Changes in the subcellular distribution of brain and heart hexokinase isoenzymes during alloxan diabetes

Changes in the subcellular distribution of hexokinase activity from three brain regions and heart were studied during alloxan induced diabetes. There was an overall decrease in the particulate hexokinase with an increase in the soluble form, after different time intervals of the onset of diabetes. Administration of insulin to the diabetic rats showed a partial counteraction of the enzyme changes. A possible regulation of brain hexokinase by metabolite changes is proposed     

Effect of 6-Aminonicotinamide on the activity of hexokinase and lactate dehydrogenase isoenzymes in regions of the rat brain

Changes in the activity of hexokinase and lactate dehydrogenase isoenzymes in the three brain regions and heart were studied in the 6-Aminonicotinamide-treated rats. Drug administration decreased the particulate hexokinase and lactate dehydrogenase activity, but increased the soluble hexokinase     

Hexokinase in developing rabbit erythroid cells

The activity and isozyme distribution of hexokinase were studied in bone marrow cells from normal and anemic rabbits seperated by density centrifugation or by unit-gravity sedimentation. The specific activity of the enzyme was found to be about 150-fold higher in the basophilic erythroblasts as compared with the mature circulating erythrocytes. Mos of the falls in hexokinase activity take place whent the cell completes its final division and matures from the polychromatic stage to the orthochromatic stage. Concomitant with this strong decrease in enzyme activity, qualitative as well as quantitative changes in the hexokinase isozymic pattern become apparent. While in the basophilic and polychromatic erythroblasts the only hexokinase isozyme present is hexokinase type I, the orthochromatic cells also contain hexokinase Ib. This last isozymic form, which increases further at the reticulocyte stage, is also present in the circulating reticulocytes but not in mature red blood cells.     

Hexokinase isozyme distribution and regulatory properties in lymphoid cells

The glycolytic enzyme hexokinase is studied in cultured leukemic lymphoblasts, in normal lymphocytes and in lymphoblasts obtained by stimulation of normal lymphocytes with phytohaemagglutinin.Hexokinase activity levels in cultured lymphoblasts and in normal lymphocytes are identical, but somewhat higher levels are found in stimulated lymphocytes. Cultured leukemic lymphoblasts differ in isozyme content in comparison to the other lymphoid cells. Besides hexokinase I, which is detected in all the lymphoid cells, they are characterized by the presence of hexokinase II. The concentration of type II increases during cell growth. Another difference between leukemic lymphoblasts and mature and stimulated lymphocytes is found in the regulatory properties of hexokinase I. Hexokinase I from both normal and stimulated lymphocytes is inhibited by glucose-1,6-diphosphate. This inhibition is decreased in part by addition of inorganic phosphate. Hexokinase I from leukemic lymphocytes, however, is inhibited to a lesser extent by glucose-1,6-diphosphate. Inorganic phosphate has no effect at all on this inhibition.In accordance with these findings a different pattern in the hexokinase I region was detected in electrophoresis with several cell types. The subisozyme hexokinase Ib, which appears to be the phosphate-regulated form, is predominant in lymphocytes, whereas it is present in a minor fraction in the cultured leukemic lymphoblasts. In these cells hexokinase Ic predominates.     

Changes in Hexokinase Isoenzymes in Regions of Rat Brain During Insulin-Induced Hypoglycemia

Activities of hexokinase isoenzymes were determined during insulin-induced hypoglycemia in soluble and total particulate fractions from three regions of rat brain. Type I hexokinase isoenzyme activity showed a small decrease in both soluble and particulate fractions from the cerebral hemispheres. In cerebellum and brain stem, however, Type I isoenzyme showed a decrease only in the soluble fraction. A significant increase was observed in hexokinase Type II isoenzyme from both the fractions, in all the three brain regions 1 h after insulin administration.     

