Quantitative enzyme histochemistry of rat foetal brain and trigeminal ganglion

Summary The increasing concern and the efforts in determining neurological effects in offsprings resulting from maternal exposure to xenobiotics are faced with several difficulties in monitoring damage to the central nervous system. In this paper, the efficiency of several enzyme histochemical reactions for analysing the forebrain and the trigeminal ganglia of rat foetuses are reported. Brains of 20-day-old Sprague-Dawley rat foetuses were frozen and analysed for 18 enzymes that had previously been used to monitor initial injury caused by toxic compounds in liver and other organs. Eight enzymes appeared suitable as histochemical markers for the functional integrity of different areas in brain and ganglia of rats exposed to xenobiotics. They were lactate, malate, glycerophosphate (NAD-linked), succinate, aldehyde and glucose 6-phosphate dehydrogenases, -glycerophosphate-menadione oxidoreductase and cytochromec oxidase. The activities of the enzymes were determined by microphotometry and the arrangement of absorbances of the enzyme final reaction products into appropriate analytical tables is proposed as an efficient procedure for data analysis.Abbreviations AcChE acetylcholinesterase - AldDH aldehyde dehydrogenase - ALKPase alkaline phosphatase - 5AMPase adenosine monophosphatase - ATPase Mg²⁺ dependent adenosine triphosphatase - CytOx cytochromec oxidase - GAPDH glyceraldehyde phosphate dehydrogenase - GIDH glutamate dehydrogenase - GLPDH glycerophosphate: NAD oxidoreductase - CPODH glycerophosphate:menadione oxidoreductase - G6Pase glucose-6-phosphatase - G6PDH glucose-6-phosphate dehydrogenase - IDH lactate dehydrogenase - MaDH malate dehydrogenase - MAO monoamine oxidase - NADPH, DH, NADPH tetrazolium oxidoreductase - SuDH succinate dehydrogenase - 6PGDH 6-phosphogluconate dehydrogenase     

G6PD Ferrara I has the same two mutations as G6PD A(-) but a distinct biochemical phenotype

The cloning and sequencing of the normal glucose-6-phosphate dehydrogenase (G6PD) gene has led to the study of the molecular defects that determine enzymatic variants. In this paper, we describe the mutations responsible for the Ferrara I variant in an Italian man with a family history of favism, from the Po delta. Nucleotide sequencing of this variant showed a GA mutation at nucleotide 202 in exon IV causing a ValMet amino acid exchange, and a second AG mutation at nucleotide 376 in exon V causing an AsnAsp amino acid substitution. Although on the basis of its biochemical properties this variant was classified as G6PD Ferrara I, it has the same two mutations as G6PD A(-), which is common in American and African blacks, and as the sporadic Italian G6PD Matera. The mutation at nucleotide 202 was confirmed by NlaIII digestion of a polymerase chain reaction amplified DNA fragment spanning 109 bp of exon IV. The 109-bp mutated amplified sequence is not distinguishable from the normal sequence in single strand conformation polymorphism analysis.     

Physiology of polyol formation by free and immobilized cells of the osmotolerant yeast Pichia farinosa

Summary Some physiological data of cells of Pichia farinosa immobilized on sintered glass Raschig rings were compared with data from free cells. Glucose consumption and productivity of total polyols (arabitol, glycerol and erythritol) showed a simultaneous inter-lag phase. Enzymes that catalyse steps of the pentosephosphate pathway (glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, transaldolase and polyol dehydrogenase) showed a distinct increase after transfer of the cells into production medium. The activity of glycerol-3-phosphate dehydrogenase was generally low. Only alcohol dehydrogenase presented the inter-lag phase mentioned above.Offprint requests to: H.-J. Rehm     

Molecular basis of glucose-6-phosphate dehydrogenase (G6PD) deficiency in three Taiwan aboriginal tribes

We have investigated glucose-6-phosphate dehydrogenase (G6PD) deficiency in 220 unrelated aboriginal male subjects who belong to three different tribes (Saisiat, Ami, and Yami) in Taiwan. Our results show that the G6PD deficiency rates for Saisiat, Ami, and Yami people are 9.0% (6/67), 6.1% (6/99), and 0% (0/54), respectively. Among these deficiency cases, 4 of 6 (66.7%) Saisiat subjects have the 493 AG mutation and one carries the 1376 GT mutation, whereas, in Ami subjects, we found that four of six (66.7%) affected males have the 592 CT mutation and one carries the 493 AG mutation. These results contrast with our previous findings for Taiwan Chinese, in whom the 1376 GT mutation is the major mutant allele and accounts for 52.3% of the deficiency cases. This is the first report of G6PD deficiency characterized at the DNA level in Taiwan aboriginal populations.     

