Metabolic differentiation of homologous leg muscles of two aquatic birds at the level of enzymatic organization

The quantitative determination of succinic dehydrogenase (SDH), hexokinase (HK), phosphorylase, phosphofructokinase (PFK), glycerol-3-phosphate dehydrogenase (G-3-PDH) and lactate dehydrogenase (LDH) was carried out in the homologous leg muscles of two aquatic Birds. It appears that the leg muscle fibres of the coot, a surface swimmer are more oxidative in nature and appear to utilize glucose as source of energy. The leg muscles of the dabchick, a diving Bird, on the other hand, seem to depend on glycogen as source of energy. The relative activity levels of HK, phosphorylase and PFK support the accepted r?le of glycogen as primary substrate of carbohydrate catabolism in the leg muscles. The ratio of G-3-PDH/LDH in the leg muscles revealed that glycerol 3-phosphate cycle appears to be insufficient to account for the major part of NADH oxidation. However, the LDH activity is quite high in all the muscles. These results led us to believe that glycerol 3-phosphate cycle may function during rest, when the rate of glycolysis will be low.     

Alterations in dopaminergic receptors in Huntington's disease.

To detect variations in dopaminergic receptors and cholinergic activity in regions of postmortem Huntington's diseased brains, ³H-spiroperidol binding assays and choline acetyltransferase (ChAc) activities were carried out. A significant reduction in ³H-spiroperidol binding in the caudate nucleus, putamen and frontal cortex of choreic brains was detected which appeared to be due to a decrease in the total number of binding sites rather than to a decrease in affinity of ³H-spiroperidol for the dopaminergic receptor. In choreic brains, there were also significant reductions in ChAc activity in the caudate nucleus and putamen. The decreases of both ³H-spiroperidol binding and ChAc activity in the neostriatum suggest that the dopaminergic receptors are localized postsynaptically on cholinergic interneurons. Dopaminergic receptor alterations in the basal ganglia may be one of the causes of the abnormal motor movements found in HD while alterations of these receptors in the frontal cortex may be associated with the neuronal degeneration found in that area of choreic brains.     

Glycolytic enzymes in the premolt field crab Paratelphusa hydrodromus (Milne-Edwards) (Crustacea)

The quantitative assay of hexokinase (HK), phosphorylase, phosphofructokinase (PFK), glucose 6-phosphate dehydrogenase (G-6-PDH), glycerol 3-phosphate dehydrogenase (G-3 PDH) and lactate dehydrogenase (LDH) revealed that coxal muscles compared to hepatopancreas contained higher activities of all the enzymes investigated. It appears that the coxal muscles of the premolt field crab has carbohydrate-based fuel economy. The hepatopancreas is a rich source of lipid and very poor source of glycogen. The activity of G-6-PDH is moderately high in the hepatopancreas. It seems that in this lipogenic tissue conversion of G-6-P to triose phosphate occurs predominately via pentose-phosphate pathway thus generating NADPH for lipogenesis. The relative G-3PDH ad LDH activities in hepatopancreas and coxal muscles led us to believe that the reconversion of NAD from NADH in hepatopancreas nd muscle flexor is effected by glycerol 3-phosphate shuttle, whereas in muscle extensor it is achieved by both G-3PDH and LDH activities.     

Huntington's disease: regional alteration in muscarinic cholinergic receptor binding in human brain.

Huntington's Disease, an autosomal dominant neurological disorder, is characterized by diffuse neuronal degeneration particularly in the basal ganglia and cerebral cortex. The purpose of this study was to examine various discrete regions of choreic and control brains for alterations in muscarinic cholinergic receptor binding and choline acetyltransferase (ChAc) activity. Nine postmortem brains, three from patients with Huntington's Disease and six controls, were dissected into 17 discrete regions. Each regional homogenate was assayed for muscarinic receptor concentration by measuring specific membrane binding of [³H]-QNB, a potent muscarinic antagonist which selectively labels brain muscarinic receptors. Aliquots from each brain region were also assayed for ChAc activity. Of significance was the marked reduction in specific [³H]-QNB receptor binding in the caudate nucleus, putamen and globus pallidus of choreic brain while no significant alterations were detected in other brain regions. Significant decreases in ChAc activity were found in the caudate nucleus, putamen, and globus pallidus with no alterations in ChAc activity in the rest of the brain regions examined. The tissues were chosen such that protein levels were similar in both choreic and normal brain samples. The apparent reduction in the number of muscarinic cholinergic receptors in the choreic brains suggests that treatment with cholinomimetic drugs might be beneficial in Huntington's Disease.     

