Glyceraldehyde-3-phosphate dehydrogenase from Tetrahymena pyriformis: enzyme purification and characterization of a gapC gene with primitive eukaryotic features.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC.1.2.1.12) was purified to electrophoretic homogeneity from an amicronucleated strain of the ciliate Tetrahymena pyriformis using a three-step procedure. The native enzyme is an homotetramer of 145 kDa exhibiting absolute specificity for NAD. In its catalytic properties it is similar to other glycolytic GAPDHs. Chromatofocusing analysis showed the presence of only one basic GAPDH isoform with an isoelectric point of 8.8. Western blots using a monospecific polyclonal antibody raised against the T. pyriformis GAPDH showed a single 36-kDa band corresponding to the enzyme subunit in the cytosolic protein fraction of this strain and the closely related species, both from the class Oligohymenophorea, Paramecium tetraurelia. No bands were immunodetected in the ciliate Colpoda inflata (class Colpodea) and in the diverse eukaryotes and eubacteria tested. A 0.5-kb DNA fragment which corresponds to an internal region of a gapC gene was generated by polymerase chain reaction using cDNA of T. pyriformis as template. This gene codes for a basic GAPDH protein with eukaryotic-diplomonad signatures and exhibits a codon usage biased in the manner typical for T. pyriformis genes. Southern blots performed both under homologous and heterologous conditions using this amplified cDNA fragment as a probe, indicated that it should be the only gapC gene present in the macronuclear genome of this ciliate, its expression being confirmed by Northern blot analysis. These results are discussed in connection with the peculiar genomic organization of ciliates and in the context of protist evolution.

Occurrence of a differential expression of the glyceraldehyde-3-phosphate dehydrogenase gene in muscle and liver from euthermic and induced hibernating jerboa (Jaculus orientalis).

A cDNA clone which contains the near-complete open reading frame (ORF) encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) was obtained by screening a muscle cDNA library of jerboa (Jaculus orientalis), a true hibernating rodent, with a PCR-amplified 0.5-kb genomic DNA probe from an internal region of the gene. The 1.1-kb cDNA clone consists of a 927-bp ORF which codifies for 309 aa, about 93% of the original GapC gene encoding the 36-kDa protein, and a 3'-noncoding region of 167 bp. The full-length aa sequence of GAPDH was achieved by sequencing the N-terminal region of the purified protein completing the missing part in the cDNA clone. Both nt and aa sequences exhibit a high degree of homology to other mammalian GAPDHs. The expression of the GapC gene was studied in skeletal muscle and liver of euthermic and hibernating jerboas both on the mRNA level by Northern blot hybridization using the cDNA clone as a probe and on the protein level by Western blot immunodetection using an antibody raised against muscle GAPDH. A clear decrease (about threefold) in the amount of GapC mRNA, a single 1.2-kb transcript, was observed in muscle of hibernating jerboa when compared with the same tissue from the euthermic animal. This mRNA level decrease directly correlates with a reduction in both protein amount and specific activity in crude protein extracts. In contrast, both GAPDH protein and GapC mRNA levels remained unchanged in liver from euthermic and hibernating jerboas although the enzymatic activity was also about threefold lower in the hibernating tissue. These result, together with previous data obtained from protein studies [Soukri et al. (1995) Biochim. Biophys. Acta 1243, 161-168 and (1996) 1292, 177-187] indicate that jerboa GAPDH is regulated by different mechanisms during hibernation in these tissues, that is, at transcriptional level in muscle and at posttranslational level in liver. The reduced GAPDH activity should result in both cases in a decrease of the glycolytic flux that would eventually contribute to the dramatic metabolic depression of this dormant state.

Evidence for a posttranslational covalent modification of liver glyceraldehyde-3-phosphate dehydrogenase in hibernating jerboa (Jaculus orientalis).

