Biomedical subjects

W Gevers

Publications and source records attributed to W Gevers.

99 records · Page 6Linked to original sources

PubMed1967

The regulation of phosphoenolpyruvate synthesis in pigeon liver.

1. The intracellular location and maximal activities of enzymes involved in phosphoenolpyruvate synthesis have been investigated in pigeon liver. Enolase and pyruvate kinase were cytoplasmic, and the activities were 50-60 and 180-210mumoles/min./g. dry wt. at 25 degrees respectively. Phosphoenolpyruvate carboxykinase was present exclusively, and nucleoside diphosphokinase predominantly, in the mitochondria; the particles had to be disrupted to elicit maximal activities, which were 27-33 and 400-600mumoles/min./g. dry wt. at 25 degrees respectively. The activities of all four enzymes did not change significantly during 48hr. of starvation. 2. Conditions for incubation of washed isolated mitochondria were established, to give high rates of synthesis of phosphoenolpyruvate, linear with time and proportional to mitochondrial concentration. Inorganic phosphate and added adenine nucleotides were stimulatory, whereas added Mg(2+) inhibited, partly owing to activation of contaminant pyruvate kinase. Phosphoenolpyruvate formation occurred from oxaloacetate, malate, fumarate, succinate, alpha-oxoglutarate and citrate, in decreasing order of effectiveness. 3. The steady-state ATP/ADP ratio of mitochondrial suspensions was decreased in the presence of added 2.5mm-Mg(2+) (owing to stimulation of adenylate kinase and possibly of an adenosine triphosphatase), 0.5mm-Ca(2+) or 0.4mm-dinitrophenol. In each case the rate of substrate removal and oxygen uptake was increased, whereas phosphoenolpyruvate synthesis was inhibited. Citrate formation was enhanced, owing to de-inhibition of citrate synthase. These effects were not primarily related to changes in the oxaloacetate concentration. 4. Both phosphoenolpyruvate carboxykinase and nucleoside diphosphokinase were active within the atractylosidesensitive barrier to the mitochondrial metabolism of added adenine nucleotides. There was no correlation between the rate of substrate-level phosphorylation associated with the oxidation of alpha-oxoglutarate, and the synthesis of phosphoenolpyruvate. 5. The results suggest that phosphoenolpyruvate formation in pigeon-liver mitochondria is regulated partly by the phosphorylation state of the adenine and guanine nucleotides, and partly by variations in the oxaloacetate concentration, all in the mitochondrial matrix. 6. Phosphoenolpyruvate is assumed to be the metabolite transported from the mitochondria to the cytoplasm during gluconeogenesis from oxaloacetate in pigeon liver.

Animals↗

PubMed1966

The effects of adenine nucleotides on carbohydrate metabolism in pigeon-liver homogenates.

1. The effects of added AMP on carbohydrate metabolism were investigated in pigeon-liver homogenates, which can degrade glucose and synthesize it from lactate. Suitable experimental conditions were established for studying such effects, including the addition of P(i) (20mm) to stabilize adenine nucleotides and supplementation with NAD(+) (0.5mm). 2. Lactate increased the rate of oxygen consumption and kept the concentration of ATP high and that of AMP relatively low. 3. Added AMP (1.25-5mm) raised the net rate of carbohydrate removal and inhibited the net formation of glucose from lactate, as well as the incorporation of lactate into glucose. These effects were accompanied by a fall in the concentrations of hexose 6-phosphates and a rise in those of fructose diphosphate and triose phosphates. When the activity of glyceraldehyde 3-phosphate dehydrogenase was limited experimentally by a low concentration of NAD(+) or when it was blocked by iodoacetate, the accumulations of fructose diphosphate and triose phosphates were large and accounted for most of the carbohydrate degraded in the presence of AMP. 4. AMP also inhibited the conversion of pyruvate into phosphoenolpyruvate. Data on the concentrations of pyruvate, phosphoenolpyruvate and intermediates of the tricarboxylic acid cycle, as well as on isotope distribution, suggest that the effect was due to inhibition of phosphoenolpyruvate carboxykinase. 5. The results indicate that in the homogenates phosphofructokinase and fructose diphosphatase, controlled in their activity by adenine nucleotides and other cell constituents, are enzymes which regulate the direction of carbohydrate metabolism (degradation or synthesis) in the liver. 6. It is suggested that active transport of adenine nucleotides, citrate, Mg(2+), Ca(2+), P(i) and other cell constituents may play a role in regulating the activity of enzymes which are affected by these substances. 7. A procedure is described for generating alkali in a closed manometer vessel, by mixing mercuric oxide and a solution of sodium iodide, for use in a method for measuring the oxygen consumption at physiological bicarbonate concentrations.

