Quantitation of CaP2+ fluxes in chick calvaria

This report utilizes an Ussing-type apparatus to quantitative the unidirectional fluxes of Ca²⁺ across the periosteal and endosteal membranes of frontal bones derived from calvaria of 20-day chick embryos. The influx was found to be proportional to the concentration of Ca²⁺ and equal to 0.31 μmoles/cm² per h at a concentration of ultrafiltrable Ca²⁺ of 1.75 mM. There were observable differences in the influx measured from periosteal or endosteal sides. The influx was found to be inversely proportional to decreasing temperature and increasing viscosity. The influx increased to 150% of the control flux when the incubation medium contained iodoacetate at a concentration of 1 mM and increased to 200% of control flux when the endosteum or periosteum was removed. These characteristics support the view that the influx is a passive flow with the integrity of cellular layer a controlling factor. The endosteal efflux was greater by a factor of 2 when compared to the periosteal efflux at 37 °C. When the temperature was reduced to 6 °C the endosteal to periosteal efflux ratio decreased to 1.26 indicating a temperature-sensitive component in the endosteal efflux.     

Analysis of Glycerol-3H Transport in the Frog Oocyte by Extractive and Radioautographic Techniques

The efflux of glycerol-³H from mature R. pipiens oocytes was studied by extractive analysis and by quantitative radioautography using techniques suitable for diffusible solutes. Extractive analysis was used to determine the total cellular concentration of tracer, and radioautography, regional intracellular concentrations, at equilibrium and as a function of efflux time, t_E. The efflux was resolvable into four kinetic fractions: cytoplasmic fast and slow fractions, and nuclear fast and slow fractions. The fast fractions represent freely diffusible glycerol in the two compartments; the solvent space accessible to glycerol is unity in the nucleus (germinal vesicle), but only 0.73 in the cytoplasm. The efflux of both fast fractions from the cell is determined by the permeability of the cortical membrane, with neither the nuclear membrane nor diffusion in the cytoplasm detectably slowing the flux. The permeability at 13.6°C is 2.2 x 10^-5 cm/sec. The slow fractions leave the cell at about one-tenth the rate of the fast; the interpretation is that these fractions represent glycerol bound to impermeant cellular constituents. The size of these constituents is below the radioautographic resolution; in the cytoplasm, they appear not to be the yolk platelets. The extent of binding is about fourfold greater, per milliliter of compartment water, in the cytoplasm than in the germinal vesicle.     

Glycerol permeability of rye leaf protoplasts as affected by temperature and electroporation

The permeability of rye leaf protoplasts to glycerol was determined using 1,3-¹⁴C glycerol and liquid scintillation spectrometry. Estimates were 1.0×10⁻⁸ m s⁻¹ at 0°C and 4.1×10⁻⁸ m s⁻¹ at 22 and 31°C. The activation energy for glycerol permeability was 32.8 kJ/mol. The effect of electroporation on glycerol uptake was also explored. Treatments were performed with a field strength of 100 V/cm and an exponential decay constant of 5.8 ms. At 22 °C, electroporation affected the rate and extent of glycerol permeation, causing an increase in the intercept of the glycerol uptake curve and a decrease in the slope. Electroporation had no significant effect on glycerol uptake when performed at 0°C, when the cells were electroporated at 0°C then warmed to 31 °C, or when the cells were electroporated at 22 °C then cooled to 0°C. The results at 22°C were consistent with an influx of glycerol during electroporation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Amino acid accumulation in frog muscle: I. Steady-state glycine accumulation at o °C

Glycine is not significantly metabolized by frog muscle maintained at o °C in vitro. Nevertheless, in this preparation steady-state levels of [¹⁴C]glycine as high as 20 times the external concentration are attained after 3–6 days at o °C. The concentration gradient at the steady state depends on the external concentration, being highest at low external concentrations (approx. 0.1 mM) and reversed at external concentrations above 10 mM.A plot of the steady-state cellular levels of glycine vs the external concentration reveal linear and saturable components. The linear fraction has an average distribution ratio of 0.54 indicating that glycine is partially excluded from the muscle water at this temperature.Efflux of labeled glycine at o °C from previously loaded frog muscle follows first-order kinetics. The rate constant increases with increasing concentrations of glycine in the external medium (efflux facilitation).The steady-state results are shown to be consistent with an adsorption model for amino acid accumulation as well as a model in which amino acid enters the cell via a carrier and exits via a bidirectional leakage pathway. A model in which efflux proceeds through the carrier does not fit the data. This indicates that an alternative to exchange diffusion is needed to explain the observed efflux facilitation.     

