Cold-sensitive enzymes

HMG-CoA reductase, the enzyme that catalyses the formation of mevalonate [MVA, (2)] from HMG by an irreversible reaction that is considered rate-limiting with respect to the formation of cholesterol, has received much attention. Details of the purification of the enzyme from chicken liver and baker s yeast are available,15 and the solubilized enzyme from rat liver microsomes is readily and reversibly inactivated at temperatures below 19°C.16 Cold-inactivation is an uncommon phenomenon, and all the enzymes that have been found to exhibit this behaviour have been soluble proteins. Native HMG-CoA reductase is a particulate enzyme that is probably bound to protein or lipid of the microsomal membrane, although it is not known whether the solubilized enzyme contains a lipid component. Microsomal reductase is not cold-sensitive, and the cold-inactivation of the solubilized enzyme can be completely prevented by addition to the preparation of NADP+ or (more effectively) of NAD PH.17... 

Once soluble, the membrane-bound enzymes are much like cytoplasmic enzymes and can be purified using similar methods. Excellent descriptions of such methods can be found in ref. [36]. These methods are only limited by circumstances such as the salt dependence of the proteins from extreme halophiles and the cold-sensitivity exhibited by some membrane-bound enzymes when solubilized. 

The ELISA kits are based on the use of selective antibodies raised against specific PAHs, which are attached to a solid matrix support. These are combined with sensitive enzyme reactions (see Section 6.4.4). The kits provide high selectivity and can provide an immediate answer on site, but the disadvantages are that they can be difficult to use in the less than ideal conditions on site and they can only usually give a range of concentrations, e.g. >50, 20-50, 1-20 or <1 mg/kg of total PAHs and mainly rely on a manual rapid cold shake extraction technique which may only extract a small fraction of the PAHs in some samples. In addition, the test kits can prove quite expensive for a screening analysis. 

A third allelic variety is one which produces an arginine requirement for growth. Mutants of this type have been found in E. coli [77] and Salmonella [76a]. In the latter case, the auxotrophic phenotype is expressed only at low temperatures. The mutant enzyme has one-sixth the activity of the wild-type enzyme and is remarkedly altered in its allosteric properties. At 20°, as compared with 37°, the inhibition by UMP is increased, and activation by ornithine is decreased. The result is a cold-sensitive arginine auxotroph. A fourth form is one in which growth is inhibited by arginine and restored by uracil [76a,78]. Although the enzyme activity of this mutant type is considerably reduced, the exact nature of the regulatory alteration is yet to be determined. Table III summarizes the different allelic forms that have been obtained by mutational events in the pyrA locus. 

The cold-sensitive hisW mutant ceases growth immediately when shifted to 20°C. This implies that the product of the hisW gene is essential for cell growth. To date, all cold-sensitive mutations have been traced to mutant enzymes, rather than to nucleic acids [111, 112]. While... 

Missense mutant proteins/enzymes may have full, partially impaired, or null (biological) activities depending on the location of the mutation. When the functional impairment manifests itself only at a higher or lower than normal temperature that is considered optimal for the wild-type enzyme, the mutation is called temperature sensitive (ts). Although most ts mutants are sensitive to higher than normal temperatures, some ts mutants are cold sensitive. When an amino acid codon is changed to a termination codon, it is called a nonsense mutation. Nonsense mutations result in incomplete or truncated polypeptides of varying sizes and functional profiles. 

In the case of cold-induced aggregation and gelation, two different types of gel microstructure, namely filamentous and particulate (Figure 2.1), have been obtained by adding different concentrations of a ferrous salt to solutions of pre-denatured p-lactoglobulin (the major whey protein). This substantial difference in microstructure turns out to have a major impact on the iron delivery, due to the different sensitivities of the structures to the relevant environmental conditions, such as pH and the presence of digestive enzymes. In particular, the filamentous gel micro-... 

Chillproofers are added to freshly brewed beers to prevent the formation of haze upon cold storage. Papain-based enzymes have been used as chillproofers and their addition have to be monitored carefully, as overdosage results in flat beer. According to Skerritt et al (146), enzymatic methods currently used lack sensitivity or are slow and tedious. They have developed a polyclonal antibody-based kit for determination of these chillproofers. 

Enzymes are remarkably efficient catalysts, but they are also labile (sensitive) to such factors as heat and cold, changes in pH, and various specific inhibitors. In the first experiment of this chapter you will have an opportunity to observe the biosynthesis of ethanol and to test the effects of various agents on the enzyme system. 

Vitamin D is a fat soluble vitamin derived from cholesterol. In the human epidermis (skin), sunlight spontaneously oxidizes cholesterol to 7-dehydrocholesterol (Fig. 10.10a). The 7-dehydrocholesterol leaks into the blood where it isomerizes to cholecalciferol (vitamin D3, Fig. 10.10b and c). Cholecalciferol is enzymatically hydroxylated at C25 in the liver (25-cholecalciferol) and then passes to the kidney where another enzyme is activated by parathyroid hormone to hydroxylate it at Cl, forming calcitriol (Fig. lO.lOd). The kidney hydroxylase is sensitive to feedback inhibition. As the amount of calcitriol increases, it binds to the hydroxylase and alters the specificity of the kidney enzyme. Additional 25-cholecal-ciferol is hydroxylated to 24,25-dihydroxycholecalciferol (inactive calcitriol) instead of 1,25 dihydroxycholecalciferol (calcitriol). Other vitamin D derivatives that can be converted to calcitriol are obtained enzymatically from cholesterol in other vertebrates. The most common of these are vitamin D3 (lamisterol) and D2 (ergosterol) from cold-water fish such as cod, where their presence keeps membranes fluid at low body temperatures 10-20°C. 

The sensor exhibited optimal sensitivity at pH 7.4 (Fig. 67). In spite of the low enzyme loading, a functional stability of 4-6 weeks was assured by storing the sensor overnight in a cold solution containing EDTA and mercaptoethanol. 

Temperature and pressure extremes require different strategies. Cellular lipids, proteins and nucleic acids are sensitive to high temperatures. Hyperthermophile bacteria have ether lipids instead of the more hydrolysis sensitive ester lipids in mesophiles [13]. Enzymes from hyperthermophiles show an unusual thermostability in the laboratory, and an important aspect of protein chemistry research is to find out the stabilizing principles. Crude cell extracts of hyperthermophiles show the presence of heat inducible proteins, called chaperones, which assist in the folding of proteins during cellular synthesis. Molecular details for cold adaptation of enzymes have been reported but are less extensively studied [14]. 

The secretion of the active substance by the leucocytes is not instantaneous it takes place slowly, and is hindered by cold. It appears very sensitive, as we have seen, to the influence of the wail of the vessel in which the blood is preserved. Blood, in contact with paraffin or vaseline, curdles more slowly than if it touches glass or porcelain. It is very probable that the phenomenon of molecular adhesion enters to accelerate the enzymic secretion, but it is necessary to observe that this explanation of the part played by the wall in the coagulation of blood is not exclusive. Bordet and Gengou, in experiments which we shall describe later, have, in fact, shown that plasma, deprived of every cell, is likewise much slower to coagulate in a parafiined vessel than in a glass vessel. It appears that other factors, which we shall study later, enter into this phenomenon. 

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