Solute compatibility with enzyme function and structure: rationales for the selection of osmotic agents and end-products of anaerobic metabolism in marine invertebrates.

The major nitrogenous osmolytes present in the cells of marine invertebrates, notably the free amino acids glycine, alanine and proline, and trimethylamine oxide and betaine, are highly compatible with proper enzyme function and structure. These nitrogenous osmolytes display either non-perturbing or, in some cases, favorable effects on enzyme-substrate and enzyme-cofactor complex formation, catalytic velocity and protein structural stability. In contrast, inorganic salts (KCl and NaCl) and certain of the free amino acids which play only a minor osmotic role, e.g., arginine and lysine, have strongly perturbing effects on one or more of these enzymic parameters. The compatible nitrogenous solutes therefore are suitable for use at high (several tenths molar) concentrations and at widely varying concentrations in osmo-conforming species. Certain nitrogenous solutes, especially trimethylamine oxide, betaine and glutamate, offset some of the perturbing effects of inorganic ions on enzyme function. The selective accumulation of osmolytes thus involves not only the concentration of non-perturbing solutes, but also a balanced accumulation of solutes with opposing effects on enzymes. The selection of end-products of anaerobic metabolism also appears to be based, in part, on considerations of solute compatibility with enzyme function. Octopine is a non-perturbing solute, whereas arginine, which is condensed with pyruvate to form octopine, is very strongly perturbing. Succinate has marked stabilizing effects on protein structure. We conclude that the composition of the intracellular fluids of marine invertebrates reflects selection for osmolytes and end-products whose net effects create a cellular microenvironment which is conducive to optimal enzyme function and structure. The accumulation of compatible solutes may preclude the necessity for widespread changes in protein structure in adapting to concentrated or highly variable osmotic environments.     

Effects of solute hydrophobicity and phospholipid composition on permeability of composite membranes containing phospholipids

Permeabilities of several solutes through the composite membranes containing phospholipids have been measured. They were inversely proportional to the content of the phospholipids in the membrane. Both the permeability of solutes and the degree of permeability change around the phase transition temperature of the phospholipids for the hydrophobic solutes such as n-butanol and salicylamide were larger than those for the hydrophilic solutes such as amino acids and pyridoxine. These results suggest thatthe permeation path of hydrophobic solutes is different from that of hydrophilic ones. The addition of phosphatidyl ethanolamine, phosphatidyl serine, or phosphatidic acid to the composite membrane influenced the solute permeability due to the introduced negative charge and/or the change in the molecular packing of phospholipid.     

水生生物的紫外防护剂——类菌胞素氨基酸

类菌胞素氨基酸(mycosporine-like amino acids, MAAs)是一大类以环己烯酮为基本骨架, 与不同类型氨基酸通过缩合作用形成的水溶性物质。它能在真菌、海洋细菌、蓝藻和真核藻类细胞中合成, 并在海生无脊椎动物和鱼类等生物体内积累, 广泛存在于多种水生生物中。它具有紫外光防护、渗透、繁殖调节及发育保护等功能。本文主要介绍有关类菌胞素氨基酸的分布、结构、生物合成和积累以及生理功能的研究进展。     

水生生物的紫外光防护剂--类菌胞素氨基酸

类菌胞素氨基酸（mycosporine—like amino acids,MAAs）是一大类以环己烯酮为基本骨架,与不同类型氨基酸通过缩合作用形成的水溶性物质。它能在真菌、海洋细菌、蓝藻和真核藻类细胞中合成,并在海生无脊椎动物和鱼类等生物体内积累,广泛存在于多种水生生物中。它具有紫外光防护、渗透、繁殖调节及发育保护等功能。本文主要介绍有关类菌胞素氨基酸的分布、结构、生物合成和积累以及生理功能的研究进展。     

Metabolism of amino acids during hyposmotic adaptation in the whiteleg shrimp, Litopenaeus vannamei