Glycerol and dihydroxyacetone dissimilation in Desulfovibrio strains

During growth on glycerol two marine Desulfovibrio strains that can grow on an unusually broad range of substrates contained high activities of glycerol kinase, NAD(P)-independent glycerol 3-phosphate dehydrogenase and the other enzymes necessary for the conversion of dihydroxyacetone phosphate to pyruvate. Glycerol dehydrogenase and a specific dihydroxyacetone kinase were absent. During growth on dihydroxyacetone, glycerol kinase is involved in the initial conversion of this compound to dihydroxyacetone phosphate which is then further metabolized. Some kinetic properties of the partially purified glycerol kinase were determined. The role of NAD as electron carrier in the energy metabolism during growth of these strains on glycerol and dihydroxyacetone is discussed.Glycerol also supported growth of three out of four classical Desulfovibrio strains tested. D. vulgaris strain Hildenborough grew slowly on glycerol and contained glycerol kinase, glycerol 3-phosphate dehydrogenase and enzymes for the dissimilation of dihydroxyacetone phosphate. In D. gigas which did not grow on glycerol the enzymes glycerol kinase and glycerol 3-phosphate dehydrogenase were absent in lactate-grown cells.Abbreviations DHA dihydroxyacetone - DHAP dihydroxyacetone phosphate - G3P glycerol 3-phosphate - GAP glyceraldehyde 3-phosphate - 3-PGA 3-phosphoglycerate - 2-PGA 2-phosphoglycerate - 2,3-DPGA 2,3-diphosphoglycerate - PEP phosphoenolpyruvate - DH dehydrogenase - GK glycerol kinase - DHAK dihydroxyacetone kinase - TIM triosephosphate isomerase - PGK 3-phosphoglycerate kinase - PK pyruvate kinase - LDH lactate dehydrogenase - DTT dithiotreitol - HEPES 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid - PIPES piperazine-1,1-bis(2-ethane sulfonic acid) - BV²⁺/BV⁺ oxidized/reduced benzylviologen - PMS phenazine methosulfate - DCPIP 2,6-dichlorophenolindophenol - MTT 3-(4,5-dimethylthiazol-2-yl)-2,4-diphenyltetrazolium bromide     

Hydrolysis of G6P by a microsomal aspecific phosphatase and glucose phosphorylation by a low K m hexokinase in the digestive gland of the crab Carcinus maenas: variations during the moult cycle

Summary The hydrolysis of glucose-6-phospate in the digestive gland of the crab Carcinus maenas is carried out by an aspecific phosphatase. This enzyme possesses the following features: (1) insensitivity to acid treatment; (2) absence of inhibition when exposed to citrate at low pH; (3) similar affinity for G6P as the acid phosphatase for Na--glycerophosphate (K _m 2.3 and 2.0 mM, respectively). Glucose-6-phosphate and Na--glycerophate hydrolysis reactions seem to be catalysed by the same enzyme, since both activities exhibit the same distribution in a subcellular fractionation of the gland. Furthermore, as these activities are principally recovered in the subcellular fraction enriched in calcospherites (or calcium phosphate granules), it is proposed that the aspecific G6P-phosphohydrolase could play a major role in the formation of these granules. The phosphorylation of glucose is made by two low K _m hexokinases (230 and 64 M, respectively). As their level of activity shows significant changes over the moult cycle, these enzymes could be considered as having a regulatory role in the storage of glucose in the digestive gland.Abbreviations Acid Pase aspecific acid phosphatase - ATP adenosine triphosphate - DTT dithiothreitol - EDTA ethylenediaminetetra-acetate - G calcium phosphate granules fraction - G6P glucose-6-phosphate - G6Pase hepatic glucose-6-phosphatase - G6PDH glucose-6-phosphate dehydrogenase - K _m Michaelis-Menten constant - MI mitochondria and intermediate postmitochondrial particles - N nuclei fraction - NADH nicotineamide adenine dinucleotide - P microsome fraction - Pi inorganic phosphate - PMSF phenylmethylsulphonylfluoride - STI soybean trypsin inhibitor - glyP Na--glycerophosphate - T_1,2,3 transport protein 1,2,3 - TCA trichloroacetic acid     

