Improved protease activity of Pixian broad bean paste with cocultivation of Aspergillus oryzae QM-6 and Aspergillus niger QH-3

BACKGROUND: The preparation of broad bean koji is a key process in the production of Pixian broad bean paste (PBP). Protease is essential for the degradation of proteins during PBP fermentation. To obtain broad bean koji with high protease activity using the cocultivated strains of Aspergillus oryzae QM-6 (A. oryzae QM-6) and Aspergillus niger QH-3 (A. niger QH-3), the optimization of acid and neutral protease activities was carried out using Box­Behnken design with response surface methodology (RSM). RESULTS: The optimum conditions were found to be as follows: inoculation proportion (X1), 3:1 (A. oryzae QM-6: A. niger QH-3, w/w); culture temperature (X2), 33°C; inoculum size (X3), 0.5% (w/w); incubation time (X4), 5 d. The acid and neutral protease activities were 605.2 ± 12.4 U/g and 1582.9 ± 23.7 U/g, respectively, which were in good agreement with the predicted values. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles revealed that the broad bean koji extracellular proteins in the case of cocultivation were richer compared to those in the case of A. oryzae QM-6 or A. niger QH-3 strain only. In addition, the free amino acids (FAAs) in the fermentation product were 55% higher in the cocultivation process than in that involving only A. oryzae QM-6, further confirming the diversity of proteases in the fermentation products. CONCLUSIONS: The optimal conditions of koji-making in PBP were obtained using RSM. The cocultivation of A. oryzae and A. niger increases the overall enzyme activities in the culture medium and the FAAs content, which would thus have potential application in the PBP industry.

Isolation and gene expression analysis of Phospholipase C in response to abiotic stresses from Vigna radiata (L.) Wilczek.

Phosphatidylinositol (PtdIns) is a major phospholipid in eukaryotic cells. Many studies have revealed that the phosphoinositide (PI) signaling pathway plays an important role in plant growth and development. Phospholipase C (PLC) is reported to have a crucial role in the PI pathway. This work focuses on the isolation and investigation of PLC in response to abiotic stress factors in green gram. The PLC cDNA, designated VrPLC, encoding a protein of 591 amino acids was cloned and expressed in E. coli.The predicted isoelectric point (pI) and molecular weight were 5.96 and 67.3 kDa, respectively. The tertiary structure of the PLC was also predicted and found to be mainly composed of random coils. In addition, VrPLC expression analysis was performed under environmental stress and the results showed that the expression of VrPLC was rapidly induced in an abscisic acid independent manner in response to drought and salt stress. PLC expression was found to be up-regulated by SA and down-regulated by wound in leaf tissues; however, there was no significant difference in the expression of PLC in plants subjected to high temperature and H2O2. Our results suggest that a close link/relationship between PLC expression and stress responses in green gram.

Role of H2O2 and cell wall monoamine oxidases in germination of Vigna radiata seeds.

Plant cell wall expresses monoamine oxidases (MAOs) that catalyze oxidation of secreted amines and produce H2O2 in the process. The H2O2, so produced is used by cell wall peroxidases for lignification of cell wall or for plant defense. The natural substrates for these MAOs are elusive, but polyamines and certain catecholamines have been proposed as candidates. Reactive oxygen species are also known to act as signaling molecules controlling plant metabolism. Mungbean (Vigna radiata) has long served as the plant model of choice while studying molecular programs followed during germination and seed development. In this study, we tested the effect of externally added MAO substrates epinephrine and H2O2 on storage protein mobilization in germinating seeds of Vigna radiata. The seeds were imbibed in the presence of 50 M epinephrine and 10 M H2O2. These low concentrations of the two compounds were used to exclude direct effects on proteolysis and were arrived at after testing a range of the two and choosing the most effective concentration. These seeds showed 11% and 7% decrease in fresh weight respectively, indicating greater storage mobilization and a corresponding 19% and 46% increase in axis length as compared to untreated seeds. Soluble protein in seeds treated with epinephrine and H2O2 decreased significantly by 34% and 33% as compared to untreated seeds. Electrophoretic analysis of seed proteins revealed a startling and selective depletion of storage proteins in treated seeds. The results indicated a clear involvement of H2O2 in storage protein mobilization in the cotyledons. We propose that H2O2 generated within cell walls of seeds serves as a signaling molecule guiding germination events, including protein reserve mobilization.

