Subunit structure of three glyceraldehyde 3-phosphate dehydrogenases of some flowering plants.

A procedure is described for the purification of three glyceraldehyde phosphate dehydrogenases from a batch of beet leaves. Glyceraldehyde 3-phosphate:NADP⁺ reductase, nonphosphorylating (EC 1.2.1.9) has been purified over 1500-fold. The M_r of this enzyme is 190,000 and its subunits have an M_r of 53,000, suggesting a tetramer as the active form. Its pI is 6.0. Cytosolic glyceraldehyde 3-phosphate dehydrogenase, NAD dependent (EC 1.2.1.12), has an M_r of 145,000 and subunits of M_r 37,000. It is dissociated to inactive dimers by ATP, whereas NAD⁺ in the presence of reductant promotes its reactivation. The amino acid composition is related to glyceraldehyde 3-phosphate dehydrogenases from animal sources and is most similar to pea seed glyceraldehyde 3-phosphate dehydrogenase. The enzyme exhibits a range of pI values from 5 to 7, but a second electrofocusing in the presence of dithioerythritol results in a single main form with pI 5.33, consistent with the behavior in polyacrylamide and cellulose acetate gel electrophoresis. Chloroplast NAD(P)-glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.13) has been obtained from beet, pea, Ranunculus, Arum, and maize leaves. The stable form is an oligomer of about 800,000 M_r (±10%), while a minor, possibly damaged fraction elutes as a retarded peak from agarose columns. The M_r 800,000 form is reversibly dissociated to protomers of M_r 160,000 by NADP⁺, with increase of apparent NADP-dependent activity. Two subunits are present in similar amounts in all association states and after all treatments: α with M_r 36,000, and β with M_r 41,000. The form found in density gradient ultracentrifugation has an M_r of 390,000. Isoelectric points of the various forms lie between pH 4.1 and 4.7 for all species, with a main peak usually at pI 4.45. The amino acid composition of beet chloroplast glyceraldehyde phosphate dehydrogenase is not closely related to that of beet leaf NAD-dependent glyceraldehyde 3-phosphate dehydrogenase.     

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.     

The investigation of substrate-induced changes in subunit interactions in glyceraldehyde 3-phosphate dehydrogenases by measurement of the kinetics and thermodynamics of subunit exchange.

1. Lactate oxidase from Mycobacterium smegmatis is completely resolved into free flavin and apoenzyme by treatment with acid (NH4)2SO4. 2. Reconstitution involves rapid binding of FMN, but the recovery of enzyme activity was slower and appeared to be biphasic. 3. The preparation of the holoenzyme obtained differs from the native enzyme in specific activity, extinction coefficients and mobility on disc-gel electrophoresis. 4. Dialysis of this reconstituted enzyme in 0.1 M-sodium phosphate buffer, pH 7.0, at 0 degrees C for 1 week yields a preparation which closely resembles the native enzyme.     

The hybridization of glyceraldehyde 3-phosphate dehydrogenases from rabbit muscle and yeast. Kinetics and thermodynamics of the reaction and isolation of the hybrid

A kinetic and thermodynamic study was made of the formation of the hybrid (R(2)Y(2)) glyceraldehyde 3-phosphate dehydrogenase from the yeast (Y(4)) and rabbit (R(4)) enzymes. The values of the thermodynamic parameters for the equilibrium between R(4), Y(4) and R(2)Y(2) suggest that the R(2)-R(2) and Y(2)-Y(2) interactions are similar. However, the failure to observe the RY(3) and R(3)Y hybrids is interpreted in terms of differences at the interfaces of the R-R and Y-Y interactions (the glyceraldehyde 3-phosphate dehydrogenase molecule being regarded as a dimer of dimers). The kinetics of formation of the R(2)Y(2) hybrid were studied and a model was proposed to account for the results. Best-fit values for the rate constants of the individual steps were evaluated by computer simulation, and the rate-limiting steps were identified as the dissociation of tetramers to dimers. It is proposed that the cleavage plane for dissociation of the tetramers corresponds to the region of low electron density through the centre of the molecule in the X-ray-crystallographic structure for human glyceraldehyde 3-phosphate dehydrogenase (Watson et al., 1972), which is probably the plane containing the Q and R axes in the lobster enzyme (Buehner et al., 1974). The R(2)Y(2) hybrid was isolated in milligram amounts by ion-exchange chromatography and its rate of reversion to the native enzyme was shown to be consistent with the kinetic model proposed from the hybrid-formation experiments.     

