Cold sensitive proteins

Because temperature shifts may also influence the packing quality, the temperature should not be changed during the chromatographic step and the packing of the column should be done at the operation temperature. To prevent the denaturation of sensitive proteins, the chromatography is carried out in a cold chamber (or cabinet). For this purpose the column packing has to be performed at the same ambient temperature (store the gel before use at the same temperature ). 

What could be the signal for the induction of the cold shock proteins It has been observed that shifting E. coli cells from 37 to 5 °C results in an accumulation of 70S monosomes with a concomitant decrease in the number of polysomes [129]. Further, it has been shown that a cold shock response is induced when ribosomal function is inhibited, e.g. by cold-sensitive ribosomal mutations [121] or by certain antibiotics such as chloramphenicol [94]. These data indicate that the physiological signal for the induction of the cold shock response is inhibition of translation caused by the abrupt shift to lower temperature. Then, the cold shock proteins RbfA, CsdA and IF2 associate with the 70S ribosomes to convert the cold-sensitive nontranslatable ribosomes into cold-resistant translatable ribosomes. This in turn results in an increase in cellular protein synthesis and growth of the cells. 

It has been suggested that brown adipose tissue may also function to convert excess dietary fat into heat and thereby to resist obesity.k m Mice lacking the gene for the mitochondrial uncoupling protein are cold-sensitive but not obese. However, other proteins, homologous to UCP1, have been discovered. They may partially compensate for the loss.m,h... 

The discoveries of Csp s and trigger factor may represent the tip of a large iceberg. In view of the pervasive effects of low temperature on the structures of all classes of macromolecules, it is reasonable to conjecture that many more types of proteins will be discovered whose roles are to offset the effects of cold shock on the cell. Some of these molecules may be expressed constitutively and may be part of the normal machinery of the cell. For example, certain ribosomal proteins are thought to function as RNA chaperones, and if present in sufficient amounts, these proteins may allow the cell to cope with the effects of cold shock on the structures of certain classes of RNAs. In yeast, a constitutively expressed ribosomal protein has helicase activity, and mutation in the gene encoding the protein confers on the cells a cold-sensitive phenotype (Schmid and Linder, 1992). Perhaps the apparent absence of cold-induced RNA chaperones in eukaryotic cells is... 

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. 

Enerback S, Jacobsson A, Simpson EM, et al. Mice lacking mitochondrial uncoupling protein are cold-sensitive but not obese. Nature 1997 387 90-94. 

Lhoest, J. Colson, C. (1981). Cold-sensitive ribosome assembly in an . colt mutant lacking a single methyl group in ribosomal protein L3. Eur. J. Biochem. 121, 33-37. 

Adachi, Y., Yanagida, M. (1989). Higher order chromosome structure is affected by cold-sensitive mutations in a Schizosaccharomyces pombe gene crml, which encodes a 115-kD protein preferentially localized in the nucleus and its periphery. 

The degree of association of muscle P. depends on its concentration. At 7 mg protein/ml it exists as an active hexameric aggregate M, 2 x 10 ) at 0.5 mg protein/ml as an active monomer (M, 340,000). In 6 M guanidinium chloride and 0.1 M mercaptoethanoi, the latter dissociates into its 4 inactive chains (M, 80,000). Similar behavior has been described for the octameric erythrocyte P. (Af, 500,000). Chicken liver P. (M, of the smallest active form 400,000) is cold-sensitive it dissociates reversibly at 0°C into four inactive protomers M, 100,000), each consisting of two identical chains. Clostridium species contain a P. of M, 144,000 (4 subunits, M, 35,000). 

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. 

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