Grãos latino-americanos tradicionais: compostos polifenólicos, capacidade antioxidante e potencial anti-hiperglicêmico e anti-hipertensivo in vitro / Traditional latin-american grains: polyphenolic compounds, antioxidant capacity and anti-hyperglycemia and anti-hypertension potential in vitro

A incidência de doenças crônicas não transmissíveis como a diabetes tipo 2 e complicações cardiovasculares tem aumentado significativamente, e tem-se associado principalmente às mudanças nos hábitos alimentares tradicionais. O objetivo do presente estudo foi caracterizar diferentes cultivares de feijão, lupino e grãos da região dos Andes quanto a seus compostos fenólicos antioxidantes, capacidade antioxidante e potencial anti-hiperglicêmico e anti-hipertensivo in vitro. Dependendo do tipo de cultivar, o feijão é uma fonte promissora de taninos condensados, antocianinas, e flavonóis; enquanto que o lupino andino destacou-se pela presença de isoflavonas. Após o tratamento térmico, o feijão e lupino andino inibiram significativamente a enzima conversora da angiotensina 1, relevante na prevenção da hipertensão, enquanto o milho roxo andino inibiu a α-glicosidase, relevante na prevenção da hiperglicemia. Uma combinação apropriada de grãos tradicionais como parte da dieta poderia contribuir na modulação dos níveis de glicose e na prevenção das complicações relacionadas ao desequilíbrio óxido-redução...

Genetic diversity in some perennial plant species with-in short distances.

Distinct morphophysiological variations observed for over 2 years with-in short distances among four perennial plants indicated genetic diversity among the lines growing at three places. The isozyme and SDS polyacrylamide gel banding patterns as genetic markers were used to investigate four perennial species, namely Dalbergia sissoo Roxb., Delonix regia (Boj.) Refin., Cassia fistula L. and Calotropis procera R. Br. Plant materials collected from three locations (Agra, Gwalior and Lucknow) differing in climo-edaphic variables were examined for 4 enzyme systems, viz., esterase, polyphenol oxidase, peroxidase and superoxide dismutase (EST, PPO, PRX and SOD). Among the four isozymes SOD and PRX revealed best discriminating power. Protein banding patterns as well as zymogram revealed that Dalbergia sissoo growing at Gwalior was closer to that of Agra; Delonix regia depicted highest similarity between Lucknow and Agra and Calotropis procera of Lucknow location was more closer to Gwalior than Agra. The results confirm genetic diversity in the species as a means of adaptation to differing climo-edaphic variables.

Photosynthetic acclimation to elevated CO2 in relation to Rubisco gene expression in three C3 species.

Wheat (Triticum aestivum L. var. DL 1266-5), sunflower (Helianthus annuus L. var. MSFH 17) and mungbean [Vigna radiata (L.) Wilczek var. P 9072] were grown in field under atmospheric (360 +/- 10 cm3 m(-3), AC) and elevated (650 +/- 50 cm3 m(-3), EC) CO2 concentrations in open top chambers for entire period of growth and development. Photosynthetic acclimation to elevated CO2 was examined by comparing photosynthesis rate (Pn), Pn/Ci curves, leaf contents of RuBP carboxylase/oxygenase (Rubisco), change in the transcripts of Rubisco small subunit (SSU) gene and leaf carbohydrate constituents in AC and EC grown plants. The study indicated that photosynthetic acclimation to elevated CO2 concentration in wheat occurred because of down regulation of Rubisco, through limitation imposed on Rubisco SSU gene expression, as a consequence of sugar accumulation in the leaves. Leaf starch accumulators, sunflower and mungbean, showed no down regulation of Pn under EC. The Rubisco contents (%) in leaf soluble protein and rbcS transcript levels were not significantly affected in EC plants compared to AC plants of sunflower and mungbean. The study indicated that accumulation of excess assimilates in the leaves as starch was less inhibitory to Pn and would, therefore, be an important trait for sustenance of Pn not only under EC, but also under AC, where Pn inhibited by end products.

