Browsing by Author "P.K. Ambasht"

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Accumulation of partly folded states in the equilibrium unfolding of ervatamin A: Spectroscopic description of the native, intermediate, and unfolded states
(2007) Sreedevi Nallamsetty; Vikash K. Dubey; Monu Pande; P.K. Ambasht; M.V. Jagannadham
Ervatamin A, a cysteine proteases from Ervatamia coronaria, has been used as model system to examine structure-function relationship by equilibrium unfolding methods. Ervatamin A belongs to α+β class of proteins and exhibit stability towards temperature and chemical denaturants. Acid induced unfolding of ervatamin A was incomplete with respect to the structural content of the enzyme. Between pH 0.5 and 2.0, the enzyme is predominantly in β-sheet conformation and shows a strong ANS binding suggesting the existence of a partially unfolded intermediate state (IA state). Surprisingly, high concentrations of GuHCl required to unfold this state and the transition mid points GuHCl induced unfolding curves are significantly higher. GuHCl induced unfolding of ervatamin A at pH 3.0 as well as at pH 4.0 is complex and cannot be satisfactorily fit to a two-state model for unfolding. Besides, a strong ANS binding to the protein is observed at low concentration of GuHCl, indicating the presence of intermediate in the unfolding pathway. On the other hand, even in the presence of urea (8 M) the enzyme retains all the activity as well as structural parameters at neutral pH. However, the protein is susceptible to urea unfolding at pH 3.0 and below. Urea induced unfolding of ervatamin A at pH 3.0 is cooperative and the transitions curves obtained by different probes are and non-coincidental. Temperature denaturation of ervatamin A in IA state is non-cooperative, contrary to the cooperativity seen with native protein, suggesting the presence of two parts in the molecular structure of ervatamin A may be domains, with different stability that unfolds in steps. Careful inspection of biophysical properties of intermediate states populated in urea and GuHCl (IUG state) induced unfolding suggests all these three intermediates are identical and populated in different conditions. However, the properties of the intermediate (IA state) identified at pH ∼1.5 are different from those of the IUG state. © 2007 Elsevier Masson SAS. All rights reserved.
An Assay Procedure for Determining the Rate of an Enzyme Reaction Lacking an Optical Signal: Validity of Coupled Enzyme Assays
(Elsevier Ltd, 1996) O.P. Malhotra; P.K. Ambasht; Prakash Prabhakar; Ashish K. Lal; Arvind M. Kayastha
[No abstract available]
Plant pyruvate kinase
(2002) P.K. Ambasht; Arvind M. Kayastha
Pyruvate kinase is an important enzyme of glycolytic pathway that also functions in providing carbon skeleton for fatty acid biosynthesis. It has been purified to near homogeneity from Ricinus communis, Selenastrum minutum, Cynodon dactylon, Brassica campestris and B. napus, and characterised. Partially purified preparations are reported from several other sources. A phosphoenolpyruvate (PEP) phosphatase accompanies pyruvate kinase. In plants, two isozymes of pyruvate kinase are reported, namely cytosolic and plastidic. Isoforms of cytosolic pyruvate kinase have also been reported from spinach. In most cases pyruvate kinase is a tetrameric protein and the molecular mass lies between 200 to 250 kDa. The pH optimum is in the range of 6.2 to 7.5. It requires both Mg2+ and K+ for maximum activity. ATP, citrate, and oxalate inhibit pyruvate kinase in most cases. A sequential compulsory ordered mechanism of binding of substrates to the enzyme has been proposed.
Purification, characterisation and steady state kinetic properties of cytosolic pyruvate kinase free of phosphoenol pyruvate phosphatase activity from germinating mung beans (Vigna radiata L.)
(1996) P.K. Ambasht; O.P. Malhotra; Arvind M. Kayastha
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°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.
Regulatory properties and active site groups of cytosolic mung bean pyruvate kinase
(1997) P.K. Ambasht; O.P. Malhotra; Arvind M. Kayastha
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 α-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 μg/ml) and rose bengal (5 and 10 μg/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.