Rabbit Muscle Enzyme Research Articles

Isolation and partial characterization of rat muscle fructose biphosphatase

Fructose bisphosphatase ( d-fructose-1,6-biphosphate 1-phosphohydrolase, EC 3.1.3.11) has been isolated in homogeneous form from rat muscle by a simple and convenient procedure, including adsorption on carboxymethylcellulose and substrate elution. The resultant enzyme preparation has a specific activity comparable to that of the enzymes isolated from rabbit liver, rabbit muscle and rat liver. The native relative molecular mass of the enzyme was estimated by sedimentation equilibrium centrifugation to be approx. 138 000, and the enzyme appears to be a tetramer containing subunits of M r approx. 34 500. The amino acid composition is distinctly different from that of the rabbit muscle, rabbit liver and rat liver enzymes. The purified enzyme contains no tryptophan and has a blocked amino terminal.

Nucleotide sequence and high-level expression of the major Escherichia coli phosphofructokinase.

The gene for the major phosphofructokinase enzyme in Escherichia coli, pfkA, has been sequenced. Comparison of the amino acid sequence with other phosphofructokinases showed that this enzyme is related to the Bacillus stearothermophilus and rabbit muscle enzymes, but is different from the second, minor phosphofructokinase found in E. coli. The region which has been sequenced comprises the complete pfkA--tpi interval on the E. coli genetic map. Two other genes have been identified from the nucleotide sequence: a gene for a periplasmic sulphate-binding protein, sbp, and for a membrane-bound enzyme, CDP-diglyceride hydrolase, cdh. This establishes the complete gene arrangement in this region as pfkA-sbp-cdh-tpi. The pfkA gene has been subcloned into a high-copy-number plasmid under the control of a strong, chimaeric promoter which arose as an artefact in the construction of the plasmid gene bank from which the original pfkA recombinant was isolated. A specialised recombinant has been constructed which carries a 1.4 X 10(3)-nucleotide insert containing just the pfkA gene flanked by two HindIII recognition sites providing a simple system for the recloning of this gene into different vectors. This recombinant expresses the enzyme at high levels (40-50% of total cell protein is active, soluble phosphofructokinase). This expression system is now being used to study the enzyme using 'reverse genetics'.

Rat skeletal muscle glycogen synthase: Phosphorylation of the purified enzyme by cAMP-dependent and -independent protein kinases

Glycogen synthase was purified to near homogeneity from rat skeletal muscle, and was found to resemble the rabbit skeletal muscle enzyme in several respects. An apparent molecular weight ( M app) of 86,000 was estimated from the electrophoretic mobility of the subunit on polyacrylamide gels in the presence of sodium dodecyl sulfate. Limited proteolysis of the rat synthase with trypsin resulted in the formation of species with M apps equal to 75,000, 69,000, and 67,000. The enzyme could be phosphorylated by cAMP-dependent protein kinase, phosphorylase kinase, and the cAMP-independent protein kinases, PC 0.7 and F A GSK-3 . Essentially all of the phosphorylation observed occurred on serines located in two cyanogen bromide fragments, denoted CB-1 ( M app = 13,000) and CB-2 ( M app = 22,000). F A GSK-3 and cAMP-dependent protein kinase phosphorylated sites in both fragments. Phosphate introduced by phosphorylase kinase was located exclusively in CB-1, and that incorporated with PC 0.7 was found in CB-2. Phosphorylation by F A GSK-3 reduced the electrophoretic mobility of the subunit, introduced heterogeneity into CB-2, and was synergistic with phosphorylation by PC 0.7. To separate phosphorylation sites more completely, samples of glycogen synthase were subjected to extensive proteolysis using trypsin, followed by reverse-phase liquid chromatography. When phosphorylated by the same kinases, the pattern of fragments obtained with rat and rabbit skeletal muscle glycogen synthase were almost identical. The results presented provide strong evidence that the subunit of rat skeletal muscle glycogen synthase has at least five phosphorylation sites that are very similar, if not identical, to sites present on the rabbit muscle enzyme.