Adenine Nucleotides↗

PubMedDate not supplied

The hypoxic, low-flow perfused rat heart: characterization as a model of global ischaemia.

In the study of the mechanism of mitochondrial damage in myocardial ischaemia, production of a small animal model of global ischaemia proved to be necessary. Thus, the effects of coronary flow rate, oxygen availability and substrate availability were studied on the functional capacity of mitochondria isolated from perfused rat hearts. The hypoxic, low-flow perfused heart eventually chosen was evaluated as a model of myocardial ischaemia by measuring tissue contents of high energy phosphate compounds and glycolytic intermediates and by assessing the effects of exogenous substrates on the above parameters.

Adenosine Diphosphate↗

PubMedDate not supplied

Substrate effects on mitochondrial function and tissue lipids in low-flow hypoxia of isolated perfused rat hearts.

A possible causal relationship between tissue FFA contents and the depression in mitochondrial oxidative phosphorylation in myocardial ischaemia has been suggested. To test this hypothesis, the effects of different substrates added to the perfusates of hypoxic, low-flow perfused hearts were examined on oxidative phosphorylation catalysed by mitochondria isolated from such tissue. In an additional series of experiments tissue neutral glyceride and FFA levels were analysed and correlated with changes in mitochondrial function. Mitochondria isolated from hearts with a high tissue FFA content exhibited the lowest ADP/O ratios, RCI and QO2 values. On the other hand, mitochondria isolated from hearts with reduced FFA contents, performed significantly better with respect to these parameters of mitochondrial function studied.

Animals↗

PubMedDate not supplied

Biochemical aspects of cell death.

Studies of cell injury and death at the molecular and cell-biological level will help to provide methods for the reconstruction of the course of events between the occurrence of primary pathology and the time of investigation. The general role of component turnover, energy metabolism and lysosomes in cellular injury is briefly described.

Adenosine Triphosphate↗

PubMedDate not supplied

Low density lipoprotein metabolism in cultured fibroblasts from a new group of patients presenting clinically with homozygous familial hypercholesterolemia.

The metabolism of low density lipoproteins (LDL) was studied in cultured fibroblasts obtained from five local patients diagnosed, on the basis of clinical features and serum cholesterol concentrations, as having the homozygous form of familial hypercholesterolemia. LDL receptor function was assessed by measuring the binding, internalization, and degradation of 125I-labeled LDL, and by measuring the stimulation of cellular acyl-CoA cholesterol acyltransferase (ACAT) activity which followed exposure to LDL. Fibroblasts from two cases (CF and GM) showed receptor activities which were approximately 10% of the values obtained with normal cells, while ACAT stimulation by LDL was very low. These two patients were classified as homozygous for a receptor-defective abnormality. However, fibroblasts from the other three patients (JG, ES, and TT) showed greater than 25% of normal receptor activity, as assessed by 125I-LDL binding and catabolism. ACAT stimulation by LDL in cells from JG and ES was within the range of values shown by cells previously characterized as heterozygous for a receptor-negative mutation. Cells from ES behaved atypically: a low, but nonsaturable, activation by LDL was evident. ACAT stimulation by LDL was normal in cells from TT. Receptor activities of the cells from the available parents, assessed on the basis of LDL binding and degradation or of ACAT stimulation, were not clearly distinguishable from those of normal cells. These results add to the growing evidence of genetic heterogeneity underlying the clinical picture associated with familial hypercholesterolemia in different geographical distributions.

Adolescent↗

PubMedDate not supplied

In vitro metabolism of LDL labeled with a nondegradable cholesteryl ester analogue.