The penaeid prawn, Litopenaeus vannamei, was employed to investigate intracellular isosmotic regulation in situations where invertebrates encounter hyposmosis. Hemolymph osmolality was first analyzed to confirm osmoregulatory conditions in the experimental animals, followed by analysis of amino acids in muscle and hemolymph using high-performance liquid chromatography. Total muscle amino acid levels decreased when hemolymph osmolality was extremely low, whereas glycine and l-serine levels increased in the hemolymph. These results suggest that tissue amino acids were released into the hemolymph to lower the osmolality of the tissues for purposes of low-salinity adaptation. Next, oxygen consumption and ammonia excretion rates were examined, and the O/N ratio was determined. Oxygen consumption levels and ammonia excretion rates increased, and the O/N ratio decreased when the animals were exposed to low salinity. These results suggest that amino acids were abundantly consumed as an energy source when animals were exposed to low salinity. To confirm the consumption of particular amino acids, the specific activity of l-serine ammonia lyase was also examined. Specific activity was highest when l-serine levels in the hemolymph were highest. Thus, it appears that l-serine levels increased under hyposmotic conditions due to the consumption of l-serine as an energy source. It was concluded that particular amino acids as osmolytes are likely metabolized as energy sources and consumed for purposes of hyposmotic adaptation.     

Kinetic and Thermodynamic Aspects of Sodium-Coupled Amino Acid Transport by Marine Invertebrates

SYNOPSIS. Marine invertebrates absorb amino acids directly acrosstheir external body surfaces. This absorption process occursvia carrier-mediated transport systems which, recent evidencesuggests, may be sodium-dependent. Steady-state amino acid gradientsare maintained at levels exceeding 10³–10⁶ times thatof the external environment. Examination of the standing gradientsof total free amino acids, Na, and K in seven invertebratessuggests that an Na/amino acid cotransport model, or an Na/K/aminoacid cotransport model can account for amino acid gradientsof this magnitude. However, the Na : amino acid coupling coefficientsmust be 2 or 3 depending on factors such as membrane potentialand the intracellular Na activities. Evidence from studies ofL-alanine transport in the integument of the polychaete Glyceradibranchiata is presented showing that, for this case, the Na: alanine coupling coefficient is 3. It is concluded that themodels presented are plausible and readily testable explanationsfor the observed amino acid gradients in marine invertebrates.     

Osmotic adjustment in the mycelial ascomyceteNeocosmospora vasinfecta (E. F. Smith)

Osmotic adjustment in the ascomyceteNeocosmospora vasinfecta was investigated by determining intramycelial water, mycelial solutes, and total mycelial osmolality. Major organic and inorganic solutes as well as proline and glycine betaine were determined under conditions of osmotic stress and shock, imposed by 0.5M KCl. Comparison to glucose as a nonelectrolytic osmoticum was also made. Results quantitatively implicated the polyhydric alcohols as the osmotic adjusters. Changes in amino acids were due to growth and were not osmoregulatory in nature. The osmoticum was not utilized for osmotic adjustment. The growth ofN. vasinfecta in the presence of KCl indicated that this organism is moderately sensitive to osmotic stress.     

The Saltiness of the Sea Breaks DNA in Marine Invertebrates: Possible Implications for Animal Evolution

More than 97 percent of the world's water is ocean and its average osmolality of 1000 mosmol/kg is much higher than the 300 mosmol/kg found in most of the intercellular fluids of vertebrates. Many marine invertebrates are osmoconformers, meaning that the osmolality of their extracellular fluid is the same as that of seawater. We report here that marine invertebrates from diverse phyla have numerous DNA breaks in their cells while they are exposed to normal seawater containing high NaCl, but that the DNA breaks decrease or disappear when the animals are acclimated to the same water diluted to 300 mosmol/kg. We speculate that, since DNA breaks cause mutations, salinity might have important background effects on the rate and course of evolution.     

