Levels Of Glyceraldehyde-3-phosphate Dehydrogenase Research Articles

Differential expression of GAPDH and beta3-actin in growing collateral arteries.

Housekeeping genes like glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-actin are often used as internal standards for quantitative RNA analysis. In our study we analyzed the relative expression level of GAPDH and beta-actin as well as of the 18S rRNA and the Poly (A)+ RNA in growing collateral arteries in a rabbit model of arteriogenesis which is not associated with ischemia. Relative quantitation of the housekeeping genes displayed a significant upregulation of the beta-actin- and GAPDH mRNA during the first 24 h of vessel growth. For day 3 our results revealed an even stronger upregulation of the beta-actin mRNA (140%) but a significant downregulation of the GAPDH mRNA (50% of control). The 18S rRNA, however, showed for the same periods only minor alterations compared to the Poly (A)+ RNA. From these results we conclude that the 18S rRNA, but not the GAPDH- or beta-actin mRNA is an appropriate internal control for relative quantitation of gene expression under conditions of cell proliferation in growing vessels.

A novel protein complex distinct from mismatch repair binds thioguanylated DNA.

To elucidate molecular mechanism(s) of cellular response to mercaptopurine, a widely used antileukemic agent, we assessed mercaptopurine (MP) sensitivity in mismatch repair (MMR) proficient and MMR deficient human acute lymphoblastic leukemia (ALL) cells. Sensitivity to thiopurine cytotoxicity was not dependent on MMR (i.e., MutSalpha) competence among six cell lines tested. Using electrophoretic mobility shift assay analysis, we found that the incubation of nuclear extracts from ALL cells with synthetic 34-mer DNA duplexes containing deoxythioguanosine (G(S)) within either G(S).T or G(S).C pairs, resulted in formation of a DNA-protein complex distinct from the DNA-MutSalpha complex and unaffected by ATP. Isolation and sequence analysis of proteins involved in this DNA-protein complex identified glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a component. Western blot analysis of nuclear extracts from a panel of human lymphoblastic leukemia cell lines revealed markedly different basal levels of GAPDH in nuclei, which was significantly related to thiopurine sensitivity (p = 0.001). Confocal analysis revealed markedly different intracellular distribution of GAPDH between nucleus and cytosol in six human ALL cell lines. Redistribution of GAPDH from cytosol to nucleus was evident after MP treatment. These findings indicate that a new DNA-protein complex containing GAPDH and distinct from known MMR protein-DNA complexes binds directly to thioguanylated DNA, suggesting that this may act as a sensor of structural alterations in DNA and serve as an interface between these DNA modifications and apoptosis.

Glyceraldehyde-3-phosphate Dehydrogenase Is Required for Vesicular Transport in the Early Secretory Pathway

Protein transport in the early secretory pathway requires Rab2 GTPase. This protein promotes the recruitment of soluble components that participate in protein sorting and recycling from pre-Golgi intermediates (vesicular tubular clusters (VTCs)). We previously reported that a constitutively activated form of Rab2 (Q65L) as well as Rab2 wild type promoted vesicle formation from VTCs. These vesicles contained Rab2, beta-COP, p53/gp58, and protein kinase Ciota/lambda but lacked anterograde-directed cargo. To identify other candidate Rab2 effectors, the polypeptide composition of the vesicles was further analyzed. We found that vesicles released in response to Rab2 also contained the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). To study the relationship of this enzyme to Rab2 function, we performed a quantitative binding assay to measure recruitment of GAPDH to membrane when incubated with Rab2. Rab2-treated microsomes showed a 5-10-fold increase in the level of membrane-associated GAPDH. We generated an affinity-purified anti-GAPDH polyclonal to study the biochemical role of GAPDH in the early secretory pathway. The antibody arrests transport of a reporter molecule in an assay that reconstitutes ER to Golgi traffic. Furthermore, the affinity-purified antibody blocked the ability of Rab2 to recruit GAPDH to membrane. However, the antibody did not interfere with Rab2 stimulated vesicle release. These data suggest that GAPDH is required for ER to Golgi transport. We propose that membranes incubated with anti-GAPDH and Rab2 form "dead end" vesicles that are unable to transport and fuse with the acceptor compartment.

