Freezing and cellular metabolism in the gall fly larva,Eurosta solidaginis

Summary The effects of extracellular freezing on intracellular metabolism were monitored over both a short (9 h) and long (12 weeks) time course using the freeze tolerant larvae of the gall fly,Eurosta solidaginis.The process of freezing, monitored over the short time course, had no effect upon cellular energy levels (adenylates, arginine phosphate) but initiated a rise in glucose-6-P and lactate levels. This suggests that freezing initiates a shift towards glycolysis as the predominant mode of energy production. The process of thawing at 3°C (after 24 h at –16°C) also had no effect, even transient, on cellular energy levels demonstrating that thawing and the rapid redistribution of water and solutes which must accompany it does not disrupt cellular metabolism. During thawing accumulated lactate was quickly cleared with a t 1/2 of 20–30 min.Long term freezing at –16°C had dramatic effects on energy metabolism. Freezing for up to 1 week had minimal effects with only a small drop in arginine phosphate reserves and an increase in lactate content noted. Between 1 and 2 weeks of freezing, however, larvae showed strong signs of energy stress. The arginine phosphate pool fell from 75% to 30% of control levels, ATP content dropped by 50% and energy charge dropped to 0.75. This state, with continued lactate accumulation, was maintained through 4 weeks of freezing. Between 6 and 12 weeks of freezing energy stress became even greater. Phosphagen and ATP contents dropped to 5 and 25% of control values and energy charge decreased to about 0.50. Despite this stress, however, 94% of larvae survived 12 weeks of freezing with an 86% hatch rate of adults. The data demonstrate that the larvae can survive prolonged periods of winter freezing drawing upon glycolysis and phosphagen reserves to supply the continued basal energy demands of the cell.     

Kinetic regulation of the mitochondrial glycerol-3-phosphate dehydrogenase by the external NADH dehydrogenase in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, the two most important systems for conveying excess cytosolic NADH to the mitochondrial respiratory chain are external NADH dehydrogenase (Nde1p/Nde2p) and the glycerol-3-phosphate dehydrogenase shuttle. In the latter system, NADH is oxidized to NAD+ and dihydroxyacetone phosphate is reduced to glycerol 3-phosphate by the cytosolic Gpd1p; glycerol 3-phosphate gives two electrons to the respiratory chain via mitochondrial glycerol-3-phosphate dehydrogenase (Gut2p)-regenerating dihydroxyacetone phosphate. Both Nde1p/Nde2p and Gut2p are located in the inner mitochondrial membrane with catalytic sites facing the intermembranal space. In this study, we showed kinetic interactions between these two enzymes. First, deletion of either one of the external dehydrogenases caused an increase in the efficiency of the remaining enzyme. Second, the activation of NADH dehydrogenase inhibited the Gut2p in such a manner that, at a saturating concentration of NADH, glycerol 3-phosphate is not used as respiratory substrate. This effect was not a consequence of a direct action of NADH on Gut2p activity because both NADH dehydrogenase and its substrate were needed for Gut2p inhibition. This kinetic regulation of the activity of an enzyme as a function of the rate of another having a similar physiological function may be allowed by their association into the same supramolecular complex in the inner membrane. The physiological consequences of this regulation are discussed.     

Developmental regulation of glycerol-3-phosphate dehydrogenase synthesis inDrosophila

