scholarly journals Affinity of extracellular phosphatases for ELF97 phosphate in aquatic environments

2007 ◽  
Vol 58 (5) ◽  
pp. 454 ◽  
Author(s):  
Jiří Nedoma ◽  
France Van Wambeke ◽  
Alena Štrojsová ◽  
Martina Štrojsová ◽  
Solange Duhamel
Keyword(s):  
Pure Culture ◽  
Marine Bacterium ◽  
Aquatic Environments ◽  
Kinetic Properties ◽  
Michaelis Constant ◽  
Lower Affinity ◽  
Saturation Kinetics ◽  
Elf97 Phosphate ◽  
Hydrolysis Of ◽  
Phosphatase Substrate

Recently, the phosphatase substrate ELF97 phosphate (ELFP) has been employed to study the presence of extracellular phosphatases in different plankton populations in natural aquatic environments. Kinetic properties of ELFP hydrolysis by natural extracellular phosphatases are, however, mostly unknown. We indirectly studied the affinity of extracellular phosphatases for ELFP in different aquatic environments through its ability to inhibit the hydrolysis of 4-methylumbelliferyl phosphate (4MUP). Values of inhibition constants, Ki, which correspond to the concentrations necessary for half saturation of phosphatases by ELFP, were lowest (0.18–4.5 µmol L–1) in the oligotrophic Mediterranean Sea. We found higher values (i.e. lower affinity) in oligo- to mesotrophic acidified lakes (5.2–14 µmol L–1), in a eutrophic reservoir (13–35 µmol L–1) and in a pure culture of the marine bacterium Alteromonas infernus (29 µmol L–1). ELFP had a pronounced effect on the parameter KM (Michaelis constant) of 4MUP saturation kinetics, while its effect on the parameter Vmax was low. This behaviour is compatible with the assumption of competitive interaction between 4MUP and ELFP. Our experiments indicated that the assay ELFP concentration in the detection kit used was 250–500 µmol L–1 (after the recommended dilution to a ratio of 1:20), which would ensure >99% saturation of extracellular phosphatases in marine environments and >90% saturation in the studied fresh waters.

scholarly journals Kinetic properties of microtubule-activated 13 S and 21 S dynein ATPases251. Evidence for allosteric behaviour associated with the inner row and outer row dynein arms

1986 ◽  
Vol 83 (1) ◽  
pp. 251-267
Author(s):  
F.D. Warner ◽  
J.H. McIlvain
Keyword(s):  
Atpase Activity ◽  
Kinetic Parameters ◽  
Turnover Rate ◽  
Kinetic Properties ◽  
Significant Shift ◽  
Subunit Interactions ◽  
Michaelis Constant ◽  
Saturation Kinetics ◽  
Dynein Arms ◽  
Fixed Quantity

The 13 S and 21 S dynein ATPases from Tetrahymena cilia rebind to extracted doublet microtubules as inner row and outer row arms. Rebinding is accompanied by four- to ninefold activation of the ATPase activity. The soluble (microtubule-free) forms of the two dyneins exhibit simple saturation kinetics (h = 1.0) with Vmax much less than mumol Pi mg-1 min-1 and Km = 20–40 microM-ATP. Mixing a fixed quantity of free dynein with increasing concentrations of extracted doublets results in systematic increases in all three kinetic parameters for each dynein. At infinite concentrations of doublets and ATP, each enzyme undergoes a significant shift to sigmoid saturation kinetics (h = 2–3), Vmax increases to a turnover rate of about 90 mol ATP per mol Es-1 and the Michaelis constant increases to much greater than 100 microM-ATP. These data suggest that both enzymes are allosteric and can be interpreted in terms of positive cooperativity relative to a minimum of two or three interacting sites. It is less clear whether this cooperativity is related to subunit interactions within the 21 S or 13 S particles, or to subunit interactions between adjacent particles (arms) on the microtubule lattice.