Differential expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ß actin and hypoxanthine phosphoribosyltransferase (HPRT) in postnatal rabbit sclera

Purpose. GAPDH, ß-actin, HPRT and 18S rRNA are constitutively expressed in all mammalian cells. In accordance with the nature of invariant control, these genes have been used to standardize genes of interest in expression studies. Recent studies have suggested that GAPDH, ß-actin and HPRT in special situations may come under temporary regulatory control, but that 18S rRNA may be more likely to remain constitutive. However, little is known about the quantitative expression of these genes in fibroblasts and in particular during early postnatal development, a time of rapid changes in cell metabolism. In this study we have examined the differential expression of these genes in association with scleral development from an early postnatal age up to young adult status. Methods. GAPDH, ß-actin, HPRT, and 18S rRNA gene expression were analyzed in the rabbit sclera from 1 day to 8 weeks postnatally by real-time, comparative PCR. Results. Real-time PCR analysis showed that the expression levels of GAPDH, ß-actin, and HPRT were higher in the first postnatal week and then declined. However, from 2 to 8 weeks, the mRNA levels of these three genes underwent significant variations (P < 0.01) in their levels of expression. In contrast, the expression level of 18S rRNA showed no significant variation (P = 0.5) over this time period. Conclusions. The present study shows that GAPDH, ß actin and HPRT gene were differentially expressed in early postnatal scleral development. It also suggests that these gene products could be implicated in the developmental process and have a crucial role in the early postnatal period. This study demonstrates that 18S rRNA may be preferable to normalize genes of interest in studies of early development.

Progressive association of a "soluble" glycolytic enzyme with the detergent-insoluble cytoskeleton during in vitro morphogenesis of MDCK epithelial cells.

In MDCK epithelial cells, cell contact at confluency initiates a protracted process of morphogenesis during which several proteins known to bind the cytoskeleton become progressively associated with the detergent-resistant cell fraction and distributed to their characteristic polarized domains. Using extraction protocols that identify this tight cytoskeletal linkage, here we show a similar but slower, time-dependent enrichment in the detergent resistant fraction of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a highly abundant glycolytic enzyme that is traditionally considered soluble. Similar enrichment did not occur for two other glycolytic enzymes, phosphoglycerate mutase or lactate dehydrogenase. Insoluble GAPDH was not homogeneously distributed in the cytoplasm but rather displayed several discrete patterns that varied within and among MDCK cells. It also localized prominently to a few nuclei in the phenotypically heterogeneous cells of late confluency cultures. Disruptors of cytoskeletal filaments were relatively ineffective in the postconfluent epithelial monolayers, although use of disrupting agents implicated actin as the cytoplasmic filament that tethers insoluble GAPDH. Catalytic activity could be demonstrated in the insoluble fraction of GAPDH from postconfluent cultures, but only after release by mechanical disruption of insoluble extracts. Treatment of postconfluent cells with agents that deplete ATP diminished the fraction of cytoskeletally associated GAPDH, and levels of insoluble GAPDH were restored with ATP repletion, suggesting that ATP levels may regulate cytoskeletal linkage and thereby local enzyme activity. We conclude that the highly abundant and ubiquitous enzyme GAPDH becomes progressively enriched in detergent stable subcellular compartments during the process of epithelial morphogenesis. The process that produces GAPDH compartments is slow, suggesting that epithelial cells just at confluency, when they are typically analyzed, have not yet maximized the organizational state that can be attained in monolayer culture.