Increase in NADH-glutamate dehydrogenase activity by mercury in excised bean leaf segments.

Application of Hg to excised bean leaf segments increased the glutamate dehydrogenase (NADH-GDH) activity substantially. However, specific activity of the enzyme decreased at lower concentration of Hg, and increased to lesser extent at higher concentration of Hg. Mercury supply increased the glutamate synthase (NADH-GOGAT) activity also. Mercury supply increased the NADH-GDH activity in the presence of NH4NO3, but to a lesser extent than in the absence of NH4NO3. The specific activity of the enzyme decreased considerably at lower concentration of Hg, but increased significantly at higher concentration of Hg. An increase in NADH-GOGAT activity was observed in the presence of NH4NO3, but specific activity of the enzyme decreased marginally. Increase in GDH activity due to Hg remained unaffected by the supply of sucrose, but was reduced by glutamine and glutathione and enhanced by Al. The glutamate dehydrogenase (+Hg enzyme) from mercury treated leaf segments had higher value of S0.5 for NADH than the enzyme (-Hg enzyme) from material not treated with mercury indicating that Hg binding to enzyme prevented NADH binding to the enzyme possibly at thiol groups. However, + Hg enzyme has more reactivity, as apparent Vmax value was higher for it. It has been suggested that Hg activates the NADH-GDH enzyme in the bean leaf segments by binding to thiol groups of protein and pronounced increase in activity by Hg suggests a possible role of enzyme under Hg-stress.

Secondary stem anatomy and uses of four drought-deciduous species of a tropical dry forest in México

Wood and bark anatomy and histochemistry of Acacia bilimekii Humb. & Bonpl., Acacia cochliacantha Mcbride., Conzatia multiflora (Rob) Stand. and Guazuma ulmifolia Lam.are described from stem samples collected in a tropical dry forest (Morelos,Mexico). Enzyme activities were tested in tangential, radial and transverse cuts of fresh material. Histochemistry and stem anatomy were studied on similar cuts previously softened in a solution of water-glicerol-PEG. Our results show that the anatomical patterns of bark and wood, as well as the histochemical patterns and specific gravity, are influenced by water accessibility and climate; these patterns could guarantee mechanical and anti-infection strategies to support extreme conditions. Enzyme cytochemistry reveals biochemical activities probably related to lipid utilization routes for the lignification processes and for synthesis of extractives; these results suggest that the formation and maturation of woody tissue is very active at the beginning of the rainy season. These species are widely used by the local population. Traditional uses include firewood, dead and live fences, fodder, construction, supporting stakes, handcrafts, farming tools, extraction of tanning products, and medicine. There is no relationship between use and abundance. Alternative uses are proposed according to a density index

Se estudió la anatomía e histoquímica del tallo secundario de Acacia bilimekii, Acacia cochliacantha, Conzatia multiflora y Guazuma ulmifolia. Las muestras de tallo se colectaron en una selva baja caducifolia del estado de Morelos, México. La actividad enzimática se estudió en cortes frescos de caras tangenciales, radiales y transversales. La anatomía e histoquímica se hizo en cortes similares de muestras previamente ablandadas con una mezcla de agua-glicerol-PEG. Los resultados muestran que el patrón anatómico de la corteza y madera, así como las características histoquímicas no enzimáticas están relacionados con el acceso al agua y el clima; estos patrones garantizan que las estrategias mecánicas de resistencia al deterioro les permitan sobrevivir a condiciones extremas. Los resultados de la histoquímica y la citoquímica enzimática sugieren que la lignificación y la síntesis de extractivos a partir de los lípidos de reserva se encuentra activa desde el principio de la estación de lluvias. Se sugieren usos potenciales para las especies estudiads de acuerdo con las densidades relativas

Cysteine proteases in nodulation and nitrogen fixation.

