Mitochondrial physiology in the major arbovirus vector Aedes aegypti: substrate preferences and sexual differences define respiratory capacity and superoxide production.

Adult females of Aedes aegypti are facultative blood sucking insects and vectors of Dengue and yellow fever viruses. Insect dispersal plays a central role in disease transmission and the extremely high energy demand posed by flight is accomplished by a very efficient oxidative phosphorylation process, which take place within flight muscle mitochondria. These organelles play a central role in energy metabolism, interconnecting nutrient oxidation to ATP synthesis, but also represent an important site of cellular superoxide production. Given the importance of mitochondria to cell physiology, and the potential contributions of this organelle for A. aegypti biology and vectorial capacity, here, we conducted a systematic assessment of mitochondrial physiology in flight muscle of young adult A. aegypti fed exclusively with sugar. This was carried out by determining the activities of mitochondrial enzymes, the substrate preferences to sustain respiration, the mitochondrial bioenergetic efficiency and capacity, in both mitochondria-enriched preparations and mechanically permeabilized flight muscle in both sexes. We also determined the substrates preferences to promote mitochondrial superoxide generation and the main sites where it is produced within this organelle. We observed that respiration in A. aegypti mitochondria was essentially driven by complex I and glycerol 3 phosphate dehydrogenase substrates, which promoted distinct mitochondrial bioenergetic capacities, but with preserved efficiencies. Respiration mediated by proline oxidation in female mitochondria was strikingly higher than in males. Mitochondrial superoxide production was essentially mediated through proline and glycerol 3 phosphate oxidation, which took place at sites other than complex I. Finally, differences in mitochondrial superoxide production among sexes were only observed in male oxidizing glycerol 3 phosphate, exhibiting higher rates than in female. Together, these data represent a significant step towards the understanding of fundamental mitochondrial processes in A. aegypti, with potential implications for its physiology and vectorial capacity.

Low-protein, high-carbohydrate diet increases glucose uptake and fatty acid synthesis in brown adipose tissue of rats.

OBJECTIVE: The aim of this study was to evaluate glucose uptake and the contribution of glucose to fatty acid (FA) synthesis and the glycerol-3-phosphate (G3P) of triacylglycerol synthesis by interscapular brown adipose tissue (IBAT) of low-protein, high-carbohydrate (LPHC) diet-fed rats. METHODS: LPHC (6% protein; 74% carbohydrate) or control (17% protein; 63% carbohydrate) diets were administered to rats (∼ 100 g) for 15 d. Total FA and G3P synthesis and the synthesis of FA and G3P from glucose were evaluated in vivo by (3)H2O and (14)C-glucose. Sympathetic neural contribution for FA synthesis was evaluated by comparing the synthesis in denervated (7 d before) IBAT with that of the contralateral innervated side. The insulin signaling and ß3 adrenergic receptor (ß3-AR) contents, as well as others, were determined by Western blot (Student's t test or analysis of variance; P ≤ 0.05). RESULTS: Total FA synthesis in IBAT was 133% higher in the LPHC group and was reduced 85% and 70% by denervation for the LPHC and control groups, respectively. Glucose uptake was 3.5-fold higher in the IBAT of LPHC rats than in that of the control rats, and the contribution of glucose to the total FA synthesis increased by 12% in control rats compared with 18% in LPHC rats. The LPHC diet increased the G3P generation from glucose by 270% and the insulin receptor content and the p-AKT insulin stimulation in IBAT by 120% and reduced the ß3-AR content by 50%. CONCLUSIONS: The LPHC diet stimulated glucose uptake, both the total rates and the rates derived from glucose-dependent FA and G3P synthesis, by increasing the insulin sensitivity and the sympathetic flux, despite a reduction in the ß3-AR content.

Trypanosoma rangeli: an alkaline ecto-phosphatase activity is involved with survival and growth of the parasite.