Rat brain glycolysis regulation by estradiol-17 beta.

The effect of estradiol-17 beta on the activities of glycolytic enzymes from female rat brain was studied. The following enzymes were examined: hexokinase (HK, EC 2.7.1.1), phosphofructokinase (PFK, EC 2.7.1.11), aldolase (EC 4.1.2.13), glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), phosphoglycerate kinase (EC 2.7.2.3), phosphoglycerate mutase (EC 2.7.5.3), enolase (EC 4.2.1.11) and pyruvate kinase (PK, EC 2.7.1.40). The activities of HK (soluble and membrane-bound), PFK and PK were increased after 4 h of hormone treatment, while the others remained constant. The changes in activity were not seen in the presence of actinomycin D. The significant rise of the activities of the key glycolytic enzymes was also observed in the cell culture of mouse neuroblastoma C1300 treated with hormone. Only three of the studied isozymes, namely, HKII, B4 and K4 were found to be estradiol-sensitive for HK, PFK and PK, respectively. The results obtained suggest that rat brain glycolysis regulation by estradiol is carried out in neurons due to definite isozymes induction.     

Histochemical studies on the submandibular ganglia of marmosets (Callithrix jacchus and Callithrix penecillata).

The histochemistry of the neural cells was studied in the submandibular ganglia of 5 Callithrix jacchus (3 males and 2 females) and 4 Callithrix penicillata (2 males and 2 females). These cells contain neutral mucopolysaccharides, nucleoproteins and lipidic materia, but are apparently devoid of glycogen. It is impossible to demonstrate in them any reactivity for UDPG-GT, phosphorylases, ATPase at pH 6.3, leucine aminopeptidase and alanyl aminopeptidas. The reaction for the other searched enzymes was as follows: weak (F-1,6-P Ald and cytochrome oxidase), weak to moderate (ADH, 6-P-GDH, ICDH, SDH, MDH, alpha-GPDH and beta-OHBDH), moderate (G-6-PDH, F-1,6-PA, LDH and GDH), moderate to strong (ATPase at pH 7.4, nonspecific esterase and acid phosphatase) and strong (G-6-PA, NADH2,-TR, NADPH2-TR, ATPase at pH 8.5 and 9.4 and alkaline phosphatase).     

Effect of thyroid hormones on the glycolytic enzyme activity in brain areas of the rat

The glycolytic metabolism through the key enzymes, hexokinase, phosphofructokinase, pyruvate kinase and lactate dehydrogenase, have been studied in the brain areas: anterior cortex, amygdala, hypothalamus, septum and hippocampus in adult rats with pharmacologically induced hyperthyroidism. The oxidative metabolism of glucose is accelerated in most brain areas by treatment with high doses of T3, as is shown by the increase in HK activity, approaching normality on reducing the dose. This decrease can also by observed in the PFK activity through the effect of assayed doses of thyroxine. The anterior cortex is the only brain area that does not show significant variations of PK activity through the effects of treatment with thyroid hormones. On the other hand, a general inhibition of the glycolytic anaerobic pathway by treatment with T3 was observed.     