The cooperative behavior of yeast D-glyceraldehyde-3-phosphate. Dehydrogenase as studied by the formation of the fluorescent NAD derivative.

The ultraviolet irradiation of the yeast D-glyceraldehyde-3-phosphate dehydrogenase carboxymethylated at the active site Cys residues, as with the rabbit muscle enzyme, led to the formation of a fluorescent NAD derivative with an emission maximum at 410 nm. Similar results were obtained with the enzyme selectively carboxymethylated at only 2 of its 4 active site Cys residues. The binding of NAD+ to both the carboxymethylated enzymes is non-cooperative or only weakly negatively cooperative when determined by NAD+ quenching of the intrinsic protein fluorescence. However, determinations of the amount of fluorescent NAD derivative formed under different NAD+ concentrations show that both the carboxymethylated enzymes appeared to bind NAD+ with positive cooperativity as in the case of the binding of NAD+ to the native apoenzyme. This seems to suggest that the spatial positioning of the nicotinamide moiety at the active site of the irradiated enzyme resembles more closely that of the nicotinamide ring in the native holoenzyme as compared to the carboxymethylated enzymes.

Interactions and spatial arrangement of spin-labeled NAD+ bound to glyceraldehyde-3-phosphate dehydrogenase. Comparison of EPR and X-ray modeling data.

The spatial arrangement of coenzyme NAD+ in remote and adjacent binding sites in various stoichiometric complexes with tetrameric glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle was examined via EPR spectroscopy. An adenosine N6-15N,2H17 spin-labeled derivative of coenzyme NAD+ (SL-NAD+) was chemically synthesized for this work. The spectral simplifications and narrow line widths afforded by 15N and 2H substitution enabled experimental EPR spectra to be deconvoluted into their three component spectra: (a) unbound coenzyme, (b) bound coenzyme without adjacent site occupied, and (c) bound coenzyme with adjacent site occupied. Binding of SL-NAD+ in adjacent active centers of R axis-related subunits resulted in resolved dipolar interactions which characterized intersubunit distances. Binding to distant subunits related by the P and Q axes gave no dipolar interaction. Once the first NAD+ site was occupied, EPR spectra at various stoichiometries provided evidence for nonpreferential spatial binding of SL-NAD+ to the three unoccupied sites. EPR spectral simulations indicated a separation of 12.8 A for the unpaired electrons of spin label moieties of R axis-related coenzymes. Molecular modeling based on x-ray crystallographic data predicted 11-13 A. The angles and distance relating to interacting spin-labels were calculated from atomic coordinates based on molecular modeling of both anti-anti and anti-syn (adenine-ribose) conformations of SL-NAD+. Computer-generated line shapes indicated best agreement with experimental EPR results when the anti-anti geometry was employed. Comparison of EPR spectra from soluble and ammonium sulfate-precipitated enzymes indicated that the NAD+-binding domains are positioned equivalently in the two physical states. Since the observed dipolar line shapes are critically dependent on the distance and geometry relating to the interacting SL-NAD+, these data provide direct evidence for a high degree of conservation of quaternary structure of the enzyme in the hydrated crystalline state. Studies on the enzyme isolated from human erythrocytes also indicated a close correlation with the rabbit muscle enzyme in both the arrangement of NAD+-binding domains and negative cooperativity of coenzyme binding.

ESTIMATION OF THE RATE OF GLYCEROL 3-PHOSPHATE DEHYDROGENASE EVOLUTION IN HIGHER VERTEBRATES.