Adsorption and release of amino acids from epilithic biofilms in streams

SUMMARY. I. Stream sediment with a natural biofilm generally adsorbed more glycine, aspartic acid, and lysine than sterile sediment. Lysine was adsorbed to a greater extent than the other two amino acids.
2. In the presence of Ca²⁺, adsorption of aspartic acid increased and adsorption of lysine decreased. Glycine was not affected.
3. Stream sediment preferentially removed amino acids with a positive charge or a polar R group from maple leaf leachate.
4. It is estimated that stream invertebrates may have access to a maximum of 7.3% of the amino acids present in epilithic biofilms. More amino acids were available from biofilms exposed to leaf leachate.     

Some aspects of the ecophysiology of Tubificoides benedii and ultrastructural observations on endocuticular bacteria

Tubificoides benedii is regularly found in sulphide-rich sediments with extremely low oxygen tensions and can tolerate anaerobic conditions for several days. Although the anaerobic energy production of marine invertebrates has been well studied, almost nothing is known about the anaerobic metabolism of marine oligochaetes. Preliminary results after measuring end-products during anaerobic incubation show that in contrast to all previously examined marine facultative anaerobe invertebrates T. benedii degrades malate during anaerobiosis. Also, the concentration of free amino acids is extremely low for a marine organism. Low levels of free amino acids could be concomitant with malate utilization: the utilization of the amino acid aspartate (as observed in all other examined marine invertebrates) seems to be excluded by the low concentrations of aspartate and other amino acids in T. benedii.The physiological lab studies were supplemented by ecological investigations in the field and laboratory on the vertical distribution of T. benedii. 90% of the population was always found within the first few cm below the sediment surface. Aquarium observations showed that the posterior end of the worm projects above the sediment surface, where it slowly waves back and forth. This behavior points towards an intestinal respiration. The described orientation, an intestinal respiration and anaerobic energy production could be advantageous in sulphide-rich sediments where O₂ only penetrates a few mm into the sediment. The worm can easily inhabit the first three to four cm by holding its tail in the upper oxygenated sediment and water. Here it would be able to feed on the rich quantities of bacteria at the anoxic-oxic interface and yet still keep up an aerobic metabolism. In addition, its ability to produce energy anaerobically would allow T. benedii to dwell in deeper anoxic sediments for limited periods of time or to survive complete O₂ absence that could develop during low tide.The posterior ends of T. benedii found in a sulphide-rich habitat in the German Wadden Sea were covered with filamentous epibacteria (Dubilier, 1986). Electron microscopy showed that the bacteria were anchored in the cuticle. The association is apparently not pathogenic whereas positive forms of interaction can be envisioned.     

A fast and sensitive method for measuring picomole levels of total free amino acids in very small amounts of biological tissues

Summary. In the present study we describe a simple and fast method to measure the concentration of total free amino acids in very small amounts of biological tissues. The procedure described here is based on the reaction of free amino acids with o-phthaldialdehyde (OPA) in the presence of a reducing agent, β-mercaptoethanol (MET), to give a complex which can be measured by fluorescence. It is a very rapid process and has the same reliability as the conventional ninhydrin method of Moore and Stein but is about 500 times more sensitive. The sensitivity of the new protocol is such to permit the determination with high reliability of very small amounts of free amino acids at picomole levels, either in a standard amino acid mixture or in biological tissues, without chromatographic separation of the amino acids. It is particularly useful when the amount of the sample is very low, e.g. on a single pituitary or pineal gland of small animals or on single cells, such as oocytes or eggs, as well as single ganglions or axons of marine invertebrates. Received September 22, 1999 Accepted July 5, 2000     

Recent Progress in the Study of "Die Ernahrung der Wassertiere und der Stoffhaushalt der Gewasser"

SYNOPSIS. It is now possible to provide direct evidence fornet removal of significant amounts of specific amino acids fromnaturally occurring dissolved organic material by a marine invertebrate.This demonstration serves to focus attention and invite reconsiderationof the large body of less direct evidence supporting the generaloccurrence of this capacity among soft-bodied marine invertebrates.     