Enzymatic Hydrolysis of PET: Structural Diversity and Kinetic Properties of Cutinases from Thermobifida

2014 ◽  
Vol 31 ◽  
pp. S88
Author(s):  
Altijana Hromic ◽  
Doris Ribitsch ◽  
Andrzej Lyskowski ◽  
Georg Steinkellner ◽  
Helmut Schwab ◽  
...  
Keyword(s):  
Enzymatic Hydrolysis ◽  
Structural Diversity ◽  
Kinetic Properties ◽  
Hydrolysis Of

scholarly journals Inhibition of Amino Acid Transport in Rabbit Intestine by p-Chloromercuriphenyl Sulfonic Acid

1973 ◽  
Vol 62 (2) ◽  
pp. 131-146 ◽  
Author(s):  
John F. Schaeffer ◽  
Robert L. Preston ◽  
Peter F. Curran
Keyword(s):  
Transport System ◽  
Conformational Changes ◽  
Marked Reduction ◽  
Sulfhydryl Group ◽  
Major Effect ◽  
Acid Transport ◽  
Michaelis Constant ◽  
Saturation Kinetics ◽  
Rabbit Intestine ◽  
Transport Site

Influx of phenylalanine across the brush border of rabbit intestine is markedly reduced by treatment with 5 mM p-chloromercuriphenyl sulfonate (PCMBS). The effect is rapidly and completely reversed by dithiothreitol. Phenylalanine influx into PCMBS-treated tissue can be competitively inhibited by other neutral amino acids and follows saturation kinetics. PCMBS causes an increase in the apparent Michaelis constant from the value observed in control tissue but does not alter the maximal influx significantly. Treatment of the tissue with PCMBS leads to a significant reduction in the Na-sensitivity of the transport, and a number of results indicate that the major effect of the reagent is to cause a marked reduction in the affinity of the transport system for Na. The transport system can be partially protected against reaction with PCMBS by phenylalanine and tryptophan but not by methionine or norleucine. The results suggest that PCMBS reacts with a sulfhydryl group in the region of the transport site and may alter conformational changes associated with the binding of substrates.

P-Nitrophenol-alpha-D-glucopyranoside as substrate for measurement of maltase activity in human semen.

1978 ◽  
Vol 24 (2) ◽  
pp. 208-211 ◽  
Author(s):  
P Chapdelaine ◽  
R R Tremblay ◽  
J Y Dubé
Keyword(s):  
Seminal Plasma ◽  
Reduced Glutathione ◽  
Rapid Determination ◽  
Potassium Phosphate ◽  
Potassium Phosphate Buffer ◽  
Michaelis Constant ◽  
Maltase Activity ◽  
Constant Rate ◽  
Hydrolysis Of

Abstract Hitherto, seminal plasma maltase has been measured with maltose as substrate; this method is time consuming and lacks specificity. The use of a synthetic substrate, p-nitrophenol-alpha-D-glucopyranoside, allows accurate and rapid determination of this activity. When maltase is added to the incubation medium (the substrate and reduced glutathione in potassium phosphate buffer, pH 6.8), maintained at 37 degrees C, hydrolysis of the original substrate to p-nitrophenol goes at a constant rate during 4 h. Under optimal conditions of incubation, the Michaelis constant of the reaction, calculated by the Hanes method, was 2.92 +/- 0.84 (SD) X 10(-3) for six different semen samples. Isomaltase appeared to be absent from seminal plasma. The enzyme is stable to freezing and slow thawing and can be stored for at least 26 days at -80 degrees C. Its molecular weight is 259 000. Tris(hydroxymethyl)aminomethane (pH 6.8) exerts a noncompetitive inhibition on the enzyme activity. In 68 men 23 to 45 years old, whose semen analyses were normal, the seminal plasma maltase activity was 467 +/- 135 (SD) mU/g of protein. It was generally decreased in patients with infertility disorders.

scholarly journals Active-site modification of native and mutant forms of inosine 5′-monophosphate dehydrogenase from Escherichia coli K12

1980 ◽  
Vol 191 (2) ◽  
pp. 533-541 ◽  
Author(s):  
Harry J. Gilbert ◽  
William T. Drabble
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Enzyme Inactivation ◽  
Enzyme Hydrolysis ◽  
Thiol Groups ◽  
Imp Dehydrogenase ◽  
Nitrobenzoic Acid ◽  
Saturation Kinetics ◽  
Mutant Forms ◽  
Hydrolysis Of

