Change In Affinity Of Enzyme Research Articles

Effect of individual plasma components on the performance of a glucose enzyme electrode based on redox polymer mediation of a flavin adenine dinucleotide-dependent glucose dehydrogenase

The performance of glucose enzyme electrodes, consisting of crosslinked flavin adenine dinucleotide glucose dehydrogenase (FADGDH), an osmium redox polymer and multi-walled carbon nanotubes on graphite electrodes, was tested in phosphate buffered saline, artificial plasma and the individual components of artificial plasma to assess the effect of each component on current response and operational stability of the response to better understand the decrease in electrode performance observed in blood. Electrodes tested in artificial plasma show a significant decrease in current response in 5 mM glucose, and operational stability of the response in 100 mM glucose, compared to electrodes tested in buffer. The lowest current response for the enzyme electrodes was observed in the presence of physiological level of uric acid although the largest alteration to enzyme affinity, as estimated from the apparent Michaelis-Menten constant, occurred upon addition of physiological level of sodium bicarbonate. The operational stability observed in the presence of uric acid was the lowest of all components tested, with only 46% of initial current response after 12 h, and was comparable to the 27% of current remaining after 12 h for electrodes operating in artificial plasma. The effect of uric acid on glucose oxidation by enzyme electrodes prepared using both glucose oxidase (GOx) and a recombinant cellobiose dehydrogenase (CDH) was assessed. The maximum current decreased for both FADGDH and GOx enzyme electrodes in the presence of uric acid, with no significant change to the enzyme affinity, suggesting non-competitive inhibition. The CDH based electrodes provided highest stability of current signal in buffer, with 86% of the initial signal present after 12 h, but display significant change in enzyme affinity, maximum current and operational stability, dropping to only 33%, in the presence of uric acid. In contrast the operational stability of the GOx-based enzyme electrodes was unaffected by the presence of physiological level of uric acid. As uric acid and sodium bicarbonate are present in blood, these results highlight the importance of enzyme selection for in vivo biosensing and biofuel cell applications. Further work is required to understand the mechanism of uric acid inhibition on each of the enzymes.

Changes of renin-angiotensin system-related aminopeptidases in early stage Alzheimer's disease

Activities of aminopeptidases A, B, and N (ApA, ApB & ApN) and insulin-regulated aminopeptidase (IRAP) have been seen to be decreased amongst patients with Alzheimer's disease (AD). All of these enzymes are involved with the brain renin-angiotensin system which is believed to be involved with learning and memory. This study aimed to explore the time course and the mechanisms underlying these changes.Serum samples were collected from 45 AD patients at the start of the study, and again 13months later (n=37). The control group was 22 healthy, older, adults. Enzyme activity was determined at two substrate concentrations to allow Michaelis-Menten analysis of the enzyme activity.The results indicated that there was decreased activity of ApA, ApB and ApN amongst AD patients but no difference in serum IRAP activity. There were no associations between enzyme activity and age, gender nor scores on psychomotor tests.Consideration of the data for the two time points for AD patients showed that the changes in ApB occurred at an early stage of the disease and persisted, whilst those of ApA and ApN only became apparent at later stages of the disease. Although differences in Michaelis-Menten parameters were not statistically significant, consideration of the values suggested that the decrease in ApB activity may be a result of changes in enzyme protein conformation, whilst that of ApN may be a consequence of decreased enzyme expression. Importantly, the different time courses of the effects and the differential changes in enzyme affinity and expression indicated that the observed changes with progression of AD were not a ‘class effect’ for serum aminopeptidases but were idiosyncratic for the individual enzymes.

Characterization of platelet-activating factor acetylhydrolase in human bronchoalveolar lavage.