Among proteins which have been examined for polymorphism in natural populations, sn-glycerol 3-phosphate dehydrogenase (E.C. 1.1.1.8) is one of the least polymorphic (Ayala et al., 1974; Collier and Maclntyre, 1977, 1978; Lakovaara et al., 1977; Coyne et al., 1979; Johnson, 1979). These studies have also indicated that the enzyme, also known as aXGPDH, is evolving rather slowly. It is possible that limited polymorphism is correlated with slow rates of amino acid replacement (Skibinski and Ward, 1982); however, for most enzymes whose natural polymorphism has been well-studied there are very few amino acid sequence data. Such is the case for glycerol 3-phosphate dehydrogenase. The only published sequence is for the rabbit muscle enzyme (Otto et al., 1977, 1980). The structure of the Drosophila enzyme is under study (Niesel et al., 1982). Glycerol 3-phosphate dehydrogenase is a cytosolic enzyme present in most tissues of all eukaryotes. In vertebrates amounts of this enzyme are particularly high in liver and white skeletal muscle, but relatively low in heart, brain, and most neoplastic tissues (Young and Pace, 1958). This enzyme is particularly concentrated in the flight muscles of insects such as bees and flies (Crabtree and Newsholme, 1972) where the enzyme

Purification and Comparative Characterization of an Enolase from Spinach

An enolase has been purified to apparent homogeneity, as measured by gel electrophoresis, some 400-fold from spinach (Spinacia oleracea). This is the first plant enolase that has been purified to homogeneity. At moderate ionic strengths, the 5,5-dithio-bis-2-(nitrobenzoate) (DTNB)-or parachloromercuribenzoate-reacted enzyme elutes from a Bio-Gel P-200 column with somewhat greater volumes than the yeast enzyme (M(r) = 93,000) indicating a greater size. Its elution volume from Ultrogel in 50% ammonium sulfate, however, suggests it exists as an active monomer (M(r) = 47,000). Sodium dodecyl sulfate-gel electrophoresis indicates the subunit molecular weight is 50,000 +/- 3,000, like that of yeast enolase.The enzyme contains 23 +/- 4 half-cystines per mole of subunit. Titrations with DTNB in guanidine hydrochloride or nondenaturing media indicate that most of these, if not all, are in the reduced state. Reaction of one or more of the sulfhydryls with DTNB or parachloromercuribenzoate stabilizes the enzyme.The kinetic parameters of the reaction catalyzed by spinach enolase, as well as the inhibitions by transition metal ions and fluoride, are similar to those properties of the yeast and rabbit muscle enzymes.

The regulatory properties of yeast pyruvate kinase.

The kinetics of pyruvate kinase from Saccharomyces cerevisiae were studied in assays at pH 6.2 where the relationships between the initial velocities of the catalysed reaction and the concentrations of the substrates ADP, phosphoenolpyruvate and Mg2+ are non-hyperbolic. The findings were represented empirically by the exponential model for a regulatory enzyme. The analysis shows that ADP, phosphoenolpyruvate and Mg2+ display positive homotropic interaction in their binding behaviour with (calculated) Hill slopes at half-saturation equal to 1.06, 2.35 and 3.11 respectively [Ainsworth (1977) J. Theor. Biol. 68, 391-413]. The direct heterotropic interaction between ADP and phosphoenolpyruvate is small and negative, but the overall interaction between these substrates becomes positive when their positive interactions with Mg2+ are taken into account. The heterotropic interactions of the substrates, though smaller in magnitude, are comparable with those revealed by the rabbit muscle enzyme [Ainsworth, Kinderlerer & Gregory (1983) Biochem. J. 209, 401-411], and it is suggested that they have a common origin in charge interactions within the active site.

コイ筋肉ホスホリラーゼｂキナーゼの精製とその触媒的性質

Procedures are described for purification of phosphorylase b kinase (EC. 2.7.1.38.) form carp muscle. The phosphorylase b kinases separated into two active fractions (Peak I and Peak II) on phospho-cellulose column chromatography by the linear gradient of KCl have similar properties with pH-depedency of activity, effect of Ca2+ on activity and Km values for ATP and phosphorylase b. Both kinases have a high ratio of activities at pH 6.8/pH 8.2 of 0.3-0.4 in comparison with a ratio of 0.05-0.08 for rabbit muscle enzyme.

