ABSTRACT
Effect of Na+ ions on yeast invertase activity has been studied as a function of pH. At acidic pH, Na+ (5-100 mM) had no effect but invertase activity was inhibited (38-44%) by Na+ ions (100 mM) with an increase in pH (6.8 and 8.0). Kinetic analysis revealed that invertase inhibition by Na+ ions was non-competitive and reversible in nature. Value of K(m) remained unaltered (33.3 mM) in presence of Na+ (20-100 mM) while Vmax decreased by 21-44% under these conditions. Value of Ki was of the order of 85 mM. Mechanism of observed inhibition of invertase activity as a consequence of Na+ ions interactions at the active site of the enzyme has been described.
Subject(s)
Glycoside Hydrolases/antagonists & inhibitors , Hydrogen-Ion Concentration , Immune Sera , Kinetics , Saccharomyces cerevisiae/enzymology , Sodium/metabolism , beta-FructofuranosidaseABSTRACT
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.
Subject(s)
Binding Sites , Cations/pharmacology , Enzyme Inhibitors/pharmacology , Enzyme Stability , Fabaceae/enzymology , Glycoside Hydrolases/antagonists & inhibitors , Hydrogen-Ion Concentration , Kinetics , Plants, Medicinal , Sulfhydryl Reagents/pharmacology , Thermodynamics , beta-FructofuranosidaseABSTRACT
A ceramide glycanase (CGase activity has been characterized from lactating rat mammary tissue which cleaves the glycosidic bond between sphingosine and the glucose chain of a glycosphingolipid (GSL) thus liberating the intact oligosaccharide chain from a GSL. The majority (65%) of the hydrolase activity was detected in the supernatant fraction when the rat mammary tissue homogenate was centrifuged at 100,000 x g. Attempts to purify the enzyme indicated that the CGase protein is of hydrophobic nature as it binds to hydrophobic columns. The enzyme has been partially purified using hydrophobic columns in tandem. The partially purified protein was found to be immunoreactive to the antibody raised against the purified clam CGase. The immunostained band corresponded to a 64 kDa protein as also found with the clam enzyme. This immuno cross-reactivity indicated probable structural similarities between CGase proteins isolated from widely separated species in the evolutionary tree. The rat CGase was found to have a specific detergent requirement for optimal activity, and the pH optimum was found to be between 5 and 6. The enzyme activity is partially heat stable. It is not a divalent cation requiring enzyme; however, the activity is totally inhibited in the presence of mercury, indicative of a sulfhydryl group in the active site of the enzyme. The rat mammary CGase activity is inhibited in the presence of both D- and L-PPMP (1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol. HCl), homologs of PDMP (1-phenyl-2-decanoylamino-3-morpholino-1-propanol. HCl), a well-known inhibitor of GlcT-1 (Ceramide: UDP-Glc Glucosyltransferase), an enzyme in the glycolipid synthetic pathway. The inhibition seems to be of a competitive nature and the same type of inhibition is also observed with clam CGase. The CGase activity was found to be highest in lactating tissue compared to the activity found in either pregnant or post-lactating rat mammary tissues. Tissue survey indicated the presence of high levels of CGase in lactating rat liver, uterus, and ovary; moderate activity was detected in kidney and spleen. Both virgin and male rat mammary tissue also indicated a basic level of CGase activity. However, newborn spleen and mammary tissue showed a comparable level of activity to that found in lactating rat tissues. This report is mainly concerned with the characterization of CGase activity from a mammalian source and its importance in cellular processes.