Glyceraldehyde-3-phosphate dehydrogenase expression during apoptosis and proliferation of rat ventral prostate.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional enzyme known to play a critical role in neuronal apoptosis. We undertook the current studies to determine whether GAPDH also plays a role in prostate epithelial cell apoptosis in response to androgen deprivation. To do so, we analyzed GAPDH staining by immunohistochemistry during castration-induced involution and androgen-induced regeneration of rat ventral prostate. We found that GAPDH was undetectable in secretory epithelial cells at baseline and that staining did not increase in the epithelium during the period of peak apoptosis from 1 to 3 days after castration. However, GAPDH levels did increase within nuclei of some basal epithelial cells 5 days after castration and within the cytoplasm of all secretory epithelial cells 7 days after castration. GAPDH was also abundant within the cytoplasm of secretory epithelial cells during the period of maximal cell proliferation from 2 to 3 days after androgen replacement and was clearly apparent within nuclei of some epithelial cells 4 days after androgen replacement. Our studies suggest that GAPDH plays multiple roles during prostate epithelial cell apoptosis and proliferation.

Pyruvate Metabolism in Lactococcus lactis Is Dependent upon Glyceraldehyde-3-phosphate Dehydrogenase Activity

Modification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity from Lactococcus lactis was undertaken during batch fermentation on lactose, by adding various concentrations of iodoacetate (IAA), a compound which specifically inhibits GAPDH at low concentrations, to the culture medium. As IAA concentration is increased, GAPDH activity diminishes, provoking a decrease of both the glycolytic flux and the specific growth rate. This control exerted at the level of GAPDH was due partially to IAA covalent fixation but also to the modified NADH/NAD+ ratio. The mechanism of inhibition by NADH/NAD+ was studied in detail with the purified enzyme and various kinetic parameters were determined. Moreover, when GAPDH activity became limiting, the triose phosphate pool increased resulting in the inhibition of pyruvate formate lyase activity, while the lactate dehydrogenase is activated by the high NADH/NAD+ ratio. Thus, modifying the GAPDH activity provokes a shift from mixed-acid to homolactic metabolism, confirming the important role of this enzyme in controlling both the flux through glycolysis and the orientation of pyruvate catabolism.

Differential Effects of Ethanol on Insulin‐Signaling Through the Insulin Receptor Substrate‐1

Insulin stimulation increases cell proliferation and energy metabolism by activating the insulin receptor substrate I (IRS‐1)‐signaling pathways. This downstream signaling is mediated by interactions of specific tyrosyl phosphorylated (PY) IRS‐1 motifs with SH2‐containing molecules such as growth‐factor receptor‐bound protein 2 (Grb2) and Syp. Ethanol inhibits insulin‐stimulated tyrosyl phosphorylation of IRS‐1 and DNA synthesis. This study explores the roles of the Grb2‐ and Syp‐binding motifs of IRS‐1 in relation to the inhibitory effects of ethanol on insulin‐stimulated DNA synthesis, proliferating cell nuclear antigen (PENA) and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) expression, and activation of mitogen‐activated protein kinase (MAPK), which is known to be essential for cell proliferation. NIH3T3 cells were stably transfected with wild‐type IRS‐1, or IRS‐1 mutated at the Grb2 (IRS‐lΔGrb2), Syp (IRS‐lΔSyp), or Grb2 and Syp (IRS‐lΔGrb2ΔSyp)‐ binding sites. Cells transfected with IRS‐1 had increased levels of DNA synthesis, PCNA, GAPDH, and activated MAPK. The IRS‐lΔGrb2 transfectants were highly responsive to insulin stimulation, achieving levels of GAPDH, PCNA, and activated MAPK that were higher than control. In contrast, the IRS‐IΔSyp and IRS‐lΔGrb2ΔSyp transfectants had reduced levels of DNA synthesis, PCNA, and activated MAPK. Ethanol exposure decreased insulin‐stimulated DNA synthesis, PCNA, GAPDH, and activated MAPK levels in all clones, but the wild‐type IRS‐1 transfectants were relatively resistant, and the IRS‐IΔGrb2 transfectants were extraordinarily sensitive to these inhibitory effects of ethanol. The findings suggest that insulin‐stimulated DNA synthesis and PCNA expression are mediated through the Syp‐binding domain, whereas GAPDH expression and MAPK activation are modulated through both the Grb2 and Syp motifs of IRS‐1. In addition, ethanol exposure may preferentially inhibit downstream signaling that requires interaction between Syp and PY‐IRS‐1.