Changes of Activity and Isozyme Pattern of Phosphofructokinase in the Brains of Patients with Alzheimer's Disease

Abstract: A severe reduction of the in vivo cerebral glucose consumption rate is generally found in patients with Alzheimer's disease. In postmortem studies changes in the activities of key regulatory glycolytic enzymes, including 6-phosphofructokinase (PFK), have been reported in Alzheimer's disease brains, but the results obtained so far are inconsistent and controversial. We reevaluated the activity of PFK in brain tissue from clinically and neuropathologically confirmed cases of Alzheimer's disease using optimized tissue disintegration and assay methods and determined the PFK isozyme pattern. PFK activity in brains from patients with Alzheimer's disease was significantly increased in frontal and temporal cortex and unchanged in the other brain areas studied when compared with control brains. All three PFK isozymes were detected in each of the brain areas studied. In brains of Alzheimer's disease patients the level of the C-type PFK was slightly reduced at the expense of the M- and L-type subunits. The data presented do not support the results of other groups, which reported up to a 90% reduction of PFK activity in Alzheimer's disease. In contrast, the data presented clearly rule out the suggestion that changes of PFK activity might be one of the causes for the reduced glucose consumption in Alzheimer's disease brains.     

Trypsinization of chick glial cells before seeding: Effects on energy metabolism enzymes and glutamine synthetase

In order to test the possible involvement of surface proteins on some metabolical aspects of chick glial cell differentiation in culture, perturbations were induced on the glial cell surface membrane by limited trypsinization before seeding. The developmental changes of enzymes involved in the energy metabolism of the cell: malate dehydrogenase (MDH), glutamate dehydrogenase (GDH), hexokinase (HK), lactate dehydrogenase (LDH), enolase as well as glutamine synthetase (GS) were determined in trypsin treated cells and controls. The total protein and DNA content per dish was higher in treated cells than in controls, however the protein ratio towards DNA remained unchanged. The levels of GS, GDH, LDH, and enolase activities were significantly enhanced after trypsin treatment of the cells compared to controls. The enhanced value of total LDH activity is essentially the result of the increase of M subunit containing isoenzymes. Considering that a higher level of GS activity characterizes some maturation of the glial cells (as observed during the maturation of the chick brain) it is apparent that modifications of cell surface located factors, by trypsin treatment, induce differentiation phenomena at the functional state of the glial cells in culture. This may indicate that interactions located at the cell surface are involved in the modulation of key enzymes of the energy metabolism pathway.     

Modifications in energy metabolism during the development of chick glial cells and neurons in culture

Developmental changes in lactate dehydrogenase (LDH), enolase, hexokinase (HK), malate dehydrogenase (MDH), and glutamate dehydrogenase (GDH) activities were measured in cultures of pure neurons and glial cells prepared from brains of chick embryos (8 day-old for neurons, 14 day-old for glial cells) as a function of cellular development with time in culture. The modifications observed in culture were compared to those measured in brain extracts during the development of the nervous tissue in the chick embryo and during the post-hatching period. A significant increase of MDH, GDH, LDH, and enolase activities are observed in neurons between 3 and 6 days of culture, whereas simultaneously a decrease of HK values occurs. In the embryonic brain between 11 and 14 days of incubation, which would correspond for the neuronal cultures to day 3 through 6, modifications of MDH, GDH, HK, and enolase levels are similar to those observed in neurons in culture. Only the increase of LDH activity is less pronounced in vivo than in cultivated cells. The evolution of the tested enzymatic activities in the brain of the chick during the period between 7 days before and 10 days after hatching is quite similar to that observed in cultivated glial cells (prepared from 14 day-old embryos) between 6 and 18 days of culture. All tested activities increased in comparable proportions. The modifications of the enzymatic profile indicate that some maturation phenomena affecting energy metabolism of neuronal and glial elements in culture, are quite similar to those occuring in the total nervous tissue. A relationship between the development of the energy metabolism of the brain and differentiation processes affecting neuroblasts and the glial-forming cells is discussed.     