The aim of this work was to investigate whether an alkaline ecto-phosphatase activity is present in the surface of Trypanosoma rangeli. Intact short epimastigote forms were assayed for ecto-phosphatase activity to study kinetics and modulators using ß-glycerophosphate (ß-GP) and p-nitrophenyl phosphate (pNPP) as substrates. Its role in parasite development and differentiation was also studied. Competition assays using different proportions of ß-GP and pNPP evidenced the existence of independent and non-interacting alkaline and acid phosphatases. Hydrolysis of ß-GP increased progressively with pH, whereas the opposite was evident using pNPP. The alkaline enzyme was inhibited by levamisole in a non-competitive fashion. The Ca(2+) present in the reaction medium was enough for full activity. Pretreatment with PI-PLC decreased the alkaline but not the acid phosphatase evidence that the former is catalyzed by a GPI-anchored enzyme, with potential intracellular signaling ability. ß-GP supported the growth and differentiation of T. rangeli to the same extent as high orthophosphate (Pi). Levamisole at the IC50 spared significantly parasite growth when ß-GP was the sole source of Pi and stopped it in the absence of ß-GP, indicating that the alkaline enzyme can utilize phosphate monoesters present in serum. These results demonstrate the existence of an alkaline ecto-phosphatase in T. rangeli with selective requirements and sensitivity to inhibitors that participates in key metabolic processes in the parasite life cycle.

A low-protein, high-carbohydrate diet increases de novo fatty acid synthesis from glycerol and glycerokinase content in the liver of growing rats.

We had previously shown that adipose tissue increased in rats fed a low-protein, high-carbohydrate (LPHC) diet (6% protein, 74% carbohydrate) without a simultaneous increase in the de novo fatty acids (FA) synthesis. In addition, impairment in insulin signaling in adipose tissues was observed in these rats. For this study, we hypothesized that the insulin signaling pathway is preserved in the livers from these rats, which contributes to an increase in liver lipogenesis and, consequently, an increase in the weight of the adipose tissue. We also hypothesized that glycerol from triacylglycerol is an important substrate for FA synthesis. Our results showed that administration of the LPHC diet induced an increase in the in vivo rate of total FA synthesis (150%) as well as FA synthesis from glucose (270%) in the liver. There were also increased rates of [U-¹4C]glycerol incorporation into glyceride-FA (15-fold), accompanied by increased glycerokinase content (30%) compared with livers of rats fed the control diet. The LPHC diet did not change the glycerol-3-phosphate generation from either glucose or glyceroneogenesis. There was an increase in the insulin sensitivity in liver from LPHC-fed rats, as evidenced by increases in IR(ß) (35%) levels and serine/threonine protein kinase (AKT) levels (75%), and basal (95%) and insulin-stimulated AKT phosphorylation (105%) levels. The LPHC diet also induced an increase in the liver sterol regulatory element-binding protein-1c content (50%). In summary, these data confirmed the hypothesis that lipogenesis and insulin signaling are increased in the livers of LPHC-fed rats and that glycerol is important not only for FA esterification but also for FA synthesis.

Addition of bone morphogenetic protein type 2 to ascorbate and β-glycerophosphate supplementation did not enhance osteogenic differentiation of human adipose-derived stem cells

