Inactivation curve

The catalytic rate of hydrogenase adsorbed on the graphite electrode was measured by potential step chronoamperometry, in which cnrrent is monitored throughout a fixed sequence of potentials. This allows for direct observation of hydrogen oxidation activity at a particular potential over a period of time. Figures 5.14 and 5.15 show how chronoamperometry can be used to study the kinetics of reductive activation and oxidative inactivation respectively. A series of oxidative inactivation curves from several experiments like that shown in Fig. 5.14, showing the effect of pFl on oxidative inactivation, are shown in Fig. 5.15. The kinetics of the reactivation process can be... 

The thermal inactivation curve of the enzyme in 0.15 M acetate buffer-0.01 M ethylenediaminetetraacetate (EDTA), pH 5.0, showed that a 50% inactivation was obtained by heating for 20 min at 56° 11). 

Versteeg (8) tested the heat stability of the purified PEs in orange juice, pH 4.0, and found that PEII was the least stable, being completely inactivated at 55°C (131°F) PEI was inactivated at 65°C (149°F), and HM-PE at 85°C (185°F). Heat inactivation curves of a crude preparation of orange PE in orange juice indicated that about 5% of the total PE activity was due to HM-PE, 60% to PEI and 30% to PEII. 

The potassium channels present in neuronal membranes could also be affected by phenothiazine derivatives. In the study performed by Ogata et al. [255] it was shown that CPZ (9) interfered with several types of potassium channels present in membranes of neurons of the newborn rat cultured dorsal root ganglia. Reversible reduction of the amplitude was found for transient and delayed rectified K+ currents, while inward rectified K+ current remained unaffected by CPZ (9). The block of delayed rectified K+ current by CPZ (9) was, however, less potent than block of the transient one. The hyper-polarizing shift of the steady-state inactivation curve for transient K+ current indicated that CPZ (9) binds preferably to the channels in the inactivated state. 

The GFPuv inactivation curves were considered first-order models represented by... 

Fig. 1. (opposite page) Inactivation curves for GFPuv in (A) acetate buffer at pH 6.0, (B) phosphate buffer at 85°C and different pH values, (C)Tris-HCl at pH 8.5. The D-values were determined from the reciprocal of the slope of the linear portion of the GFPuv inactivation curves, considered first-order models. 

N-bromoacetamide, which is less reactive than NBS at pH 4.0 and consequently requires more time to oxidize tryptophan residues, similar results were obtained (Fig. 27, curve B). Extrapolation of the initial steep portion of the inactivation curve obtained with NBA (Fig. 26) gives a value for tryptophan destruction of about 1 mole per mole of protein. This might suggest the association of one of the seven tryptophan residues present in the chymotrypsin molecule with the maintenance of catalytic activity. A similar involvement of one tryptophan residue in the reaction of diisopro-pylphosphorofluoridate with a-chymotrypsin is suggested by comparison of... 

A notable heat-inactivation curve resulted when aliquots of a Bio-Gel-filtered sample were heated for 5 minutes at different temperatures and were then assayed at 30° (Fig. 6). Activity was progressively lost above 55° inactivation was total at 90°. These facts are somewhat surprising, because the polymer was twice submitted to heating at 80° for 5 minutes prior to Bio-Gel treatment, and the resulting material was yet active. Oshima suggests that a stabilizing factor may have been... 

Cummins At least from my evidence concerning closed state inactivation of Na channels (Cummins et al 1998), it might be that the inactivation is dependent on the channels opening for the peripheral neuronal channels, as opposed to skeletal muscle channels, which are more likely to inactivate from the closed states. This may be why you see such a concordance between the Hinf (steady-state inactivation curve) and the voltage-dependence of the persistent current the peripheral neuronal Na channels may have to open if they are going to inactivate. 

Gold There were just two voltage traces illustrated and they were from different resting membrane potentials. These cells have a number of K currents that are subject to steady-state inactivation where the steep part of the inactivation curve coincides with resting membrane potential. The result is that differences in available current may explain the apparent differences in after-hyperpolarization. 

B.aman In whole cell recordings from Purkinje cells over time, the inactivation curve tends to shift to more negative voltages. 

We determined the synergistic effect of 0.5 pM EPA on 15 pAf carbamazepine in CAl neurons from healthy rats, in CAl neurons isolated from the hippocampus surgically removed from patients with intractable temporal lobe epilepsy, and in neocortical pyramidal neurons from the same patients (for details on methods, see Vreugdenhil Wadman, 1999). Figure 7B shows a clear relation between the shift in the inactivation curve induced by 15 iM carbamazepine and the additional shift induced on top of 0.5 pM EPA. This suggests that the effect of a therapeutically relevant dose of carbamazepine can be boosted by subthreshold levels of PUFAs. 

Fig. 6C shows that, when the sodium inactivation curve is shifted by 10 mV towards more positive potentials to account for the properties of some regions of the axons such as the site of initiation of the nerve impulse, the equations predict that the same small insecticide concentration that would normally only increase the afterpotential (lower trace) induces repetitive activity (upper trace). 

Fig.6 Computer reconstruction of nerve membrane activity based on the existence in the membrane of a small proportion (A) of modified sodium channels. In panels A and B, this proportion was varied from zero to 0.048 (i.e. 4.8 % of modified channels) to reproduce the membrane potential changes observed in strongly poisoned insects. In panel C, repetitive firing was obtained by shifting the sodium inactivation curve towards more positive potentials. Tau p x 200 indicates that, for these examples, the time constant of activation of the modified channels was 100 times slower than that of the normal channels.

Kinetic analyses of the peak sodium current have revealed that the time constants of activation (rm), inactivation (r ), and the initial phase of the tail current ( tail) are not affected by tetramethrin (14,15). However, in the presence of tetramethrin the slow component of tail current and the slow steady-state inactivation curve were both shifted in the hyperpolarizing direction. The amplitude of the slow tail current was dose-dependent, whereas its time constant was not. These observations led to the suggestion that tetramethrin modifies a population of sodium channels to give rise to slow activation and inactivation kinetics. The normal peak transient current in tetramethrin simply represents the activity of the unmodified channels. 

Kaplan [66] investigated the persistence of VACV and determined inactivation curves at temperature ranges between 50°C-60°C. The results from this study demonstrated a rapid fall of infectivity followed by a complete inactivation at a much slower rate. VACV can be inactivated by pasteurization at 60°C in a variety of protein solutions [90], Approximately a 2 logi0 inactivation of VACV was observed after 1 h pasteurization in alphai-proteinase inhibitor solution within 3 h of exposure, no active VACV was detectable. Similarly, in human plasma protein solution, VACV was reduced by 4 logio within 30 min, and by 2 h vims activity was undetectable. 

An unfolded globular protein generally is very susceptible to proteolytic cleavage, if a suitable protease is present in the active state. Such a situation can readily produce inactivation curves like those in Figure 7.11b, where the fast inactivation between 45 and 55°C would be due to proteolysis, until also the protease has attained the unfolded, i.e., inactive, state. One example is the autodigestion at intermediate temperatures shown by several proteolytic enzymes an example is in Figure 7.11c. 

The rates of heating and cooling may significantly affect the shape of the inactivation curves, especially in the case just mentioned. Another cause may be slow reactivation (refolding) as exemplified in Figure 7.10b in such cases, also the time elapsed between cooling and estimation of residual activity plays a part. 

---