Phosphofructokinase from Ascaris suum. Purification and properties.

A rapid and efficient procedure has been developed to purify phosphofructokinase from the muscle of the parasitic roundworm, Ascaris suum. The procedure can be accomplished in 1 day with a 420-fold purification and a 60% yield. The enzyme was shown to be homogeneous by two-dimensional electrophoresis, Sepharose 6B column chromatography, and high performance liquid chromatography utilizing a size exclusion column. The subunit molecular weight of the enzyme was found to be 95,000 by electrophoresis in the presence of sodium dodecyl sulfate. In solutions of low ionic strength, the native enzyme aggregated to species of higher molecular weight than did the rabbit muscle phosphofructokinase. In the presence of 0.2 M (NH4)2SO4, the minimum native molecular weight was determined to be 398,000 by high performance liquid chromatography and Sepharose 6B column chromatography. Therefore, the enzyme appears to be a tetramer with identical or near-identical subunits. The apparent isoelectric point of the enzyme was shown to be 7.3 to 7.4 by both column and gel isoelectric focusing. Amino acid analysis revealed a lower number of the aromatic residues Phe, Tyr, and Trp than in the rabbit muscle enzyme and this is in agreement with the lower extinction coefficient of E1%280 nm = 6.5. Analysis of the purified enzyme revealed 7.4 +/- 0.6 mol of phosphate/mol of enzyme.

38] Fructose-bisphosphate aldolase from human erythrocytes

Publisher Summary This chapter describes an assay for fructose-bisphosphate aldolase from human erythrocytes that is based on the oxidation of NADH by dihydroxyacetone phosphate in a coupled system containing triosephosphate isomerase and α-glycerophosphate dehydrogenase. The purification process involves washing of erythrocytes and hemolysis, batch process, column chromatography, and reverse ammonium sulfate fractionation. Isoelectric focusing reveals the presence of one form of aldolase in the erythrocyte with an isoelectric point of 8.9. Sedimentation equilibrium ultracentrifugation and gel filtration chromatography reveals a native molecular weight of 158,000. The enzyme, like other mammalian aldolases, appears to be a tetramer with identical or near identical subunits. The amino acid composition is very similar to that of the human heart and rabbit muscle enzymes. The human erythrocyte aldolase appears to be a type A isozyme or muscle-type aldolase.

54] Glyceraldehyde-3-phosphate dehydrogenase from pig liver

This chapter describes the assay method of glyceraldehyde-3-phosphate dehydrogenase isolated from the pig liver. Liver glyceraldehyde-3- phosphate dehydrogenase (GAPD) operates in the backward direction when conditions require gluconeogenesis. With the rabbit muscle enzyme, the backward reaction is strongly inhibited by nicotinamide adenine dinucleotide (NAD), whereas the liver enzyme is only weakly inhibited by NAD. The pig liver enzyme is highly soluble in metal ion and high salt concentrations; a major step is the initial precipitation of large amounts of unwanted proteins with ZnCl 2 . The reaction velocity shows a decrease with time, unless precautions are taken to avoid: (a) product inhibition by nicotinamide adenine dinucleotide dehydrogenase (NADH); (b) product inhibition by 1,3-diphosphoglycerate; and (c) the deterioration of glyceraldehyde-3-P with time. The use of arsenate in the assay, in place of phosphate, avoids the inhibition by 1,3-diphosphoglycerate. The steps involved in the purification of GAPD are: (1) homogenization in ZnCl 2 solutions, (2) ammonium sulfate fractionation, (3) chromatography on diethylaminoethyl (DEAE)-Sephadex, and (4) crystallization. Sodium dodecyl sulfate polyacrylamide gel electrophoresis experiments show a single protein band, indicating that the enzyme is pure and consists of similar subunits.