Unusual organic osmolytes in deep-sea animals: adaptations to hydrostatic pressure and other perturbants

Shallow-living marine invertebrates use free amino acids as cellular osmolytes, while most teleosts use almost no organic osmolytes. Recently we found unusual osmolyte compositions in deep-sea animals. Trimethylamine N-oxide (TMAO) increases with depth in muscles of some teleosts, skates, and crustaceans (up to 300 mmol/kg at 2900 m). Other deep-sea animals had high levels of (1). scyllo-inositol in echinoderms, gastropods, and polychaetes, (2). that polyol plus beta-alanine and betaine in octopods, (3). hypotaurine, N-methyltaurine, and unidentified methylamines in vestimentiferans from hydrothermal vents and cold seeps, and (4). a depth-correlated serine-phosphate osmolyte in vesicomyid clams from trench seeps. We hypothesize that some of these solutes counteract effects of hydrostatic pressure. With lactate dehydrogenase, actin, and pyruvate kinase, 250 mM TMAO (but not glycine) protected both ligand binding and protein stability against pressure. To test TMAO in living cells, we grew yeast under pressure. After 1 h at 71 MPa, 3.5 h at 71 MPa, and 17 h at 30 MPa, 150 mM TMAO generally doubled the number of cells that formed colonies. Sulfur-based osmolytes which are not correlated with depth, such as hypotaurine and thiotaurine, are probably involved in sulfide metabolism and detoxification. Thus deep-sea osmolytes may have at least two other roles beyond acting as simple compatible osmotica.     

Volume-regulatory Amino Acid Release from the Protozoan Parasite Crithidia luciliae

The unicellular protozoan parasite, Crithidia luciliae, responded to osmotic swelling by undergoing a regulatory volume decrease. This process was accompanied by the efflux of amino acids (predominantly alanine, proline and glycine). The relative loss of the electroneutral amino acids proline, valine, alanine and glycine was greater than that for the anionic amino acid, glutamate; there was negligible loss of the cationic amino acids, lysine, arginine and ornithine. The characteristics of amino acid release were investigated using a radiolabeled form of the nonmetabolized alanine analogue α-aminoisobutyrate. α-Aminoisobutyrate efflux was activated within a few seconds of a reduction of the osmolality, and inactivated rapidly (again within a few seconds) on restoration of isotonicity. The initial rate of efflux of α-aminoisobutyrate from cells in hypotonic medium was unaffected by the extracellular amino acid concentration. Hypotonically activated α-aminoisobutyrate efflux (as well as the associated regulatory volume decrease) was inhibited by the sulfhydryl reagent N-ethylmaleimide but was not inhibited by a range of anion transport blockers. As in the efflux experiments, unidirectional influx rates for α-aminoisobutyrate increased markedly following reduction of the osmolality, consistent with the swelling-activated amino acid release mechanism allowing the flux of solutes in both directions. Hypotonically activated α-aminoisobutyrate influx showed no tendency to saturate up to an extracellular concentration of 50 mm. The functional characteristics of the amino acid release mechanism are those of a channel, with a preference for electroneutral and anionic amino acids over cationic amino acids. However, the pharmacology of the system differs from that of the anion-selective channels that are thought to mediate the volume-regulatory efflux of organic osmolytes from vertebrate cells. Received: 13 May 1996/Revised: 9 July 1996     

Gas-liquid chromatography and enzymatic determination of alanopine and strombine in tissues of marine invertebrates