Differential effects of ethanol on insulin-signaling through the insulin receptor substrate-1.

Insulin stimulation increases cell proliferation and energy metabolism by activating the insulin receptor substrate I (IRS-1)-signaling pathways. This downstream signaling is mediated by interactions of specific tyrosyl phosphorylated (PY) IRS-1 motifs with SH2-containing molecules such as growth-factor receptor-bound protein 2 (Grb2) and Syp. Ethanol inhibits insulin-stimulated tyrosyl phosphorylation of IRS-1 and DNA synthesis. This study explores the roles of the Grb2- and Syp-binding motifs of IRS-1 in relation to the inhibitory effects of ethanol on insulin-stimulated DNA synthesis, proliferating cell nuclear antigen (PCNA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression, and activation of mitogen-activated protein kinase (MAPK), which is known to be essential for cell proliferation. NIH3T3 cells were stably transfected with wild-type IRS-1, or IRS-1 mutated at the Grb2 (IRS-1deltaGrb2), Syp (IRS-1deltaSyp), or Grb2 and Syp (IRS-1deltaGrb2deltaSyp)- binding sites. Cells transfected with IRS-1 had increased levels of DNA synthesis, PCNA, GAPDH, and activated MAPK. The IRS-1deltaGrb2 transfectants were highly responsive to insulin stimulation, achieving levels of GAPDH, PCNA, and activated MAPK that were higher than control. In contrast, the IRS-1deltaSyp and IRS-1deltaGrb2deltaSyp transfectants had reduced levels of DNA synthesis, PCNA, and activated MAPK. Ethanol exposure decreased insulin-stimulated DNA synthesis, PCNA, GAPDH, and activated MAPK levels in all clones, but the wild-type IRS-1 transfectants were relatively resistant, and the IRS-1deltaGrb2 transfectants were extraordinarily sensitive to these inhibitory effects of ethanol. The findings suggest that insulin-stimulated DNA synthesis and PCNA expression are mediated through the Syp-binding domain, whereas GAPDH expression and MAPK activation are modulated through both the Grb2 and Syp motifs of IRS-1. In addition, ethanol exposure may preferentially inhibit downstream signaling that requires interaction between Syp and PY-IRS-1.

Regulation of glyceraldehyde-3-phosphate dehydrogenase in differentiating HD3 cells

Red blood cells usually replenish their ATP pools by glycolysis, fueled by glucose imported via the cell membrane. Mature red cells of some species (e.g. pig, chicken) have, however, been reported to show very low glucose transport. The subject of this study was the possible dependency of the level of a key glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAD) on glucose transporter activity during the maturation of chicken red cells. The chicken pronormoblast cell line, HD3, was used as a model system. These cells contain higher levels of GAD and glucose transporter activities than normal chicken bone marrow cells, but reduce their levels during maturation. In an attempt to assess whether the decrease in GAD activity is regulated by the glucose transport, the chicken GLUT3 expressed under the control of viral promoter was introduced into HD3 cells by retroviral infection (pDOL-cGT3). Upon cell differentiation and maturation, both cells with and without the exogenous transporter decreased GAD activity. Butyric acid did not affect the regulation of GAD activity upon differentiation. These results show that the development of chicken red cells is manifested by reduction of their GAD activity and that this is not affected by their sugar transporter activity. The very low GAD activity in embryonic chicken red cells is thus due to a loss of this activity at an early stage in their development. Because of the very low glucose transport and GAD activities in mature chicken red cells, rates of glycolysis are likely to be low and suggesting an alternative pathway for ATP production in these cells.

