Inactivation temperature dependence

Anandamide is inactivated in two steps, first by transport inside the cell and subsequently by intracellular enzymatic hydrolysis. The transport of anandamide inside the cell is a carrier-mediated activity, having been shown to be a saturable, time- and temperature-dependent process that involves some protein with high affinity and specificity for anandamide (Beltramo, 1997). This transport process, unlike that of classical neurotransmitters, is Na+-independent and driven only by the concentration gradient of anandamide (Piomelli, 1998). Although the anandamide transporter protein has not been cloned yet, its well characterized activity is known to be inhibited by specific transporter inhibitors. Reuptake of 2-AG is probably mediated by the same facilitating mechanism (Di Marzo, 1999a,b Piomelli, 1999). 

A study on mechanistic aspects of di-ir-methane rearrangements has been published recently [72]. The kinetic modeling of temperature-dependent datasets from photoreactions of 1,3-diphenylpropene and several of its 3-substituted derivatives 127a-127d (structures 127 and 128) show that the singlet excited state decays via two inactivated processes, fluorescence and intersystem crossing, and two activated processes, trans-cis isomerization and phenyl-vinyl bridging. The latter activated process yields a biradical intermediate that partitions between forma-... 

Another method which may become a useful technique for selective inactivation of cellulases in enzyme mixtures is the use of selective heat inactivation. While establishing the thermostability properties of crude xylanases from a fungal strain Y-94, Mitsuishi et al. (80) observed differential heat labilities of the cellulase and xylanase activities in the culture filtrate. After an incubation period of 20 minutes at 65°C, the xylanase activity was reduced by 5-10% whereas the Avicelase and /3-glucosidase activities were reduced by 100% and 60%, respectively. We have observed a similar temperature dependency of xylanase and cellulase activities in T. auranti-acus. As indicated in Figure 2, treatment of the culture filtrate at 70°C for 20 minutes resulted in less than a 5% loss in xylanase activity whereas cellulase activities were reduced by 40-50%. A similar effect has also been observed for the xylanases and cellulase enzymes produced in culture filtrates from T. harzianum (93). Further work in the area of heat treatments may improve the effectiveness of cellulase inactivation. Since the cellulase activities of some enzyme preparations can be more rapidly inactivated on... 

Fig. 9.10-5. Temperature dependence of E.coli inactivation, 15 min at 2 kbar (adapted from (181)...

L1364H- IXLCSNB reduced current density, reduced channel expression (temperature dependent), increased rate of inactivation, increased rate of recovery from inactivation (Hoda et al., 2006)... 

Measurement of the inactivation rates of AMDH were performed under various conditions of salt types, salt concentrations, temperatures, and buffers (Mevarech and Neumann, 1977 Pundak et al, 1981 Zaccai et al., 1986b, 1989 Hecht andjaenicke, 1989a). It was found that (1) the inactivation process is of first order (which means that only one active form of the enzyme exists), (2) the rate constant for inactivation increases as the salt concentration decreases, (3) the temperature dependence of the rate constants of inactivation depends on type of salt, and (4) the dependence of the rate constants on salt type follows the Hofmeister series (von Hippel and Schleich, 1969), being lower for salting-out salts. The different models for the role of the salts in the stabilization of the AMDH will be discussed in Section IV,G. 

Peroxidase is used as a convenient index of lipase activity. The inactivation temperature for lipases and associated enzymes is dependent on the moisture content. At 4% moisture, inactivation temperature for lipoxygenase is 40°C, lipase is 55°C, and peroxidase is 70°C (28). 

Measurements of the temperature dependence of enzymatic activity suggested the presence of an intermediate step in enzyme inactivation. The proposed mechanism involves a reversible change to an inactive state, which precedes the final irreversible inactivation step [21-23]. This mechanism could now be refined based on experiments with single enzymes, which detected the intermediate steps directly. This more detailed information allowed the establishment of a tentative model for a-chymotrypsin inactivation (Fig. 25.2d). 

If one measures the enzyme activity as a function of pH or temperature using a soluble substrate, such as hydroxyethyl cellulose, one obtains curves characteristic of many enzymes. The temperature curve follows an Arrhenius dependence at temperatures leading up to the optimum, then drops sharply at inactivating temperatures (Fig. 1). The pH curve is roughly a bell shape, with the optimum spanning 1 to 3 pH units (Fig. 2). This exercise gives a first estimate of the pH and temperature curves, but often the behavior in specific applications is quite different. 