Gas-liquid chromatography (GLC) and enzymatic assays were developed for quantitating the imino acids, alanopine and strombine, alternate products of anaerobic glycolysis (replacing lactate) in the tissues of many marine invertebrates. For GLC analysis, d-strombine (2-methyliminodiacetic acid) and meso-alanopine (2,2′-iminodipropionic acid) were chromatographeo as N-trifluoroacetyl isobutyl esters. Modifications of techniques used for GLC analysis of amino acids were required to overcome steric hindrance in the acylation reaction caused by the presence of imino, rather than amino, groups. Both imino acids were separated from each other and from all amino acids by GLC. Detection limit of the technique was 0.05 μg imino acid. Enzymatic determination of imino acids made use of the alanopine-specific alanopine dehydrogenase (ADH) purified from the periwinkle, Littorina littorea, and the strombine/alanopine utilizing strombine dehydrogenase (SDH) from the clam, Mercenaria mercenaria, with assay conditions: 300 mm hydrazine buffer, pH 9.0, 5 mm NAD, and 0.3 unit ADH or 1.0 unit SDH. Enzymatic determinations of mixtures of alanopine and strombine in tissue samples required a dual analysis using both enzymes. Production of alanopine and strombine during anoxic stress in two species of marine molluscs was quantitated.     

Principles of cell volume regulation

Cell volume is determined by the content of osmotically active solute (cell osmoles) and the osmolarity of the extracellular fluid. Cell osmoles consist of non-diffusible and diffusible solutes. A large fraction of the diffusible cation content balances negative charges on the non-diffusible solutes. The content of diffusible solutes is determined by the electrochemical gradients driving them across the plasma membrane and the availability and activity of transport pathways in the membrane. The classical view that the sodium pump offsets passive leaks must be modified to accommodate the contributions of a number of secondary active transport processes, as well as to allow for changes in cell nondiffusible osmoles and in their net negative charge. The behaviour of cells in anisosmotic media is often different from that predicted for a perfect osmometer. In many cases this is a consequence of changes in cell osmole content. However, caution is required in extrapolating from in vitro responses of isolated cells to large, acutely induced changes in medium osmolality to the responses of tissues in vivo to more subtle changes in extracellular osmolality.     

Free amino acids in some sponges

Most invertebrates, particularly those of marine origin, have relatively high concentrations of free amino acids which are considered an important constituent of their osmoregulatory mechanisms [1]. Very little information is available on the free amino acid distribution in Porifera [2,3]. Common amino acids in some sponges were recognised by paper chromatography by Inskip and Cassidy [4] and Ackermann et al. [5,6] included a few sponges in their survey of the occurence of nitrogen compounds in marine invertebrates. More recently Bergquist and Hartman [7] surveyed semiquantitatively the distribution of free amino acids in several sponges. In the present paper we report on the amino acid composition of 12 species of sponges belonging to the class Demospongiae as a part of a study on the metabolites of Porifera [8]. Fresh sponges were extracted with aqueous ethanol. The organic solvent was removed and the aqueous solution, after removal of the ether soluble compounds, was separated into cationic, anionic and neutral fractions by ion-exchange chromatography. The cation fraction was analysed for amino acids using an automatic amino acid analyser. The results, which are presented in Table 1, show that all species of sponges examined have a similar composition in common amino acids. Glycine almost always appears as the dominant protein amino acid, followed by high concentrations of alanine and glutamic acid, whereas relatively lower concentrations of basic amino acids are present. In Axinella cannabina, Chondrosia reniformis, Chondrilla nucula, Cliona viridis and Hymeniacidon sanguinea, glycine represents more than 77% of the total amino acids. The high percentage of free glycine (90.4%) in Chondrosia reniformis is noteworthy. The anionic and the neutral fractions were examined for sulfur-containing amino acids using PC. Taurine (Table 2) was detected in all the Porifera examined; this is in agreement with previous observations [5–7]. N-Methyltaurine was identified in some of the species examined, whereas neither N,N-dimethyltaurine nor N,N,N-trimethyltaurine were found.     