The cysteine proteinases or cysteine endopeptidases (EC 3.4.22) are known to occur widely in plant cells. They are involved in almost all aspects of plant growth and development including germination, circadian rhythms, senescence and programmed cell death. They are also involved in mediating plant cell responses to environmental stress (such as water stress, salinity, low temperature, wounding, ethylene, and oxidative conditions) and plant-microbe interactions (including nodulation). In the development and function of legume root nodules, cysteine proteases could be involved in several important processes:-(i) a defence response to root invasion by microorganisms; (ii) protein turnover required during the formation of new tissue; (iii) cellular homeostasis and metabolism; (iv) adaptation of host cells to physiological stresses; (v) control of nodule senescence. Because of their central importance to plant physiology, cysteine proteases could serve as important targets for the study of nodule development and functioning at the molecular level. Because of their widespread occurrence in nodulating plants they could also serve as candidate genes for targeted plant breeding programmes.

Purification and partial characterization of Phaseolus vulgaris seed aminopeptidase

The aminopeptidase activity of Phaseolus vulgaris seeds was measured using L-Leu-p-nitroanilide and the L-aminoacyl-ß-naphthylamides of Leu, Ala, Arg and Met. A single peak of aminopeptidase activity on Leu-ß-naphthylamide was eluted at 750 µS after gradient elution chromatography on DEAE-cellulose of the supernatant of a crude seed extract. The effluent containing enzyme activity was applied to a Superdex 200 column and only one peak of aminopeptidase activity was obtained. SDS-polyacrylamide gel electrophoresis (10 per cent) presented only one protein band with molecular mass of 31 kDa under reducing and nonreducing conditions. The aminopeptidase has an optimum pH of 7.0 for activity on all substrates tested and the highest Vmax/KM ratio for L-Leu-ß-naphthylamide. The enzyme activity was increased 40 per cent by 0.15 M NaCl, inhibited 94 per cent by 2.0 mM Zn2+, inhibited 91 per cent by sodium p-hydroxymercuribenzoate and inhibited 45 per cent by 0.7 mM o-phenanthroline and 30 µM EDTA. Mercaptoethanol (3.3 mM), dithioerythritol (1.7 mM), Ala, Arg, Leu and Met (70 µM), p-nitroaniline (0.25 mM) and ß-naphthylamine (0.53 mM) had no effect on enzyme activity when assayed with 0.56 mM of substrate. Bestatin (20 µM) inhibited 18 per cent the enzyme activity. The aminopeptidase activity in the seeds decayed 50 per cent after two months when stored at 4oC and room temperature. The enzyme is leucyl aminopeptidase metal- and thiol group-dependent.

Nitrate levels and stages of growth in hypernodulating mutants of Lupinus Albus. II.Enzymatic activity and transport of N in the xylem SAP