40] Glycerol dehydrogenase from rabbit muscle

Glycerol dehydrogenase activity is found in several mammalian tissues. This chapter describes the assay method of glycerol dehydrogenase isolated from the rabbit muscle. The enzyme catalyzes the nicotinamide adenine dinucleotide phosphate kinase (NADP + )-linked reduction of a wide variety of aldoses and aliphatic and aromatic aldehydes. It is similar to both NADP L-hexonate dehydrogenase and aldose reductase. Glycerol dehydrogenase activity from the rabbit muscle is because of two enzymes, both of which are different from aldehyde reductase. One of the rabbit muscle enzymes is immunologically identical with aldose reductase from the rabbit lens. Glycerol dehydrogenase activity is assayed spectrophotometrically by determining the decrease in the absorbance of nicotinamide adenine dinucleotide phosphate dehydrogenase (NADPH) at 340 nm. The reaction is carried out at 25°C in a Beckman 25 double-beam recording spectrophotometer. The scale is expanded to give a full scale expansion of 0.25. The chapter discusses the steps involved in the purification procedure of glycerol dehydrogenase. It also presents the diethylaminoethyl (DEAE)-Sephacel chromatography of glycerol dehydrogenases obtained from rabbits.

Age-related effects in coenzyme binding patterns of rat muscle glyceraldehyde-3-phosphate dehydrogenase.

The binding of NAD+ and of its fluorescent analogue, nicotinamide 1,N6-ethenoadenine dinucleotide, to glyceraldehyde-3-phosphate dehydrogenase purified from the muscles of young and old rats was studied in detail. Binding of the natural coenzyme was followed both by spectrophotometric titration of the extrinsic absorption band of the enzyme-NAD+ complex and from the degree of quenching of fluorescence of the protein. Binding of the coenzyme analogue was monitored by using the large enhancement in its fluorescence upon forming the complex with the enzyme. Both dinucleotides showed strong negative cooperativity in binding to the enzyme, similar to that displayed in their association with the rabbit muscle enzyme. The enzyme purified from old rats displayed a markedly reduced affinity toward the two dinucleotides, compared with the enzyme isolated from young animals. The various dissociation constants of both dinucleotides from the enzyme from young rats were remarkably similar to the corresponding constants in the rabbit muscle enzyme. The degree of negative cooperativity (i.e., the ratio between the dissociation constants from high- and low-affinity binding sites) in the "young" and "old" enzyme forms was not very different. It was concluded from these results that while modifications in the subunits take place upon aging, the intersubunit interaction is not significantly affected. Increasing concentrations of ATP were found to cause a gradual decrease in the negative cooperativity of NAD+ binding, which completely disappeared in the presence of 5 mM ATP. The observation that all four binding sites of the old enzyme display the same affinity toward NAD+ when the negative cooperativity is removed excludes the possibility that this enzyme form is a mixture of native and inactive species. The different dissociation constants of NAD+ from young and old enzyme forms in the presence of 5 mM ATP also demonstrate the occurrence of age-related modifications in the structure of the individual subunits.

Structure and reactivity relationship in glyceraldehyde-3-phosphate dehydrogenase. Dinitrophenylation of cysteine residues of yeast and rabbit muscle enzymes.