Effect of exercise on glycolytic enzymes of zucker fatty rats

Summary The effect of fatigue (running to exhaustion) on the Vmax activity of the key glycolytic enzymes measured at saturating substrate concentrations in muscles, liver and brain of sedentary and trained (running on a treadmill one h/day at 20 m/min, five days/week for six months) female Zucker fatty rats and their lean littermates was investigated. In the sedentary rats, fatigue increased the activity of phosphofructokinase (PFK) in the red vastus muscle by 82% in lean, and 120% in obese rats. In the trained rats, fatigue increased PFK activity by 28% in the white vastus muscle of lean rats. In the lean animals, hexokinase (HK) activity was decreased by 26% in the red vastus of sedentary rats, and by 29% in the white vastus of trained rats upon fatiguing. Pyruvate kinase (PK) activity was also decreased by 29% in the white vastus of fatigued lean animals. Training by itself had no effect on the activity of glycolytic enzymes, except PK activity which was increased by 27% in the cortex of the lean animals. It is concluded that in the Zucker rat, these glycolytic enzymes may play a differential role in regulating glycolysis during exercise and fatigue; the extent of their involvement differs depending upon the type of tissue studied and exercise. In view of the reported short half-life (7–17 h) of PFK and its covalent modification, it is suggested that the total content and/or phosphorylation status of the enzyme may be affected in animals subjected to long-term fatigue.Abbreviations PFK Phosphofructokinase (EC 2.7.1.11) - PK Pyruvate Kinase (EC 2.7.1.40) - HK Hexokinase (EC 2.7.1.1) - LSC Lean Sedentary Control - LTC Lean Trained Control - LSF Lean Sedentary Fatigued - LTF Lean Trained Fatigued - OSC Obese Sedentary Control - OTC Obese Trained Control - OSF Obese Sedentary Fatigued - OTF Obese Trained Fatigued     

Energy metabolism in the granulation tissue of diabetic rats during cutaneous wound healing

The skin cells chiefly depend on carbohydrate metabolism for their energy requirement during cutaneous wound healing. Since the glucose metabolism is greatly hampered in diabetes and this might affect wound repair process. This prompted us to investigate the intermediate steps of energy metabolism by measuring enzyme activities in the wound tissues of normal and streptozotocin-induced diabetic rats following excision-type of cutaneous injury. The activities of key regulatory enzymes namely hexokinase (HK), phosphofructokinase (PFK), lactate dehydrogenase (LDH), citrate synthase (CS) and glucose-6 phosphate dehydrogenase (G6PD) have been monitored in the granulation tissues of normal and diabetic rats at different time points (2, 7, 14 and 21 days) of postwounding. Interestingly, a significant alteration in all these enzyme activities was observed in diabetic rats. The activity of PFK was increased but HK, LDH and CS showed a decreased activity in the wound tissue of diabetics as compared to normal rats. However G6PD exhibited an elevated activity only at early stage of healing in diabetic rats. Thus, the results suggest that significant alterations in the activities of energy metabolizing enzymes in the wound tissue of diabetic rats may affect the energy availability for cellular activity needed for repair process and this may perhaps be one of the factor responsible for impaired healing in these subjects. (Mol Cell Biochem 270: 71–77, 2005)     

Variation in levels of enzymes related to energy metabolism in alternative developmental pathways of Blastocladiella emersonii.

The activities of phosphofructokinase (PFK), fructose diphosphatase (FDP), nicotinamide adenine dinucleotide (NAD) and NAD phosphate (NADP)-linked isocitrate dehydrogenases (IDHNAD, IDHNADP), two NAD-linked glutamate dehydrogenases (GDH1, GDH2), and isocitrate lyase were studied during the development of the two phenotypes, ordinary colorless and resistant sporangia (OC and RS plants), of water mold Blastocladiella emersonii in synchronized liquid cultures. The OC plants had a generation time of about 12 h, whereas the RS plants required 3.5 days to reach maturity. All the enzymes were present throughout the development of both phenotypes. In zoospores, PFK, FDP, and GDH2 were localized in the cytosol. The IDHNADP activity was distributed with two-thirds in the soluble and one-third in the particulate fraction. GDH1 and IDHNAD showed the same distribution and were predominantly present in the particulate fraction, presumably in the mitochondria. Isocitrate lyase was found in the particulate fraction. The enzyme levels changed considerably during development. FDP and IDHNADP varied in a parallel manner. Similarly, the three enzymes PFK, IDHNAD and GDH1 showed parallel variations. The activity patterns for all enzymes were different for the OC and RS pathways. Isocitrate lyase exhibited the largest changes in activity during development. Thus, during OC plant formation, its activity decreased by a factor of 20. GDH2 varied similarly to PFK and IDHNADP during OC plant development, whereas it behaved like isocitrate lyase during RS plant development. The ratios between anabolic and catabolic enzymes were higher in mature plants than in zoospores and higher in RS plants than in OC plants. The results indicate that the variations in the enzyme levels are secondary to the critical changes involved in the transition from one developmental pathway to the other.     