Platelet-activating factor (PAF) is a mediator produced in human airways during acute and chronic inflammatory lung diseases. The levels of PAF are regulated by acetylhydrolase (AH), the enzyme that converts PAF to lyso-PAF. To determine whether AH was present in human bronchoalveolar lavage (BAL) fluid, BAL was obtained from normal donors (n = 18) and from adult patients with mild bronchial asthma (n = 15) or with lung fibrosis (n = 15). AH activity was consistently found in the cell-free BAL fluid. BAL-AH is an enzyme different from secretory phospholipase A2 and from plasma AH and erythrocyte AH. Furthermore, BAL-AH is inhibited as much as 95% by exposure to an oxygen radical-generating system (xanthine/xanthine oxidase). BAL-AH is significantly correlated with the number of BAL macrophages (rs = 0.63; p < 0.02). In addition, BAL macrophages release AH both spontaneously and after stimulation with tumor necrosis factor-alpha (TNF-alpha) (100 ng/ml). BAL-AH activity in patients with bronchial asthma (1.32 +/- 0.18 pmol of PAF converted to lyso-PAF/min) is significantly lower than that in normal donors (2.25 +/- 0.26 pmol/min; p < 0.001). In contrast, BAL-AH activity in patients with lung fibrosis (6.13 +/- 0.81 pmol/min) is higher than that found in normal donors (p < 0.01). The variations in BAL-AH activity in patients with bronchial asthma or lung fibrosis are due to a reduction and to an increase, respectively, in the number of active molecules rather than to changes in enzyme affinity. These data demonstrate that human BAL fluid contains an extracellular AH activity that inactivates PAF released in the airways. BAL-AH is secreted by alveolar macrophages and is highly sensitive to oxygen radical-induced damage. The secretion and inactivation of BAL-AH may influence the levels of this enzyme in BAL fluid during acute and chronic inflammatory lung diseases and, ultimately, regulate the proinflammatory activities of PAF in these disorders.

Increased gall-bladder prostanoid synthesis after bile-duct ligation in the rabbit is secondary to new enzyme formation.

Ligation of the common bile duct (BDL) in the male rabbit resulted in increased gall-bladder microsomal total cyclo-oxygenase activity with prostaglandin E2 (PGE2) and 6-oxoprostaglandin F1 alpha [6-oxo-PGF1 alpha, stable metabolite of prostaglandin I2 (PGI2; prostacyclin)] as the major prostanoids synthesized after 24 and 72 h. Kinetic analysis of gallbladder microsomal membrane fractions incubated with increasing levels of [14C]arachidonic acid indicated that BDL for 24 and 72 h did not change substrate affinity (apparent Km) but markedly increased the rate of conversion (apparent Vmax.) suggesting the presence of more total enzyme responsible for synthesis of 6-oxo-PGF1 alpha and PGE2. BDL for 24 and 72 h significantly increased gall-bladder tissue slice basal release of 6-oxo-PGF1 alpha, but not PGE2, when compared with the controls. Gall-bladder slice release of PGE2 was 3-fold less than 6-oxo-PGF1 alpha in the control gall-bladder slices. Immunoblot analysis of 72 h BDL gall-bladder microsomal membrane fractions showed a slight increase in cyclo-oxygenase content and a 5-fold increase in the content of prostacyclin synthase as compared with the control. These data suggest that the BDL-stimulated total gall-bladder cyclo-oxygenase activity was the result of an increase in the level of specific prostaglandin-synthetic enzymes, in particular prostacyclin synthase, and not from a change in enzyme affinity.

Halothane metabolism. Impairment of hepatic omega-oxidation of leukotrienes in vivo and in vitro.

Omega-oxidation of leukotrienes is the initial step of hepatic degradation and thus inactivation of these proinflammatory mediators. Omega-oxidation is followed by beta-oxidation of leukotrienes from the omega-end. After exposure of rats to a single dose of the anesthetic agent halothane, a transient decrease in leukotriene omega-oxidation was induced both in vivo and in vitro. In untreated rats, 44.1 +/- 6.0% of N-[3H]acetylleukotriene E4 injected intravenously was recovered unchanged in bile collected for 60 min in vivo; 46.5 +/- 3.0% was recovered as omega-/beta-oxidation products, of which 24.7 +/- 4.5% were associated with beta-oxidation products only (mean +/- SEM; n = 5). In rats receiving a single dose of halothane 18 h before the experiment, recovery of unchanged N-[3H]acetylleukotriene E4 was significantly increased to 79.8 +/- 4.8%, while the fraction of omega-/beta-oxidation products decreased to 9.0 +/- 1.7% (n = 5); 90 h after exposure to halothane, N-[3H]acetylleukotriene E4 recovery decreased to 30.0 +/- 3.0% and omega-/beta-oxidation products amounted to 49.1 +/- 3.8%; the fraction of beta-oxidation products was significantly increased to 43.1 +/- 3.4% (n = 5). Ten days after exposure of rats to halothane, the recoveries of N-[3H]acetylleukotriene E4, of omega-/beta-oxidation products, and of beta-oxidation products alone, returned to almost normal values. Microsomal fractions obtained from rat hepatocytes catalyzed the NADPH- and O2-dependent leukotriene omega-oxidation in vitro. The formation of omega-hydroxy-metabolites of leukotriene B4, leukotriene E4, and N-acetylleukotriene E4 was decreased by 50% in microsomal fractions obtained from rats 18 h and 90 h after halothane treatment, and returned back to control levels in microsomal fractions obtained 10 days after halothane treatment. The Km value of leukotriene B4 omega-oxidation revealed no significant change in enzyme affinity towards leukotriene B4; in contrast, as reflected by the reduction of the Vmax value by 65%, a decrease in the amount of the active enzyme in microsomes obtained from rats 18 h after halothane treatment was observed. Halothane-metabolism-dependent trifluoroacetylation of hepatic proteins may mediate this process. Thus, the time course of the density on immunoblots of trifluoroacetylated protein adducts paralleled that of the transient decrease in leukotriene omega-oxidation. In contrast to its omega-oxidation, leukotriene B4 synthesis from 5-hydroperoxyeicosatetraenoate was not inhibited in hepatocyte homogenates obtained from rats pretreated with halothane. The data suggest that metabolism of halothane causes a transient derangement of hepatic leukotriene homeostasis in vivo.

