Thermolabile enzymes in progeria and Werner syndrome: evidence contrary to the protein error hypothesis

To test the hypothesis that widespread errors in protein synthesis underlie diseases with features resembling premature aging, we examined the thermostability of two erythrocyte enzymes in three unrelated progeria families and in two Werner syndrome patients. Unlike previous reports, no increased heat-labile component of glucose-6-phosphate dehydrogenase (G6PD) or 6-phosphogluconate dehydrogenase (6PGD) was found. Our results do not support the protein error hypothesis. Our data raise questions regarding the usefulness of thermolabile enzyme level as a proposed marker for progeria or Werner syndrome.     

The Sedimentation Pattern of D-Glucose-6-Phosphate Dehydrogenase from Bovine Fetal and Adult Erythrocytes

Erythrocytes from bovine fetuses contain about 2.4 times higher D-glucose-6-phosphate dehydrogenase activities than erythrocytes from adult cows and bulls. Studying whether this is due to the existence of a special fetal type of enzyme or an increased amount of enzyme in fetal erythrocytes, the sedimentation coefficients of the enzymes have been estimated by s-zonal ultracentrifugation, and compared to normal and deficient human erythrocyte D-glucose-6-phosphate dehydrogenase, s-zonal ultracentrifugations have been performed with a computer optimized isokinetic sucrose gradient. The mainlines in the program used for calculation of sedimentation coefficients are described. Bovine fetal and adult erythrocyte D-glucose-6-phosphate dehydrogenase was found to have the same sedimentation coefficient of 7.4 S which is different from the sedimentation coefficient of 6.4 S of both human types of the enzyme. The sedimentation coefficients of 6-phospho-D-gluconate dehydrogenase from bovine fetal, bovine adult and human erythrocytes were 6 S for all three types of this enzyme. By cellulose acetate electrophoresis bovine fetal and adult D-glucose-6-phosphate dehydrogenase show the same mobility, again differing from the normal and deficient human type. The results of these experiments show that bovine fetal and adult erythrocytic D-glucose-6-phosphate dehydrogenase with respects to molecular parameters are closely related and perhaps identical enzymes.     

Age-related changes in oxidized proteins

We have previously described the oxidative inactivation of several key metabolic enzymes by a variety of mixed function oxidation systems. Because many of the enzymes which are inactivated have been shown by others to accumulate as inactive or less active forms during cellular aging, we have examined the levels of oxidatively modified proteins in two model systems used for studies on aging. The results show that levels of oxidatively modified proteins increase with age in circulating erythrocytes, and this change is correlated with the loss of marker enzyme activity. Our studies also show that in cultured fibroblasts from normal donors the levels of oxidatively modified proteins increase only after the age of 60. However, the levels of oxidatively modified proteins in fibroblasts from individuals with progeria or Werner's syndrome are significantly higher than age-matched controls. Moreover, treatment of glucose-6-phosphate dehydrogenase with a mixed function oxidation system leads to oxidative modification and increased heat lability of the enzyme. Taken together these results suggest that loss of functional enzyme activity and increased heat lability of enzymes during aging may be due in part to oxidative modification by mixed function oxidation systems.     

Simultaneous preparation from human placenta of several enzymes of glucose metabolism

A procedure for the simultaneous purification to homogeneity of hexokinase, phosphoglucomutase 1 and 2, aldolase, phosphoglucose isomerase and glucose-6-phosphate dehydrogenase from human origin has been developed. Human placenta homogenate was first chromatographed on DE-52 column which retains hexokinase and glucose-6-phosphate dehydrogenase while the other enzymes are recovered in the unabsorbed protein fraction. The other steps in the purification involve Matrex gel and specific affinity chromatography for the DE-52 retained enzymes and phosphocellulose and Matrex gel chromatography for the other enzymes. All the enzymes mentioned were obtained in one week, with recoveries from 14 percent for glucose-6-phosphate dehydrogenase to 75 percent for hexokinase. Thus, the procedures utilized seem to be useful in obtaining large amounts of enzymes in a a homogeneous form from an easily available human tissue.     

Decreased enzymic protection and increased sensitivity to oxidative damage in erythrocytes as a function of cell and donor aging.