麦红吸浆虫不同滞育期四种糖代谢酶活力分析

海藻糖是麦红吸浆虫Sitodiplosis mosellana (Gehin)滞育期间储藏能量的主要物质,为弄清其在滞育期的积累机理,本文测定了麦红吸浆虫滞育前后和滞育期糖原磷酸化酶（Gpase）、己糖激酶（HK）、磷酸果糖激酶（PFK）和醛缩酶（ALD）这4种糖代谢酶活力的变化。结果表明: 麦红吸浆虫滞育前后这些糖代谢酶活力明显不同,滞育后Gpase 活力显著提高,糖酵解有关酶HK,PFK和ALD活力降低;滞育解除后,Gpase 活力降低,HK,PFK和ALD活力升高。滞育期间,4种糖代谢酶的活力均与滞育发育有关;同时Gpase 和PFK的活力也与环境温度有关,即夏、冬季高于春、秋季;同期不同滞育状态幼虫比较,裸露幼虫HK,PFK和ALD活力总是略高于结茧幼虫,Gpase则相反。滞育当年与第2年同期幼虫4种糖代谢酶的活力无显著差异。     

Effect of copper on liver key enzymes of anaerobic glucose metabolism from freshwater tropical fish Prochilodus lineatus

We investigated the effect of copper on liver key enzymes of the anaerobic glucose metabolism (hexokinase, HK; phosphofructokinase, PFK; pyruvate kinase, PK; lactate dehydrogenase, LDH) as well as of the pentose pathway (glycose-6-phosphate dehydrogenase, G6PDH) from the fish Prochilodus lineatus. The fish were acclimated at either 20 degrees C or 30 degrees C at pH 7.0, transferred to water at pH 4.5 or 8.0, and exposed to 96 h-CL(50) copper concentrations. Copper accumulation in liver was higher in fish acclimated at 20 degrees C and maintained in water pH 8.0. Three-way analysis of variance revealed a significant effect of temperature on all enzymes, a significant effect of pH on all enzymes except for PK, and a significant effect of copper on only PFK, and LDH in pH 4.5 at 20 degrees C and, at 30 degrees C, on PFK and PK at pH 4.5 and 8.0, HK at pH 4.5 and G6PDH at pH 8.0. There were significant interactions between treatments for many enzymes. These changes suggest that the activity of enzymes in question is modified by a change in ambient water. At least at 30 degrees C, the overall reduction in the glycolytic enzyme activities of copper-exposed fish seems to reduce energy availability via glucose metabolism, thereby contributing to enhance copper toxic effects.     

Studies of γ-glutamyltransferase activity in the brain tissue post mortem

Our experiments showed that the activity of -glutamyltransferase (-GT) did not remarkably change in homogenates of mouse, rat, and bovine brains during the first four days post mortem. In the course of that period, the brain microvessels also retained their -GT activity. -GT of microvessels from bovine brain cortex, solubilized with sodium deoxycholate, was eluted in the void volume V_o when chromatographed on a Sephadex G-200 column with the detergent Triton X-100. In human post mortem brains, the specific activity of -GT in choroid plexi was found to be about five times higher than that in the cerebral cortex, white matter, basal ganglia, pons, and cerebellum but about four times lower than that in the microvessels obtained from the studied brain regions. Our findings suggest that it is possible to study the components of the blood-brain barrier on material from deceased subjects.     