The temperature-mediated metabolism of 1-acyl-lysophosphatidyl glycerol in cerulenin-treated cultures of Bacillus megaterium.

When [U-¹⁴C]palmitate was added to a culture of $\text{B. megaterium}$ that had been grown at 35°, transferred to 20° and treated with cerulenin, label was initially incorporated into lysophosphatidyl glycerol. The labeled lyso derivative, in turn, was converted to phosphatidyl glycerol, apparently by esterification of the 2-position with endogenous acyl groups. Labeled lysophosphatidyl glycerol synthesis at 20° was observed only when a culture was treated with cerulenin prior to the addition of [U-¹⁴C]palmitate. When [U-¹⁴C]palmitate was added before cerulenin, labeled lysophosphatidyl glycerol formation was not detected. When chloramphenicol was added with cerulenin at the time of culture transfer from 35° to 20°, the synthesis of lysophosphatidyl glycerol was unaffected but the rate of its esterification to phosphatidyl glycerol was significantly retarded. Transfer of such a culture back to 35° resulted in a marked acceleration in the rate of conversion of lysophosphatidyl glycerol to phosphatidyl glycerol.     

Phloem Unloading in Stem Parts Parasitized by Cuscuta: The Release of 14C and K+ to the Free Space at 0°C and 25°C

By washing out ¹⁴C-solutes or K⁺ from the free space of stem segments of Vicia faba L. and Urtica dioica L., efflux from stem tissues was measured. At 25°C the efflux of ¹⁴C or K⁺ from non-parasitized stem parts was found to be comparable with the pattern known for parenchyma cells. The efflux pattern from parasitized stem parts kept at 0°C was also a normal one, but at 25°C a more complicated pattern was found. After addition of metabolic inhibitors, however, the efflux pattern appeared to be comparable with the pattern found in 0°C experiments. Also, after washing stem segments for several hours in water at 0°C, Phloem unloading was shown to start unimpeded after transfer to 25°C. Stem parts parasitized by Cuscuta can be regarded as a model system for studying the fundamental processes underlying movement out of sieve tubes in strong sink regions.     

Permeability of the human erythrocyte to glycerol in 1 and 2 m solutions at 0 or 20 °C

There are increasing numbers of exceptions to a central tenet in cryobiology that low-molecular-weight protective solutes such as glycerol must permeate cells in high concentration in order to protect them from freezing injury. To test this supposition, it is necessary to determine the amount of solute that has permeated a cell prior to freezing. The amount in human red cells was estimated from the flux equation $\text{ds}\text{dt}\text{= P}{}_{\mathrm{<mtext>\gamma </mtext>}}\text{A}$[(activity external solute) — (activity internal solute)]. Solving the equation required knowledge of P_γ the permeability constant for the solute. Estimates of P_γ for glycerol were made in two ways: (i) by measuring the time to 50% hemolysis of human red cells suspended in 1 or 2 m solutions of glycerol that were hypotonic with respect to NaCl, and (ii) by measuring the time required for red cells in 1 or 2 m solutions of glycerol in isotonic saline-buffer to undergo osmotic shock upon tenfold dilution with isotonic saline-buffer. The measurements were made at 0 and 20 °C. The values of P_γ were about 2.5 × 10^?4 cm/min at 20 °C and about 0.9 × 10^?4 cm/min at 0 °C. The difference corresponds to an activation energy of 7.2 kcal/mole. These values of P_γ are 100 to 600 times higher than those for glycerol permeation in the bovine erythrocyte. The values of P were relatively unaffected by whether calculations were based on classical or irreversible thermodynamics and by the choice of concentration units in the flux equations. Calculations of the kinetics of glycerol entry using these P values showed that the concentration of intracellular glycerol reaches 90% of equilibrium in 1.2 min at 0 °C and in 0.6 min at 20 °C. The osmolal ratio of intracellular glycerol to intracellular nonpermeating solutes reaches 90% of equilibrium in 7 min at 0 °C and in 3.2 min at 20 °C.     