Comparison and correlation of binding mode of ATP in the kinase domains of Hexokinase family

Hexokinases (HKs) are the enzymes that catalyses the ATP dependent phosphorylation of Hexose sugars to Hexose-6-Phosphate (Hex-6-P). There exist four different forms of HKs namely HK-I, HK-II, HK-III and HK-IV and all of them share a common ATP binding site core surrounded by more variable sequence that determine substrate affinities. Although they share a common binding site but they differ in their kinetic functions, hence the present study is aimed to analyze the binding mode of ATP. The analysis revealed that the four ATP binding domains are showing 13 identical, 7 similar and 6 dissimilar residues with similar structural conformation. Molecular docking of ATP into the kinase domains using Molecular Operating Environment (MOE) soft ware tool clearly showed the variation in the binding mode of ATP with variable docking scores. This probably explains the variable phosphorylation rates among hexokinases family.     

Correlation of Hexokinase Content and Basal Energy Metabolism in Discrete Regions of Rat Brain

Brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) levels in seven regions of rat brain were estimated by photometric measurement of immunofluorescence in cryostat-cut sections. When compared with basal rates of glucose metabolism in these regions, estimated by the 6-[14C]glucose method, a significant correlation was observed. Thus, hexokinase content reflects metabolic energy demands.     

Effects of alloxan-induced diabetes on somatostatin binding to cytosolic components of rabbit gastric fundic mucosa

Diabetes was induced by administration of alloxan (150 mg/Kg) to 24 h-fasted rabbits. Somatostatinlike immunoreactivity (SLI) and cytosolic binding sites for somatostatin in gastric fundic mucosa were studied using radiolabelled Tyr¹¹-somatostatin. Three months after the onset of the disease, the specific binding of somatostatin to these sites was seen to be significantly lower, due to a reduction in the number (but not the affinity) of specific somatostatin binding sites of high-affinity and a disappearance of the specific, somatostatin binding sites of low-affinity. These changes were associated with an increase in the SLI concentration in both gastric fundic mucosa and plasma.     

Effect of Hypo- and Hyperthyroidism on Hexokinase in the Developing Cerebellum of the Rat

Abstract: Total hexokinase levels (units/g tissue) have been measured during postnatal development of the cerebellum in control, hypothyroid, and hyperthyroid rats. In addition, distribution of hexokinase in the developing cerebellum has been observed with an immunofluorescence method. Hypothyroidism delays the normally observed postnatal increase in total hexokinase activity, whereas hyperthyroidism accelerates the increase. In normal animals, hexokinase levels in maturing Purkinje cells pass through a transient increase, with maximal levels at approximately 8 days postnatally followed by rapid decline to relatively low levels by 12 days; hypothyroidism delays this transient increase and subsequent decline, but hyperthyroidism does not appear to affect markedly the timing of this phenomenon. Cerebellar glomeruli are relatively enriched in hexokinase content, as judged by their intense fluorescence. Hypothyroidism delays the development of intensely stained glomeruli. Hyperthyroidism did not appear to cause precocious increase in numbers of glomeruli but may have increased the rate at which the hexokinase was assimilated by newly formed glomeruli. The effects of hypo- and hyperthyroidism on total cerebellar hexokinase levels are interpreted in terms of the effect of thyroid hormone on the biochemical maturation of synaptic structures rich in hexokinase.     

Polyphenol oxidase isoenzymes in avocado

Avocado polyphenol oxidase (PPO) was precipitated mainly in the 30–90% saturated ammonium sulfate fraction. The 40–75% saturated ammonium sulfate fraction (the partially purified enzyme) had the highest specific activity in the cultivars Lerman, Horeshim and Fuerte. The PPO was active towards o-dihydroxyphenols. Six active enzymes (a–f) were detected with D,L-DOPA, 4-methylcatechol, catechol, caffeic acid or chlorogenic acid. Band e was the most active in all cases. More isoenzyme bands (fast-moving) were observed with caffeic acid than with 4-methylcatechol. Furthermore, the isoenzyme patterns of the partially purified extracts of the cultivars could be distinguished with respect to caffeic acid.     