The acute influence of ingested thyroid hormones on hepatic deiodination pathways in the rainbow trout, Oncorhynchus mykiss

Juvenile rainbow trout were fed once daily with trout pellets supplemented with l-thyroxine (T 4) or 3,5,3′-triiodo- l-thyronine (T 3) and the effects on plasma T 4 and T 3 levels and hepatic 5′-monodeiodinase (5′D) activity determined after 1, 2, or 3 days. In all cases T 3 (12 ppm) elevated plasma T 3 and caused a significant reduction in the functional level ( V max) of 5′D with no change in enzyme affinity ( K m ). After 3 daily meals (3 ppm T 3), 5′D activity fell to 52% of control levels. In most instances plasma T 4 was increased. In contrast, ingestion of T 4 for 3 days at levels up to 48 ppm did not modify hepatic 5′D. This may reflect either insensitivity of the 5′D system to T 4 or poor T 4 uptake from the gut, as plasma T 4 levels were influenced to a small extent by T 4 ingestion. HPLC analyses showed that dietary T 3 supplements (0, 3, 6, or 12 ppm) for 3 days acted in a dose-dependent manner, not only to suppress T 3 formation from T 4 by outer-ring deiodination, but also to promote inner-ring deiodination of T 4 to 3,3′,5′-triiodo- l-thyronine (reverse T 3) and outer-ring deiodination of T 3. In conclusion, the present data indicate that in the face of a T 3 challenge there is a rapidly responding hepatic autoregulation of T 3 production, achieved by a complex coordinated regulation of several different iodothyronine deiodination pathways.

In Vitro Reactivation of Rat Cortical Tryptophan Hydroxylase Following In Vivo Inactivation by Methylenedioxymethamphetamine

The activity of tryptophan hydroxylase (EC 1.14.16.4) from rat brain was significantly decreased 1 h following a single systemic injection of 3,4-methylenedioxymethamphetamine (MDMA) when assessed ex vivo by radioenzymatic assay or in vivo by the quantitation of 5-hydroxytryptophan accumulation following central L-aromatic amino acid decarboxylase inhibition. Recovery of enzymatic activity in vivo, which occurred within 24 h of low-dose MDMA treatment, appeared not to involve synthesis of new enzyme protein, because the return of enzymatic activity was not prevented by prior cycloheximide. Acutely MDMA-depressed cortical tryptophan hydroxylase activity could be completely restored in vitro by a prolonged (20-24 h) anaerobic incubation in the presence of dithiothreitol and Fe2+ at 25 degrees C; partial reconstitution occurred when 2-mercapto-ethanol was substituted for dithiothreitol. Cortical tryptophan hydroxylase acutely inactivated by methamphetamine or p-chloroamphetamine could be similarly reactivated. MDMA-inactivated cortical tryptophan hydroxylase derived from rats killed later than 3 days after drug treatment could not be significantly reactivated under the conditions described above, indicating the development of irreversible enzymatic damage. Kinetic analysis of enzyme reactivation revealed an approximate doubling of enzyme Vmax with no change in enzyme affinity for either substrate, tryptophan, or pterin cofactor. These studies suggest that MDMA and its congeners inactivate central tryptophan hydroxylase by inducing oxidation of key enzyme sulfhydryl groups. The reactivation capacity of drug-inactivated enzyme at various times after MDMA treatment may provide a means of assessing the development of MDMA-induced neurotoxicity.