Studies on the low temperature induced biogenesis of glycerol by adult Protophormia terranovae

Physiological and biochemical changes accompanying cold stress in the diapausing adult arctic blowfly, Protophormia terranovae, have been observed. In the laboratory, this insect survives prolonged periods at temperatures in the range of ?1°C to +4°C. Concentrations of free glycerol in excess of 10% of fresh body weight have been measured and the rate of its synthesis is greater at +1°C to +4°C than at ?1°C to 0°C. Under these conditions Protophormia also undergoes significant weight loss (up to 58% over 39 days) presumably in part due to dehydration. Its respiration rate decreases as expected when first shifted from 20°C to 0°C but the rate declines an additional 70% after exposure to 0°C for 24 hr. This lowest rate, which is then maintained, when considered with the initial faster one suggests positive thermal modulation is coupled to inverse thermal compensation during cold stress. This was not observed with nondiapausing Protophormia.Increments in free glycerol are accompanied by decreases in the insect's total glycogen reserves but upon rewarming, they return to pre-cold stress levels. While pre-stress glycogen stores are insufficient to provide for most of the free glycerol which accumulates, ingested carbohydrate present in the crop provides sufficient quantities. Studies with [¹⁴C] glucose indicate it is also metabolically active at low temperature.Neutral glyceride glycerol cannot contribute to net synthesis of free glycerol in significant amounts since the steady state concentrations present in pre-cold stressed insects decrease only slightly during cold stress. Furthermore, the specific radioactivity of acyl glyceride glycerol labelled in vivo with 2-[³H] glycerol before cold stress, remains unchanged during hibernation indicating that acyl glycerides are not turning over glycerol units produced by catabolism of hexose. The results of these studies argue that carbohydrate and not lipid glycerol is the source of the free glycerol which accumulates in Protophormia at low temperatures. The relationship of the above results to possible mechanisms which should permit glycerol accumulation under aerobic conditions are discussed.     

Freezing injury to erythrocytes. I. Freezing patterns and post-thaw hemolysis.

The extent of hemolysis of human red blood cells suspended in different concentrations of glycerol and frozen at various cooling rates was investigated on the basis of morphological observation in the frozen state. Hemolysis of the cells in the absence of glycerol showed a V-shaped curve in terms of cooling rates. There was 70% hemolysis at an optimal cooling rate of approximately 10³ °C/min and 100% hemolysis at all other rates tested. Morphologically, a lower than optimal cooling rate resulted in cellular shrinkage, while a higher than optimal rate resulted in the formation of intracellular ice.The cryoprotective effect of glycerol was dependent upon its concentration and on the cooling rate. Samples frozen at 10³ and 10⁴ °C/min showed freezing patterns which differed from cell to cell. The size of intraand extracellular ice particles became smaller, and there was less shrinkage or deformation of cells as the rate of cooling and concentration of glycerol were increased.There was some correlation between the morphology of frozen cells and the extent of post-thaw hemolysis, but the minimum size of intracellular ice crystals which might cause hemolysis could not be estimated. As a cryotechnique for electron microscopy, the addition of 30% glycerol and ultrarapid freezing at 10⁵ °C/min are minimum requirements for the inhibition of ice formation and the prevention of the corresponding artifacts in erythrocytes.     

