Big Chemical Encyclopedia

Chemical substances, components, reactions, process design ...

Articles Figures Tables About

Myristoylated protein preparation

One consideration not discussed above is the importance of the quahty and stability of the GAP and Arfl GTP for the quantitative characterization of GAP activity. We have described assays with conditions and time scales ensuring stability of the proteins being examined. The purifications described in this chapter, in Chapter 10 (Preparation of Myristoylated Arfl and Arf6), in Randazzo et al. (2000) and in Randazzo et ai, (1992) yield proteins relatively free of interfering contaminants. However, because of variability between proteins, the stability and purity of the protein preparations should be assessed when examining other Arf GAPs. [Pg.161]

The myristoylated Arf preparations described in this chapter have been used in a number of in vitro assays including GAP, GEF, and effector binding assays. The proteins are useful when efficient loading with GTP is needed. More critically, some Arf activities, such as recruitment of vesicle coat protein, are highly dependent on myristoylation. In these cases, use of a partially myristoylated Arf, particularly with preparation-to-preparation differences in the extent of myristoylation, can confound results and their clear interpretation. The methods described in this chapter require only one additional column beyond that required for purification of nonmyristoylated or partially myristoylated Arf and provide homogeneously myristoylated Arf preparations. [Pg.171]

Posttranslational modification reactions prepare polypeptides to serve their specific functions and direct them to specific cellular or extracellular locations. Examples of these modifications include proteolytic processing (e.g., removal of signal proteins), glycosylation, methylation, phosphorylation, hydroxylation, lipophilic modifications (e.g., N-myristoylation and prenylation), and disulfide bond formation. [Pg.737]

Fig. 2. Detection of myristoylated and palmitoylated proteins by metabolic labeling with co-alkynyl fatty acids. Cellular extracts were prepared from MDCK cells treated with the different co-alkynyl fatty acid probes (100 jiM) for 24 h. The proteome was subjected to click reaction with biotin-azide, separated by SDS-PAGE and detected by streptavidin-linked horseradish peroxidase. The blot in (b) represents an experiment done at the same time as in (a) except that it has been treated with hydroxylamine. Lanes 1,Alk-C10 (/r=6) 2,Alk-C11 (/ =7) 3,Alk-C13 (n=9) 4,Alk-C14 (n=10) 5,Alk-C16 (/ = 12) 6, Alk-C18 (/7=14). The DMSO lane serves as a background control for labeling with alkyne fatly acids. Fig. 2. Detection of myristoylated and palmitoylated proteins by metabolic labeling with co-alkynyl fatty acids. Cellular extracts were prepared from MDCK cells treated with the different co-alkynyl fatty acid probes (100 jiM) for 24 h. The proteome was subjected to click reaction with biotin-azide, separated by SDS-PAGE and detected by streptavidin-linked horseradish peroxidase. The blot in (b) represents an experiment done at the same time as in (a) except that it has been treated with hydroxylamine. Lanes 1,Alk-C10 (/r=6) 2,Alk-C11 (/ =7) 3,Alk-C13 (n=9) 4,Alk-C14 (n=10) 5,Alk-C16 (/ = 12) 6, Alk-C18 (/7=14). The DMSO lane serves as a background control for labeling with alkyne fatly acids.
Several types of Arf preparations with different levels of myristoylation have been described in the literature. Arf purified from mammalian tissue is 100% myristoylated. However, it is difficult to obtain a single Arf isoform and the purification from mammalian tissues is labor intensive. Expressing Arf as a recombinant protein in bacteria does not yield m) -istoylated protein unless N-myristoyl transferase is coexpressed (since bacteria do not have the enzyme). However, myristoylation has not been efficient in this system, with only a small fraction (5-15%) of the Arf being... [Pg.165]

Randazzo, P. A., and Fales, H. M. (2002). Preparation of myristoylated Arfl and Arf6 proteins. Methods Mol. Biol 189,169-179. [Pg.194]


See other pages where Myristoylated protein preparation is mentioned: [Pg.21]    [Pg.164]    [Pg.182]    [Pg.978]    [Pg.310]    [Pg.331]    [Pg.140]    [Pg.578]    [Pg.155]    [Pg.166]    [Pg.170]    [Pg.170]    [Pg.262]    [Pg.364]    [Pg.365]    [Pg.392]    [Pg.238]    [Pg.312]   


SEARCH



Myristoyl

Myristoylated protein preparation expression

Protein myristoylated

Protein myristoylation

Protein preparation

Proteins protein preparation

© 2024 chempedia.info