Bone morphogenetic protein type 2 (BMP-2) is a potent local factor, which promotes bone formation and has been used as an osteogenic supplement for mesenchymal stem cells. OBJECTIVES: This study evaluated the effect of a recombinant BMP-2 as well as the endogenous BMP-4 and BMP-7 in the osteogenic differentiation of adipose-derived stem cells (ASCs) in medium supplemented with ascorbate and β-glycerophosphate. MATERIAL AND METHODS: Human ASCs were treated with osteogenic medium in the presence (ASCs+OM+BMP-2) or absence (ASCs+OM) of BMP-2. The alkaline phosphatase (ALP) activity was determined and the extracellular matrix mineralization was evaluated by Von Kossa staining and calcium quantification. The expressions of BMP-4, BMP-7, Smad1, Smad4, and phosphorylated Smad1/5/8 were analyzed by western blotting. Relative mRNA expressions of Smad1, BMP receptor type II (BMPR-II), osteonectin, and osteocalcin were evaluated by qPCR. Results: ASCs+OM demonstrated the highest expression of BMP-4 and BMP-7 at days 21 and 7, respectively, the highest levels of BMPR-II mRNA expression at day 28, and the highest levels of Smad1 mRNA at days 14 and 28. ASCs+OM+BMP-2 demonstrated the highest levels of Smad1 mRNA expression at days 1, 7, and 21, the highest expression of Smad1 at day 7, the highest expression of Smad4 at day 14, the highest ALP activity at days 14 and 21, and expression of phosphorylated Smad1/5/8 at day 7. ASCs+OM and ASCs+OM+BMP2 showed similar ALP activity at days 7 and 28, similar osteonectin and osteocalcin mRNA expression at all time periods, and similar calcium depositions at all time periods. CONCLUSIONS: We concluded that human ASCs expressed endogenous BMP-4 and BMP-7. Moreover, the supplementation of ASCs with BMP-2 did not increase the level of osteogenic markers in the initial (ALP activity), intermediate (osteonectin and osteocalcin), or final (calcium deposition) phases, suggesting that the exogenous addition of BMP-2 did not improve the in vitro osteogenesis process of human ASCs.

Triacylglycerol biosynthesis occurs via the glycerol-3-phosphate pathway in the insect Rhodnius prolixus.

Although triacylglycerol (TAG) stores play a critical role in organisms, mechanisms underlying TAG synthesis are poorly understood in invertebrates. In mammals, the synthesis of glycerolipids, including TAG, diacylglycerol (DAG) and phospholipids (PL), occurs predominantly by the glycerol-3-phosphate (G3P) pathway in most cell types, except for in enterocytes. In these cells, the monoacylglycerol (MAG) pathway accounts for the majority of glycerolipid production. The insect Rhodnius prolixus, a vector of Chagas' disease, exhibits a high capacity to produce glycerolipids in the midgut after a blood meal, providing substrates that are transferred to other organs, such as the fat body, which is specialized in TAG production and storage. In this report, the genes required for TAG synthesis were identified in the R. prolixus genome. The genomic data indicated that TAG is synthesized by the G3P pathway, which is the sole pathway for TAG synthesis in this organism. Furthermore, transcription of both the RpGpat and RpDgat genes were upregulated in a diverse number of organs at moments of highest lipid production. In the midgut and fat body, in vitro synthesis of glycerolipids required G3P, but not MAG, as the initial substrate. These results indicate that the G3P pathway is the only route for TAG synthesis in R. prolixus, and its regulation at the transcriptional level can be a determinant of glycerolipid synthesis and TAG formation in insect organs.

Comparative phytohormone profiles, lipid kinase and lipid phosphatase activities in barley aleurone, coleoptile, and root tissues.

We analyzed lipid kinase and lipid phosphatase activities and determined endogenous phytohormone levels by liquid chromatography-tandem mass spectrometry in root and coleoptile tissues following germination of barley (Hordeum vulgare) seeds. The enzymes showing highest activity in aleurone cells were diacylglycerol kinase (DAG-k, EC 2.7.1.107) and phosphatidate kinase (PA-k). The ratio of gibberellins (GAs) to abscisic acid (ABA) was 2-fold higher in aleurone than in coleoptile or root tissues. In coleoptiles, phosphatidylinositol 4-kinase (PI4-k, EC 2.7.1.67) showed the highest enzyme activity, and jasmonic acid (JA) level was higher than in aleurone. In roots, activities of PI4-k, DAG-k, and PA-k were similar, and salicylic acid (SA) showed the highest concentration. In the assays to evaluate the hydrolysis of DGPP (diacylglycerol pyrophosphate) and PA (phosphatidic acid) we observed that PA hydrolysis by LPPs (lipid phosphate phosphatases) was not modified; however, the diacylglycerol pyrophosphate phosphatase (DGPPase) was strikingly higher in coleoptile and root tissues than to aleurone. Relevance of these findings in terms of signaling responses and seedling growth is discussed.

