Characterization of glyceraldehyde-3-phosphate dehydrogenase as a novel transferrin receptor

A majority of cells obtain of transferrin (Tf) bound iron via transferrin receptor 1 (TfR1) or by transferrin receptor 2 (TfR2) in hepatocytes. Our study establishes that cells are capable of acquiring transferrin iron by an alternate pathway via GAPDH.These findings demonstrate that upon iron depletion, GAPDH functions as a preferred receptor for transferrin rather than TfR1 in some but not all cell types. We utilized CHO-TRVb cells that do not express TfR1 or TfR2 as a model system. A knockdown of GAPDH in these cells resulted in a decrease of not only transferrin binding but also associated iron uptake. The current study also demonstrates that, unlike TfR1 and TfR2 which are localized to a specific membrane fraction, GAPDH is located in both the detergent soluble and lipid raft fractions of the cell membrane. Further, transferrin uptake by GAPDH occurs by more than one mechanism namely clathrin mediated endocytosis, lipid raft endocytosis and macropinocytosis. By determining the kinetics of this pathway it appears that GAPDH-Tf uptake is a low affinity, high capacity, recycling pathway wherein transferrin is catabolised. Our findings provide an explanation for the detailed role of GAPDH mediated transferrin uptake as an alternate route by which cells acquire iron.     

Characterization of a nitric-oxide-catalysed ADP-ribosylation of glyceraldehyde-3-phosphate dehydrogenase.

Auto-ADP-ribosylation of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GraPDH) has recently been demonstrated to be dramatically stimulated in the presence of nitric oxide. In order to obtain insight into the sequence of events leading to ADP-ribosylation of GraPDH, we studied the target amino acid, the nucleotide cofactor requirement, pH dependency and the stoichiometry of the reaction. Basal as well as stimulated ADP-ribose transfer is inhibited by the SH-group alkylating reagent, N-ethylmaleimide. Furthermore, the radiolabel of auto-[32P]ADP-ribosylated GraPDH is removed by treatment with HgCl2, suggesting an ADP-ribose-cysteine bond. Several indirect and direct mechanistic considerations point to NAD+ as the only cofactor for the ADP-ribosylation reaction, excluding the possibility of a reaction sequence involving a NAD-glycohydrolase(s) followed by nonenzymatic ADP-ribose transfer to GraPDH. Optimal ADP-ribosylations were carried out at alkaline pH values using 10 microM free NAD+ as the sole nucleotide cofactor. Bovine serum albumin with an S-nitrosylated SH group can serve as a model of ADP-ribose transfer from NAD+ and suggests that the nitric-oxide-modified SH group (S-nitrosylated SH group) is a prerequisite for the reaction.     

Mechanism of glyceraldehyde-3-phosphate transfer from aldolase to glyceraldehyde-3-phosphate dehydrogenase

The catalytic interaction of glyceraldehyde-3-phosphate dehydrogenase with glyceraldehyde 3-phosphate has been examined by transient-state kinetic methods. The results confirm previous reports that the apparent Km for oxidative phosphorylation of glyceraldehyde 3-phosphate decreases at least 50-fold when the substrate is generated in a coupled reaction system through the action of aldolase on fructose 1,6-bisphosphate, but lend no support to the proposal that glyceraldehyde 3-phosphate is directly transferred between the two enzymes without prior release to the reaction medium. A theoretical analysis is presented which shows that the kinetic behaviour of the coupled two-enzyme system is compatible in all respects tested with a free-diffusion mechanism for the transfer of glyceraldehyde 3-phosphate from the producing enzyme to the consuming one.     

Sturgeon glyceraldehyde-3-phosphate dehydrogenase.

The formation of binary complexes between sturgeon apoglyceralddhyde-3-phosphate dehydrogenase, coenzymes (NAD+ and NADH) and substrates (phosphate, glyceraldehyde 3-phosphate and 1,3-bisphosphoglycerate) has been studied spectrophotometrically and spectrofluorometrica-ly. Coenzyme binding to the apoenzyme can be characterized by several distinct spectroscopic properties: (a) the low intensity absorption band centered at 360 nm which is specific of NAD+ binding (Racker band); (b) the quenching of the enzyme fluorescence upon coenzyme binding; (c) the quenching of the fluorescence of the dihydronicotinamide moiety of the reduced coenzyme (NADH); (D) the hypochromicity and the red shift of the absorption band of NADH centered at 338 nm; (e) the coenzyme-induced difference spectra in the enzyme absorbance region. The analysis of these spectroscopic properties shows that up to four molecules of coenzyme are bound per molecule of enzyme tetramer. In every case, each successively bound coenzyme molecule contributes identically to the total observed change. Two classes of binding sites are apparent at lower temperatures for NAD+ Binding. Similarly, the binding of NADH seems to involve two distinct classes of binding sites. The excitation fluorescence spectra of NADH in the binary complex shows a component centered at 260 nm as in aqueous solution. This is consistent with a "folded" conformation of the reduced coenzyme in the binary complex, contradictory to crystallographic results. Possible reasons for this discrepancy are discussed. Binding of phosphorylated substrates and orthophosphate induce similar difference spectra in the enzyme absorbance region. No anticooperativity is detectable in the binding of glyceraldehyde 3-phosphate. These results are discussed in light of recent crystallographic studies on glyceraldehyde-3-phosphate dehydrogenases.     