Oxidative events in neuronal and glial cell-enriched fractions of rat cerebral cortex

The aim of this work was to investigate how neurons and glial cells separated from rat brain cortex respond to “in vitro” oxidative stress induced by incubation of the cellular fractions in the presence of prooxidant mixtures; in addition, the endogenous enzymatic antioxidant capacity of the purified fractions was investigated. Neuronal and glial cell-enriched fractions were obtained from rat cerebral cortex following passages of the tissue through meshes and centrifugations. The following parameters were evaluated: antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSHPx), and glucose-6-phosphate dehydrogenase (G6PDH); lipid peroxidation products (TBARS) prior to (basal) and after (iron-stimulated) incubation with a mixture of iron and ascorbic acid; intracellular production of reactive oxygen species (ROS) using a fluorescent probe, dichlorofluorescin-diacetate, in basal, iron-stimulated, and menadione stimulated conditions. SOD and GSHPx activities showed no significant changes between neurons and glia, whereas CAT and G6PDH activities were found to be significantly lower in glia than in neurons. TBARS levels were significantly lower in the glial fraction than in neurons, both in basal and iron-stimulated conditions. ROS production showed no differences between neurons and glia in both basal and menadione-stimulated conditions. Iron-stimulation produced a marked increase in ROS production, limited to the neuronal fraction, with the glial values being similar to the basal ones. Our conclusion is that glia and neurons isolated from rat cerebral cortex show a similar pattern of the most important antioxidant enzymes and of their basal ROS production, whereas glia is more resistant in “oxidative stress” conditions.     

Comparative studies on lipogenic enzyme activities in the liver of human and some animal species

1. The activities of enzymes involved in fatty acid synthesis in the human liver (sample taken during abdominal surgery) and in the livers of some animals were studied. 2. Fatty acid synthase, ATP-citrate lyase and malic enzyme activities were found to be from 4 to 70-fold lower in human liver than in rat or bird livers. 3. The activities of hexose monophosphate shunt dehydrogenases in human liver were from half to almost equal to the corresponding activities in birds, but much lower than in rat liver. 4. The activities of all enzymes listed above in human and beef liver were very similar (except fatty acid synthase which was undetectable in the beef liver). 5. Very high activity of NADP-linked isocitrate dehydrogenase was found in livers of all species tested. 6. These results are discussed in relation to the role of the human liver in lipogenesis. 7. The activities of the enzymes generating NADPH in human liver taken during abdominal surgery were similar to the activities observed in the tissue obtained post mortem. 8. This suggested that post mortem tissue may be used as a reliable human material for some enzyme assays. 9. Thus we also examined the activity of malic enzyme in post mortem human kidney cortex, heart, skeletal muscle and brain. 10. Relatively high activity of NADP-linked malic enzyme has been observed in human brain.     

Effect of 6-Hydroxydopamine Lesions of the Medial Prefrontal Cortex on Neurotransmitter Systems in Subcortical Sites in the Rat

The effect of lesions of the catecholamine nerve terminals in the medial prefrontal cortex of the rat on neurotransmitter mechanisms within the basal ganglia has been investigated. Bilateral 6-hydroxydopamine lesions were stereotaxically placed in the dopamine-rich (DA) area of th frontal cortex. Animals were pretreated with desmethylimipramine to block the uptake of neurotoxin into noradrenergic (NA) terminals and to make it more selective for DA terminals. The lesion produced a selective reduction of both NA and DA from the medial prefrontal cortex, a result related to falls in tyrosine hydroxylase activity at this site. Lesioned animals showed enhanced DA turnover and utilisation in striatal and limbic regions. There was no change in subcortical tyrosine hydroxylase activity. In addition there were significant falls in other putative neurotransmitters within basal sites, including 5-hydroxytryptamine and GABA. Decreased activity of the neurotransmitter-synthesizing enzyme glutamate decarboxylase and choline acetyltransferase was also recorded in certain regions of the basal ganglia. The results suggest that frontal cortical catecholamine systems may serve to regulate various neurotransmitter mechanisms in the basal ganglia.