Erythrocytes from young and old rats were separated into four age fractions by density-gradient centrifugation. The specific activities per cell were determined for glucose-6-phosphate dehydrogenase (EC 1.1.1.49), glutathione peroxidase (EC 1.11.1.9), glutathione reductase (EC 1.6.4.2) and catalase (EC 1.11.1.6). Decreased specific activities were observed with increasing cell age for all four enzymes in both young and old animals. In addition, significant differences in the activities of these enzymes were observed between cells of the same age fraction from young and old donors. Susceptibility of fractionated erythrocytes to oxidative attack in vitro generated by incubation with xanthine/xanthine oxidase increased with both cell and animal age. The amount of membrane-lipid peroxidation also increased with cell and animal aging, as measured by both thiobarbituric acid and fluorescent chromolipid assays. Increases of 2-3-fold in the contents of lipid peroxides were observed between the youngest and oldest age fractions of young rats. Lipid peroxide contents in young cells of old animals were equal to those in old erythrocytes from young rats and increased by 30% with cell aging in the old donors. These results suggest that the extent of enzymic protection against oxidative and peroxidative damage decreases with erythrocyte aging. More importantly, enzymic protection in cells of old rats is considerably decreased already in the early stages of their lifespan.     

Oxidant/antioxidant status in blood of patients with malignant breast tumour and benign breast disease

The aim of this study was to investigate the alterations in lipid peroxidation and antioxidant enzyme defences in the blood of patients with malignant breast tumour and benign breast disease. Forty patients with malignant breast tumour, 20 patients with benign breast disease and also 20 healthy control subjects were recruited for the study. Malondialdehyde levels in plasma and erythrocytes, and the activities of erythrocyte CuZn-superoxide dismutase, catalase, glutathione peroxidase and glucose-6-phosphate dehydrogenase were measured. Malondialdehyde levels were higher in patients with both benign breast disease and malignant breast tumour compared with control subjects. The activities of all antioxidant enzymes were higher in patients with malignant breast tumour, while only glutathione peroxidase and CuZn-superoxide dismutase activities were higher in patients with benign breast disease. Except for glucose-6-phosphate dehydrogenase, the antioxidant enzymes studied correlated positively with the malondialdehyde levels in patients with malignant breast tumour. On the other hand, only glucose-6-phosphate dehydrogenase activity was increased by the level of malignancy. The activity increases in erythrocyte antioxidant enzymes may be a compensatory upregulation in response to increased oxidative stress especially in patients with malignant breast tumour.     

Age-related changes in glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in the subcellular fractions from the rat brain and the effect of dimethylaminoethanol

The activity of pentose phosphate pathway enzymes (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) was measured in the cytosol and the particulate fractions (mitochondrial-synaptosomal and microsomal) from the cerebrum and the cerebellum of the rats aged 1, 2, 3, 6, 9 and 12 months. The results showed that the two enzymes occurred both in cytosol and particulate fractions. Both the enzymes were higher in the particulate fractions from cerebellum than in the same fractions from cerebrum. In both regions of the brain, particulate fraction enzymes showed an age-related decline in their activity, but the cytosol fraction enzymes remained unchanged in all the age groups. Dimethylaminoethanol, an important molecular constituent of some antiageing drugs, increased the activity of these enzymes in a dose dependent manner only in the particulate fractions.     

Partial purification and some properties of human erythrocyte prostaglandin 9-ketoreductase and 15-hydroxyprostaglandin dehydrogenase.

Human erythrocytes were found to contain two prostaglandin metabolizing enzymes: a prostaglandin E 9-ketoreductase catalyzing the reduction of prostaglandin E₂ to form prostaglandin F_2α and a 15-hydroxyprostaglandin dehydrogenase that catalyzes the oxidation of prostaglandin F_2α to form 15-ketoprostaglandin F_2α. Both enzymes are found in the cytoplasmic fraction of erythrocytes and both enzymes use the triphosphopyridine nucleotides as cofactors more effectively than the diphosphopyridine nucleotides. These two enzymes were partially purified from erythrocyte homogenates and some of their properties were studied.     

Thermal properties of enzymes from Bacillus flavothermus,grown between 34 and 70°C

The activity and stability of several enzymes from the facultative thermophile Bacillus flavothermus, grown within the mesophilic and thermophilic region at 34 degrees C, 43 degrees C, 52 degrees C and 70 degrees C, have been examined. While the temperature optima and maxima of all enzymes tested were found to remain unchanged at all growth temperatures, it was demonstrated that the heat stability of the proteins increased with ten perature, however, not uniformly for all enzymes. One exception was acetate kinase and the intrinsic stability of pyruvate kinase was found to increase only slightly. With all other proteins tested (alanine dehydrogenase, isocitric dehydrogenase and glucose-6-phosphate dehydrogenase, glutamate-oxalacetate and glutamate-pyruvate transaminase and myokinase) the intrinsic stability was found to increase to about 55 degrees C, but stayed unaltered at higher growth temperatures. Except for acetate kinase and myokinase, the enzymes could be stabilized by their respective substrates and the heat stability of the ES-complexes was found also to depend on the growth temperature of the cells. These data lead to the conclusion that the enzymes undergo a transition from heat-labile to thermostable within the growth temperature range between 44 degrees C and 51 degrees C while the thermal characteristics are not changed below and beyond this crucial region.     