A novel single-base mutation in the glucose 6-phosphate dehydrogenase gene is associated with chronic non-spherocytic haemolytic anaemia

More then 80 variants of glucose-6-phosphate dehydrogenase (G6PD) are associated with chronic non-spherocytic haemolytic anaemia (CNSHA); however, the molecular basis of this association is not fully understood. We have used the polymerase chain reaction and nucleotide sequence analysis to characterize a new G6PD variant, which we designate as G6PD Bari, in a G6PD-deficient boy affected by CNSHA. A single mutation leading to an amino-acid substitution was detected in the G6PD coding region, viz. a CT transition at position 1187 predicting leucine at residue 396 in the enzyme; proline is invariably present in evolutionary distant G6PD molecules at this position. Inheritance in the patient's family was demonstrated by the polymerase chain reaction followed by diagnostic restriction enzyme analysis. The CT transition responsible for G6PD Bari maps close to several other mutations previously identified in G6PD variants associated with CNSHA.     

Molecular heterogeneity underlying the G6PD Mediterranean phenotype

Summary As part of a study aiming to define the molecular basis of glucose-6-phosphate dehydrogenase (G6PD) deficiency, we analysed a sample from a Portugese boy with a family history of favism. Although the biochemical properties of red-cell G6PD from this subject were similar to those of the common variant G6PD Mediterranean, the corresponding mutation (563 CT) was not present. Instead, polymerase chain reaction (PCR) amplification and sequencing of the entire gene detected a CT transition at nucleotide 592 in exon VI, changing an arginine residue to a cysteine residue only 10 amino acids downstream from the Mediterranean mutation. Single-strand conformation polymorphism analysis of a PCR-amplified DNA fragment spanning exons VI and VII of the G6PD gene has detected the same mutation, confirmed by sequencing, in a G6PD-deficient patient from Southern Italy. We name this new variant G6PD Coimbra.     

Metabolic correlates to glycerol biosynthesis in a freeze-avoiding insect,Epiblema scudderiana

Summary The course of glycerol biosynthesis, initiated by exposure to –4°C, was monitored in larvae of the goldenrod gall moth,Epiblema scudderiana, and accompanying changes in the levels of intermediates of glycolysis, adenylates, glycogen, glucose, fructose-2,6-bisphosphate, and fermentative end products were characterized. Production of cryoprotectant was initiated within 6 h after a switch from +16° to –4°C, with halfmaximal levels reached in 30 h and maximal content, 450–500 mol/g wet weight, achieved after 4 days. Changes in the levels of intermediates of the synthetic pathway within 2 h at –4°C indicated that the regulatory sites involved glycogen phosphorylase, phosphofructokinase, and glycerol-3-phosphatase. A rapid increase in fructose-2,6-bisphosphate, an activator of phosphofructokinase and inhibitor of fructose-1,6-bisphosphatase, appeared to have a role in maintaining flux in the direction of glycerol biosynthesis. Analysis of metabolite changes as glycerol production slowed suggested that the inhibitory restriction of the regulatory enzymes was slightly out of phase. Inhibition at the glycerol-3-phosphatase locus apparently occurred first and resulted in a build-up of glycolytic intermediates and an overflow accumulation of glucose. Glucose inhibition of phosphorylase, stimulating the conversion of the activea to the inactiveb forms, appears to be the mechanism that shuts off phosphorylase function, counteracting the effects of low temperature that are the basis of the initial enzyme activation. Equivalent experiments carried out under a nitrogen gas atmosphere suggested that the metabolic make-up of the larvae in autumn is one that obligately routes carbohydrate flux through the hexose monophosphate shunt. The consequence of this is that fermentative ATP production during anoxia is linked to the accumulation of large amounts of glycerol as the only means of maintaining redox balance.Abbreviations G6P glucose-6-phosphate - F6P fructose-6-phosphate - F1, 6P fructose-1,6-bisphosphate - F2,6P ₂ fructose-2,6-bisphosphate - G3P grycerol-3-phosphate - DHAP dinydroxyacetonephosphate - GAP glyceraldehyde-3-phosphate - PEP phosphoenolpyruvate - PFK phosphofructokinase - FBPase fructose-1,6-bisphosphatase - PK pyruvate kinase     