Effect of environmental temperature on the kinetic properties of goldfish brain choline acetyltransferase.

1. Michaelis constants of goldfish brain choline acetyltransferase were found to depend on the concentration of the second substrate present and on the temperature to which the fish had been adapted. 2. Primary plots constructed from results obtained with enzyme prepared from cold-adapted or warm-adapted fish indicated that synthesis of acetylcholine took place by a sequential mechanism. 3. The affinity of choline acetyltransferase for acetyl-CoA was about 100 times that for choline irrespective of whether the enzyme had been prepared from warm-adapted or cold-adapted fish. 4. The maximum rate at which choline acetyltransferase synthesized acetylcholine and the energy of activation for this synthesis remained independent of the previous environmental temperature of the fish. 5. The affinity of choline acetyltransferase for choline and acetyl-CoA showed a complex dependence on temperature. The affinity of the enzyme from cold-adapted fish for substrates increased as the incubation temperature was lowered, whereas that of the enzyme from warm-adapted fish first increased and then decreased. 6. The maximum affinity of choline acetyltransferase for both substrates, from both cold-adapted and warm-adapted fish, occurred at temperatures that corresponded approximately to the respective environmental temperatures of the fish. 7. These changes in enzyme affinity for substrates are not thought to be due to the presence of isoenzymes. Their adaptive significance is unknown, but it could be connected with the maintenance of the enzyme in a stable form.

Effect of diphenylhydantoin on synaptosome sodium-potassium-ATPase.

Previous studies have demonstrated that electrically induced seizures in rat result in an increased brain intracellular sodium which can be decreased by treatment with sodium diphenylhydantoin (DPH). The correlation of cation transport with membrane-oriented sodium-potassium-adenosine triphosphatase (Na-K-ATPase) prompted an investigation of the effect of DPH upon ATPase enzyme activity.Rat cerebral cortical synaptosomes isolated in Ficoll gradients were employed as the source for Na-K-ATPase. With 50 mM Na, 10 mM K, 7.5 mM Mg, and 1.8 mM ATP, the specific activity of the preparation was 70 mumoles P(i) released/mg synaptosomal protein per 30 min. The ionic and substrate concentrations yielding one-half maximal velocity were 0.5 mM K, 5 mM Na, and 8.5 x 10(-5) M ATP, respectively. At 50 mM Na and 0.2 mM K, DPH produced an average of 92% stimulation of P(i) release above control. The ratio of Na:K rather than the absolute levels of the ions was critical in determining the effect of DPH. DPH produced significant stimulation of enzyme activity under conditions of a high Na:K ratio (25-50:1). At ratios of 5-10:1, DPH produced little or no effect, and at low Na:K ratios (less than 5:1), DPH was inhibitory. Under all ionic conditions examined, DPH produced no apparent change in enzyme affinity for ATP. Assuming the proposed association of Na-K-ATPase with cation transport in brain, the data suggest the possibility that DPH may control seizures by its stimulation of Na-K-ATPase activity.

Glutamic decarboxyiase of ergot, Claviceps purpurea.

Anderson, John A. (Oregon State University, Corvallis), Vernon H. Cheldelin, and Tsoo E. King. Glutamic decarboxylase of ergot, Claviceps purpurea. J. Bacteriol. 82:354-358. 1961.-l-Glutamic acid is the only naturally occurring amino acid which can be decarboxylated by cell-free extracts of Claviceps purpurea. This decarboxylase was partially purified and the properties of the enzyme studied. The specific activity of the purified preparation was 111 muliters per 10 min per mg of protein. The products formed, stability, inhibition, stimulation of activity with pyridoxal phosphate, and pH activity curve were typical of l-glutamic decarboxylase in Escherichia coli and other microorganisms. The substrate constants at pH 4.6, 5.25, and 5.65 were 0.0169 m, 0.0174 m, and 0.0139 m, respectively. The respective maximal velocities at these pH values were 104, 104, and 90 muliters per 10 min. The pH optimum was 4.8 to 5.2. The enzyme was unstable below pH 4.5 and it was suggested that the fall in activity at the lower end of the pH curve was due to inactivation of the enzyme. The decrease in activity above pH 5.2 did not appear to be due to a change in affinity of enzyme for substrate but to a change of the enzyme-substrate complex into an inactive form.