Stress-70 proteins ATPase activity

A substantial number of observations have demonstrated that the stress-70 proteins bind denatured proteins, as well as some short peptides, and that ATP hydrolysis (possibly with concomitant release of Pj or ADP) results in the release of bound peptides (de Silva et ai, 1990 Hendershot, 1990 Hurtley 1989 Kassenbrock 1988 Palleros etai, 1991). Conversely, bindingof peptides to stress-70 proteins induces ATPase activity above basal levels peptide-induced ATPase activity is often used as a facile in vitro assay for stress-70 protein activity. Schematically, this activity can be subdivided into (1) ATP binding and hydrolysis, followed by product release (P and ADP), (2) peptide binding and release, and (3) a mechanism of coupling peptide binding/release and nucleotide hydrolysis/product release. 

The turnover rate of ATP by stress-70 proteins is relatively slow, and is enhanced severalfold by the binding of peptides or by the action of accessory proteins. In the absence of peptides, the E. coli dnaK protein has been reported to have basal ATPase rates in the range of 0.15 (mol ATP/mol dnaK min measured at 37°C, pH 8.1) (McCarty and Walker, 1991) to 0.21 (mol ATP/mol dnaK min measured at 30°C, pH 8.8) (Liberek et ai, 1991a). The basal ATPase rate of dnaK rises steeply as a function of temperature, increasing 70-fold from 20 to 53°C. Above 53°C, the ATPase activity drops precipitously, possibly due to the onset... 

Hence, although the specific numerical values reported for ATPase rates of stress-70 proteins show some variation, possibly attributable to differences in specific protein preparation and assay procedures utilized by different individuals, the consensus scheme that these data show is that in the absence of substrate peptide, the stress-70 proteins have a low basal ATPase rate, typically found to be 0.01-0.03 (mol ATP/mol stress-70 protein min). This can be enhanced severalfold either by binding of peptides or denatured proteins, or (as demonstrated by the effect of grpE and dnaJ proteins on dnaK) by the action of ancillary proteins. To the extent that the observations on HSC70 can be generalized to other members of the stress-70 protein family, peptide binding appears to relieve the attenuation of ATPase activity and allow it to proceed at the rate characteristic of the ATPase fragment of the protein alone. 

A biologic reason for the abundance of nonlamellar lipids in membranes is that they possess the ability to modulate the activities of membrane proteins (15, 16). It has been recognized that membranes exist in a state of curvature frustration, which may be sufficiently large to have significant effect on certain protein conformations (17). Many examples show that the lipid bilayer elastic curvature stress indeed couples to conformational changes of membrane proteins (15, 18, 19). Protein kinase C is one such example of an enzyme activated by lipids that exhibit a propensity for nonlamellar phase formation (20). The activity of Ca " -ATPase from sarcoplasmic reticulum membranes also strongly correlates with the occurrence of nonbilayer lipids in the membrane and increases with the increase of their amount. It is noteworthy that the protein activity does not depend on the chemical structure of the lipids but only on their phase propensity thus specific binding interactions are ruled out. The list of proteins with activities that depend on the phase properties... 

Members of the hspVO family of stress proteins bind the GSL 3 sulfogalactosyl ceramide (SGC) (207). The binding site is in the N-terminal ATPase domain (208). Adamantyl SGC, similarly generated via fatty acid replacement, similarly has proven water soluble and is an effective inhibitor of hsp70-SGC binding (200). Adamantyl SGC has been shown to inhibit hsp70 ATPase activity (209) in vitro and therefore may modulate its chaperone function in cells. Such an effect also has therapeutic potential (210). 

Some of these environmental aspects are obvious, such as anthropogenic sources of chemicals that disrupt the structure of delicate epithelia involved in solute transport, or directly inhibit the solute transporting proteins (e.g. Cu inhibition of Na+K+ ATPase [82]). The general effects of environmental stress are also well known, since many of the hormones released into the blood during stressful situations will alter the activity of ion transporters (e.g. corticosteroids, catecholamine [14]). 

Another activity associated with a stress-70 protein is the in vitro disassembly of clathrin cages into triskelions (see Section I V,D). Rothman and colleagues isolated and characterized a protein from bovine brain that facilitated the disassembly of clathrin cages in a reaction that required hydrolysis of ATP they referred to the protein as a clathrin-uncoating ATPase (Schlossman et ai, 1984). Subsequently, it was realized that this protein was a constitutively expressed member of the stress-70 family (HSC70) (Chappell et al 1986). 

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