Addition of bone morphogenetic protein type 2 to ascorbate and ß-glycerophosphate supplementation did not enhance osteogenic differentiation of human adipose-derived stem cells.

UNLABELLED: Bone morphogenetic protein type 2 (BMP-2) is a potent local factor, which promotes bone formation and has been used as an osteogenic supplement for mesenchymal stem cells. OBJECTIVES: This study evaluated the effect of a recombinant BMP-2 as well as the endogenous BMP-4 and BMP-7 in the osteogenic differentiation of adipose-derived stem cells (ASCs) in medium supplemented with ascorbate and ß-glycerophosphate. MATERIAL AND METHODS: Human ASCs were treated with osteogenic medium in the presence (ASCs+OM+BMP-2) or absence (ASCs+OM) of BMP-2. The alkaline phosphatase (ALP) activity was determined and the extracellular matrix mineralization was evaluated by Von Kossa staining and calcium quantification. The expressions of BMP-4, BMP-7, Smad1, Smad4, and phosphorylated Smad1/5/8 were analyzed by western blotting. Relative mRNA expressions of Smad1, BMP receptor type II (BMPR-II), osteonectin, and osteocalcin were evaluated by qPCR. RESULTS: ASCs+OM demonstrated the highest expression of BMP-4 and BMP-7 at days 21 and 7, respectively, the highest levels of BMPR-II mRNA expression at day 28, and the highest levels of Smad1 mRNA at days 14 and 28. ASCs+OM+BMP-2 demonstrated the highest levels of Smad1 mRNA expression at days 1, 7, and 21, the highest expression of Smad1 at day 7, the highest expression of Smad4 at day 14, the highest ALP activity at days 14 and 21, and expression of phosphorylated Smad1/5/8 at day 7. ASCs+OM and ASCs+OM+BMP2 showed similar ALP activity at days 7 and 28, similar osteonectin and osteocalcin mRNA expression at all time periods, and similar calcium depositions at all time periods. CONCLUSIONS: We concluded that human ASCs expressed endogenous BMP-4 and BMP-7. Moreover, the supplementation of ASCs with BMP-2 did not increase the level of osteogenic markers in the initial (ALP activity), intermediate (osteonectin and osteocalcin), or final (calcium deposition) phases, suggesting that the exogenous addition of BMP-2 did not improve the in vitro osteogenesis process of human ASCs.

Thyroid hormone contributes to the hypolipidemic effect of polyunsaturated fatty acids from fish oil: in vivo evidence for cross talking mechanisms.

n-3 polyunsaturated fatty acids (n-3 PUFA) from fish oil (FO) exert important lipid-lowering effects, an effect also ascribed to thyroid hormones (TH) and TH receptor ß1 (TRß1)-specific agonists. n-3 PUFA effects are mediated by nuclear receptors, such as peroxisome proliferator-activated receptors (PPAR) and others. In this study, we investigated a role for TH signaling in n-3 PUFA effects. Euthyroid and hypothyroid adult rats (methimazole-treated for 5 weeks) received FO or soybean oil (control) by oral administration for 3 weeks. In euthyroid rats, FO treatment reduced serum triglycerides and cholesterol, diminished body fat, and increased protein content of the animals. In addition, FO-treated rats exhibited higher liver expression of TRß1 and mitochondrial α-glycerophosphate dehydrogenase (mGPD), at protein and mRNA levels, but no alteration of glutathione S-transferase or type 1 deiodinase. In hypothyroid condition, FO induced reduction in serum cholesterol and increase in body protein content, but lost the ability to reduce triglycerides and body fat, and to induce TRß1 and mGDP expression. FO did not change PPARα liver abundance regardless of thyroid state; however, hypothyroidism led to a marked increase in PPARα liver content but did not alter TRß1 or TRα expression. The data suggest that part of the effect of n-3 PUFA from FO on lipid metabolism is dependent on TH signaling in specific steps and together with the marked upregulation of PPARα in liver of hypothyroid rats suggest important in vivo consequences of the cross-talking between those fatty acids and TH pathways in liver metabolism.

