N.K. Nagradova

Determination of sepharose-bound protein with coomassie brilliant blue G-250

The colorimetric procedure of Bradford (M.M. Bradford, 1976, Anal. Biochem. 72, 248-254) was found to be convenient for determining the content of a protein immobilized on Sepharose. Being simple, sensitive, and rapid, this method appears very useful in studies involving multiple analyses of immobilized protein species present at low concentrations.

Study of the properties of phosphorylating D-glyceraldehyde-3-phosphate dehydrogenase

The properties of the active center of phosphorylating D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are considered with emphasis on the structure of anion-binding sites and their role in catalysis. The results of studies on the molecular mechanism of the effect of NAD+ on the enzyme conformation are discussed. Experimental evidence is presented supporting the idea that negative cooperativity of NAD+ binding and half-of-the-sites reactivity exhibited by GAPDH are generated by different mechanisms. Data obtained with rabbit muscle and Escherichia coli GAPDH point to preexisting asymmetry in these tetramers. Structural determinants that can control the transition of the tetramer from the symmetric to the asymmetric state were found.

Cold inactivation of glyceraldehyde-phosphate dehydrogenase from rat skeletal muscle

Inactivation of apo-glyceraldehyde-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase(phosphorylating) (EC 1.2.1.12) from rat skeletal muscle at 4 degrees C in 0.15 M NaC1, 5 mM EDTA, 4 mM 2-mercaptoethanol pH 7.2 is a first-order reaction. The rate constant of inactivation depends on protein concentration. With one molecule of NAD bound per tetrameric enzyme, a 50 per cent loss in activity is observed and the rate constant of inactivation becomes independent of the protein concentration over a 30-fold range. Two moles of NAD bound per mole of enzyme fully protect it against inactivation. NADH affords a cooperative effect on enzyme structure similar to that of NAD. Inactivation of 7.8 S apoenzyme is reflected in its dissociation into 4.8-S dimers. In the case of enzyme-NAD1 complex, no direct relationship between the extent of inactivation and dissociation is observed, suggesting that these two processes do not occur simultaneously; we may say that dissociation is slower than inactivation. A mechanism in which the rate-limiting step for inactivation is a conformational change in the tetramer occurring prior to dissociation and affecting only the structure of the non-liganded dimer, is consistent with the experimental observations. Inorganic phosphate protects apoenzyme against inactivation. Its effect is shown to be due to the anion binding at specific sites on the protein with a dissociation constant of 2.6 plus or minus 0.4 mM. The NaC1-induced cold inactivation of glyceraldehyde-phosphate dehydrogenase is fully reversible at 25 degrees C in the presence of 20 mM dithiothreitol and 50 mM inorganic phosphate. The rate of reactivation is independent of protein concentration. Inactivated enzyme retains the ability to bind specific antibodies produced in rabbits, but diminishes its precipitating capability.

Phosphorylation of D‐glyceraldehyde‐3‐phosphate dehydrogenase by Ca2+/calmodulin‐dependent protein kinase II

Rabbit muscle D‐glyceraldehyde‐3‐phosphate dehydrogenase was shown to serve as a substrate for Ca2+/calmodulin‐dependent protein kinase II with a K m of 0.33 μM and a V max of 2.63 μmol · min−1 · mg−1 at pH 7.5 and 30°C. In the absence of calmodulin, the V max was halved and K m unchanged. 0.99 mol of phosphate was incorporated per tetrameric molecule of D‐glyceraldehyde‐3‐phosphate dehydrogenase under the experimental conditions employed.

Association of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase The biochemical and electron-microscopic evidence

Rabbit muscle glyceraldehyde‐3‐phosphate dehydrogenase covalently bound to Sepharose was shown to form a complex with soluble 3‐phosphoglycerate kinase. The strength of the association appeared to depend upon the functional state of both enzymes. The holoform of the dehydrogenase exhibited a lower affinity for the kinase than the enzyme‐3‐phosphoglycerol·NADH complex. The substrate‐free 3‐phosphoglycerate kinase associated much stronger with the acylated dehydrogenase than the kinase in complex with 1,3‐diphosphoglycerate. Electron‐microscopic evidence for the association of the soluble acyl‐glyceraldehyde‐3‐phosphate dehydrogenase·NADH complex and 3‐phosphoglycerate kinase was also obtained.

Essential arginine residues in d-glyceraldehyde-3-phosphate dehydrogenase

Two arginyl residues per subunit of yeast D-glyceraldehyde-3-phoshphate dehydrogenase were modified by treatment with butanedione without significant changes in the compostion of other amino acid residues. The modified enzyme displays no dehydrogenase activity. It retains the capacity for interacting with the coenzyme NAD, but binds it less firmly than does the native enzyme. The molar absorbance of the enzyme-NAD complex is markedly reduced and the reactivity of the active-center SH groups is changed in the modified enzyme. The native and modified enzymes show identical fluorescence spectra, absorbance and CD spectra.