Effect of oxidized glutathione on some enzymes of erythrocytes and its relation to erythrocytic enzyme activity and electrophoretic mobility

1. Oxidized glutathione reacts or interacts with some erythrocytic enzymes (glucose 6-phosphate dehydrogenase, EC 1.1.1.49, aspartate aminotransferase, EC 2.6.1.10) but not with some others (lactate dehydrogenase, EC 1.1.1.27). 2. GSSG does not diminish the activity of any of these enzymes and is therefore not responsible for the decreased enzyme activities associated with older erythrocytes. 3. It may be that the reaction of aspartate aminotransferase with GSSG is the cause for the more rapid anodic electrophoretic mobility of this enzyme derived from human erythrocytes when compared with the mobility of the same enzyme from other human tissues. 4. A reinterpretation of some related, previously published, data with regard to the electrophoretic mobility of the above-mentioned enzymes from young and old erythrocytes is presented.     

Dehydrogenases of the pentose phosphate pathway in rat liver peroxisomes

Subcellular distribution of pentose-phosphate cycle enzymes in rat liver was investigated, using differential and isopycnic centrifugation. The activities of the NADP+-dependent dehydrogenases of the pentose-phosphate pathway (glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase) were detected in the purified peroxisomal fraction as well as in the cytosol. Both dehydrogenases were localized in the peroxisomal matrix. Chronic administration of the hypolipidemic drug clofibrate (ethyl-alpha-p-chlorophenoxyisobutyrate) caused a 1.5-2.5-fold increase in the amount of glucose-6-phosphate and phosphogluconate dehydrogenases in the purified peroxisomes. Clofibrate decreased the phosphogluconate dehydrogenase, but did not alter glucose-6-phosphate dehydrogenase activity in the cytosolic fraction. The results obtained indicate that the enzymes of the non-oxidative segment of the pentose cycle (transketolase, transaldolase, triosephosphate isomerase and glucose-phosphate isomerase) are present only in a soluble form in the cytosol, but not in the peroxisomes or other particles, and that ionogenic interaction of the enzymes with the mitochondrial and other membranes takes place during homogenization of the tissue in 0.25 M sucrose. Similar to catalase, glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase are present in the intact peroxisomes in a latent form. The enzymes have Km values for their substrates in the millimolar range (0.2 mM for glucose-6-phosphate and 0.10-0.12 mM for 6-phosphogluconate). NADP+, but not NAD+, serves as a coenzyme for both enzymes. Glucose-6-phosphate dehydrogenase was inhibited by palmitoyl-CoA, and to a lesser extent by NADPH. Peroxisomal glucose-6-phosphate and phosphogluconate dehydrogenases have molecular mass of 280 kDa and 96 kDa, respectively. The putative functional role of pentose-phosphate cycle dehydrogenases in rat liver peroxisomes is discussed.     

Erythrocyte antioxidant enzymes in toxicological evaluation of commonly used organophosphate pesticides

Erythrocytes are excellent models for the study of interactions of xenobiotics with biomembranes. Present work is designed to study the in vitro effects of some organophosphates (ethion, chlorpyrifos, dimethoate and monocrotophos) on rat erythrocytes. Treatment of erythrocytes with organophosphates resulted in decreased erythrocyte glucose-6-phosphate dehydrogenase (G-6-PD) activity, whereas activities of glutathione-s-transferase (GST) and glutathione reductase (GR) were increased. Reduced Glutathione (GSH) content of RBCs was decreased after treatment with the pesticides. Increased activities of GST and GR were due to induction of natural defense mechanism of erythrocytes against the toxicity of the pesticides. Membrane bound enzymes like acetylcholinesterase (AChE), Na(+)-K(+)-ATPase and Ca(2+)-ATPase were also inhibited. Altered activities of these enzymes along with decreased GSH content indicate increased oxidative stress in erythrocytes after treatment with organophosphates.     