The very sharp crystalline formation of the C. acidi-urici ferredoxin relative to that from C. pasteurianum has been correlated with their different amino acid compositions, in particular with the extra proline at site 16 (294). It is hence interesting that the kinetics of heat-inactivation at 70 °C indicates a higher stability for the former. Similarly, the aerobic reaction of the C. acidi-urici and C. tartarivorum ferredoxins with o-phenanthroline is faster for the most thermolabile of the two. This was reflected clearly in the temperature dependence of the reaction (285). Along these lines, Gillard et al. (295) have reported that iron is removed from native Peptostreptococcus elsdenii and from C. pasteurianum ferredoxins by o-phenanthroline while this ferrous chelator was found inactive on the C. acidi-urici protein. The magnitude of these differences needs further substantiation since other authors have claimed that o-phenanthroline can sequester iron from the C. acidi-urici protein as well (296). 

The effectiveness of irradiation in inactivating dry RNase depends on the temperature, as shown by Fluke (34), He determined the doses needed to reduce the activity to 37% of its original value (D37) at temperatures from —160 to 182°C. Arrhenius-type plots of D37 1 vs. T1 are found to be nonlinear and were interpreted in terms of a temperature-independent term and two temperature-dependent terms. The inactivation becomes relatively effective above about 60°C. 

The effect of temperature on enzymatic reactions is very complex. If the enzyme structure would remain unchanged as the temperature is increa.sed, the rate would probably follow the Arrhenius temperature dependence. However, as the temperature increases, the enzyme can unfold and/or become denatured and lose its catalytic activity. Consequently, as the temperature increase.s, the reaction rate, -fj, increases up to a maximum with increasing temperature and then decreases as the temperature is increased further. The descending part of this curve is called temperature inactivation or thermal denaiurizing. Figure 7-9 shows an example of this optimum in enzyme activity. ... 

Even when mechanisms and pathways are unknown, it is sometimes possible to use Arrhenius or empirical equations to determine apparent rate constants at other temperatures. Apparent first-order rate constants for a-chymotrypsin and bromelain inactivation exhibited linear Arrhenius plots (Fig. 21 1).879 Apparent rate constants for kallikrein inactivation calculated according to the reaction scheme in Eq. (5.1) resulted in the temperature dependence seen in Fig. 212.850 For inactivation of various peptide and protein pharmaceuticals... 

The radiation inactivation process involves Q, the average amount of radiation energy required to inactivate one mole of protein (Kepner and Macey, 1968). Q for radical formation, one of the steps leading to protein inactivation, is temperature dependent (Muller, 1962). Beauregard and Potier (1985) proposed that the effect of temperature on Q is directly responsible for the temperature dependence of protein inactivation, as revealed by the variation of D37, since the number of hits (or ionizations) per unit mass of the sample is temperature independent (Lea 1955 Augenstein and Mason, 1962). Thus the efficiency of a hit to result effectively in the inactivation of a molecule depends on the irradiation temperature. From Kepner and Macey (1968) and Jung (1984), we rewrite equation (4) to take the temperature into account ... 

The exact temperature-sensitive physical and chemical steps in the chain of events involved in protein inactivation after a hit are largely unknown. Ionization itself is not temperature dependent because of the large amount of energy deposited (Lea, 1955 Augenstein and Mason,... 

Beauregard G, Potier M. Temperature dependence of the radiation inactivation of proteins. Anal Biochem 1985 150 117-120. 

Table III shows the results obtained with two crystalline preparations of the polysaccharide. Single crystals and a "quench precipitate" (prepared by quickly chilling a polymer solution in ice to precipitate the polysaccharide with minimal crystallinity) were compared. The extent of degradation of lamellar crystals is highly temperature dependent, with the total fraction of polymer digested at any temperature being finite and quite reproducible from one batch of crystals to another. Incubation up to 20 hours causes no additional decrease in turbidity nor do the crystals inactivate the enzyme. The plateau in absorbance is evidence of a "two region" model of crystal morphology, i.e., one with both accessible and inaccessible zones. The extent of digestion at 20°C of the lamellar crystalline material is to be compared with that of the "quench precipitate" form at the same temperature.

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