The specific activity of D-glyceraldehyde-3-phosphate (G3P) dehydrogenase (phosphorylating) (GPDH, EC 1.2.1.12) found in liver of induced hibernating jerboa (Jaculus orientalis) was 2-3-fold lower than in the euthermic animal. However, the comparative analysis of the soluble protein fraction of these tissues by SDS-PAGE and Western blotting showed no significant changes in the intensity of the 36 kDa protein band of the GPDH subunit. After using the same purification procedure, the GPDH from liver hibernating jerboa exhibited lower values for both apparent optimal temperature and specific activity than the enzyme from the euthermic animal. Similar non-linear Arrhenius plots were obtained, but the Ea values calculated for the GPDH from hibernating tissue were higher. Although in both purified enzyme preparations four isoelectric GPDH isoforms were resolved by chromatofocusing, those of hibernating liver exhibited more acidic pI values (pI 7.3-6.1) than the hepatic isoforms of euthermic animals (pI 8.7-8.1). However, all liver GPDH isoforms exhibited similar native and subunit molecular masses and cross-reacted with an antibody raised against muscle GPDH. The comparison of the kinetic parameters of both purified preparations and the main isoforms isolated from euthermic and hibernating tissues showed the decreased catalytic efficiency of hibernating enzyme being exclusively due to a lower Vmax for both substrates G3P and NAD+. Phosphodiesterase treatment of cell-free extracts increased GPDH activity in the case of hibernating liver only. The pI of the main isoform purified from this tissue, about 6.9, changed after this treatment to an alkaline value (pI 8.44) similar to those of the euthermic GPDH isoforms. Differential ultraviolet absorption spectra of these isoforms indicated that a substance absorbing at 260 nm, that was released by the phosphodiesterase digestion, was present in the enzyme of hibernating tissue. Incubation of purified GPDH with the NO-releasing agent sodium nitroprussite produced under conditions that promote mono-ADP-ribosylation a dramatic decrease of activity (up to 60%) of both euthermic and phosphodiesterase-treated hibernating preparations but only a marginal inhibition of the hibernating enzyme. These data suggest that the liver GPDH of hibernating jerboa exhibits a posttranslational covalent modification, being probably a mono-ADP-ribosylation. The resulting inhibition of enzyme activity could contribute to the wide depression of the glycolytic metabolic flow associated with mammalian hibernation.

Characterization of muscle glyceraldehyde-3-phosphate dehydrogenase isoforms from euthermic and induced hibernating Jaculus orientalis.

The specific activity of D-glyceraldehyde-3-phosphate (G3P) dehydrogenase (phosphorylating) (GPDH, EC 1.2.1.12) found in skeletal muscle of induced hibernating jerboa (Jaculus orientalis) was 3-4-fold lower than in the euthermic animal. The comparative analysis of the soluble protein fraction of these tissues by SDS-PAGE and Western blotting showed a significant decrease in the intensity of a protein band of about 36 kDa, the GPDH subunit, in hibernating jerboa. After using the same purification procedure, the GPDH from muscle of hibernating jerboa exhibited lower values for both apparent optimal temperature and specific activity than the enzyme from the euthermic animal. Non-linear Arrhenius plots were obtained in both cases, but the Ea values calculated for the GPDH from hibernating tissue were higher. Although in both purified enzyme preparations three isoelectric GPDH isoforms, exhibiting pI values in the range 8.2-7.5, were resolved by chromatofocusing, clear differences were observed in these preparations concerning the relative contribution to the total enzymatic activity of the two main isoforms, named GPDH I (pI values, 8.1-8.2) and GPDH II (pI values, 7.8-7.9). Thus, whereas GPDH I was the major isoform purified from euthermic muscle, accounting for more than 90% of the total activity, the amount of activity due to GPDH II reached up to 65% in preparations of hibernating jerboa. All isoforms exhibited similar native and subunit molecular masses and cross-reacted with an anti-GPDH antibody raised against the GPDH I. However, the two muscle GPDH isoforms prevailing under hibernating conditions exhibited a decreased catalytic efficiency when compared with the corresponding major isoforms purified from euthermic animals, as indicated by their different specific activities and kinetic parameters, i.e. relatively high Km and low Vmax values. Since the glycolytic flow has been found to be widely reduced in skeletal muscle of induced hibernating jerboa, the changes in the GPDH isoforms described in the present study could provide a molecular basis to explain some of the metabolic changes associated with mammalian hibernation.