Dinitrophenylation of rabbit muscle and yeast glyceraldehyde-3-phosphate dehydrogenases modifies only SH groups. The rabbit muscle apoenzyme loses 75% of its original activity upon dinitrophenylation of two SH groups per tetramer whereas the yeast apoenzyme is totally inactivated under the same conditions. Dinitrophenylation of the active-site cysteine-149 of rabbit muscle and yeast holoenzymes results in an loss of activity corresponding to a 'half-of-the-sites' and a 'full-sites' reactivity, respectively. Determination of the sulphydryl content of the modified enzymes shows an unmasking of the cysteine residues of the dinitrophenylated rabbit muscle apoenzyme which is not observed for the yeast protein. However, conformational changes are revealed for both dinitrophenylated apoenzymes by differential absorption spectroscopy or by limited proteolysis. Sulphydryl group unmasking is not observed after modification of the cysteine residues of the rabbit muscle holoenzyme but it does occur when dinitrophenylation is performed in the presence of two moles NAD+/mole rabbit muscle enzyme. Although the apoenzyme is sensitive to an induced conformational change, our results favour symmetrical structures for both yeast apo and holo enzymes. The bis-dinitrophenylated rabbit muscle apoenzyme presents all the characteristics of an asymmetrical structure; however, it is not possible to deduce whether this symmetry is due to the chemical modification or whether it preexists in the native apoenzyme. The results of the dinitrophenylation of the rabbit holoenzyme, however, indicate that this enzyme possesses an asymmetrical structure.

Molecular symmetry and metastable states of enzymes exhibiting half-of-the-sites reactivity.

Half-of-the-sites reactivity in oligomeric enzymes has generally been accepted as evidence for structural asymmetry between subunits. However, we show that the symmetric two-state allosteric model [Monod, J., Wyman, J., & Changeux, J.-P. (1965) J. Mol. Biol. 12, 88-118] is quantitatively consistent with half-of-the-sites reactivity data for several hexameric and tetrameric enzymes. Specifically, the time courses for both the modification and the inactivation of glutamate dehydrogenase by glutamyl alpha-chloromethyl ketone and uridine diphosphoglucose dehydrogenase by 5-(iodoacetamidoethyl)aminonaphthalene-1-sulfonic acid are fit with just five parameters for each enzyme-modifier pair. In the case of glyceraldehyde-3-phosphate dehydrogenase, the time courses for modification of the yeast enzyme by iodoacetic acid and the rabbit-muscle enzyme by 3,3,3-trifluorobromoacetone are fit with the same model, and parameter values from these fits are used to generate theoretical inactivation curves which are found to agree well with the experimentally measured inactivation. We conclude that half-of-the-sites reactivity, if it is not an artifact of residual heterogeneity, could be a kinetic phenomenon related to metastability of partially modified states of a symmetric oligomer and that asymmetry between subunits should therefore not necessarily be inferred from such behavior. If similar metastability occurs in substrate binding, it may play a significant role in mechanism of catalysis and control. In such cases, the virtual inaccessibility of the substrate binding equilibrium would preclude conventional quasi-equilibrium models for the enzyme kinetics.

Structural basis for the difference of the regulatory properties between potato and rabbit muscle phosphrylases. The NH2-terminal sequence of the potato enzyme.

alpha-Glucan phosphorylases (EC 2.4.1.1) from potato tuber and rabbit muscle are similar in some of the structural and kinetic properties, despite the difference in the regulation of enzyme activity. The NH2-terminal region in the rabbit muscle enzyme is important for both allosteric and covalent controls. In this paper, the NH2-terminal 104-residue sequence of the potato enzyme was determined to compare it with that of the rabbit muscle enzyme. Edman degradations of the whole protein revealed the terminal sequence of Thr-Leu-X-Glu-X-X-His-His-. Using this sequence as a marker, the NH2-terminal 81-residue peptide was isolated from the CNBr-treated protein and sequenced. The sequence of this peptide was extended further by the 23-residue sequence of the other two CNBr peptides by considering the sequence similarity between the potato and rabbit muscle enzymes. The combined results of the present and previous studies (Nakano, K., Wakabayashi, S., Hase, T., Matsubara, H. and Fukui, T. (1978) J. Biochem. (Tokyo) 83, 1085-1094 and Nakano, K., Fukui, T., and Matsubara, H. (1980) J. Biochem. (Tokyo) 87, 919-927) indicate that the potato and rabbit muscle enzymes are highly homologous except for the NH2-terminal 33-residue region which is very different. The potato enzyme has 2 more residues attached to the NH2-terminal region. The remarkable dissimilarity in the structure of this region provides a basis for the difference of the regulatory properties between the two enzymes. It is conceivable that phosphorylases existed originally as a large catalytically active molecule, and that the regulatory mechanism was then formed within the molecule during the course of evolution.