Changes in the non-structural carbohydrate content of cotton (Gossypium spp.) fibres at different stages of development

The neutral sugars (glucose, fructose, and sucrose) and the sugar phosphates (glucose 6-phosphate, glucose 1-phosphate and fructose 6-phosphate) soluble in hot aqueous 80% methanol from the fibres of cotton — Gossypium arboreum L., G. barbadense L., and G. hirsutum L. — were determined at various stages of fibre development. In addition, the (13)--D-glucan content was measured and in the case of G. arboreum the rate of (13)--D-glucan and cellulose synthesis was determined with [¹⁴C]sucrose as the precursor. For each of the species a similar chronology was obtained for the changes in content of the various non-structural carbohydrates. At the early stages of secondary wall formation, glucose and fructose exhibited a maximum which was closely followed by a maximum in the (13)--D-glucan content and in the sugar phosphates. On the other hand, the sucrose content increased regularly until fibre maturity. The rates of synthesis of (13)--D-glucan and of cellulose were highest following the maximum in the (13)--D-glucan content, when the latter was being depleted.Abbreviations DMSO dimethyl-sulphoxide - DPA days post anthesis - UDP-glucose uridinediphosphoglucose     

Molecular analysis of G6PD variants in northern Italy: a study on the population from the Ferrara district

In the Ferrara district, an area south of the Po delta, four different variants of glucose-6-phosphate dehydrogenase (G6PD;E.C.1.1.49) have been described as a result of biochemical characterization of the enzyme protein: one was G6PD Mediterranean (G6PD Med) and three were local variants named Ferrara I, II, and III. The Ferrara I variant was recently analysed at the DNA level and shown to correspond to G6PD A^376G/202A, while the mutations causing the variants II and III, still remain unknown. We analysed the G6PD coding region of 18 apparently unrelated G6PD deficient subjects, whose families have lived in the Ferrara district for at least three generations: 12 subjects had G6PD Med^563T/1311T, 3, G6PD Santamaria^376G/542T and 2, G6PD A-^376G/202A. In one subject we found a new mutation, a GA transition at nucleotide 242 causing an ArgHis amino acid replacement at position 81. We named this new variant G6PD Lagosanto^{242 A}. Phenotypically the enzyme has nearly normal kinetic properties and appears different from the variants Ferrara II and III.     

Quantitative histochemistry of the sorbitol pathway in glomeruli and small arteries of human diabetic kidney.

Recent evidence has suggested a role for the polyol pathway in pathogenesis of cell damage in diabetes Glucose may be phosphorylated to glucose-6-phosphate via hexokinase and enter glycolysis or reduced to sorbitol via aldose reductase to enter the polyol pathway. The poorly diffusible sorbitol is converted via sorbitol dehydrogenase to fructose. Hexokinase, aldose reductase and sorbitol dehydrogenase activities were measured in glomeruli (G) and small arteries (SA) taken from normal and diabetic human kidneys, Hexokinase in diabetic G was 1688, which was significantly decreased from normal, 3147 mmoles/kg-1/h-1. Alodse reductase was significantly elevated in diabetic G,56-6, compared to normal G,10-8 mmoles/kg-1/h-1. In contrast, sorbitol dehydrogenase was significantly depressed in diabetic G, 3-7 VERSUs 10-9 mmoles/kg-1/h-1. The enzymatic changes observed in diabetic G would facilitate accumulation of sorbitol and therefore could contribute to the progression of glomerulosclerosis. The activity of hexokinase was also significantly reduced in SA, whereas aldose reductase and sorbitol dehydrogenase were unchanged.     