SODIUM-DEPENDENT EFFLUX AND EXCHANGE OF GABA IN SYNAPTOSOMES

Abstract— The influx and efflux of [³H]GABA were investigated in synaptosomes. Two efflux components were detected. The first, termed spontaneous efflux, was not affected by the external sodium chloride concentration. The second, termed GABA-stimulated efflux, was observed when low levels of GABA were added to the incubation medium and was found to require external sodium chloride. The rate of spontaneous efflux at 0°C was about 37 per cent of the rate at 27°C but both GABA-stimulated efflux and GABA influx were completely inhibited at 0°C. The stimulation of efflux by external GABA followed simple Michaelis–Menten kinetics with respect to external GABA. The concentration of external GABA required for half-maximal stimulation was 4·9 ± 1·4 μm and the V_max for efflux was 1·0 ± 0·6 nmol. min^-1.mg^-1 of protein. A similar stimulation of efflux was observed with GABA analogue l -2,4-diamino-butyric acid which is a competitive inhibitor of influx. The concentration of external l -2,4-diaminobutyric acid required for half-maximal stimulation of efflux was 51 ± 12 μm and the V_max for efflux was 0·8 ± 0·5 nmol.min^-1.mg^-1 of protein. Since the sodium-dependency, temperature sensitivity, and kinetic properties of the GABA-stimulated efflux system were similar to the influx system, GABA-stimulated efflux was attributed to carrier-mediated exchange diffusion. Measurement of efflux and influx in the same preparation showed there was a net efflux when total fluxes were considered and that the exchange ratio (influx to GABA-stimulated efflux) was 0·9 when carrier-mediated fluxes were considered. The effect of the temperature of the fluid used to rinse synaptosomes collected on filters in influx experiments was investigated. There was no detectable difference in measured values of influx between samples rinsed with cold fluid (0°C) and warm fluid (27°C). The endogenous GABA content of synaptosomes was found to be 20·3 ± 2·5 nmol GABA per mg of protein. From this value, the cytoplasmic concentration of GABA in synaptosomes was estimated to be a maximum of 40 mm . About 5 per cent of total cerebral cortical GABA was found in the synaptosomal fraction.     

Activation of K-Cl Cotransport by Mild Warming in Guinea Pig Red Cells

Unidirectional, ouabain-insensitive K⁺ influx rose steeply with warming at temperatures above 37°C in guinea pig erythrocytes incubated in isotonic medium. The only component of ouabain-insensitive K⁺ influx to show the same steep rise was K-Cl cotransport (Q₁₀ of 10 between 37 and 41°C); Na-K-Cl cotransport remained constant or declined and residual K⁺ influx in hypertonic medium with ouabain and bumetanide rose only gradually. Similar results were obtained for unidirectional K⁺ efflux. Thermal activation of K-Cl cotransport-mediated K⁺ influx was fully dependent on the presence of chloride in the medium; none occurred with nitrate replacing chloride. The increase of K⁺ influx through K-Cl cotransport from 37 to 41°C was blocked by calyculin A, a phosphatase inhibitor. The Q₁₀ of K-Cl cotransport fully activated by hydroxylamine and hypotonicity was about 2. The time course of K⁺ entry showed an immediate transition to a higher rate when cells were instantly warmed from 37 to 41°C, but there was a 7-min time lag in returning to a lower rate when cells were cooled from 41 to 37°C. These results indicate that the steepness of the response of K-Cl cotransport to mild warming is due to altered regulation of the transporter. Total unidirectional K⁺ influx was equal to total unidirectional K⁺ efflux at 37–45°C, but K⁺ influx exceeded K⁺ efflux at 41°C when K-Cl cotransport was inhibited by calyculin or prevented by hypertonic incubation. The net loss of K⁺ that results from the thermal activation of isosomotic K-Cl cotransport reported here would offset a tendency for cell swelling that could arise with warming through an imbalance of pump and leak for Na⁺ or for K⁺. Received: 1 November 1997/Revised: 5 March 1998     

Freezing-induced cellular and membrane dehydration in the presence of cryoprotective agents