Role of calpains in diabetes mellitus-induced cataractogenesis: A mini review

Premature visual impairment due to lens opacification is a debilitating characteristic of untreated diabetes. Lens opacification is primarily due to the insolubilization of crystallins, proteins essential for lens optical properties, and recent studies have suggested that a major cause of this insolubilization may be the unregulated proteolysis of crystallins by calpains. These are intracellular cysteine proteases whose activation requires the presence of calcium (Ca²⁺) and elevated levels of lens Ca²⁺ is a condition associated with both diabetic cataractogenesis and other forms of the disorder. A number of calpains have been identified in the lens, including calpain 2, calpain 10 and two isozymes of calpain 3:Lp82 and Lp85. The use of animal hereditary cataract models have suggested that calpain 2 and/or Lp82 may be the major calpains involved in murine cataractogenesis with contributions from calpain 10 and Lp85. However, calpain 2 appears to be the major calpain involved in murine diabetic cataractogenesis and the strongest candidate of the calpains for a role in human types of cataractogenesis. Here, we present an overview of recent evidence on which these observations are based with an emphasis on the ability of calpains to proteolyse lens crystallins and calpain structural features, which appear to be involved in the Ca²⁺-mediated activation of these enzymes. (Mol Cell Biochem 261: 151–159, 2004)     

Localization of Hexokinase in Neural Tissue: Immunofluorescence Studies on the Developing Cerebellum and Retina of the Rat

Abstract: Antiserum against purified rat brain hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) has been used in a study of the distribution of hexokinase during the postnatal development of rat cerebellum and retina. The cells of the external germinal layer of the cerebellum exhibit little or no fluorescence. The Purkinje cells exhibit a transient increase in hexokinase levels between 2 and 8 days postnatally, followed by a precipitous decrease (8–12 days) to the relatively low levels found in the mature Purkinje cell. Development of the intensely fluorescent cerebellar glomeruli in the granule cell layer is readily followed during the 3rd and 4th weeks postnatally. With respect to postnatal changes in hexokinase distribution of the retina, perhaps most notable is the observation that even the cytoplasmic protrusions which represent the precursors of the photoreceptor segments are richly endowed with hexokinase. Biochemical differentiation of the photoreceptor segments into hexokinase-rich inner segments and hexokinase-poor outer segments is readily observed long before the growth of the photoreceptor segments has been completed.     

Thymidine kinase isoenzymes in human acute monocytic leukemia

Summary Two thymidine kinase isoenzymes, TK 3 and TK 4, from mononuclear leucocytes from a patient with acute monocytic leukemia, were purified and characterized in regard to the molecular weights and kinetic properties.The molecular weights of TK 3 and TK 4 were 60 000 and 45 000, respectively. In the presence of 2 mM ATP, the molecular weight of TK 3 increased to 200 000, whereas the molecular weight of TK 4 was unchanged.Studies of the kinetic properties showed clear differences between TK 3 and TK 4. With thymidine as substrate, TK 3 showed biphasic kinetics with a K_m of 22 µM, and TK 4 showed Michaelis-Menten kinetics with a K_m of 0.33 µM With ATP as substrate, TK 3 showed Michaelis-Menten kinetics with a K_m of 100 µM, and TK 4 showed biphasic kinetics with a Km of 3.5 µM. With dTTP as inhibitor, TK 3 showed cooperative inhibition kinetics, and TK 4 showed non-cooperative competitive inhibition kinetics. The dTTP concentration at 50% inhibition was 75 µM for TK 3 but 380 µM for TK 4.Comparison of the molecular weights and the kinetic properties of TK 3 and TK 4 with the corresponding data previously obtained for TK 1 and TK 2 from normal human lymphocytes indicate the existence of four thymidine kinase isoenzymes in human leucocytes.     