Methods have been developed to measure the synthesis of glycerol-3-phosphate dehydrogenase (GPDH) during the development of Drosophila melanogaster. In emerged adult flies, GPDH is a principal component of protein synthesis, comprising between 1 and 2% of the protein synthetic effort. This high relative rate of protein synthesis continues throughout adult life during a period of stable enzyme concentration. Therefore, it is evident that GPDH undergoes continual turnover. Analysis of GPDH synthesis in the adult segments reveals that this enzyme is synthesized in head, thorax, and abdomen. In 5-day-old flies, the relative rates of GPDH synthesis in the thorax and abdomen are similar. However, the concentration of GPDH in the thorax greatly exceeds that found in the abdomen. Therefore, it appears that the turnover rate of GPDH in the abdomen must be greater than the turnover rate of GPDH in the GPDH-containing cells (flight muscle) of the thorax. GPDH represents between 0.5 and 0.9% of the protein synthetic effort of larvae. The principle GPDH-containing tissue of larvae is fat body. The turnover of GPDH in larvae is similar to that in adult abdomen. This may be related to the concurrent presence of GPDH isozyme-3 in both tissues. Our studies indicate that the cell type-specific control of GPDH occurs at several levels.     

Enzyme activity profiles in an overwintering population of freeze-tolerant larvae of the gall fly,Eurosta solidaginis

The activity of some enzymes of intermediary metabolism, including enzymes of glycolysis, the hexose monophosphate shunt, and polyol cryoprotectant synthesis, were measured in freeze-tolerant Eurosta solidaginis larvae over a winter season and upon entry into pupation. Flexible metabolic rearrangement was observed concurrently with acclimatization and development. Profiles of enzyme activities related to the metabolism of the cryoprotectant glycerol indicated that fall biosynthesis may occur from two possible pathways: 1. glyceraldehyde-phosphate glyceraldehyde glycerol, using glyceraldehyde phosphatase and NADPH-linked polyol dehydrogenase, or 2. dihydroxyacetonephosphate glycerol-3-phosphate glycerol, using glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase. Clearance of glycerol in the spring appeared to occur by a novel route through the action of polyol dehydrogenase and glyceraldehyde kinase. Profiles of enzyme activities associated with sorbitol metabolism suggested that this polyol cryoprotectant was synthesized from glucose-6-phosphate through the action of glucose-6-phosphatase and NADPH-linked polyol dehydrogenase. Removal of sorbitol in the spring appeared to occur through the action of sorbitol dehydrogenase and hexokinase. Glycogen phosphorylase activation ensured the required flow of carbon into the synthesis of both glycerol and sorbitol. Little change was seen in the activity of glycolytic or hexose monophosphate shunt enzymes over the winter. Increased activity of the -glycerophosphate shuttle in the spring, indicated by greatly increased glycerol-3-phosphate dehydrogenase activity, may be key to removal and oxidation of reducing equivalents generated from polyol cryoprotectan catabolism.Abbreviations 6PGDH 6-Phosphogluconate dehydrogenase - DHAP dihydroxy acetone phosphate - F6P fructose-6-phosphate - F6Pase fructose-6-phospha-tase - FBPase fructose-bisphosphatase - G3P glycerol-3-phosphate - G3Pase glycerol-3-phosphate phophatase - G3PDH glycerol-3-phosphate dehydrogenase - G6P glucose-6-phosphate - G6Pase glucose-6-phosphatase - G6PDH glucose-6-phosphate dehydrogenase - GAK glyceraldehyde kinase - GAP glyceraldehyde-3-phosphate - GAPase glyceraldehyde-3-phosphatase - GAPDH glyceraldehyde-3-phosphate dehydrogenase - GDH glycerol dehydrogenase - GPase glycogen phosphorylase - HMS hexose monophosphate shunt - LDH lactate dehydrogenase - NADP-IDH NADP⁺-dependent isocitrate dehydrogenase - PDHald polyol dehydrogenase, glyceraldehyde activity - PDHgluc polyol dehydrogenase, glucose activity - PFK phosphofructokinase - PGI phosphoglucoisomerase - PGK phosphoglycerate kinase - PGM phosphoglucomutase - PK pyruvate kinase - PMSF phenylmethylsulfonylfluoride - SoDH sorbitol dehydrogenase - V _max maximal enzyme activity - ww wet weight     

Cold winter microenvironments conserve energy and improve overwintering survival and potential fecundity of the goldenrod gall fly, Eurosta solidaginis