Abstract

FTIR and cryomicroscopy have been used to study mouse embryonic fibroblast cells (3T3) during freezing in the absence and presence of DMSO and glycerol. The results show that cell volume changes as observed by cryomicroscopy typically end at temperatures above ?15°C, whereas membrane phase changes may continue until temperatures as low as ?30°C. This implies that cellular dehydration precedes dehydration of the bound water surrounding the phospholipid head groups. Both DMSO and glycerol increase the membrane hydraulic permeability at subzero temperature and reduce the activation energy for water transport. Cryoprotective agents facilitate dehydration to continue at low subzero temperatures thereby decreasing the incidence of intracellular ice formation. The increased subzero membrane hydraulic permeability likely plays an important role in the cryoprotective action of DMSO and glycerol. In the presence of DMSO water permeability was found to be greater compared to that in the presence of glycerol. Two temperature regimes were identified in an Arrhenius plot of the membrane hydraulic permeability. The activation energy for water transport at temperature ranging from 0 to ?10°C was found to be greater than that below ?10°C. The non-linear Arrhenius behavior of Lp has been implemented in the water transport model to simulate cell volume changes during freezing. At a cooling rate of 1°C min^-1, ～5% of the initial osmotically active water volume is trapped inside the cells at ?30°C.     

Apparent cooperative effects in acetylcholine receptor-mediated ion flux electroplax membrane preparations.

The kinetics of acetylcholine receptor-mediated flux of ²²sodium ions from microsacs has been measured in the presence of activators (carbamylcholine and decamethonium) and an inhibitor (d-tubocurarine) of neural transmission. The dependence of the first-order rate constant, k_obs, for ²²sodium ion efflux on either decamethonium or carbamylcholine concentration does not exhibit cooperativity. The apparent cooperativity observed by Kasai and Changeux in dose-response curves for ²²sodium flux from the same preparation is adequately accounted for by the contribution which efflux from non-excitable microsacs, the main component of the preparation, makes to the measurements. d-Tubocurarine was found to be a non-competitive inhibitor of decamethonium-activated ²²sodium efflux. The results of the kinetic measurements are in agreement with equilibrium measurements of the interaction of decamethonium with the same microsac preparation, i.e. adherence to a classic Langmuir binding isotherm and separate binding sites for activators and inhibitors of neural activity. The results indicate a direct relationship between ligand binding and receptor-mediated ion flux. How these two processes contribute to electrophysiological measurements is not apparent.     

Characterization of Thermotoga maritima glycerol dehydrogenase for the enzymatic production of dihydroxyacetone

NAD-dependent Thermotoga maritima glycerol dehydrogenase (TmGlyDH) converts glycerol into dihydroxyacetone (DHA), a valuable synthetic precursor and sunless tanning agent. In this work, recombinant TmGlyDH was characterized to determine if it can be used to catalyze DHA production. The pH optima for glycerol oxidation and DHA reduction at 50 °C were 7.9 and 6.0, respectively. Under the conditions tested, TmGlyDH had a linear Arrhenius plot up to 80 °C. TmGlyDH was more thermostable than other glycerol dehydrogenases, remaining over 50 % active after 7 h at 50 °C. TmGlyDH was active on racemic 1,2-propanediol and produced (R)-1,2-propanediol from hydroxyacetone with an enantiomeric excess above 99 %, suggesting that TmGlyDH can also be used for chiral synthesis. (R)-1,2-propanediol production from hydroxyacetone was demonstrated for the first time in a one-enzyme cycling reaction using glycerol as the second substrate. Negative cooperativity was observed with glycerol and DHA, but not with the cofactor. Apparent kinetic parameters for glycerol, DHA, and NAD(H) were determined over a broad pH range. TmGlyDH showed little activity with N⁶-carboxymethyl-NAD⁺ (N⁶-CM-NAD), an NAD⁺ analog modified for easy immobilization to amino groups, but the double mutation V44A/K157G increased catalytic efficiency with N⁶-CM-NAD⁺ ten-fold. Finally, we showed for the first time that a GlyDH is active with immobilized N⁶-CM-NAD⁺, suggesting that N⁶-CM-NAD⁺ can be immobilized on an electrode to allow TmGlyDH activity in a system that reoxidizes the cofactor electrocatalytically.     