The enzymatic study and transport of N in the xylem sap was carried out with a view to observing the influence of different nitrate levels and growth stages of the plant in chemically treated mutants of Lupinus albus. Several stresses induce a reduction in plant growth, resulting in the accumulation of free amino acids, amides or ureides, not only in the shoot, but also in the roots and nodules. Although enzyme activity is decisive in avoiding products that inhibit nitrogenase by ammonium, little is known about the mechanism by wich the xylem carries these products. However, this process may be the key to the function of avoiding the accumulation of amino acids in the cells of infected nodules. The behaviour of the enzymes nitrate reductase (NR), phosphoenolpyruvate carboxylase (PEPC), glutamine synthetase (GS) and nitrogen compounds derived from fixation, such as N-Ó-amino, N-ureides and N-amide in mutant genotypes were observed. The NR enzyme was highly influenced by the application of nitrate showing much higher values than those in the non-application of nitrate, independently of genotype, being that the NR, the best evaluation period was in the tenth week. The L-62 genotype characterized with nitrate-resistance, clearly showed that the enzyme PEPC is inhibited by presence of nitrate. The L-135 genotype (nor fix) showed GS activity extremely low, thus demonstrating that GS is an enzyme highly correlated with fixation. With regard to the best growth stage for GS, Lupinus albus should be evaluated in the seventh week.

Purification and characterization of neutral invertase from chickpea nodules.

Neutral invertase from nodules of chickpea (Cicer arietinum L.) was isolated and purified by ammonium sulphate fractionation, gel filtration and DEAE-cellulose column chromatography. The purified enzyme was stable between 0 to 40 degrees C beyond which it was irreversibly denatured. Optimum temperature and pH of the enzyme were 37 degrees C and 7.0, respectively. K(m) for sucrose was 14.2 mM and Vmax was 4.8 mumole hr-1. The enzyme was inhibited by several metal ions. From the temperature effect on K(m) and Vmax values, the energy of activation (Ea), enthalpy change (delta H) and entropy change (delta S) of the enzyme were calculated to be 147 kJmol-1, -4.10 kJmol-1 and -2.33 JK-1mol-1, respectively. By employing photo-oxidation and chemical modification and by studying the effect of pH on K(m) and Vmax, the involvement of sulphydryl-, imidazole- and alpha-amino groups in the active site of the enzyme has been indicated.

Regulatory properties and active site groups of cytosolic mung bean pyruvate kinase.

Properties of mung bean pyruvate kinase were studied and the active site groups were derived. Metabolites like AMP, glucose, glucose-6-phosphate, fructose-6-phosphate, fructose-1, 6-bisphosphate, 3-phospho-glycerate, isocitrate, malate and alpha-ketoglutarate had practically no effect on pyruvate kinase activity. Alanine, serine, glutamine, methionine and GMP had a weak activating effect on the enzyme. Some metabolites such as ATP, GTP, and UMP were found to be weakly inhibitory. Moderate to strong inhibition was observed with citrate, succinate, glutamate and oxalate. Inhibition brought about by ATP and citrate when present together showed synergistic effect. Inhibition by citrate was non-competitive with respect to both PEP and ADP suggesting the presence of a regulatory site. Mung bean pyruvate kinase showed half optimal activity at pH 6.6 and 8.9 at saturating concentrations of PEP, ADP and Mg2+. Small concentrations of the SH specific reagents, namely iodoacetamide (0.1 and 0.2 mM), N-ethylmaleimide(0.05-0.1 mM) and p-chloromercuribenzoate (0.1 mM) inactivated the enzyme; single exponential loss of activity was observed in each case. Photooxidation of the enzyme in the presence of methylene blue (100 and 200 micrograms/ml) and rose bengal (5 and 10 micrograms/ml) also led to a single exponential activity decay. When the enzyme was treated with diethyl pyrocarbonate (DEP), a time dependent exponential decay in its activity was observed with a parallel increase in absorbance at 240 nm. PEP protected the enzyme against inactivation by DEP. Reagents specific for tyrosine (iodine and tetranitromethane) and tryptophan residues (N-bromosuccinimide) residues had no effect. These observations confirm that SH and imidazole groups are vital for the activity of the enzyme.

Serine proteinase from rice bean.

