can be converted into an oxidized form by incubation with oxidized glutathione. The oxidized enyzme can be reduced again by incubation with mercaptoethanol, with reduced glutathione a partial reduction of the enzyme is obtained. 2. 2. The oxidized enzyme shows a lower affinity for the substrate phosphoenol-pyruvate and for the allosteric effector fructose 1,6-diphosphate. The thermolability of the oxidized enzyme is markedly increased, compared with the freshly isolated or reduced enzyme. 3. 3. The data obtained with oxidized enzyme are discussed in relation to the data obtained with pyruvate kinase from pyruvate kinase-deficient patients. It is concluded that erythrocyte pyruvate kinase deficiency can be a consequence of an increased oxidized glutathione concentration in the red blood cell.

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.

Difference spectra, catalase- and peroxidase activities of isolated parenchymal and non-parenchymal cells from rat liver

Abstract The reduced minus oxidized difference spectra from isolated parenchymal and non-parenchymal cells from rat liver indicate that the non-parenchymal cells contain a considerable amount of peroxidase. This interpretation is favoured by the more than 30 times higher specific activity of peroxidase (EC 1.11.1.7) in the non-parenchymal cells as compared to the parenchymal cells. The catalase (EC 1.11.1.6) activity in the non-parenchymal cells is 4 times lower than in the parenchymal cells. These results are consistent with an antimicrobial function of the non-parenchymal cells in liver.

Some kinetic properties of the allosteric M-type pyruvate kinase from rat liver; influence of pH and the nature of amino acid inhibition

Abstract 1. 1. The influence of the pH on the activity of the M-type pyruvate kinase (ATP:pyruvate phosphotransferase, EC 2.7.1.40) from rat liver has been studied. The K 0.5 for the substrate phosphoenolpyruvate was pH dependent and above pH 7.25 sigmoidal curves have been obtained. Frutcose-1.6-diphosphate was able to convert these curves into a hyperbolic relationship. 2. 2. It was found that alanine acts as an allosteric inhibitor. The inhibition could be fully abolished by the addition of fructose-1,6-diphosphate. The alanine-inhibition is dependent on the pH and on the phosphoenolpyruvate concentration. 3. 3. It was concluded that most of the properties can be explained by the model of Monod, Wyman and Changeux.

On the molecular basis of pyruvate kinase deficiency II. Role of thiol groups in pyruvate kinase from pyruvate kinase-deficient patients

Abstract 1. 1. Human erythrocyte pyruvate kinase (ATP:pyruvate phosphotransferase, EC 2.7.1.40) from the class of pyruvate kinase-deficient patients, characterized by an increased affinity towards phosphoenolpyruvate and a loss of cooperative interaction towards this substrate, shows less affinity for the allosteric inhibitor ATP, when compared to pyruvate kinase from control persons. From the obtained kinetic data we can conclude that the loss of cooperativity towards phosphoenolpyruvate is a consequence of a shift in the R ⇌ T equilibrium to the R state. 2. 2. Incubation of pyruvate kinase, obtained from this class of pyruvate kinase-deficient deficient patients with mercaptoethanol, changes the abnormal kinetics into normal kinetics, as can be conclded from the change in phosphoenolpyruvate dependency and ATP inhibition. 3. 3. The effect of mercaptoethanol on the kinetics of pyruvate kinase from pyruvate kinase-deficient patients suggests that the alteration in the enzyme is a consequence of a modification of the -SH groups. It is suggested that pyruvate kinase deficiency is a secondary defect and that the process which causes the change in the -SH groups of pyruvate kinase, may also be responsible for the increased rate of haemolysis, found in these patients.

Relative contributions of parenchymal and non-parenchymal (sinusoidal) liver cells in the uptake of chylomicron remnants

The relative contributions of parenchymal cells and non-parenchymal (sinusoidal) cells to the in vivo hepatic uptake of chylomicron remnants was measured 30 min after intravenous injection into rats. The chylomicron remnants were labeled with [3H]leucine, which was almost exclusively present in apolipoprotein B. The isolated non-parenchymal cells (a mixture of Kupffer cells and endothelial cells) contained 6.7 times more apolipoprotein B radioactivity per mg cell protein than the isolated parenchymal cells. It was calculated that the contributions of non-parenchymal and parenchymal liver cells to the total hepatic uptake of chylomicron remnants are 35% and 65%, respectively.

M-type pyruvate kinase of leucocytes: An allosteric enzyme

Abstract 1. 1. The influence of pH and amino acids on the activity of pyruvate kinase (ATP: pyruvate phosphotransferase, EC 2.7.1.40) from leucocytes were studied 2. 2. The data can be explained by the model of Monod, Wyman and Changeux. It is proposed that this model is also valid for the other various types of pyruvate kinase.