Kinetics of the acid hydrolysis of heparin from its Cu (II) complex and its relation to biological activity

The acid-catalyzed hydrolysis of heparin from Cu(II) complex was studied as a function of time and temperature. Four independent calculations showed that the hydrolysis, during the 5-hr period examined, obeys the first-order kinetic law. Specific rate constants, calculated at 50°C, 57°C, 65°C, 71°C, and 80°C, were 3.3 × 10^?5 sec^?1, 6.5 × 10^?5 sec^?1, 10.4 × 10^?5 sec^?1, 15.1 × 10^?5 sec^?1, and 26.6 × 10^?5 sec^?1, respectively. Arrhenius plots of the data yielded 14.7 kcal as the energy of activation. An independent run of the self-hydrolysis of heparin at 57°C also obeyed first-order kinetics and its specific rate constant of 6.4 × 10^?5 sec^?1 is in excellent agreement with that of the hydrolysis of Cu(II)-heparin at 57°C. The anticoagulant activity of heparin and of the Cu(II)-heparin are not appreciably different. Further, the inactivation of heparin closely parallels Cu(II) release from the Cu(II) complex which in turn parallels desulfation.     

Tubulin and microtubules from bovine kidney: purification, properties, and characterization of ligand binding.

Tubulin was purified from bovine renal medulla by in vitro assembly of microtubules in the presence of dimethyl sulfoxide and glycerol. Light scattering measurements of the polymerization process demonstrate that dimethyl sulfoxide and glycerol decrease the critical concentration of tubulin required for polymerization. The minimum concentration of tubulin from bovine renal medulla is about 1% of the total soluble protein. Assembly occurs in the absence of detectable amounts of high-molecular weight proteins or τ-protein. Microtubules polymerized in the absence and presence of 10% dimethyl sulfoxide and 4 m glycerol are similar morphologically as detected by electron microscopy. Molecular weights of α- and β-tubulin from bovine renal medulla are 54,000 ± 700 and 52,000 ± 800, respectively, as determined by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. Colchicine-binding activity of renal medullary tubulin decays in an apparent first-order process which is temperature dependent. The half-time of decay in buffer is 5.1 h and addition of 5 μm vinblastine sulfate increases the half-time of decay to 10.9 h at 37 °C. Calculations based on measurements of the rate of decay of colchicine-binding activity at different temperatures indicates that vinblastine sulfate stabilizes the binding activity by decreasing the entropy of activation of the decay process. Colchicine decreases the rate of decay about 3.5-fold both in the absence and presence of vinblastine sulfate at 37 °C. Values of the apparent colchicine-binding constant, K_A, of bovine renal medullary tubulin are 5.9 × 10⁶ and 7.8 × 10⁶m^?1 at 37 °C in the absence and presence of vinblastine sulfate. Vinblastine sulfate decreases the rate of decay and increases the apparent binding constant of colchicine binding. Lumicolchicine does not affect the binding of colchicine. Podophyllotoxin apparently competitively inhibits the binding of colchicine; the apparent K_i for podophyllotoxin is 4.0 × 10^?7m at 37 °C. Thus, tubulin from bovine renal medulla has ligand-binding characteristics which exhibit differences and similarities to the corresponding characteristics of the brain tubulin. These biochemical properties of the colchicine-binding activity of bovine renal medullary tubulin support previous physiologic studies which demonstrate that microtubules are required for the function of vasopressin in mammalian kidneys.     

Permeation of Chinese hamster ovary cells by glycerol: Mechanism and kinetics

Summary When monolayer cultures of Chinese hamster ovary cells were exposed to³H-glycerol ranging in concentration from 0.1 m to 200mm, glycerol influx was found to increase in direct proportion to the extracellular concentration of glycerol. Other experiments indicated that the same relationship existed at concentrations in excess of 1.0m. Similarly, glycerol efflux was found to vary in direct proportion to the intracellular concentration of glycerol. In neither case could influx or efflux be saturated,. Glycerol influx was not affected by depletion of ATP, alkylation by parachloromercuribenzene sulfonic acid, or exposure to persantine. Altering the pH or temperature also had little effect. Attempts at demonstrating countertransport of glycerol were negative. These data indicate that glycerol probably passes through the membrane by a nonmediated process. For cells in monolayer, the kinetics of influx and efflux are biphasic. Similar biphasic kinetics are observed with cells in suspension culture. A close fit to the data may be obtained by adding together two first-order curves. The pair of curves for influx are clearly different from the pair for efflux. The fit provided by the two pairs of first-order functions suggested that glycerol might diffuse into and out of two intracellular compartments. However, the experimental data do not agree with the predicted behavior of a two-compartment system. As a result, the exact nature of the diffusion limiting steps which are described by the first-order equations remains undefined.     