Low density lipoprotein (LDL) metabolism by human skin fibroblasts was studied using LDL labeled in the neutral lipid fraction with a nonhydrolysable cholesteryl ester analogue 3H-cholesteryl linoleyl ether (3H-CLE-LDL). LDL uptake could be quantitated accurately using 3H-CLE-LDL, since the label accumulated intracellularly due to its resistance to hydrolysis. 3H-CLE-LDL was taken up via apo B,E receptor-mediated endocytosis in a manner similar to 125I-labeled LDL. This was demonstrated by similar rates of uptake of the two differently labeled LDL preparations, saturation kinetics of uptake with respect to 3H-CLE-LDL concentration, regulation of 3H-CLE-LDL uptake by procedures that up-regulate or down-regulate the number of apo B,E receptors, and negligible uptake of 3H-CLE-LDL by receptor-negative cell strains. The major difference between the handling of 125I-LDL and 3H-CLE-LDL was the finding that, while the amount of trypsin-releasable (surface) 3H-CLE increased progressively over a 24-hour experimental period, trypsin-releasable 125I-LDL reached a maximum within 30 minutes. After 24 hours of incubation, the 3H radioactivity released by brief trypsinization was three to four times higher than could be accounted for by 125I radioactivity released by a similar treatment. Possible reasons for this behavior are discussed.

Cholesterol↗

PubMedDate not supplied

Low density lipoprotein receptor mutations in South African homozygous familial hypercholesterolemic patients.

The binding and catabolism of low density lipoprotein (LDL) were studied in cultured skin fibroblasts and/or Epstein-Barr virus-transformed lymphoblastoid cells from 20 familial hypercholesterolemic (FH) homozygotes of 14 Afrikaner kindreds. Cells from available heterozygotes were also analyzed. Good resolution between the normal, heterozygote, and homozygote groups was obtained on the basis of these assays using either cell type. Results obtained with fibroblasts allowed the classification of 13 of these kindreds as being typically receptor-defective, and one kindred with two homozygotes as being receptor-negative. Fibroblasts from homozygotes of the receptor-defective class expressed LDL receptor activities which varied between 3% and 25% of normal. Where two homozygotes from the same family were available for assays (six families), both yielded similar activities. In contrast to fibroblasts, all the lymphoblastoid cells derived from FH homozygotes yielded LDL receptor activities equal to or less than 10% of normal; in a number of cases no significant 125I-LDL binding was detectable. The use of lymphoblastoid cells provides a convenient means for screening for FH at the cellular level. Our results indicate a predominance of a receptor-defective type of abnormality, which is consistent with a founder gene effect in the Afrikaner population.

Adolescent↗

PubMedDate not supplied

Retro-endocytosis of low density lipoprotein by cultured human skin fibroblasts.

A fraction of the low-density lipoprotein (LDL) internalized by cells via receptor-mediated endocytosis follows a short-circuit pathway, termed "retro-endocytosis," that results in the rapid exocytosis of ligand. Results from the current study suggest that retro-endocytosis of LDL in human fibroblasts is caused by resurfacing of endocytotic vesicles that contain both free and receptor-bound ligand, resulting in discharge of vesicular contents and in spontaneous dissociation of LDL from its receptor. The bulk of the released LDL particles had the same size, density, and immunogenic properties as native LDL, indicating that they were discharged intact. Some of the retro-endocytosed LDL was larger than native LDL, and some exhibited altered sedimentation properties. When fusion of endosomes with lysosomes was inhibited by chilling cells to 18 degrees C, the proportion of intracellular LDL subsequently released was unaffected, suggesting that retro-endocytosis does not require lysosomal participation. Furthermore, the shorter the internalization phase the greater was the proportion of LDL subsequently released, suggesting that LDL was discharged from compartments formed early in endocytosis. Retro-endocytosis of LDL was stimulated by agents that neutralize acid intracellular compartments, such as ionophores (monensin) and weak bases (chloroquine and methylamine). Monensin increased the proportion of intracellular LDL released, suggesting that it had a direct effect on retro-endocytosis. The effect of weak bases appeared to be secondary to their ability to promote cellular accumulation of undegraded LDL. Thus, retro-endocytosis of LDL becomes a major pathway when intracellular compartments fail to maintain a low pH or where the intracellular concentration of LDL reaches abnormal levels.

Cells, Cultured↗