We studied the influence of two overwintering microenvironments on survival and potential fecundity of goldenrod gall flies, Eurosta solidaginis (Fitch) (Diptera, Tephritidae). These freeze-tolerant larvae overwinter above the snow on standing goldenrod stems (elevated) or below the snow on broken stems (ground-level). When covered by snow, the ground-level larvae were well insulated and thus protected from the lowest temperatures of the winter, but, because they were warmer, they consumed more energy than their elevated counterparts. The ground-level group also experienced greater warming from the soil during sunny spring days, and their galls were less prone to drying than their elevated counterparts. By winter's end the ground-level larvae exhibited significantly lower rates of emergence (83.5% vs 93.0%) and reduced potential fecundity (274±11 eggs/female vs 336±17 eggs/female). Models of seasonal energy use indicate that these differences were due to higher metabolic rates in the ground-level microenvironment due to insulation by snow and warming from the soil, which reduced the energy available for morphological development and egg production in the spring. We conclude that colder winter microenvironments can have a strong positive effect on overwintering ectotherms, particularly those that rely on energy stores accumulated during the autumn to produce eggs in spring. The enhanced reproductive output of insects overwintering in colder microenvironments may be a selective force promoting the evolution of increased cold-hardiness.     

Structural analysis of glycerol-3-phosphate dehydrogenase from severalDrosophila species

This report describes preliminary protein structural studies of glycerol-3-phosphate dehydrogenase (alpha-GPDH) from Drosophila spp. and an important innovative feature of our enzyme purification protocol. The scheme involves the coupling of substrate (alpha-glycerophosphate) elution from CM-Sephadex and cofactor (NADH) elution from Affi-Gel blue resin. Using this method a 32.7% yield and a 111-fold purification were obtained from a D. melanogaster line carrying the alpha-GpdhS allele at the alpha-Gpdh locus. The product obtained from 0 to 3-day-old adult flies was electrophoretically homogeneous and consisted mainly of the adult alpha-GPDH-1 isozyme. The method was used to obtain alpha-GPDH protein from D. melanogaster (two lines), D. hydei, D. immigrans, and D. mercatorum. Peptide mapping revealed structural differences among the enzymes from the different species, and amino acid sequencing showed many similarities between D. melanogaster alpha-GPDH and the rabbit muscle enzyme.     

Cuticular lipids and desiccation resistance in overwintering larvae of the goldenrod gall fly, Eurosta solidaginis (Diptera: Tephritidae)

Within their gall, larvae of the goldenrod gall fly (Eurosta solidaginis) experience severe desiccating conditions as well as highly variable thermal conditions and extreme cold during winter. Through the autumn and early winter, field-collected larvae acquired markedly enhanced resistance to desiccation and freezing. At the same time, they increased their cuticular surface hydrocarbons. Hydrocarbons were the major lipid class extracted by hexane or chloroform from the cuticular surface of overwintering gall fly larvae. The major hydrocarbon classes were the 2-methylalkanes which consisted mainly of 2-methyltriacontane. 2-Methyltriacontane comprised 48-68% of the total hydrocarbons during the larval stages. Total hydrocarbons increased from 122 ng/larva in early third instar larvae collected in September to 4900 ng/larva in those collected in January. Although washing of the cuticular surface with chloroform or chloroform:methanol (2:1, v:v) caused marked increases in rates of water loss, treatment with hexane and methanol had little effect on water loss rates.     

Identification of a mitochondrial glycerol-3-phosphate dehydrogenase from Arabidopsis thaliana: evidence for a mitochondrial glycerol-3-phosphate shuttle in plants

We report molecular characterization of an Arabidopsis gene encoding a mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase (FAD-GPDH) that oxidizes glycerol-3-phosphate (G-3-P) to dihydroxyacetone phosphate. We demonstrate through in vitro targeting assays that the encoded gene product can be imported into mitochondrial membrane systems. Enzyme activity of the protein was confirmed through heterologous expression in Escherichia coli. The Arabidopsis gene is expressed throughout plant development, but at the highest level during seed germination. We also show that expression of the Arabidopsis FAD-GPDH gene is coupled to oxygen consumption and affected by ABA and stress conditions. Together with an NAD(+)-dependent GPDH, this enzyme could form a G-3-P shuttle, as previously established in other eukaryotic organisms, and links cytosolic G-3-P metabolism to carbon source utilization and energy metabolism in plants.     