IMP dehydrogenase of Escherichia coli was irreversibly inactivated by Cl-IMP (6-chloro-9-β-d-ribofuranosylpurine 5′-phosphate, 6-chloropurine ribotide). The inactivation reaction showed saturation kinetics. 6-Chloropurine riboside did not inactivate the enzyme. Inactivation by Cl-IMP was retarded by ligands that bind at the IMP-binding site. Their effectiveness was IMP>XMP>GMP»AMP. NAD+ did not protect the enzyme from modification. Inactivation of IMP dehydrogenase was accompanied by a change in λmax. of Cl-IMP from 263 to 290nm, indicating formation of a 6-alkylmercaptopurine nucleotide. The spectrum of 6-chloropurine riboside was not changed by IMP dehydrogenase. With excess Cl-IMP the increase in A290 with time was first-order. Thus it appears that Cl-IMP reacts with only one species of thiol at the IMP-binding site of the enzyme: 2–3mol of Cl-IMP were bound per mol of IMP dehydrogenase tetramer. Of ten mutant enzymes from guaB strains, six reacted with Cl-IMP at a rate similar to that for the native enzyme. The interaction was retarded by IMP. None of the mutant enzymes reacted with 6-chloropurine riboside. 5,5′-Dithiobis-(2-nitrobenzoic acid), iodoacetate, iodoacetamide and methyl methanethiosulphonate also inactivated IMP dehydrogenase. Reduced glutathione re-activated the methanethiolated enzyme, and 2-mercaptoethanol re-activated the enzyme modified by Cl-IMP. IMP did not affect the rate of re-activation of methanethiolated enzyme. Protective modification indicates that Cl-IMP, methyl methanethiosulphonate and iodoacetamide react with the same thiol groups in the enzyme. This is also suggested by the low incorporation of iodo[14C]acetamide into Cl-IMP-modified enzyme. Hydrolysis of enzyme inactivated by iodo[14C]acetamide revealed radioactivity only in S-carboxymethylcysteine. The use of Cl-IMP as a probe for the IMP-binding site of enzymes from guaB mutants is discussed, together with the possible function of the essential thiol groups.

scholarly journals Carbonic anhydrase isoenzymes in the erthrocytes and dorsolateral prostate of the rat

1969 ◽  
Vol 114 (3) ◽  
pp. 463-476 ◽  
Author(s):  
J. E. A. McIntosh
Keyword(s):  
Carbonic Anhydrase ◽  
High Activity ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Exchange Chromatography ◽  
Kinetic Properties ◽  
Efficient Catalyst ◽  
Molecular Weights ◽  
Hydrolysis Of ◽  
Low Activity

1. Three forms of the zinc-containing enzyme carbonic anhydrase (EC 4.2.1.1) were isolated from the erythrocytes of the rat and two forms from the dorsolateral prostate of the rat. Several additional minor components were observed but not isolated. Separation of the isoenzymes was achieved by ion-exchange chromatography, polyacrylamide-gel electrophoresis and isoelectric focusing. 2. The general properties of the isolated isoenzymes, their molecular weights and their contents of zinc were closely similar. As catalysts of the hydration of carbon dioxide, however, they were distinctly different. The two most abundant isoenzymes of the erythrocytes, which were found in equal proportions, differed 70-fold in specific activity, whereas the isoenzymes of the dorsolateral prostate were similar to one another and resembled the high-activity component of the erythrocytes. The inhibition of the latter by acetazolamide (5-acetamido-1-thia-3,4-diazole-2-sulphonamide) was mainly competitive, whereas in identical conditions the low-activity erythrocyte component and the dorsolateral prostate isoenzymes were non-competitively inhibited. 3. The use of chloroform–ethanol to remove haemoglobin from the rat haemolysate was found (a) to bring about changes in the kinetic properties of the soluble isoenzymes and (b) to cause the appearance of an additional isoenzyme. 4. The actions were compared of the inhibitors acetazolamide, 1,1-dimethylaminonaphthalene-5-sulphonamide and ethoxzolamide (6-ethoxybenzothiazole-2-sulphonamide) on the hydrolysis of p-nitrophenyl acetate catalysed by the isoenzymes. 5. The low-activity erythrocyte isoenzyme was an efficient catalyst of the hydrolysis of β-naphthyl acetate whereas the high-activity forms were much less active towards this ester. Neither of the isoenzymes present in the dorsolateral prostate catalysed this reaction. 6. Carbonic anhydrase in the rat dorsolateral prostate accounts for no more than 5% of the unusually high content of zinc in this organ.