Thermotropic behavior of sphingophospholipids of marine invertebrates

Thermotropic behavior of total fraction of sphingomyelin (SM) and its forms, sphingomyelin that contains acyl residues of normal fatty acids (SMN) and sphingomyelin that is Nacylated predominantly with hydroxy acids (SMH) as well as of ceramideaminoethylphosphonate (CAEP) has been studied by the method of differential scanning microcalorimetry. It was shown that chromatographically pure anhydrous sphingophospholipids isolated from different species of marine invertebrates have no phasic transitions in the entire temperature interval of scanning, from-100 to 100°C, whereas partially hydrated samples or mixtures of sphingophospholipids with glycerophospholipids (phosphatidylcholine or phosphatidylethanolamine isolated from corresponding marine invertebrates had phasic transitions in a large temperature range with thermoabsorption maxima in the areas of -40–20°C and 20–80°C. It is suggested that sphingophospholipids, as compared to marine invertebrate glycerophospholipids, have a much lower capability for self-organization, and to form regular supramolecular sphingophospholipid structures both in artificial systems and in biomembranes, such orientants as glycerophospholipids and water are necessary. Possible causes of an unusual behavior of sphingophospholipids of marine invertebrates as compared to glycerophospholipids are discussed.     

Cloning of the cDNa for a Na+/myo-inositol cotransporter, a hypertonicity stress protein.

Kidney medullary cells in situ, as well as kidney-derived Madin-Darby canine kidney (MDCK) cells accumulate nonperturbing, small organic solutes (osmolytes), including myo-inositol, when bathed in hypertonic media. Accumulation of osmolytes balances the osmolality of extracellular fluid without raising intracellular salts that would perturb cellular functions. In hypertonic media, increased myo-inositol accumulation is the result of increased activity of a Na+/myo-inositol cotransporter. We have isolated a cDNA encoding a Na+/myo-inositol cotransporter from MDCK cells using expression in Xenopus oocytes. The cDNA sequence predicts a protein of 718 amino acids with a significant amino acid sequence similarity to the Na+/D-glucose cotransporters of absorbing epithelia. Transporter mRNA is present in kidney and brain and is markedly induced in MDCK cells by medium hypertonicity, demonstrating that adaptation to hypertonic stress involves up-regulation of transporter mRNA accumulation.     

Hypotaurine, N-methyltaurine, taurine, and glycine betaine as dominant osmolytes of vestimentiferan tubeworms from hydrothermal vents and cold seeps

Organic osmolytes, solutes that regulate cell volume, occur at high levels in marine invertebrates. These are mostly free amino acids such as taurine, which are "compatible" with cell macromolecules, and methylamines such as trimethylamine oxide, which may have a nonosmotic role as a protein stabilizer, and which is higher in many deep-sea animals. To better understand nonosmotic roles of osmolytes, we used high-performance liquid chromatography and (1)H-nuclear magnetic resonance (NMR) to analyze vestimentiferans (vestimentum tissue) from unusual marine habitats. Species from deep hydrothermal vents were Riftia pachyptila of the East Pacific Rise (2,636 m) and Ridgeia piscesae of the Juan de Fuca Ridge (2,200 m). Species from cold hydrocarbon seeps were Lamellibrachia sp. and an unnamed escarpid species from subtidal sediment seeps (540 m) off Louisiana and Lamellibrachia barhami from bathyal tectonic seeps (1,800-2,000 m) off Oregon. Riftia were dominated by hypotaurine (152 mmol/kg wet wt), an antioxidant, and an unidentified solute with an NMR spectrum consistent with a methylamine. Ridgeia were dominated by betaine (N-trimethylglycine; 109 mmol/kg), hypotaurine (64 mmol/kg), and taurine (61 mmol/kg). The escarpids were dominated by taurine (138 mmol/kg) and hypotaurine (69 mmol/kg). Both Lamellibrachia populations were dominated by N-methyltaurine (209-252 mmol/kg), not previously reported as a major osmolyte, which may be involved in methane and sulfate metabolism. Trunk and plume tissue of the Oregon Lamellibrachia were nearly identical to vestimentum in osmolyte composition. The methylamines may also stabilize proteins against pressure; they were significantly higher in the three deeper-dwelling groups.