Molecular characterization of pig muscle phosphoglucose isomerase

As a corollary to X-ray crystallographic work performed by H. Muirhead, detailed studies on crystalline pig muscle phosphoglucose isomerase have been conducted to establish its basic physical and chemical properties. The enzyme species being investigated by X-ray diffraction has been determined to be isoenzyme III. Its molecular weight in the native state was found to be 132,000, its s 0 20, w value to be 7·25 S. The enzyme is composed of two subunits of equal molecular weight (66,000). Its amino acid composition is largely similar to that of rabbit muscle phosphoglucose isomerase, with the significant exception that the pig muscle isomerase contains only three sulfhydryl groups per polypeptide chain (two of them accessible to titration with p-mercuribenzoate) as compared with twice that number for the rabbit muscle enzyme. This low number of sulfhydryl groups is interpreted as being responsible for the ease with which heavy-atom, isomorphous derivatives could be prepared for the pig muscle enzyme by Shaw & Muirhead (1977).

Heavy-atom effects associated with methylmercury(II) binding to rabbit glyceraldehyde-3-phosphate dehydrogenase.

AbstractThe complex of CH3Hg(II) with the accessible cysteines of glyceraldehyde‐3‐phosphate dehydrogenase (GAPD, EC 1.2.1.12) from rabbit muscle has been studied by phosphorescence and optically detected magnetic resonance (ODMR) spectroscopy. The wavelength dependence of the phosphorescence decay kinetics has also been measured. Comparison of CH3Hg(II)–GAPD with GAPD by these methods shows that a specific optically resolved tryptophan site of GAPD is perturbed by the interaction with a nearby mercury atom. The perturbation on the luminescence and ODMR properties is typical of an external heavy‐atom effect. Based on the x‐ray diffraction structure of the lobster enzyme, it is proposed that the heavy‐atom effect results from the interaction of tryptophan‐310 with CH3Hg(II) bound to cysteine‐281 in the rabbit muscle enzyme.

Subunit interactions in glyceraldehyde-3-phosphate dehydrogenases. Their involvement in nucleotide binding and cooperativity

1. 1. The hybridization of rabbit muscle and yeast glyceraldehyde-3-phosphate dehydrogenase ( d-glyceraldehyde-3-phosphate:NAD + oxidoreductase (phosphorylating), EC 1.2.1.12) was used to study the involvement of subunit interactions in NAD + and NADH binding by these enzymes. 2. 2. In the presence of 1 mM NAD + or NADH no hybrid formation was observed with our preparations of the two enzymes. 3. 3. The inhibition by NADH of the hybrid formation is shown to be a consequence of an unfavourable equilibrium of the hybridization process in the presence of NADH. 4. 4. The inhibition of NAD + of the hybrid formation is shown to be a consequence of both a shift in the equilibrium, as in the case of NADH, and a decrease in the rate of the dissociation of the enzymes. 5. 5. The dimer of the yeast enzyme binds NAD + or NADH with equal affinity irrespective of whether it is combined with another yeast dimer in the yeast tetramer or with a rabbit muscle dimer in the hybrid. 6. 6. The binding of NAD + or NADH to the dimer of the rabbit muscle enzyme is stronger in the rabbit muscle tetramer than in the hybrid; this explains the shift in the equilibrium of the hybridization process caused by these nucleotides. 7. 7. Alkylation of the rabbit muscle enzyme with iodoacetate does not influence the hybridization process in the absence of nucleotides. 8. 8. After alkylation of the rabbit muscle enzyme NADH cannot cause a large shift in the equilibrium of the hybridization process.