Profiling the changes in signaling pathways in ascorbic acid/ß-glycerophosphate-induced osteoblastic differentiation.

Despite numerous reports on the ability of ascorbic acid and ß-glycerophosphate (AA/ß-GP) to induce osteoblast differentiation, little is known about the molecular mechanisms involved in this phenomenon. In this work, we used a peptide array containing specific consensus sequences (potential substrates) for protein kinases and traditional biochemical techniques to examine the signaling pathways modulated during AA/ß-GP-induced osteoblast differentiation. The kinomic profile obtained after 7 days of treatment with AA/ß-GP identified 18 kinase substrates with significantly enhanced or reduced phosphorylation. Peptide substrates for Akt, PI3K, PKC, BCR, ABL, PRKG1, PAK1, PAK2, ERK1, ERBB2, and SYK showed a considerable reduction in phosphorylation, whereas enhanced phosphorylation was observed in substrates for CHKB, CHKA, PKA, FAK, ATM, PKA, and VEGFR-1. These findings confirm the potential usefulness of peptide microarrays for identifying kinases known to be involved in bone development in vivo and in vitro and show that this technique can be used to investigate kinases whose function in osteoblastic differentiation is poorly understood.

A low-protein, high-carbohydrate diet increases the adipose lipid content without increasing the glycerol-3-phosphate or fatty acid content in growing rats.

The amount of triacylglycerol (TAG) that accumulates in adipose tissue depends on 2 opposing processes: lipogenesis and lipolysis. We have previously shown that the weight and lipid content of epididymal (EPI) adipose tissue increases in growing rats fed a low-protein, high-carbohydrate (LPHC) diet for 15 days. The aim of this work was to study the pathways involved in lipogenesis and lipolysis, which ultimately regulate lipid accumulation in the tissue. De novo fatty acid synthesis was evaluated in vivo and was similar for rats fed an LPHC diet or a control diet; however, the LPHC-fed rats had decreased lipoprotein lipase activity in the EPI adipose tissue, which suggests that there was a decreased uptake of fatty acids from the circulating lipoproteins. The LPHC diet did not affect synthesis of glycerol-3-phosphate (G3P) via glycolysis or glyceroneogenesis. Glycerokinase activity - i.e., the phosphorylation of glycerol from the hydrolysis of endogenous TAG to form G3P - was also not affected in LPHC-fed rats. In contrast, adipocytes from LPHC animals had a reduced lipolytic response when stimulated by norepinephrine, even though the basal adipocyte lipolytic rate was similar for both of the groups. Thus, the results suggest that the reduction of lipolytic activity stimulated by norepinephrine seems essential for the TAG increase observed in the EPI adipose tissue of LPHC animals, probably by impairment of the process of activation of lipolysis by norepinephrine.

Glyceroneogenesis and the supply of glycerol-3-phosphate for glyceride-glycerol synthesis in liver slices of fasted and diabetic rats.