Down-regulation of hepatic and renal 11β-hydroxysteroid dehydrogenase in rats with liver cirrhosis

Background & Aims: 11β-Hydroxysteroid dehydrogenase (11β-OHSD) enzymes are responsible for the interconversion of active 11β-hydroxycorticosteroids into inactive 11-ketoglucocorticosteroids and by that mechanism regulate the intracellular access of the steroids to the cognate receptor. A down-regulation of the shuttle of active to inactive glucocorticoids enhances access of glucocorticosteroids to both the glucocorticoid and the mineralocorticoid receptors. In liver cirrhosis, enhanced mineralocorticoid and glucocorticoid effects are observed. We therefore investigated the impact of liver cirrhosis after bile duct ligation on the transcription and activity of 11β-OHSD1 and 11β-OHSD2 in the corresponding tissues. Methods: Messenger RNA from 11β-OHSD1 and 11β-OHSD2 was assessed by reverse-transcription polymerase chain reaction; activity was assessed by measuring the interconversion of corticosterone to dehydrocorticosterone. The effect of bile and bile salts was determined using COS-1 cells transfected with 11β-OHSD1 or 11β-OHSD2. Results: In liver tissue, the messenger RNA ratios of 11β-OHSD1 to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels and, in kidney tissue, the ratios of 11β-OHSD2 to GAPDH levels decreased after induction of liver cirrhosis. The 11β-OHSD activities were correspondingly reduced. Bile and individual bile salts inhibited 11β-OHSD1 and 11β-OHSD2 oxidative activity in transfected COS-1 cells. Conclusions: These findings indicate that in liver cirrhosis the mineralocorticoid and glucocorticoid receptor–protecting effects by the 11β-OHSD isoenzymes are down-regulated and that by the same mechanism the glucocorticoid and mineralocorticoid effects are enhanced. GASTROENTEROLOGY 1998;114:175-184

Increased glyceraldehyde-3-phosphate dehydrogenase gene expression in late pathological stage human prostate cancer.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional enzyme best known to many investigators as a 'housekeeping' gene used as a loading control on northern blots. Prior studies, however, have shown that GAPDH RNA levels vary greatly among different prostate cancer cell lines. We undertook this study to determine the level of GAPDH gene expression within primary human prostate tumors and to determine the validity of using GAPDH as a loading control for northern analysis of human prostate cancer specimens and normal human organs. We found that GAPDH expression was significantly higher in specimens from patients with pathological stage C and D prostate cancer than those with stage B disease. Within the human prostate cancer cell lines TSUPr1, DU145, LNCaP and PC-3 and a normal neonatal prostate epithelial cell line FNC 267beta1, LNCaP cells expressed the highest level of GAPDH but all cell lines, including the normal neonate prostate cell line had very strong GAPDH gene expression. GAPDH RNA levels were highly variable among 16 normal human adult organs and four human fetal organs examined. We conclude that GAPDH RNA levels in human prostate tumors correlate with pathologic stage and that GAPDH should not be used as a loading control in northern blot experiments. Other controls, such as beta-actin or 28s ribosomal RNA, would be more suitable for such purposes.

Effect of lead nitrate on liver carbohydrate enzymes and glycogen content in the rat

Male Wistar rats were given a single i.v. injection of lead nitrate (10 mumol/100 g body wt) and were killed with matched controls 24, 48, 72 h and 20 days after the treatment. Changes of liver carbohydrate metabolism were studied histochemically testing the following parameters: glycogen content, activities of glycogen synthase (SYN), glycogen phosphorylase (PHO), glucose-6-phosphatase (G6PASE), glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In addition, gammaglutamyltransferase (GGT) activity was demonstrated. Between 24 and 48 h after lead nitrate injection there was a nearly complete loss of liver glycogen. Seventy-two hours later the polysaccharide reappeared in single hepatocytes and after 20 days the livers of the lead-treated animals not only had replenished their glycogen stores but contained even more glycogen than the matched controls. SYN and PHO activities were diminished from 24 to 72 h, but returned to control values after 20 days. G6PASE and GGT remained elevated up to 72 h before dropping to normal at 20 days after treatment. The pentose phosphate pathway enzymes G6PDH and 6PGDH showed the most remarkable changes in livers treated with lead nitrate. G6PDH was already elevated at 24 h, but only in Kupffer cells. At 48 and 72 h, when hepatocytes exhibited a highly increased mitotic rate, the levels of G6PDH, 6PGDH and GAPDH were elevated. After 20 days dehydrogenase activities were comparable to those of controls. The results of this study suggest that a single dose of lead nitrate not only stimulates proliferation of hepatocytes but also induces considerable changes in rat liver carbohydrate metabolism, especially between 24 and 72 h after administration. During that period glycogen metabolism undergoes a strong reduction, whereas gluconeogenesis and particularly the pentose phosphate pathway respond with a remarkable increase. This metabolic profile is most likely associated with lead biotransformation as well as with liver cell proliferation. It corresponds only partially to that found in preneoplastic and neoplastic liver lesions observed in chemical carcinogenesis, and is reversible, in contrast to the persistent alterations associated with neoplastic transformation.