Specific RNase isoenzymes in the human central nervous system

After inactivation of RNase inhibitor by parachloromercuribenzoate, total alkaline RNase activity was found to be two fold higher in white matter as in grey matter extracts from human brain tissue. This activity was lower in human purified myelin. Two human cerebrospinal fluid (CSF) RNase isoenzymes of group 3 (a minor one, RNase 3.1, and a major one, RNase 3.2) were found to be present in human grey and white matter extracts and in purified myelin, but absent in human serum, peripheral nerve, liver, and spleen extracts. A RNase isoenzyme similar to central nervous system (CNS) RNase 3.2 was present in human kidney extracts but it differed in its carbohydrate structure. RNase isoenzymes 3.1 and 3.2 were not found in mouse, rat, and bovine brains. Thus, RNases 3.1 and 3.2 seem specific to human CNS. RNases of group 3 are the predominant RNase isoenzymes in CSF and one of the two predominant RNase groups in brain tissue. However, the proportion of RNases of group 3 is different in CSF and in brain extracts: RNases 3.1-3.2 are the major constituents of group 3 RNases in brain tissue, while another RNase isoenzyme of group 3, RNase 3.0, which is more glycosylated than RNases 3.1-3.2, is only a minor part of RNase of group 3 in brain extracts. Conversely, RNases 3.1-3.2 are lower or equivalent to RNase 3.0 in control CSF since the ratio of RNases 3.1-3.2 to RNase 3.0 did not exceed 1.0. This ratio decreased in pathological CSF including multiple sclerosis or infectious CNS diseases that were free of transudation phenomena. In conclusion, CSF RNases 3.1-3.2 seem to originate in brain tissue and could be markers of RNA catabolism from brain cells.     

Effects of diabetes type and treatment on zinc status in diabetes mellitus

Zinc status was assessed in 53 diabetic patients: 18 insulin-dependent diabetic patients (IDDM), 22 noninsulin-dependent diabetic patients (NIDDM) treated with oral antidiabetic agents, and 13 insulin-treated, noninsulin-dependent diabetic patients (IRDM). Plasma zinc concentrations were in the usual range for healthy subjects in these three groups (15.3±0.9 μmol/L). Urinary zinc excretions were elevated in the IDDM group (18.3±4.1 μmol/24 h;p<0.01 vs normal) and in the NIDDM group (17.5±3.5 μmol/24 h;p<0.01 vs normal), but normal in the IRDM group (11.3±2.4 μmol/24 h). In 14 NIDDM patients treated with transient continuous sc insulin injections, urinary zinc decreased from 16.5±2.2 μmol/24 h before insulin treatment to 11.5±0.3 μmol/24 h after insulin treatment without any modification in plasma zinc concentrations.     

铃蟾肽对四氧嘧啶引起的大鼠糖尿病的预防作用

本工作从三个不同的层次对铃蟾肽防止胰岛 B 细胞损伤的作用进行了研究:(1)在整体水平,预先注射铃蟾肽(50μg/kg,iv)可明显抑制单独给予四氧嘧啶(200mg/kg,s.c.)引起的大鼠血糖升高和血浆胰岛素水平下降的趋势。(2)在离体胰腺灌流实验发现,在四氧嘧啶之前预灌流铃蟾肽(10~(-2)mmol/L)可使胰腺对高糖刺激产生反应性分泌;而仅以四氧嘧啶灌流时,胰腺对高糖刺激无反应。(3)在离体胰岛水平,初步研究了在四氧嘧啶引起胰岛 B 细胞功能改变时,铃蟾肽对胰岛内胰岛素、胰高血糖素和生长抑素分泌的影响。结果表明,铃蟾肽可防止四氧嘧啶引起的胰岛素和生长抑素分泌的抑制及胰高血糖素分泌的增加趋势。     