The pathways of glycerol-3-phosphate (G3P) generation for glyceride synthesis were examined in precision-cut liver slices of fasted and diabetic rats. The incorporation of 5 mM [U-(14)C]glucose into glyceride-glycerol, used to evaluate G3P generation via glycolysis, was reduced by approximately 26-36% in liver slices of fasted and diabetic rats. The glycolytic flux was reduced by approximately 60% in both groups. The incorporation of 1.0 mM [2-(14)C]pyruvate into glyceride-glycerol (glyceroneogenesis) increased approximately 50% and approximately 36% in slices of fasted and diabetic rats, respectively, which also showed a two-fold increase in the activity phosphoenolpyruvate carboxykinase. The increased incorporation of 1.0 mM [2-(14)C]pyruvate into glyceride-glycerol by slices of fasted rats was not affected by the addition of 5 mM glucose to the incubation medium. The activity of glycerokinase and the incorporation of 1 mM [U-(14)C]glycerol into glyceride-glycerol, evaluators of G3P formation by direct glycerol phosphorylation, did not differ significantly from controls in slices of the two experimental groups. Rates of incorporation of 1 mM [2-(14)C]pyruvate and [U-(14)C]glycerol into glucose of incubation medium (gluconeogenesis) were approximately 140 and approximately 20% higher in fasted and diabetic slices than in control slices. It could be estimated that glyceroneogenesis by liver slices of fasted rats contributed with approximately 20% of G3P generated for glyceride-glycerol synthesis, the glycolytic pathway with approximately 5%, and direct phosphorylation of glycerol by glycerokinase with approximately 75%. Pyruvate contributed with 54% and glycerol with 46% of gluconeogenesis. The present data indicate that glyceroneogenesis has a significant participation in the generation of G3P needed for the increased glyceride-glycerol synthesis in liver during fasting and diabetes.

Leishmania mexicana glycerol-3-phosphate dehydrogenase showed conformational changes upon binding a bi-substrate adduct.

Certain pathogenic trypanosomatids are highly dependent on glycolysis for ATP production, and hence their glycolytic enzymes, including glycerol-3-phosphate dehydrogenase (GPDH), are considered attractive drug targets. The ternary complex structure of Leishmania mexicana GPDH (LmGPDH) with dihydroxyacetone phosphate (DHAP) and NAD(+) was determined to 1.9A resolution as a further step towards understanding this enzyme's mode of action. When compared with the apo and binary complex structures, the ternary complex structure shows an 11 degrees hinge-bending motion of the C-terminal domain with respect to the N-terminal domain. In addition, residues in the C-terminal domain involved in catalysis or substrates binding show significant movements and a previously invisible five-residue loop region becomes well ordered and participates in NAD(+) binding. Unexpectedly, DHAP and NAD(+) appear to form a covalent bond, producing an adduct in the active site of LmGPDH. Modeling a ternary complex glycerol 3-phosphate (G3P) and NAD(+) with LmGPDH identified ten active site residues that are highly conserved among all GPDHs. Two lysine residues, Lys125 and Lys210, that are presumed to be critical in catalysis, were mutated resulting in greatly reduced catalytic activity. Comparison with other structurally related enzymes found by the program DALI suggested Lys210 as a key catalytic residue, which is located on a structurally conserved alpha-helix. From the results of site-directed mutagenesis, molecular modeling and comparison with related dehydrogenases, a catalytic mechanism of LmGPDH and a possible evolutionary scenario of this group of dehydrogenases are proposed.

Cytochemical analysis of acid phosphatase activity in the venom secretory cells of Bothrops jararaca.

A study of the histochemical reaction for acid phosphatase (AcPase) in venom gland secretory cells from Bothrops jararaca was done to investigate the distribution of lysosomes and related structures in stages of high- and low-protein synthesis. From this analysis, it was expected to gain insight into the cellular pathway by which AcPase is secreted into the venom. Two subtypes of AcPase reactivities were detected in the venom gland secretory cells: one was found in lysosomes and related structures and in some trans-Golgi network (TGN) elements and reacts with beta-glycerophosphate (betaGP) as substrate; the other was found in secretory vesicles, apical plasmalemma, lysosomes and related structures, and in some TGN elements, and reacts with cytidine monophosphate (CMP). The results are compatible with the possibility that there is a secretory via for AcPase in the venom gland of B. jararaca and that the elements composing this pathway are noted only when CMP is used as substrate. Large autophagosomes reactive to both betaGP and to CMP were commonly observed in the basal region of the secretory cells, and they were more abundant in the glands during the stage of low activity of protein synthesis.

Comparative study of Leishmania mexicana and Trypanosoma brucei NAD-dependent glycerol-3-phosphate dehydrogenase.