Substitution of a pentalenolactone-sensitive glyceraldehyde-3-phosphate dehydrogenase by a genetically distinct resistant isoform accompanies pentalenolactone production in Streptomyces arenae.

Pentalenolactone (PL), an antibiotic produced by Streptomyces arenae, is a potent inhibitor of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The producer strain contains different isoforms of GAPDH: a PL-sensitive enzyme on nonproduction media and a PL-insensitive enzyme on production media. After induction of PL synthesis, the sensitive GAPDH disappears parallel to the disappearance of its activity, as shown by Western (immunoblot) hybridization. The two isoenzymes exhibit little immunological cross-reactivity and differ in size, amino acid composition, and several amino acid residues of their amino termini. Two different types of plasmids from a S. arenae genomic library, named pBRPLR1 and pBRPLR2, were cloned in Escherichia coli by selection for enhanced PL resistance. Both contain a GAPDH structural gene. Plasmid pBRPLR1 increases E. coli PL tolerance 7-fold, and plasmid pBRPLR2 increases it 30-fold. GAPDH from pBRPLR1 transformants shows biphasic PL inactivation kinetics. These cells contain PL-sensitive GAPDH from both E. coli and S. arenae. GAPDH from pBRPLR2 transformants tolerates higher PL concentrations than either E. coli or S. arenae PL-sensitive GAPDH but is less resistant than S. arenae PL-insensitive GAPDH. Nondenaturing polyacrylamide electrophoresis showed this GAPDH to be a hybrid of E. coli and S. arenae PL-insensitive GAPDH. The hybrid enzyme could be purified to homogeneity. Induction of the lacZ promoter of pUC subclones of both GAPDH genes had only a small effect on raising the level of intracellular GAPDH.

The toxicology and metabolism of chlorothalonil in fish. III. Metabolism, enzymatics and detoxication in Salmo spp. and Galaxias spp.

Hepatic glutathione S-transferase (GST) activity towards chlorothalonil, tetrachloroisophthalonitrile (TCIN), was established for the five fish species: Salmo gairdneri, S. trutta, Galaxias maculatus, G. truttaceus and G. auratus. Molecular weights and hepatic activity toward 1-chloro-2,4-dinitrobenzene (CDNB) of GST were determined in all species. Low level exposure to TCIN was found to induce higher GST activity in G. maculatus, G. truttaceus and S. gairdneri. Other properties of GST enzymes investigated in these fish included TCIN binding, reaction order and the effect of acephate on activity. The effect of TCIN exposure on liver thiol and glutathione (GSH) levels in S. gairdneri and G. truttaceus was investigated. Significant decreases occurred in liver thiol levels of fish when exposed to varying concentrations of TCIN for 96 h without feeding. Liver GSH levels increased in S. gairdneri exposed to 10 μg/l over 96 h with feeding. A marked increase in GST activity was observed during 96 h exposure to 10 μg/l. No significant changes in the activity of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were observed during exposure to 10 μg/l TCIN in vivo. A transient decrease in activity was observed during exposure to 30 μg/l TCIN. The previously reported in vitro inhibition of GAPDH by TCIN was studied in the context of liver cytosolic levels of GAPDH and GSH. Hepatic GSH and GST activity levels toward 14C-TCIN and CDNB were compared in S. gairdneri, G. maculatus and G. auratus. The order of asymptotic LC 50 values for the three species agreed with that for total hepatic GST activity toward 14C-TCIN consistent with a detoxication role for GSH-TCIN conjugation. A proposal for a screening process for Australian freshwater fish based on detoxication enzyme activity is discussed.