Ascorbate-induced oxidation of glyceraldehyde-3-phosphate dehydrogenase

Oxidation of the essential cysteins of glyceraldehyde-3-phosphate dehydrogenase into the sulfenic acid derivatives was observed in the presence of ascorbate, resulting in a decrease in the dehydrogenase activity and the appearance of the acylphosphatase activity. The oxidation was promoted by EDTA, NAD(+), and phosphate, and blocked in the presence of deferoxamine. The ascorbate-induced oxidation was suppressed in the presence of catalase, suggesting the accumulation of hydrogen peroxide in the conditions employed. The data indicate the metal-mediated mechanism of the oxidation due to the presence of metal traces in the reaction medium. Physiological importance of the mildly oxidized GAPDH is discussed in terms of its ability to uncouple glycolysis and to decrease the ATP level in the cell.     

The Muridae glyceraldehyde-3-phosphate dehydrogenase family

Summary Although only one gene is known to be functional, numerous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) related sequences are scattered throughoutMus musculus andRattus rattus genomes. In this report we show that: (1) GAPDH pseudogenes are repeated to comparable extents, at least 400 copies, in 12 other Muridae species; (2) the complete, or nearly so, sequence of GAPDH messenger RNA is amplified, and a high proportion, if not all of these copies, are intronless; (3) GAPDH pseudogenes are preferentially located in heavily methylated and DNAse I-insensitive regions of chromatin; and (4) the presence of atypical GAPDH-related mRNAs in different cellular contexts raises the possibility that more than one GAPDH gene is transcribed.     

The radiolysis of glyceraldehyde-3-phosphate dehydrogenase.

The yields in molecules per 100 eV for active-site and sulphydryl loss from glyceraldehyde-3-phosphate dehydrogenase have been determined in nitrous-oxide-saturated, aerated and argon-saturated solutions. Molecular hydrogen peroxide produces a sulphenic acid product, which can be repaired by post-irradiation treatment with dithiothreitol. Comparison of the yields under various conditions showed that in aerated solutions both .OH and .O2-radicals inactivated the enzyme with an efficiency of about 26 per cent. However, the efficiency of .OH in air-free solutions was less, and inactivation by .H and eaq- did not appear to be appreciable. There is a correlation between SH loss and loss of active sites.     

Immunolocalization of glyceraldehyde-3-phosphate dehydrogenase inArabidopsis thaliana

Summary NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (NAD-dependent GAPDH) was purified to homogeneity and injected into a rabbit to induce a polyclonal antibody. The antibody was judged to be of high specificity and high affinity. This antibody was used to probe sections ofArabidopsis leaf, stem or roots which were fixed using either paraformaldehyde or a high-pressure freezing method. Our results show that the NAD-dependent GAPDH localizes in the nucleus as well as in the cytosol. In phloem tissue, the NAD-dependent GAPDH was found in companion cells but not in the sieve element.     

Structural evidence for a liver-specific glyceraldehyde-3-phosphate dehydrogenase.

The amino acid sequences near the amino termini of glyceraldehyde-3-phosphate dehydrogenase from bovine and porcine liver have been determined. Using classical peptide isolation techniques as well as automated Edman degradation, the NH₂-terminal 30 residues of the bovine liver enzyme were determined to be Val-Lys-Val-Gly-Val-Asn-Gly-Phe-Gly-Arg-Ile-Gly-Arg-Leu-Val-Thr-Arg-Ala-Ala-Phe-Asn-Ser-Gly-Lys-Val-Asp-Ile-Val-Phe-Ile. Twenty-two residues from the NH₂-terminus of the porcine liver enzyme, determined using the automated Edman degradation, were identical to the corresponding sequence from bovine liver enzyme. Both liver enzymes have Asn at position 6. The corresponding residue 6 in the muscle and yeast glyceraldehyde-3-phosphate dehydrogenases is Asp. This evidence suggests that the Asn-6 residue is specific for the liver tissues. The exchange of Asn for Asp may significantly alter the allosteric properties of muscle and liver enzymes especially the activity of the liver enzymes in gluconeogenesis.