Alterations of hemorheological parameters and tubulin content in erythrocytes from diabetic subjects

Treatment of human erythrocytes with high glucose concentrations altered the content and distributions of three tubulin isotypes, with consequent reduction of erythrocyte deformability and osmotic resistance. In erythrocytes from diabetic subjects (D erythrocytes), (i) tubulin in the membrane-associated fraction (Mem-Tub) was increased and tubulin in the sedimentable fraction (Sed-Tub) was decreased, (ii) deformability was lower than in erythrocytes from normal subjects (N erythrocytes), and (iii) detyrosinated/acetylated tubulin content was higher in the Mem-Tub fraction and tyrosinated/acetylated tubulin content was higher in the Sed-Tub fraction, in comparison with N erythrocytes. Similar properties were observed for human N erythrocytes treated with high glucose concentrations, and for erythrocytes from rats with streptozotocin-induced diabetes. In N erythrocytes, high-glucose treatment caused translocation of tubulin from the Sed-Tub to Mem-Tub fraction, thereby reducing deformability and inducing acetylation/tyrosination in the Sed-Tub fraction. The increased tubulin acetylation in these cells resulted from inhibition of deacetylase enzymes. Increased tubulin acetylation and translocation of this acetylated tubulin to the Mem-Tub fraction were both correlated with reduced osmotic resistance. Our findings suggest that (i) high glucose concentrations promote tubulin acetylation and translocation of this tubulin to the membrane, and (ii) this tubulin is involved in regulation of erythrocyte deformability and osmotic fragility.     

Radioimmunoassay and chemical properties of glucose-6-phosphate dehydrogenase and of a specific NADP(H)-binding protein (FX) from human erythrocytes

Two sensitive radioimmunoassays, based on a double-antibody technique, were developed which allow detection of nanogram amounts of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and of a so far unknown NADP(H)-binding protein present in human erythrocytes (designated FX).The two proteins isolated in homogeneous form from human erythrocytes were iodinated with ¹²⁵I by means of lactoperoxidase. Antisera to both purified proteins were raised in rabbits and sequentially adsorbed on human erythrocytes and on human serum before use. No cross-reaction between the two proteins was apparent.Hemolysates from normal as well as from glucose-6-phosphate dehydrogenase-deficient subjects were investigated for their content in both immunoreactive proteins using the two radioimmunoassay methods. This preliminary study showed significantly lowered levels of immunoreactive glucose-6-phosphate dehydrogenase in erythrocytes from subjects carrying the Mediterranean variant of this enzyme (characterized by severe deficiency of catalytic activity), compared with normal subjects. This figure was reversed as concerns the content of immunoreactive FX which was found to be twice as high in glucose-6-phosphate dehydrogenase Mediterranean erythrocytes as in normal ones.The two purified proteins were submitted to a comparative analysis of their chemical properties including NH₂-terminal residues, CNBr peptides and tryptic fingerprints. These studies revealed significant differences in the primary structures of the two proteins and therefore tend to exclude FX'x being a discrete product arising from degradation of native glucose-6-phosphate dehydrogenase. Moreover, amino axid analysis and tryptic fingerprints indicated that FX, as well as glucose-6-phosphatase dehydrogenase, is composed of very similar and possibly identical polypeptide chains.     

Red blood cell glucose metabolism in Down's syndrome

The specific activity of red blood cell glycolytic enzymes was determined in 20 Down's syndrome patients and compared with 20 normal controls. According to previous evidence, a 50% increase of phosphofructokinase and a 30% increase of glucose-6-phosphate dehydrogenase and glutathione peroxidase activity was found. Metabolic studies of the patients' erythrocytes revealed a decrease in fructose-6-phosphate and 2, 3-diphosphoglycerate concentrations, while fructose-1, 6-diphosphate and ADP both increased. Glucose utilization by intact erythrocytes from Down's syndrome patients did not differ from that of normal controls. However, addition of methylene blue or inorganic phosphate produced a higher stimulation of erythrocyte glycolysis in patients with Down's syndrome compared to controls. These metabolic abnormalities could be, at least in part, ascribed to the increased phosphofructokinase activity which is due to a gene-dosage effect.     

