Testing the Ability of Non-Methylamine Osmolytes Present in Kidney Cells to Counteract the Deleterious Effects of Urea on Structure, Stability and Function of Proteins

Abstract

Human kidney cells are under constant urea stress due to its urine concentrating mechanism. It is believed that the deleterious effect of urea is counteracted by methylamine osmolytes (glycine betaine and glycerophosphocholine) present in kidney cells. A question arises: Do the stabilizing osmolytes, non-methylamines (myo-inositol, sorbitol and taurine) present in the kidney cells also counteract the deleterious effects of urea? To answer this question, we have measured structure, thermodynamic stability (ΔG D o) and functional activity parameters (K m and k cat) of different model proteins in the presence of various concentrations of urea and each non-methylamine osmolyte alone and in combination. We observed that (i) for each protein myo-inositol provides perfect counteraction at 1∶2 ([myo-inositol]:[urea]) ratio, (ii) any concentration of sorbitol fails to refold urea denatured proteins if it is six times less than that of urea, and (iii) taurine regulates perfect counteraction in a protein specific manner; 1.5∶2.0, 1.2∶2.0 and 1.0∶2.0 ([taurine]:[urea]) ratios for RNase-A, lysozyme and α-lactalbumin, respectively.

A current perspective on the compensatory effects of urea and methylamine on protein stability and function

“…Interestingly, it has also been reported there are other counteraction systems including ureamyo-inositol and urea-taurine at least under in vitro conditions (Khan et al, 2013). Thus under cellular conditions, the cells may probably counteract urea's effect on proteins and enzymes by employing multiple counteracting systems as supported by the arguments that counteracting osmolytes act independently on proteins, i.e., none of the osmolytes affects the efficacy of the other (Khan et al, 2013;Mello and Barrick, 2003).…”

“…Thus under cellular conditions, the cells may probably counteract urea's effect on proteins and enzymes by employing multiple counteracting systems as supported by the arguments that counteracting osmolytes act independently on proteins, i.e., none of the osmolytes affects the efficacy of the other (Khan et al, 2013;Mello and Barrick, 2003).…”

“…However, in vitro studies by Ahmad and his group (Khan et al, 2013) have shown that the non-methylamine osmolytes, like myo-inositol and taurine can counteract the deleterious effects of urea on some model proteins like RNase-A, alpha-lactalbumin and lysozyme. For all proteins, myo-inositol was found to bring about perfect counteraction at 1:2 ratio (myoinositol: urea) while taurine shows protein-specific counteraction.…”

“…However, osmolytes other than methylamines must be tallied with these methylamines to achieve this ratio (Treberg et al, 2006). The urea-methylamine counteraction systems so far has been examined and found successfully working on (i) the thermal stability and functional activity of proteins/enzymes (Khan et al, 2013;Singh et al, 2007); (ii) thermal stability of Nucleic acids (tRNA) (Gluick and Yadav, 2003); (iii) the rate and extent of renaturation of acid denatured enzyme (Yancey and Somero, 1979); (iv) the reactivity of thiol groups of an enzyme (bovine glutamate dehydrogenase) (Yancey and Somero, 1979); (v) protein polymerisation (Tubulin and actin) (Hatori et al, 2014;Sackett, 1997;Tseng and Graves, 1998) and (vi) membrane fluidity (Barton et al, 1999), enzyme functional dynamics (Lactate dehydrogenase) (Zhadin and Callender, 2011) and aggregation of methane in aqueous solution (Sarma and Paul, 2012). Pertinently, it has been found that elasmobranches even during the embryo development accumulate both urea and TMAO together (Barton et al, 1999).…”

Co-evolution of proteins and solutions: protein adaptation versus cytoprotective micromolecules and their roles in marine organisms

“…Other studies showed synergistic interactions between amino acid and sugar stabilizers in thermal denaturation (Khan et al, 2013b) and between sucrose and glycine in pressure inactivation of an enzyme (Kidman and Northrop, 2005).…”