An ascorbate-mediated transmembrane-reducing system of the human erythrocyte.

Actively metabolizing human erythrocytes catalyze the extracellular reduction of ferricyanide to ferrocyanide. Because neither of these anions can enter the cell, reducing equivalents generated in the course of glycolysis must in some manner be transferred across the cell membrane, thereby resulting in ferricyanide reduction. Work described in this paper suggests that the transmembrane reduction is effected by ascorbic acid. This compound in its oxidized form (dehydroascorbate) rapidly enters the cell. Here it obtains reducing equivalents which appear to come from NADH made available at the level of glyceraldehyde 3-phosphate dehydrogenase. Once reduced, it leaves the cell as ascorbic acid and accomplishes the non-enzymatic reduction of ferricyanide.

Formaldehyde-Induced Hemolysis during Chronic Hemodialysis

We investigated an outbreak of hemolytic anemia among patients on hemodialysis shortly after the installation of a new water-filtration system. Using in vitro technics, we demonstrated that a water-soluble inhibitor of red-cell metabolism could easily be extracted from the intact filters. Subsequent experiments showed this toxic substance to be formaldehyde, a potent reducing agent whose mechanism of action involves conversion of NAD to NADH. This alteration of the redox state of the erythrocyte leads to inhibition of glycolysis at the level of glyceraldehyde 3-phosphate dehydrogenase and rapid decline in cellular energy (ATP) stores. Because formaldehyde is widely used as a preservative for dialysis equipment, the toxicity of this agent for red blood cells is of clinical importance.

Interaction between Adenosine Triphosphate and Glyceraldehyde 3-Phosphate Dehydrogenase: III. MECHANISM OF ACTION AND METABOLIC CONTROL OF THE ENZYME UNDER SIMULATED IN VIVO CONDITIONS

Abstract The inhibitory effect of ATP on glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) has been investigated under conditions simulating the intracellular milieu. The inhibition is strongly pH-dependent. With the physiological concentrations of ATP, NAD, and orthophosphate, the dehydrogenase reaction is 87 % inhibited at pH 6.8, 67 % at pH 7.4, and only 20 % at pH 8.1. ADP, adenosine 5'-monophosphate, and cyclic adenosine 3':5'-monophosphate are almost equally inhibitory but have less physiological significance since the intracellular levels are much lower than ATP. The ATP inhibition is mixed type inhibition in which ATP is nearly competitive with respect to either NAD or Pi. ATP (6 mm) increases Km for NAD from 0.06 to 0.3 mm and decreases Vmax from 91 to 65 µmoles of NADH per min per mg of protein. Similarly the Km for Pi is changed from 1.2 to 6.1 mm whereas Vmax is decreased from 89 to 61. Experiments using AMP or pyrophosphate as the component parts of ATP have also been carried out. AMP reacts competitively with respect to NAD but not Pi, whereas PPi is nearly competitive with Pi but not NAD. These data suggest that ATP simultaneously obstructs both NAD and Pi binding in the active center. ATP is noncompetitive with regard to glyceraldehyde 3-phosphate. Mg2+ or Mn2+ reduces the inhibitory effect of ATP by complexing with the nucleotide. ATP also effects a time-dependent inactivation, which is additive with the instantaneous inhibition mentioned above. This time-dependent inhibition is counteracted by NAD but not Pi. The physiological implication of ATP inhibition on the dehydrogenase is discussed in the connection with muscle physiology. ATP inhibition may in part account for the high levels of glyceraldehyde 3-phosphate dehydrogenase in the muscle, protect against excessive acidity due to lactic production, limit maximal glycolytic flux, and modify the turnover of the enzyme.