Histochemical demonstration of enzymes in rat liver during post-natal development

Summary Male and female rat liver were studied during post-natal development. A correlation was found between biochemically determined hydroxylations and enzymhisto-chemically determined NADPH-nitro-BT reductase and Naphthol-AS-D esterase. No correlation was found between glucose-6-phosphate dehydrogenase or iso-citric acid dehydrogenase activity and hydroxylations. The difference in hydroxylating capacity between male and female rats may be caused by the fact that the number of cells with hydroxylating activity in the liver lobule, as judged by the NADPH-nitro-BT reductase and Naphthol-AS-D esterase activity, is higher in male than in female rats.List of Abbreviations NADH reduced nicotinamide adenine dinucleotide - NADPH reduced nicotinamide adenine dinucleotide phosphate - G6PD glucose-6-phosphate dehydrogenase - ICD iso-citric acid dehydrogenase - G6Pase glucose-6-phosphatase - NADPH -nitro-BT red - NADPH Nitro-blue tetrazolium reductase - SDH succinic acid dehydrogenase - TCA trichloracetic acid     

The influence of freezing and freeze-drying of tissue specimens on enzyme activity

Summary In the presented study the influence of freezing and freeze-drying on enzyme activity is described. Attention is paid to 16 enzymes which can be used for quantitative enzyme histochemical techniques.With the exception of succinate dehydrogenase only, no significant inactivation during freezing and freeze-drying procedures could be demonstrated with lactate dehydrogenase, malate dehydrogenase (NAD⁺), malate dehydrogenase (decarboxylating) (NADP⁺), isocitrate dehydrogenase (NADP⁺), glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, NADH-oxydoreductase, mitochondrial glycerol-3-phosphate dehydrogenase, cytochrome c oxidase, phosphoglucomutase, glucosephosphate isomerase, glucose-6-phosphatase, acid phosphatase, -glucuronidase and non specific aryl esterase. Therefore the results supply a sound foundation for those quantitative enzyme histochemical techniques in which tissue specimens are frozen or frozen-dried before enzyme estimations are performed.     

Mutation analysis of glucose-6-phosphate dehydrogenase (G6PD) variants in Costa Rica

Summary Glucose-6-phosphate dehydrogenase (G6PD) deficiency has previously been reported among both the black and white populations of Costa Rica. All 28 G6PD A — samples were found to be of the common G6PD A-^376G/202Atype. A previously described mutation associated with nonspherocytic hemolytic anemia, G6PD Puerto Limón, was found to be due to a GA transition at nucleotide (nt) 1192, causing a glulys substitution. Mutations in this region of the G6PD molecule seem invariably to be associated with chronic hemolytic anemia. G6PD Santamaria had been described previously in two unrelated white subjects. We found that both did, indeed, have the same mutations. In this variant the AG substitution at nt 376 that is characteristic of G6PD A was present, but an AT mutation at nt 542, apparently superimposed on the ancient G6PD A mutation, resulted in an aspval substitution. Thus, the gain of a negative charge at amino acid 126 was counterbalanced by the loss of a charge at amino acid 181, giving rise to a variant with the G6PD A mutation but with normal electrophoretic mobility.     

Heterotrophic capacities of Plectonema boryanum

Acquisition of the dark heterotrophic growth capacity on glucose in Plectonema boryanum involves both adaptation and enrichment of a fast-growing genotype. The adaptation includes induction of functions involved in glucose incorporation and increase in glucose-6-phosphate dehydrogenase activity. Photosynthetic products are implicated in the control of both systems. Efficient energy conversion in the dark, as measured by cyanophage multiplication, correlates in time with the increase in potential for glucose incorporation while heterotrophic growth capacity correlates with the increase in glucose-6-phosphate dehydrogenase activity. The lower efficiency of heterotrophic growth compared to photoautotrophic growth is discussed in light of the conservation of the photosynthetic potency in the heterotrophic cells.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DTT dithiothreitol - G6P glucose-6-phosphate - NADP nicotinamide adenine dinucleotide phosphate - NTG N-methyl-N-nitro-N-nitrosoguanidine - RUDP ribulose-1,5-diphosphate - TCA trichloroacetic acid Dedicated to Prof. R. Y. Stanier on the occasion of his 60th birthday     

Histochemical demonstration of enzymes related to NADPH-dependent hydroxylating systems in rat liver after phenobarbital treatment

