Maximum Velocity Of Hydrolysis Research Articles

Using of silica particles as porogen for preparation of macroporous chitosan macrospheres suitable for enzyme immobilization

Porous chitosan macrospheres were prepared at the first time by using silica particles as porogen, and the optimal weight ratio of silica to chitosan during preparation was determined. Scanning electron microscopy micrographs showed that the support with silica as porogen (support I) had a much more porous surface structure than the support without porogen (support II). Both supports were applied to immobilize β-galactosidase from Aspergillus oryzae. Much higher specific activity and yield of galactose hydrolysis products were observed for the support I. Properties of both immobilized enzyme were determined and compared with the free enzyme, satisfactory results were obtained in thermostability, pH arid operational stability, Michaelis constants K m and in maximum velocity of hydrolysis (V m). Suggested method allow to prepare chitosan macrospheres as immobilized enzyme carrier with moreporous surface structure and more active reaction groups.

Detection of in vivo proteasome activity in a starfish oocyte using membrane-impermeant substrate.

A method was investigated for monitoring the activity of protease(s) in cytosol of a single starfish oocyte using succinyl-Phe-Leu-Arg-coumarylamido-4-methanesulfonic acid as the substrate, which was injected into the cell. After preincubation of immature oocytes with a proteasome inhibitor, N-carbobenzoxy-L-leucinyl-L-leucinyl-L-norvalinal, the initial hydrolysis of the substrate was remarkably inhibited. The inhibitor blocked 1-methyladenine-triggered cyclin degradation, which is known to be mediated by proteasome. However, calpain inhibitor E-64 did not inhibit the hydrolysis of the substrate. These results suggested that the protease activity measured by this method is mainly attributable to cytoplasmic proteasome. The hydrolysis of the substrate was partially inhibited by bestatin, suggesting that the substrate was cleaved by aminopeptidase. Thus, the initial velocity of hydrolysis of the substrate (V0) by proteasome was assayed in a living oocyte after preinjection of bestatin. The values of V0 increased gradually after 1-methyladenine addition and reached the maximum level at the time corresponding to cyclin degradation. The calculated maximum velocity of hydrolysis by a mature oocyte was approximately three times higher than that by an immature oocyte. The Michaelis-Menten constant value was also higher in mature than immature oocytes. These results suggest that proteasome-dependent proteolysis is regulated not only by ubiquitination of substrates, as is generally believed, but also by the proteasome activity itself.

Modification of the kinetic properties of Triton-solubilized rabbit brain acetylcholinesterase by allosteric effectors at low ionic strength

Gallamine and tubocurarine increased the rate of decarbamylation of carbamylated Triton-solubilized rabbit brain acetylcholinesterase and interacted synergistically with 3,3-dimethyl-1-butanol in the acceleration of decarbamylation at low ionic strength. Gallamine and tubocurarine also accelerated decarbamylation at a physiological ionic strength, but in this case the interaction with 3,3-dimethyl-1-butanol was not synergistic. Tubocurarine decreased the rate of ageing of isopropylmethyl-phosphonyl-acetylcholinesterase at low ionic strength, but gallamine and tubocurarine had no effect on ageing at a physiological ionic strength, nor did gallamine at low ionic strength. However reductions in the rate of ageing were observed for gallamine/3,3-dimethyl-1-butanol and tubocurarine/3,3-dimethyl-1-butanol mixtures at low ionic strength. Gallamine increased the maximum velocity of hydrolysis of acetylthiocholine at low ionic strength, but not at physiological ionic strength. Gallamine increased the rate of carbamylation of acetylcholinesterase by physostigmine and neostigmine at low ionic strength; however the data were not consistent with a simple model of complexing inhibition by these carbamates. Overall, the kinetic properties of Triton-solubilized rabbit brain acetylcholinesterase were less sensitive to modification by proposed allosteric effectors than bovine erythrocyte acetylcholinesterase, the allosteric properties of which have been reported previously, and millimolar concentrations of gallamine and tubocurarine were required to produce observable effects at physiological ionic strength. Further experiments are required to determine whether acetylcholinesterase is similarly insensitive to allosteric regulation in vivo.

Purification and properties of arylsulfatase of Klebsiella aerogenes identity of the enzymes formed by non-repressed and de-repressed synthesis.

Intracellular arylsulfatases from Klebsiella aerogenes W 70 cells grown in methionine medium (M enzyme) and inorganic sulfate medium containing tyramine (T enzyme) were purified respectively by fractionation with (NH4)2SO4, followed by successive chromatographies on DEAE cellulose, hydroxylapatite, Sephadex G-100 and DEAE Sephadex A-25. On polyacrylamide gel electrophoresis, the two enzymes gave single bands with the same mobilities. Molecular weights of both, determined by SDS gel electrophoresis and by Sephadex G-100 chromatography, were 47, 000 and 45, 000, respectively. Their activities were maximal at pH 7.5. The affinities of the enzymes (M and T enzymes) for their substrate (Km) and the maximum velocity of hydrolysis (Vmax) were enhanced by addition of electron withdrawing substituents. The enzymes were inhibited by inorganic phosphate, cyanide, hydroxylamine and tyramine. The inhibition by tyramine was competitive (Ki=1.0×10-4 M). These results show that the two enzymes were identical. This was confirmed by the fact that mutant strains, which were unable to synthesize arylsulfatase when grown with methionine, could also not synthesize the enzyme when grown with tyramine.