Posttranslational modifications intracellular

In the x-ray structure of rhodopsin, an amphipathic helix runs parallel to the membrane from the intracellular end of TM-VII beneath the seven-helical bundle to the other side of TM-I and TM-II. At this point, one or more Cys residues are often found and are known to be subject to a dynamic posttranslational modification with palmitic acid residues. Like the phosphorylation event, the palmitoylation process appears to be dynamically regulated by receptor occupancy and is also involved in the desensitization phenomenon. The two posttranslational modifications can influence each other. For example, the conformational constraint induced by palmitoylation may alter the accessibility of certain phosphorylation sites. Like the phosphorylation process, the functional consequences of palmitoylation also appear to vary from receptor to receptor. 

For the first group (i.e. intracellular soluble enzymes and proteins), which need no posttranslational modification and complex domain organization influencing protein folding, E. coli is the most preferred choice. However, for the other targets, alternative expression systems often provide a higher rate of success. The most common expression systems are presented in this chapter. 

Sauer A, RobinsonDG. Intracellular localization of posttranslational modifications in the synthesis of hydroxyproline-rich glycoproteins. Peptidyl hydroxylation in maize roots. Planta 1985 164 287-294. 

Disadvantages The protein expressed is accumulated within the cell matrix (intracellular) the protein does not undergo posttranslational modifications (resulting in proteins that may be structurally different or less useful to humans) the presence of lipopolysaccharides (pyrogens—microbial substances that cause fever) is likely to contaminate the product and there is a need for more extensive chromatographic purification. 

C. Intracellular Transport of the Viral Glycoproteins 1. Posttranslational Modifications... 

All of the known class A receptors are subject to posttranslational modification at one or more iV-linked glycosylation sequences, found either in the extracellular amino terminns or in the second exdacellular loop. Glycosylation is required for the expression of some GPCRs at the plasma membrane (12,13). Furthermore, many receptors, snch as rhodopsin and the dopamine receptors, are also subject to other posttranslational modifications, such as palmitoylation at the intracellular domains... 

The molecular characterization of different 5-HT receptor subtypes has simplified fhe elucidation of gene transcription, mRNA processing, and translation as well as intracellular trafficking and posttranslational modification relevant to synaptic and postreceptor signaling. Transcriptional control regions... 

Amino acid sequence analysis of the human 5-HT1B receptor has revealed consensus phosphorylation sites in all intracellular loops for PKA and PKC. Phosphorylation of the 5-HT1B receptor was demonstrated by metabolic labeling of the 5-HTib receptor-expressing Sf9 cells with [32Pi]phosphate (141). This posttranslational modification was proposed to be involved in receptor regulation such as desensitization. 

N is often limiting in the marine environment. Further, many enzymes are sensitive to cellular substrate concentrations rather than extracellular concentrations and it is difficult to measure the relevant intracellular metabohte pools. In vitro assays may affect the conformation of enzymes and the degree to which they are modified. For example, allosteric effects (see Section 1.3.3) may be modified under in vitro conditions. Many enzymes undergo posttranslational regulation wherein enzyme activity is affected by binding of activator/inactivator proteins and covalent modification of the enzyme (e.g., adenylylation, phosphorylation or carbamylation) (Ottaway, 1988). When there is posttranslational modification of enzymes, enzyme activity measured in assays may be unrelated to in vivo activity (see Section 2.2.1) and there are few ways to determine the extent of enzyme modification in nature. 

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