Summary Male rats were given 100mg phenobarbital for three days intraperitoneally. Biochemically an increase was found in activity of nitro-anisole demethylation and in the content of cytochrome P-450. Enzymhistochemically an increase in activity was noted for NADPH tetr. red., G6PD, ICD, and Naftol AS-D-esterase; a decrease was seen in G6Pase and glycogen, but no difference was found in NADH tetr. red. From these results it has been suggested that NADPH tetr. red. is directly involved in the hydroxylation chain, while G6PD and ICD are more indirectly involved.List of Abbreviations NADH nicotinamide adenine dinucleotide - NADPH nicotinamide adenine dinucleotide phosphate - NADPH tetr. red. NADPH tetrazolium reductase - G6PD glucose-6-phosphate dehydrogenase - ICD iso-citric acid dehydrogenase - G6Pase glucose-6-phosphatase - PAS periodic acid-Schiff method     

Effects of Biogenic Amines and Polypeptide Hormones on Protein Kinase A and Adenylyl Cyclase Activities in Muscles of the Mollusc Anodonta cygnea

The effects of biogenic amines, glucagon, and insulin on the cAMP-dependent protein kinase A (PKA) activity have been studied in the muscle tissue of the freshwater bivalve mollusc Anodonta cygnea. It was shown that serotonin, glucagon, and insulin both in vivo and in vitro stimulated PKA activity, whereas isoproterenol inhibited it. The stimulating effect of serotonin and inhibiting effect of isoproterenol was blocked by serotoninergic (cyproheptadine) and adrenergic (propranolol) inhibitors, which confirms specificity of the effect of biogenic amines on the PKA activity. Taking into account participation of adenylyl cyclase system in action of the above hormones, the revealed hormonal effects on the PKA activity produce metabolic effects via the following chain reaction. In the case of serotonin and glucagon: receptor G_s-protein AC cAMP PKA phosphorylation of glycogen synthase (GS) and glucose-6-phosphate dehydrogenase (G6PDH) and inhibition of their activity; in the case of isoproterenol: -adrenoreceptor G_i-protein AC inhibition decreasing PKA inhibition of phosphorylase and stimulation of GSI and G6PDH. A participation is suggested of the insulin-stimulated AC signaling system in the mechanism of the mitogenic insulin effect mediated, as shown in this work, via the PKA activation, but not of the metabolic effect of insulin.     

A new polymorphic site in the G6PD gene

Summary A polymorphic restriction site has been found in intron 11 of the gene for glucose-6-phosphate dehydrogenase (G6PD). This site is produced by a TC substitution 13 bp upstream of exon 12, producing an NlaIII restriction site. In various populations there was a strong association between a T at nt 1311 of the G6PD cDNA and the presence of the NlaIII restriction site. Among African Americans, however, the presence of a C at nt 1311 was sometimes associated with the presence of a polymorphic NlaIII site.     

Regulation of gluconeogenesis by glycerol and its phosphorylated derivatives

Glycerol, glycerol-3-phosphate (G3P), and dihydroxyacetone phosphate (DHAP) were evaluated as inhibitors of gluconeogenesis on rat liver enzymes in vitro, and for their effects on glucose formation in vivo in well-nourished and malnourished rats. DHAP was more potent as an inhibitor than G3P on fructose-1,6-diphosphatase (FDPase), phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (G6Pase). The I50 for DHAP was 2, 8, and 9 x 10(-3) M, respectively. No effect was observed on rat liver pyruvate carboxylase (PC). Glycerol was a weak inhibitor of FDPase and PEPCK, but did not inhibit PC and G6Pase. In vivo, when G3P was injected before a parenteral L-alanine (Ala) challenge, it produced a hypoglycemic effect in malnourished rats and a lesser, but noticeable, blood glucose level reduction in well-fed animals. Glycerol caused a smaller reduction in glucose formation from Ala. No comparable effects were observed after a fructose pretreatment. These results underscore the potential hypoglycemic effects of phosphorylated glycerol metabolites and identify the steps in gluconeogenesis where this action is exerted. The study also stresses the nutritional component in the glycerol intolerance syndrome, apparent from the far more severe effects observed in malnourished rats given G3P or glycerol prior to Ala.