Enzymic changes in rabbit and rat mammary gland during the lactation cycle

1. Activities of glucose 6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), isocitrate dehydrogenase (EC 1.1.1.42), malate dehydrogenase (EC 1.1.1.37), malate dehydrogenase (decarboxylating) (EC 1.1.1.40), and pyruvate carboxylase (EC 6.4.1.1) were determined in subcellular fractions of mammary gland from rabbits during pregnancy, at different stages of lactation and during weaning. The results were compared with those obtained in similar experiments with rat mammary gland. 2. Three bases of expression of the activity of enzymes in the particle-free supernatant fraction of mammary gland were compared. During lactation, activity expressed per mg. of particle-free supernatant protein (uncorrected for milk protein) correlated well with that expressed per mug. of DNA phosphorus. The disadvantages of expressing activities per g. wet wt. are discussed. 3. The major differences between the two tissues were: (a) neither malate dehydrogenase (decarboxylating) nor a soluble form of pyruvate carboxylase could be detected in rabbit mammary gland at any stage of the lactation cycle; (b) isocitrate dehydrogenase increased in activity during lactation in rabbit mammary gland, but not in that of the rat. 4. Pyruvate carboxylase in the mitochondrial fraction of rabbit mammary gland, and in both the mitochondrial and the soluble fractions of rat mammary gland, did not change in activity during lactation. 5. For each tissue, the NADP-dependent dehydrogenases studied had a high activity at all stages of the lactation cycle compared with the rate of fatty acid synthesis at mid-lactation. The significance of these results is discussed with respect to the supply of NADPH via NADH.     

Carriers of ataxia-telangiectasia gene display additional protein fraction and changes in the environment of SH groups in erythrocyte membrane

Additional protein fraction migrating slower than spectrin has been detected in erythrocyte membranes from an ataxia-telangiectasia (A-T) patient and from his mother (A-T heterozygote). In erythrocyte membranes labelled with maleimide spin label changes in signal of the weakly immobilized spin label as related to that of strongly immobilized one (w/s) were noted. In comparison to age-matched control groups the values of w/s were lower in A-T heterozygotes (ten persons) and higher in A-T homozygotes (four persons). In control persons the values of w/s increased with age, whereas in families with A-T no significant differences in this parameter were noted between children and parents. The presence of additional protein fraction in erythrocytes membranes of A-T patient and A-T heterozygote indicates that these phenotypes can be differentiated from the healthy control persons for the first time on the basis of changes detected in the erythrocytes. This change in erythrocyte membrane may explain the decrease in the w/s parameter of electron spin resonance in A-T heterozygotes. On the other hand increased values of w/s in A-T patients may be caused by disease process.     

甘蓝型油菜子油分的积累与某些生理变化关系的研究

油菜种子发育过程中,其内部的生理代谢过程发生了规律性的变化。伴随着种子的发育进程,６－磷酸葡萄糖脱氢酶、异柠檬酸裂解酶、异柠檬酸脱氢酶和琥珀酸脱氢酶的活性均有不同程度的增强。在油分旺盛合成期,６－磷酸葡萄糖脱氢酶和异柠檬酸裂解酶的活性均达到了最大值,而此时,异柠檬酸脱氢酶和琥珀酸脱氢酶的活属于匀增加较慢;在种子的不同发育时期,高含油量品系的６－磷酸葡萄糖脱氢酶和异柠檬酸裂解酶的活性均高于低含油量的     

A Particulate Phospholipid Synthesizing System from Escherichia coli B

The phosphatidate formed by glycerol 3-phosphate acyltransferase present in a particulate fraction from E. coli remained associated with the particulate material and was converted to phosphatidylethanolamine and phosphatidylglycerol by other enzymes contained in the particulate fraction. Those enzymes required a heat-labile factor for the synthesis of phosphatidylethanolamine and a heat-stable factor for the synthesis of phosphatidylglycerol. Both of these phospholipids like their precursor remained bound to the particulate fraction.     

Concentration and enzyme content of in vitro-cultured Babesia bigemina-infected erythrocytes

Clones of in vitro-cultured Babesia bigemina-infected erythrocytes were concentrated by several density gradient procedures. The density range of infected erythrocytes containing pairs of parasites was 1.077 to 1.089 g/ml, whereas the density range of infected erythrocytes containing single parasites was 1.092 to 1.100 g/ml. Three enzymes--lactate dehydrogenase, glucose-phosphate isomerase, and glutamate dehydrogenase--were found associated with infected erythrocytes. The parasite-specific enzyme and/or isoenzymes were shown to have different mobility patterns in starch gel electrophoresis from those found in the normal bovine erythrocytes. The enzyme 6-phosphogluconate dehydrogenase was not detected as a parasite-specific enzyme in B. bigemina-infected erythrocytes.