A trypsin like serine-proteinase of M(r) 16,000 Da, optimally active at pH 8.4 on N-benzoyl-arginine ethyl ester (BAEE) was purified from 4-day old germinated seeds of rice bean, Vigna umbellata (Thunb), by ammonium sulphate precipitation, gel filtration, ion-exchange chromatography and by high performance liquid chromatography (HPLC). The purity of the enzyme was checked by polyacrylamide gel electrophoresis (PAGE). The enzyme activity was studied on natural substrates like casein, haemoglobin and vicilin, a rice bean storage protein. The activity of the enzyme was completely inhibited by phenylmethylsulfonyl fluoride, but not by iodoacetamide and HgCl2, suggesting it to be a serine protease. Loss of activity in presence of EDTA was reversed by addition of Ca2+.

Isolation and subcloning of chitinase clone from chickpea genomic library.

Chickpea genomic library constructed earlier in phage lambda (EMBL-3) was screened for the presence of chitinase clone using tobacco chitinase cDNA as a probe. Positive clones obtained by primary screening of plaques (2 x 10(6)) were ascertained by secondary and tertiary screening. Presence of chitinase insert in the positive clones obtained, was further confirmed by restricting phage DNA with Sal I and then doing southern with tobacco chitinase. The insert band was eluted out and subcloned in puc 19 plasmid.

Purification, characterisation and steady state kinetic properties of cytosolic pyruvate kinase free of phosphoenol pyruvate phosphatase activity from germinating mung beans (Vigna radiata L.)

Mung bean pyruvate kinase (PK) practically free from PEP-phosphatase has been purified about 36 fold. The enzyme is irreversibly inactivated on desalting by gel filtration or dialysis (without EDTA). The inactivation is also observed in the presence of ATP, Mg2+ or thiols but is prevented by a non-proteinous, heat stable, small molecular mass factor present in the mung bean extract. Mung bean PK has a molecular mass of 210 kDa. It shows single exponential decay of activity at various temperatures (-4 to 60 degrees C). The Km of PEP and ADP are found to be 0.12 and 0.24 mM, respectively at pH 6.5, when the enzyme is saturated with the second substrate. The Km values for PEP and ADP are 0.05 and 0.16 mM, at pH 8.5 and 0.09 and 0.17 mM, respectively at pH 7.5. The optimum pH is 7.5. The enzyme shows an absolute requirement for Mg2+ (Km 0.43 mM) or Mn2+ ions (Km 0.125 mM). Potassium ions are not essential but activate the enzyme in the presence of Mg2+ or Mn2+ ions. ATP shows competitive inhibition with ADP and non-competitive with PEP. Kinetic studies at different pHs and effects of ATP suggest the formation of a ternary complex (E.ADP.PEP) by a combination of random and compulsory ordered pathways depending on the experimental conditions.

Unique structural features of red kidney bean purple acid phosphatase.

Purple acid phosphatase from red kidney beans (Phaseolus vulgaris) has been purified to homogeneity and characterized. The enzyme is a homodimer of 60 kDa subunits each containing one atom of zinc and iron in the active site. Circular dichroism spectral studies on the purified enzyme reveals that a large portion of the peptide backbone is in the unordered and beta-turn conformation. A unique feature of the red kidney bean acid phosphatase, which we have found, is that one of the two cysteines of each subunit is involved in the formation of an inter-subunit disulphide. The thiol group of the other cysteine is not necessary for the activity of the enzyme. Western blot analysis with antibodies raised against kidney bean acid phosphatase could not recognize acid phosphatases from other sources except from potato. This paper emphasizes the fact that acid phosphatases are functionally, but not structurally, conserved enzymes.

Inactivation of glyceraldehyde-3-phosphate dehydrogenase with SH-reagents and its relationship to the protein quaternary structure.