The cerulenin-induced formation of 1-acyl-lysophosphatidyl glycerol in Bacillus megaterium.

When a culture of $\text{Bacillus megaterium}$ ATCC 14581, growing at 20° and treated with the fatty acid synthesis inhibitor, cerulenin, was incubated with [U-¹⁴C]palmitate, 50% of the incorporated label was found in 1-palmitoyl-lysophosphatidyl glycerol within 5 min. Most of the remaining ¹⁴C appeared in free fatty acid and phosphatidyl glycerol. By 45 min almost all of the lyso compound had disappeared and 80% of the incorporated label was found in phosphatidyl glycerol. At 20°, in the absence of cerulenin or at 35° in either its presence or absence, no labeled lysophosphatidyl glycerol could be found at any time after [U-¹⁴C]palmitate addition. The major radioactive lipid, in these cases, was always phosphatidyl glycerol. At 20°, the palmitate of phosphatidyl glycerol but not of lysophosphatidyl glycerol was readily desaturated.     

Amino acid accumulation in frog muscle. II. Are cycloleucine fluxes consistent with an adsorption model for concentrative uptake of amino acid?

Cycloleucine accumulation by frog muscle was studied at o °C and 25 °C. At external concentrations less than 5 mM the distribution ratio of cycloleucine is higher at 0 °C than at 25 °C. At concentrations greater than 5 mM the converse is true due to apparent exclusion of cycloleucine from a larger portion of the cell water at 0 °C.The steady state data are consistent with an absortion model for amino acid accumulation. Flux studies provide a means to rule out this model if all the possible rate-limiting steps in the movement of amino acid into and out of the cell are considered. These steps include intra-cytoplasmic diffusion, desorption from cytoplasmic or membrane sites and passage through the cell membrane. The assumption is made that the rate-limiting step for influx and efflux is the same, allowing the use of either influx or efflux data to examine the model.Diffusion-limited flux is ruled out on the basis of“influx profile analysis” of the time course of cycloleucine entry at both 0 °C and 25 °C.At least 95% of all intracellular cycloleucine leaves frog muscle cells with a single exponential time course at both 0 °C. The rate constant of efflux does not vary with cellular concentration.These findings are shown to be incompatible with desorption-limited efflux. They are compatible with membrane-limited efflex only if (i) adsorption sites are located on membranes with direct access to the extracellular space and (ii) the rate constant for desorption is equal to the rate constant of membrane-limited efflux of free amino acid. It is considered unlikely that such a coincidence would occur at both 0 °C and 25 °C. Therefore, an absorption model for cycloleucine accumulation in frog muscle appears to be untenable.     

Glycerol, α-glycerophosphate and other compounds as stabilizers of alcohol dehydrogenase from yeast

The storage stability of alcohol dehydrogenase from yeast has been considerably improved by the use of additives. Glycerol is an effective cryoprotectant at ?196 and ?20°C. At 4°C, glycerol and dl-α-glycerophosphate are stabilizers, while at 30°C dl-α-glycerophosphate, 3-phosphoglyceric acid, phosphocreatine, 6-phosphogluconic acid, phosphoarginine, phosphoserine and sucrose are examples of stabilizers. While no single mechanism can be adduced, stabilization by NAD⁺ and 5′-AMP may be attributed to binding at the active centre. Cryoprotection by glycerol is attributed to maintenance of the pH within the range at which the enzyme is stable. dl-α-Glycerophosphate caused a shift of 16°C in the transition temperature of the enzyme, as measured by differential scanning calorimetry.