Copper transport and kinetics in cultured C6 rat glioma cells

1995 ◽  
Vol 269 (4) ◽  
pp. C892-C898 ◽  
Author(s):  
Y. Qian ◽  
E. Tiffany-Castiglioni ◽  
E. D. Harris
Keyword(s):  
Membrane Fraction ◽  
Glioma Cells ◽  
Maximum Velocity ◽  
C6 Cells ◽  
Michaelis Constant ◽  
Rat Glioma ◽  
Saturation Kinetics ◽  
Efflux Mechanism ◽  
Import And Export ◽  
P Type

C6 rat glioma cells accumulate and efflux 67Cu. Both processes showed saturation kinetics with increasing 67Cu concentration. The Michaelis constant (Km) for uptake was 0.63 +/- 0.14 microM; maximum velocity (Vmax) was 3.29 +/- 0.57 pmol Cu.mg protein-1.min-1. The Km for efflux was 0.15 +/- 0.06 microM; Vmax was 1.08 +/- 0.71 pmol Cu.mg protein-1.min-1. p-Chloromercuribenzoate (p-CMB) totally blocked 67Cu efflux but had no effect on Km or Vmax of uptake. Total 67Cu in the cell after 50 min was partitioned equally between particulate and soluble fractions. p-CMB-treated cells accumulated more 67Cu, but < 10% was bound to the particulate (membrane) fraction. Pb also increased 67Cu accumulation without affecting Km and Vmax of 67Cu uptake. These data suggest that carriers for import and export of Cu in C6 cells are distinct or operate in two different cellular environments. Efflux is a sulfhydryl-dependent process subject to inhibition by Pb. The data are consistent with a P-type ATPase in the efflux of Cu from cells and the potential for Pb to inhibit the efflux mechanism.

Kinetic properties of the α-lytic protease of Sorangium sp., a bacterial homologue of the pancreatopeptidases

1969 ◽  
Vol 47 (3) ◽  
pp. 305-316 ◽  
Author(s):  
H. Kaplan ◽  
D. R. Whitaker
Keyword(s):  
Methyl Ester ◽  
Catalytic Properties ◽  
Substrate Binding ◽  
Kinetic Properties ◽  
Histidine Residue ◽  
Amino Groups ◽  
Binding Properties ◽  
Histidine Residues ◽  
Support Reaction ◽  
Hydrolysis Of

The kinetics under consideration are those of a bacterial serine protease with the same "active serine" sequence as chymotrypsin, trypsin, and elastase, and with a single histidine residue in a sequence which closely matches the sequences around histidine-57 of chymotrypsin and the analogous histidine residues of trypsin and elastase. In agreement with previous evidence of an elastase-like specificity, esters of N-substituted, neutral, aliphatic L-amino acids proved to be good to excellent substrates for the α-enzyme; esters of arginine, tyrosine, and tryptophan were not hydrolyzed. The enzyme has a much higher activity than the pancreatopeptidases towards p-nitrophenyl acetate and p-nitrophenyl trimethyl acetate; the catalytic rate coefficient kc for the latter substrate is about fivefold greater than that of elastase.The catalytic properties match those of the pancreatopeptidases in the following respects. As demonstrated with N-acetyl-L-valine methyl ester as substrate, kc is dependent on an ionization with a pKa of 6.7 in water and 7.3 in H22O; Δ log (kc/Km)/ΔpH for this ionization is equal to 1.0; kc is reduced 50% when H2O is replaced by H22O. These findings are consistent with a requirement for a single unprotonated histidine residue and general basic catalysis by that residue. The burst of p-nitrophenol in hydrolyses of p-nitrophenyl trimethyl acetate is proportional to [E]0; the magnitude of the proportionality factor and the rate of attainment of a steady state are consistent with the condition [Formula: see text], as in chymotrypsin kinetics. Thus the purely catalytic properties of the α-enzyme match those of chymotrypsin very closely. These findings do not support reaction mechanisms which require two catalytically functional histidine residues for such catalysis. The substrate-binding properties of the α-enzyme differ from those of chymotrypsin in that substrate binding does not depend on ionization of an N-terminal α-amino group; Km for the hydrolysis of N-acetyl-L-valine methyl ester is constant from pH 5 to pH 10 and enzymatic activity is unaffected by acetylation of the enzyme's α- and ε-amino groups. Ks for the hydrolysis of p-nitrophenyl trimethyl acetate is appreciably greater than the Ks of elastase for this substrate.The chloromethyl ketones of glycine and valine did not inhibit the enzyme or alkylate its histidine residue.

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