Inactivation of mung bean glyceraldehyde-3-phosphate dehydrogenase (GPDH) with excess iodoacetate or N-ethylmaleimide exhibits pseudo-first order kinetics at pH 7.3 and 8.6 in the absence and presence of NAD+, suggesting that all the reactive SH groups (four per tetrameric GPDH molecule) have equivalent reactivity towards these reagents. This is similar to the D2-symmetry conformation proposed on the basis of thermal inactivation data [Malhotra and Srinivasan, Arch. Biochem. Biophys. 236, 775-781 (1985)]. With p-chloromercury benzoate (p-CMB), the inactivation of GPDH is very fast and its kinetics can be monitored at low reagent concentration only. Keeping a high molar p-CMB: enzyme ratio (= 47), the kinetics were found to be biphasic, with half of the activity being lost in a fast and the remaining in a slow phase, characteristic of C2-symmetry conformation and half site reactivity. The p-CMB inactivation could be largely reversed on the addition of excess cysteine. A comparison of these data with literature reports on this and other GPDHs reveals that all reagents having large non-polar moieties exhibit half site reactivity with this enzyme.

Effect of substrate and phosphate ions on the quaternary structure symmetry of glyceraldehyde-3-phosphate dehydrogenases of mung beans and rabbit muscle.

Effects of glyceraldehyde-3-phosphate (G-3-P) and phosphate ions on thermal inactivation of glyceraldehyde-3-phosphate dehydrogenases (GPDHs) of mung beans and rabbit muscle have been studied at different pH. In the absence of any ligand, the two enzymes show a striking similarity in the pH-dependence of the kinetics of thermal inactivation. At lower pH values both the enzymes biphasic kinetics with each phase accounting for about half of the starting activity (a C2 symmetry of the homotetrameric enzyme molecule). The kinetics change to a single exponential decay at higher pH values, a D2 symmetry [Malhotra & Srinivasan (1985) Arch. Biochem. Biphys. 236, 775-781; Malhotra & Tikoo (1991) Indian, J. Biochem. Biophys. 28, 16-21]. With each enzyme, phosphate ions are found to have no effect on the kinetic pattern at lower pH, but G-3-P brings about a change from biphasic to a single exponential decay. At higher pH values, G-3-P has no effect on the single exponential decay kinetic pattern, but phosphate ions change the same to a biphasic loss of activity with each phase accounting for about half of the starting activity. It has been concluded that with both the enzymes, G-3-P and phosphate ions have higher affinity and stabilise the D2- and C2-symmetry conformation, respectively. Binding isotherms of the two substrates for these enzymes have been described based on the ligand concentration-dependence of the changes in the rate constants and kinetic pattern of thermal inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of coenzymes on the quaternary structure conformation of glyceraldehyde-3-phosphate dehydrogenases of mung beans and rabbit muscle.

Kinetics of thermal inactivation of glyceraldehyde-3-phosphate dehydrogenases of mung beans and rabbit muscle have been studied under different pH conditions in the absence and presence of various concentrations of NAD+ and NADH. The data have been discussed with respect to the effect of the coenzymes on the quaternary structure symmetry of the two enzymes and their binding isotherms. Both the (homo-tetrameric) apo-enzymes exhibit biphasic kinetics of thermal inactivation, characteristic of C2 symmetry, at lower pH values and a single exponential decay of enzyme activity, characteristic of D2 symmetry, at higher pHs. In each case, NAD+ has no effect on the biphasic kinetic pattern of thermal inactivation at lower pH values, but NADH brings about a change to single exponential decay. At higher pH values, NADH does not affect the kinetic pattern (single exponential decay) of any enzyme, but NAD+ alters it to biphasic kinetics in each case. The data suggest that NAD+ and NADH have higher affinity for the C2 and D2 symmetry conformation, respectively. With mung beans enzyme, the effect of NAD+ on the two rate constants of biphasic inactivation at pH 7.3 is consistent with a Kdiss equal to 110 microM. The NAD(+)-dependent changes in the kinetic pattern of thermal inactivation of this enzyme at pH 8.6 suggest a positive cooperativity in the coenzyme binding (nH = 3.0). In the binding of NADH to the mung beans enzyme, a weak positive cooperativity is observed at pH 7.3.(ABSTRACT TRUNCATED AT 250 WORDS)