Immobilized flounder muscle glyceraldehyde 3-phosphate dehydrogenase

Summary Partially purified flounder muscle (Pseudopleuronectus americanus) glyceraldehyde 3-phosphate dehydrogenase was immobilized on cyanogen bromide-activated Sepharose. The catalytic properties of the immobilized preparation were studied to determine if immobilization alters the kinetic properties of the native holoenzyme. The results indicate that the pH activity profile of immobilized glyceraldehyde 3-phosphate dehydrogenase did not differ from that of the native enzyme. The Michaelis constants (Km) for NAD and glyceraldehyde 3-phosphate were somewhat altered. The enzyme stability toward various inactivation treatments in the presence and absence of NAD was characterized and compared to that of he native enzyme. When either form of the enzyme was incubated with urea at concentrations greater than 2m, inactivation occurred very rapidly. Incubation in 0.1% trypsin for 60 minutes decreased the activity of immobilized glyceraldehyde 3-phosphate dehydrogenase by 45% and of the native soluble enzyme by 70%. The immobilized enzyme also exhibited considerably more stability than the native soluble enzyme when exposed to a temperature of 50° or to 20 mm ATP. In all cases NAD either greatly reduced the rate of inactivation or completely protected the enzyme from inactivation.     

Effect of NAD on flounder muscle glyceraldehyde 3-phosphate dehydrogenase

Flounder muscle (Pseudopleuronectes americanus) glyceraldehyde-3-phosphate dehydrogenase was characterized as to its stability towards various inactivating treatments in the presence and absence of the enzyme cofactor, NAD. Incubation of a partially purified enzyme preparation at urea concentrations greater than 2 M produced a very rapid inactivation. NAD greatly reduced the rate of inactivation at all the urea concentrations tested. Incubation of each of the three major muscle enzyme forms in 0.1 percent trypsin or chymotrypsin for forty-five minutes decreased the activity of each form by 65 percent and 55 percent, respectively. NAD (5mM) afforded complete protection to each enzyme form from proteolytic digestion by these two enzymes. Exposure of each form to 50 degrees or 20 mM ATP also led to gross inactivation which could be greatly reduced if the respective incubations were performed in the presence of 5mM NAD. NAD was also found to be required for the renaturation of the unfolded urea-denatured subunits to form the active tetramer.     

Purification of rabbit muscle glyceraldehyde 3-phosphate dehydrogenase by gel filtration chromatography.

A procedure is presented for the large-scale purification of rabbit muscle glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12). Sephadex G-100 chromatography was more effective than repeated recrystallizations for removing heme impurities, hence these proteins appear to cocrystallize. The column purified enzyme has full enzymatic activity according to dehydrogenase, esterase, and acetyl phosphatase assays.     

The comparative structure of mammalian glyceraldehyde 3-phosphate dehydrogenase

1. The amino acid sequences around the thiol groups of glyceraldehyde 3-phosphate dehydrogenase from badger and monkey skeletal muscle were compared with the sequences around the thiol groups in the enzyme isolated from other organisms. 2. Preliminary evidence of the existence of isoenzymes in the badger was obtained. Only the major form, however, could be purified completely. 3. The monkey enzyme contains only three cysteine residues per polypeptide chain compared with the four found in all the other mammalian enzymes so far examined, including that of badger, and the two in yeast. The missing thiol group in monkey was identified as residue 281 in the corresponding sequence of the pig enzyme. 4. These experiments rule out any essential role for cysteine-281 in the function of the mammalian enzymes. 5. Further evidence of the remarkable conservation of amino acid sequence in this enzyme during evolution is presented and discussed.     

Thyroid hormone increases glyceraldehyde 3-phosphate dehydrogenase gene expression in rat liver

Livers from hypophysectomized rats had low levels of glyceraldehyde 3-phosphate dehydrogenase mRNA. Administration of L-triiodothyronine increased these levels over 20-fold. The peak response was seen 72 h after hormone administration. A half-maximal response was obtained with 5 micrograms of T3 per 100 g of body weight. Thus the expression of hepatic glyceraldehyde 3-phosphate dehydrogenase appears to be regulated by thyroid hormone.     

Mutually inverse rhythmic and sigmoidal changes in activity of cytoplasmic and chloroplast glyceraldehyde 3-phosphate dehydrogenases in Lemna gibba G3

Two major timing devices probably control the temporal changesin activity of cytoplasmic NAD- and chloroplast NADP-GPDs inthe long-day plant Lemna gibba G3; one is an oscillator andthe other an hourglass. The former is reset by a light-on signaland responsible for diurnal changes in activities of GPDs incontinuous light, while the latter is reset by a light-off signalto control sigmoidal changes in activities of GPDs in continuousdarkness. The activities of Cyt-NAD-GPD and Chl-NADP-GPD alwayschange mutually inversely. (Received November 17, 1977; )     

Quantitative cytochemical measurement of glyceraldehyde 3-phosphate dehydrogenase activity.

A system has been developed for the quantitative measurment of glyceraldehyde 3-phosphate dehydrogenase activity in tissue sections. An obstacle to the histochemical study of this enzyme has been the fact that the substrate, gylceraldehyde 3-phosphate, is very unstable. In the present system a stable compound, fructose 1, 6-diphosphate, is used as the primary substrate and the demonsatration of the glyceraldehyde 3-phosphate dehydrogenase activity depends on the conversion of this compound into the specific substrate by the aldolase present in the tissue. The characteristics of the dehydrogenase activity resulting from the addition of fructose 1, 6-diphosphate, resemble closely the known properties of purified glyceraldehyde 3-phosphate dehydrogenase. Use of polyvinyl alcohol in the reaction medium prevents release of enzymes from the sections, as occurs in aqueous media. Although in this study intrinsic aldolase activity was found to be adequate for the rapid conversion of fructose 1, 6-diphosphate into the specific substrate for the dehydrogenase, the use of exogenous aldolase may be of particular advantage in assessing the intergrity of the Embden-Meyerhof pathway.     

Kinetics of thermal inactivation and quaternary structure symmetry of rabbit muscle glyceraldehyde 3-phosphate dehydrogenase

Kinetics of thermal inactivation of apo-glyceraldehyde 3-phosphate dehydrogenase have been investigated under various conditions. At most pH values, the loss of enzyme activity takes place in two phases, a fast and a slow phase. The data correspond to the rate equation A = Afast.e-kfast.t + Aslow.e-kslow, where A is the observed residual activity (expressed as % of initial activity) at time t, Afast and Aslow are amplitudes (expressed as % of initial activity, so that Afast + Aslow = 100) and kfast and kslow the rate constants of the fast and slow phases, respectively. At pH 9 or below, Afast = Aslow = 50%. As pH is increased above 9, Afast increases gradually till at pH 10 or above when it accounts for the entire initial activity (single exponential decay). The rate constants of the two phases are strongly affected by the nature of the buffer, temperature and pH, but the amplitudes depend on pH alone. It has been suggested that the tetrameric enzyme exists in two conformations of different molecular symmetry, namely C2 (two pairs of sites of unequal stability, predominating at pH 9 or below) and D2 symmetry (four equivalent sites, predominating at pH 10 or above). The C2 in equilibrium D2 transformation is found to be highly cooperative with midpoint at pH 9.6.