The NAD-dependent glycerol-3-phosphate dehydrogenases (G3PDH, EC 1.1.1.8) of Trypanosoma brucei and Leishmania mexicana are thought to have different roles in carbohydrate metabolism. Here the physicochemical and kinetic properties of natural G3PDH from T. brucei with the recombinant homologue of L. mexicana which share 63% positional identity are compared. Despite their supposed different functions in energy metabolism of the parasites the two G3PDHs have remarkably similar properties, including pH optima and K(m) value for dihydroxyacetone phosphate (DHAP) and NADH in the formation of glycerol 3-phosphate (G3P) and for NAD+ and G3P in the reverse reaction. Both enzymes are subject inhibition by dihydroxyacetone phosphate at concentrations above 0.2 mM and are inhibited by the trypanocidal drugs suramin and melarsen oxide at sub-micromolar concentrations.

The dihydroxyacetonephosphate pathway for biosynthesis of ether lipids in Leishmania mexicana promastigotes.

Biosynthetic studies using both [14C]- and [32P]-labelled substrates and a cell-free system to synthesise 1-O-alkyl moieties in glycerolipids, have shown that the three initial steps in ether-lipid biosynthesis in Leishmania mexicana promastigotes resemble those described for mammals and are associated with glycosomes. Purified glycosomes were able to sequentially synthesise the first intermediates of the ether-lipid biosynthetic pathway [acyl-dihydroxyacetonephosphate (DHAP), alkyl-DHAP and acyl/alkyl-glycerol-3-phosphate (G3P)] when incubated in the presence of radiolabelled DHAP, palmitoyl-CoA, hexadecanol and NADPH. However, when glycosomes were incubated under the same conditions in the presence of radiolabelled G3P, a rapid synthesis of acyl-G3P and phosphatidic acid was observed without any formation of alkyl-G3P, suggesting that the enzyme alkyl-synthase recognises only acyl-DHAP as substrate. Both the DHAP acyltransferase (DHAP-AT) and alkyl-DHAP synthase activities were located inside glycosomes whereas the alkyl/acyl-DHAP oxidoreductase activity was associated with the cytoplasmic face of the glycosomal membrane. The G3P acyltransferase (G3P-AT) and lyso-phosphatidic acid acyltransferase activities were not found inside glycosomes. The results suggest that the DHAP-AT and G3P-AT activities are catalysed by two distinct enzymes associated with different sub-cellular compartments.

Role of water in the energy of hydrolysis of phosphoanhydride and phosphoester bonds.

The observed equilibrium constants for hydrolysis (Kobs) of a phosphoester and a phosphoanhydride bond were measured under a variety of conditions likely to alter the interactions of reactants and products with water. These included increasing the pH of the medium from 5.0 to 10.0, increasing the MgCl2 concentration form 0 to 200 mM, and decreasing the water activity of the medium by adding either dimethyl sulfoxide (50%, v/v) or polyethylene glycol 6,000-8,000 (50%, w/v). The Kobs for phosphoesters such as phosphoserine, glucose phosphate, glycerol phosphate, and ethylene glycol phosphate varied little over this wide range of conditions, the extreme values of Kobs being 12 and 200 M. In contrast, the Kobs for the phosphoanhydride bond of pyrophosphate varied from a value greater than 20,000 to 0.1 M. In totally aqueous media at a pH between 7.0 and 8.0 and in the presence of 0.5-1.0 mM MgCl2, the energy of hydrolysis of pyrophosphate was 1.2-4.0 kcal/mol greater than that of phosphoserine. However, when the water activity was decreased by adding polyethylene glycol to the medium within the same pH and MgCl2 concentration range, the energy of hydrolysis of phosphoserine became 2.0-2.5 kcal/mol greater than that of pyrophosphate. The results suggest that for phosphoesters, the solvation energies of reactants and products, unlike the case of phosphoanhydride bonds, are not the major factors in determining the energy of hydrolysis.