SUMMARY Organic osmolytes are small solutes used by cells of numerous water-stressed organisms and tissues to maintain cell volume. Similar compounds are accumulated by some organisms in anhydrobiotic, thermal and possibly pressure stresses. These solutes are amino acids and derivatives,polyols and sugars, methylamines, methylsulfonium compounds and urea. Except for urea, they are often called `compatible solutes', a term indicating lack of perturbing effects on cellular macromolecules and implying interchangeability. However, these features may not always exist, for three reasons. First, some of these solutes may have unique protective metabolic roles, such as acting as antioxidants (e.g. polyols, taurine, hypotaurine),providing redox balance (e.g. glycerol) and detoxifying sulfide (hypotaurine in animals at hydrothermal vents and seeps). Second, some of these solutes stabilize macromolecules and counteract perturbants in non-interchangeable ways. Methylamines [e.g. trimethylamine N-oxide (TMAO)] can enhance protein folding and ligand binding and counteract perturbations by urea (e.g. in elasmobranchs and mammalian kidney), inorganic ions, and hydrostatic pressure in deep-sea animals. Trehalose and proline in overwintering insects stabilize membranes at subzero temperatures. Trehalose in insects and yeast,and anionic polyols in microorganisms around hydrothermal vents, can protect proteins from denaturation by high temperatures. Third, stabilizing solutes appear to be used in nature only to counteract perturbants of macromolecules,perhaps because stabilization is detrimental in the absence of perturbation. Some of these solutes have applications in biotechnology, agriculture and medicine, including in vitro rescue of the misfolded protein of cystic fibrosis. However, caution is warranted if high levels cause overstabilization of proteins.

Intracellular fluids of marine elasmobranchs (sharks, skates and rays), holocephalans and the coelacanth contain urea at concentrations averaging 0.4m, high enough to significantly affect the structural and functional properties of many proteins. Also present in the cells of these fishes are a family of methylamine compounds, largely trimethylamine N-oxide with some betaine and sarcosine, and certain free amino acids, mainly beta-alanine and taurine, whose total concentration is approx. 0.2m. These methylamine compounds and amino acids have been found to be effective stabilizers of protein structure, and, at a 1:2 molar concentration ratio of these compounds to urea, perturbations of protein structure by urea are largely or fully offset. These counteracting effects of solutes on proteins are seen for: (1) thermal stability of protein secondary and tertiary structure (bovine ribonuclease); (2) the rate and extent of enzyme renaturation after acid denaturation (rabbit and shark lactate dehydrogenases); and (3) the reactivity of thiol groups of an enzyme (bovine glutamate dehydrogenase). Attaining osmotic equilibrium with seawater by these fishes has thus involved the selective accumulation of certain nitrogenous metabolites that individually have significant effects on protein structure, but that have virtually no net effects on proteins when these solutes are present at elasmobranch physiological concentrations. These experiments indicate that evolutionary changes in intracellular solute compositions as well as in protein amino acid sequences can have important roles in intracellular protein function.

No fish have been found in the deepest 25% of the ocean (8,400-11,000 m). This apparent absence has been attributed to hydrostatic pressure, although direct evidence is wanting because of the lack of deepest-living species to study. The common osmolyte trimethylamine N-oxide (TMAO) stabilizes proteins against pressure and increases with depth, going from 40 to 261 mmol/kg in teleost fishes from 0 to 4,850 m. TMAO accumulation with depth results in increasing internal osmolality (typically 350 mOsmol/kg in shallow species compared with seawater's 1,100 mOsmol/kg). Preliminary extrapolation of osmolalities of predicted isosmotic state at 8,000-8,500 m may indicate a possible physiological limit, as greater depths would require reversal of osmotic gradients and, thus, osmoregulatory systems. We tested this prediction by capturing five of the second-deepest known fish, the hadal snailfish (Notoliparis kermadecensis; Liparidae), from 7,000 m in the Kermadec Trench. We found their muscles to have a TMAO content of 386 ± 18 mmol/kg and osmolality of 991 ± 22 mOsmol/kg. These data fit previous extrapolations and, combined with new osmolalities from bathyal and abyssal fishes, predict isosmotic state at 8,200 m. This is previously unidentified evidence that biochemistry could constrain the depth of a large, complex taxonomic group.deep-sea | piezolyte | chemical chaperone | osmoregulation

SYNOPSIS. Organic osmolytes are small solutes used by cells of numerous waterstressed organisms and tissues to maintain cell volume. All known osmolytes are amino acids and derivatives, polyols and sugars, methylamines, and urea; unlike salt ions, most are ''compatible,'' i.e., do not perturb macromolecules. In addition, some stabilize macromolecules and are ''counteracting'' towards perturbants, e.g., methylamines can stabilize proteins and ligand binding against perturbations by urea in elasmobranchs and mammalian kidney, and (our latest findings) high hydrostatic pressure in deep-sea animals. Methylamines appear to coordinate water molecules tightly, resulting in osmolyte exclusion from hydration layers of peptide backbones. This makes unfolded protein conformations entropically unfavorable (work of Timasheff, Galinski, Bolen and coworkers). These properties have led to proposed uses in biotechnology, agriculture and medicine, including improved biochemical methods, in vitro rescue of misfolded proteins in cystic fibrosis and prion diseases (work of Welch and others), and plants engineered for drought and salt tolerance. These properties also explain some but not all of the considerable variation in osmolyte composition among species with different metabolisms and habitats, and among and within mammalian tissues in development.