Alcohol dehydrogenase isoenzymes in chickpea cotyledons

Alcohol dehydrogenase (ADH) (EC 1.1.1.1) in the cotyledons of chickpea consists of three isoenzymes, ADH-1, ADH-2 and ADH-3, in order of decreasing ele     

Insulin restores expression of adenosine kinase in streptozotocin-induced diabetes mellitus rats

The effects of extracellular Mg²⁺ on both dynamic changes of [Ca²⁺]_i and apoptosis rate were analysed. The consequences of spatial and temporal dynamic changes of intracellular Ca²⁺ on apoptosis, in thapsigargin- and the calcium-ionophore 4BrA23187-treated MCF7 cells were first determined. Both 4BrA23187 and thapsigargin induced an instant increase of intracellular Ca²⁺ concentrations ([Ca²⁺]_i) which remained quite elevated (> 150 nM) and lasted for several hours. [Ca²⁺]_i increases were equivalent in the cytosol and the nucleus. The treatments that induced apoptosis in MCF7 cells were systematically associated with high and sustained [Ca²⁺]_i (150 nM) for several hours. The initial [Ca²⁺]_i increase was not determinant in the events triggering apoptosis. Thapsigargin-mediated apoptosis and [Ca²⁺]_i rise were abrogated when cells were pretreated with the calcium chelator BAPTA. The role of the extracellular Mg²⁺ concentration has been studied in thapsigargin treated cells. High (10 mM) extracellular Mg²⁺, caused an increase in basal [Mg²⁺]_i from 0.8 ± 0.3 to 1.6 ± 0.5 mM. As compared to 1.4 mM extracellular Mg²⁺, 1 M thapsigargin induces, in 10 mM Mg²⁺, a reduced percentage from 22 to 11% of fragmented nuclei, a lower sustained [Ca²⁺]_i and a lower Ca²⁺ influx through the plasma membrane. In conclusion, the cell death induced by thapsigargin was dependent on high and sustained [Ca²⁺]_i which was inhibited by high extracellular and intracellular Mg²⁺.     

Glutamate dehydrogenase isoenzymes in Ricinus communis seedlings

Castor bean seedling glutamate dehydrogenase isoenzymes are not artifacts. The isoenzymes have different salting out properties and they utilize NAPD to differing extents, but they have the same isoelectric point of pH 6·2. Tissue specific patterns occur but the patterns are the same between genotypes. The GDH isoenzymes are probably of functional significance in castor bean seedlings.     

Antigenic relationships between petunia peroxidase a and specific peroxidase isoenzymes in other Solanaceae

Summary A highly specific rabbit antiserum raised against peroxidase (PRXa) from petunia (Petunia hybrida) was used to investigate the antigenic relatedness of peroxidases in the Solanaceae. After SDS-PAGE of crude leaf extracts from a large number of species of this family, immunoblotting revealed that cross-reacting protein bands were present in all species tested. In order to determine whether these protein bands represent peroxidases, the peroxidase isoenzymes in thorn apple (Datura stramonium L.), tobacco (Nicotiana tabacum L.), sweet pepper (Capsicum annuum L.), potato (Solanum tuberosum L.), and tomato (Lycopersicon esculentum Mill.) were further analyzed. Immunoblots obtained after native PAGE revealed that the antiserum only recognized fast-moving peroxidase isoenzymes that are localized in the apoplast. Despite their serological relatedness, these peroxidases differed with respect to heat stability and apparent molecular weight. Differences in avidity for the petunia PRXa antiserum were suggested by immunoprecipitation with antibodies bound to protein A-Sepharose. The antiserum did not react with peroxidases from horseradish (Armoracea rusticana Gaertn., Mey and Scherb), turnip (Brassica napus L.), African marigold (Tagetes cresta L.), maize (Zea mays L.), and oats (Avena sativa L.). Apparently, the Solanaceae contain orthologous genes encoding the fast-moving anionic peroxidases homologous to petunia PRXa.