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.     

Folding domains and intramolecular ionic interactions of lysine residues in glyceraldehyde 3-phosphate dehydrogenase.

1. Treatment with methyl acetimidate was used to probe the topography of several tetrameric glyceraldehyde 3-phosphate dehydrogenases, in particular the holoenzymes from rabbit muscle and Bacillus stearothermophilus. During the course of the reaction with the rabbit muscle enzyme, the number of amino groups fell rapidly from the starting value of 27 per subunit to a value of approx. five per subunit. This number could be lowered further to values between one and two per subunit by a second treatment with methyl acetimidate. The enzyme remained tetrameric throughout and retained 50% of its initial catalytic activity at the end of the experiment. 2. Use of methyl [1-14C]acetimidate and small-scale methods of protein chemistry showed that only one amino group per subunit, that of lysine-306, was completely unavailable for reaction with imido ester in the native enzyme. This results is consistent with the structure of the highly homologous glyceraldehyde 3-phosphate dehydrogenase of lobster muscle deduced from X-ray-crystallographic analysis, since lysine-306 can be seen to form an intrachain ion-pair with aspartic acid-241 in the hydrophobic environment of a subunit-subunit interface. 3. Several other amino groups in the rabbit muscle enzyme that reacted only slowly with the reagent were also identified chemically. These were found to be located entirely in the C-terminal half of the polypeptides chain, which comprises a folding domain associated with catalytic activity and subunit contact in the three-dimensional structure. Slow reaction of these 'surface' amino groups with methyl acetimidate is attributed to intramolecular ionic interactions of the amino groups with neighbouring side-chain carboxyl groups, a conclusion that is compatible with the reported three-dimensional structure and with the dependence of the reaction of ionic stength. 4. Very similar results were obtained with the enzymes from B. stearothermophilus and from ox muscle and ox liver, supporting the view that the ion-pair involving lysine-306 and aspartic acid-241 will be a common structural feature in glyceraldehyde-3-phosphate dehydrogenases. The B. stearothermophilus enzyme was fully active after modification. 5. No differences could be detected between the enzymes from ox muscle and ox liver, in accord with other evidence that points to the identify of these enzymes. 6. The pattern of slowly reacting amino groups in the enzyme from B. stearothermophilus, although similar to that of the mammalian enzymes, indicated one or two additional intramolecular ionic interactions of lysine residues that might contribute to the thermal stability of this enzyme.     

Iodination of glyceraldehyde 3-phosphate dehydrogenase

1. A high degree of homology in the positions of tyrosine residues in glyceraldehyde 3-phosphate dehydrogenase from lobster and pig muscle, and from yeast, prompted an examination of the reactivity of tyrosine residues in the enzyme. 2. Iodination of the enzyme from lobster muscle with low concentrations of potassium tri-[(125)I]-iodide led to the identification of tyrosine residues of differing reactivity. Tyrosine-46 appeared to be the most reactive in the native enzyme. 3. When the monocarboxymethylated enzyme was briefly treated with small amounts of iodine, iodination could be confined almost entirely to tyrosine-46 in the lobster enzyme; tyrosine-39 or tyrosine-42, or both, were also beginning to react. 4. These three tyrosine residues were also those that reacted most readily in the carboxymethylated pig and yeast enzymes. 5. The difficulties in attaining specific reaction of the native enzyme are considered. 6. The differences between our results and those of other workers are discussed.     

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.