Isoenzymic forms of NAD-linked glycerol-3-phosphate dehydrogenase from rabbit brain.

Two major enzyme forms of cytosolic NAD-linked glycerol-3-phosphate dehydrogenase in rabbit brain have been purified to apparent homogeneity. One major enzyme form designated I6.5 exhibits an iso-electric point at pH 6.5, and is indistinguishable from the major form I6.5 found in other tissues. The other major form, designated I5.9, has an isolectric point at pH 5.9, and by amino acid analysis is shown to be a true isoenzyme distinct from form I6.5. Form I5.9 appears to be closely related to or identical with the major enzyme characteristic of heart. Neither the brain enzyme form I5.9 nor the major heart isoenzyme are inhibited by antiserum to the muscle enzyme. Because of the high apparent Km for NADH, it is postulated that the brain isoenzyme I5.9 serves to maintain glycolysis when NADH levels rise under relatively anaerobic conditions especially during fetal and neonatal development.     

Solubilization and immunochemical identification of mitochondrial glycerol-3-phosphate dehydrogenase

Lysophosphatidylcholine (contrary to Lubrol WX, Triton X-100, digitonine and deoxycholate) solubilizes hamster brown fat mitochondrial glycerol-3-phosphate dehydrogenase without inactivation. Optimal ratio of lysophosphatidylcholine and membrane protein for solubilization of the enzyme was found to be 0.25 mg of lysophosphatidylcholine per mg protein. The activity of solubilized enzyme, however, was not affected by low concentrations of Lubrol WX, Triton X-100, digitonine, Zwittergent TM 314. Deoxycholate exhibited a pronounced inactivating effect. One-dimensional immunoelectrophoresis of the solubilized membrane proteins revealed 10 protein bands, 3-4 of which exhibited the enzyme activity. Two-dimensional immunoelectrophoresis revealed only a single main band of glycerol-3-phosphate dehydrogenase. This technique thus appears to be the best means for the identification of glycerol-3-phosphate dehydrogenase in the mixture of solubilized membrane proteins and for concentration of the enzyme activity in one major precipitating band.     

The localization of glycerol-3-phosphate dehydrogenase inEscherichia coli

Summary Starved cells ofEscherichia coli are dependent on an exogenous source of energy. It was of interest to ask whether compounds that are commonly used to supply energy must themselves be transported or whether they can be utilized on the outer portion of the cytoplasmic membrane. The utilization of glycerol-3-phosphate an energy source is totally dependent on the membrane-bound glycerol-3-phosphate dehydrogenase. In the present report glycerol-3-phosphate was used as the energy source for uptake of amino acids. A mutant was constructed which is unable to transport this ester and the starved mutant could not drive the uptake of glutamine with glycerol-3-phosphate. It is concluded that the enzyme is located on the internal surface of the membrane in intactE. coli cells. Further evidence was obtained by showing that no glycerol-3-phosphate dehydrogenase activity could be measured in either intact cells or spheroplasts using ferricyanide as electron acceptor, due to its impermeability. The activity could be measured after destruction of the membrane permeability barrier by toluenization. With membrane vesicles prepared according to Kaback's procedure nearly half of the dehydrogenase activity was accessible to ferricyanide as well as to impermeable competitive inhibitors of the enzyme. Partial inversion during preparation of vesicles is the most probable explanation for the results.A protion of this work was presented at the Miami Winter Symposia on the Molecular Basis of Biological Transprot, 1972.