D-GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE PURIFIED FROM RABBIT MUSCLE CONTAINS PHOSPHOTYROSINE

Homogeneous preparations of d‐glyceraldehyde‐3‐phosphate dehydrogenase purified from rabbit muscle were found to contain 0.2–0.7 moles of covalently bound phosphate per mole of the enzyme. With the use of anti‐phosphotyrosine antibodies, evidence was obtained that the enzyme is phosphorylated at tyrosine residues.

Evidence for the stabilizing effect of antibodies on the subunit association of glyceraldehyde-3-phosphate dehydrogenase.

Abstract Glyceraldehyde-3-phosphate dehydrogenases isolated from bakers yeast and from rat skeletal muscle were shown to have no common antigenic determinants revealed with antibodies elicited in rabbits. Fab fragments of rabbit anti-yeast enzyme antibodies have no effect on the dehydrogenase activity of the antigen. This is consistent with the results previously obtained with a combination of the rat muscle enzyme with specific antibodies. Tetrameric yeast apo-glyceraldehyde-3-phosphate dehydrogenase immobilized on the solid support by means of interaction with specific antibodies covalently bound to the matrix retains the full catalytic activity and is capable of dissociating into dimers at low temperature in the presence of ATP. The dissociation is accompanied by the inactivation of dimers which become solubilized, whereas those bound to the matrix remain active even in the cold in the presence of ATP. The tetrameric molecule of yeast glyceraldehyde-3-phosphate dehydrogenase, covalently bound to CNBractivated Sepharose via one subunit, is completely protected from dissociation by three molecules of specific antibodies bound per tetramer. The binding of four molecules of Fab fragments of specific antibodies per tetramer protects the rat muscle apo-glyceraldehyde-3-phosphate dehydrogenase from NaCl-induced cold inactivation and from thermoinactivation in solution. The binding of two molecules of anti-rat muscle enzyme antibodies to a hybrid tetramer that consists of a yeast dimer covalently bound to Sepharose, and of a rat muscle dimer, prevents the dissociation of the immobilized molecule (that is, solubilization of the rat muscle dimer). The gel-filtration pattern of the complexes of the rat muscle apoenzyme with Fab fragments of specific antibodies points to a shift of the equilibrium between the tetrameric and dimeric forms of the enzyme in solution towards the tetrameric form. On the basis of these observations, we conclude that specific antibodies strengthen subunit interactions in the oligomer by means of stabilization of the native structure of individual subunits.

Half-of-the-sites reactivity of rat skeletal muscle d-glyceraldehyde-3-phosphate dehydrogenase

Abstract Apo-glyceraldehyde-3-phosphate dehydrogenase ( d -Glyceraldehyde-3-phosphate:NAD + oxidoreductase (phosphorylating), EC 1.2.1.12) from rat skeletal muscle is inactivated by stoichiometric amounts of 1-fluoro-2,4-dinitrobenzene (FDNB) in a time-dependent reaction. The maximal loss of activity attained in the presence of 1 and 2 equivalents of FDNB per mol of the tetrameric enzyme corresponds to 25% and 50% of the control, respectively. Further increase in FDNB concentration results in no additional loss of activity, and only two sub-units per tetramer remain modified in the presence of an excess of the reagent. Full inactivation can however be achieved by addition of stoichiometric amounts of DTNB, which readily modifies the SH groups of the two active centers apparently inaccessible to FDNB. This type of the half-of-the-sites reactivity is unusual and differs appreciably from the previously described effect of FDNB on glyceraldehyde-3-phosphate dehydrogenases from other sources. Namely, modification by FDNB of two subunits of the tetrameric molecules of the enzymes from rabbit skeletal muscle or from bakers yeast is known to inactivate the neighbouring subunits, whereas in the case of the rat muscle enzyme, the active centers remaining free are fully active and prevented from being modified by FDNB. Apo-glyceraldehyde-3-phoshate dehydrogenase from rat skeletal muscle covalently bound to CNBr-activated Sepharose 4B, exhibits the half-of-the sites reactivity of the type characteristic for the soluble enzyme. The half-of-the- sites effect is retained in an immobilized dimeric form of the dehydrogenase prepared by ADP-induced dissociation of the matrix-bound tetrameric enzyme. These results demostrate that the non-equivalence among the active centers is conserved in the isolated dimeric species of glyceraldehyde-3-phosphate dehydrogenase, and suggest that the tetrameric structure is not a prerequisite for the half-of-the-sites effect.

A convenient method for the estimation of Sepharose-bound protein.

Abstract Different methods have been developed for the determination of the amount of protein immobilized on solid supports. The most frequently used procedure is quantitative amino acid analysis after acid hydrolysis of the protein-carrier conjugate. The application of less time-consuming spectrophotometric methods to the measurement of the amount of protein bound to agarose beads was delineated by Koelsch et al. (1). In this report a modified procedure for the direct spectrophotometric protein determination with the intact gel beads is described. It is based on the measurement of optical density of the suspension of immobilized protein in polyethylene glycol. Due to the high sensitivity and reproducibility of the method, it is applicable to the measurement of small amounts of Sepharose-bound protein.