Search PubMed⌕ Search

Biomedical subjects

J H Crowe

Publications and source records attributed to J H Crowe.

At least 91 records · Page 5Linked to original sources
PubMed1987

Infrared spectroscopic evidence for a conformational alteration of plant plasma membranes upon exposure to the growth hormone analog, 2,4-dichlorophenoxyacetic acid.

Infrared spectroscopy of highly purified fractions of plasma membrane vesicles from hypocotyls of etiolated soybean (Glycine max L.) seedlings revealed changes in bands assigned to proteins and phospholipids upon exposure to the growth hormone analog, 2,4-dichlorophenoxyacetic acid (2,4-D). The changes included a concentration dependent broadening of amide I absorbance and a change in the absorbance ratios of amide I and amide II indicative of a change in protein conformation. Band broadening of amide I was observed at 2,4-D concentrations as low as 10(-8) M, and the optimal 2,4-D concentration to evoke the change was 1 microM whereas the amide peak ratios (amide II/amide I) declined steadily over the range of concentrations (10(-8) to 10(-3)M) tested. An alteration in hydrocarbon chains (CH2 scissoring) was seen only at 1 mM (10(-3) M) 2,4-D. In contrast, the vibrational frequency of the choline stretch declined proportionally over the range 10(-6) to 10(-3). The findings provide evidence for a conformational change in the plasma membrane in response to the hormone demonstrable in a cell-free system.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1987

Stabilization of phosphofructokinase with sugars during freeze-drying: characterization of enhanced protection in the presence of divalent cations.

Phosphofructokinase purified from rabbit skeletal muscle is fully inactivated after freeze-drying and dissolution. The addition of trehalose or maltose to the enzyme solution prior to freeze-drying results in a recovery of up to 80% of the original activity. Slightly less stabilization is imparted by sucrose, whereas glucose and galactose at concentrations up to 500 mM are relatively ineffective at protecting phosphofructokinase. Addition of ionic zinc to enzyme-sugar mixtures prior to freeze-drying greatly enhances the stabilization imparted by the above sugars. This effect is not simply due to the summation of the individual protective capacities of zinc and the sugar. Zinc alone affords no protection, but a high degree of stabilization is achieved when zinc is added to a sugar solution, even when the sugar is at a concentration at which, by itself, it is totally ineffective. In the presence of a constant sugar concentration (100 mM), freeze-dry stabilization of phosphofructokinase is increased as the concentration of zinc is increased. When the zinc concentration is held constant (0.9 mM) and the sugar concentration varied, the maximum stabilization is noted with less than 200 mM sugar. At higher solute concentrations the degree of enhancement decreases such that with 500 mM sugar the addition of zinc results in only a slight increase in protection. Several other organic solutes (proline, 4-hydroxyproline, glycine, trimethylamine N-oxide, glycerol and myo-inositol) that afford cryoprotection to phosphofructokinase, an effect enhanced by the addition of zinc, do not stabilize the enzyme during freeze-drying, even if zinc is present. The addition of ionic copper, cadmium, nickel, cobalt, calcium and manganese to trehalose-phosphofructokinase solutions prior to freeze-drying also increases the percentage of activity recovered after dissolution. Magnesium is ineffective in this respect.

Animals↗
PubMed1987

Modes of interaction of cryoprotectants with membrane phospholipids during freezing.

The abilities of a variety of compounds to inhibit liposome fusion during freeze/thaw were assessed by resonance energy transfer. Small unilamellar vesicles have been frozen according to three different protocols. Membrane intermixing was seen to be relatively independent of freezing protocol except when glycerol, dimethyl sulfoxide (DMSO), or sarcosine was used as the cryoprotectant. Low concentrations of polyvinylpyrolidone or 4-hydroxyproline enhanced fusion of liposomes, whereas high concentrations of these compounds had no effect. Glycerol, DMSO, proline, betaine, and sarcosine reduced fusion, but only when their concentrations were greater than 1 M. The most effective cryoprotectants were trehalose and sucrose, which both reduced fusion to minimal levels at concentrations of only 0.2 M. We have also used europium to probe the modes of interaction of these compounds with phospholipids. Europium, which is known to bind to the phosphate headgroup, maximized fusion in liposomes subjected to freeze/thaw. This "europium-induced" fusion was progressively reduced by the presence of increasing sucrose, trehalose, or glycerol, suggesting a competition for the headgroup. However, the presence of proline, betaine, or sarcosine did not reduce europium-induced fusion, suggesting that these compounds do not compete for the headgroup. Substitution of polar side chains on the hydrophobic regions of proline or sarcosine eliminate their cryoprotective properties, suggesting that these compounds interact with the acyl chains of the bilayer.

Chemical Phenomena↗
PubMed1987

Stabilization of phosphofructokinase during air-drying with sugars and sugar/transition metal mixtures.

Phosphofructokinase (PFK) purified from rabbit skeletal muscle is fully inactivated after air-drying and rehydration. The addition of trehalose, maltose, or sucrose to the enzyme solution prior to rapid drying results in a recovery of almost 70% of the original activity, whereas about 30% is recovered during slow drying. Similar stabilization is seen with up to 200 mM lactose, but at higher concentrations the sugar comes out of solution during drying, and there is a dramatic drop in the activity recovered. Glucose at concentrations up to 500 mM is ineffective at protecting air-dried PFK. Addition of ionic zinc to enzyme-sugar mixtures prior to drying greatly enhances the stabilization imparted by the above sugars, but zinc alone affords no protection. Several other organic solutes (proline, glycine, trimethylamine N-oxide, glycerol, and myo-inositol) that afford cryoprotection to PFK, an effect enhanced by the addition of zinc, do not stabilize the enzyme during air-drying, even if zinc is present. The addition of ionic copper, cobalt, or nickel to trehalose-PFK solution prior to rapid drying results in a large increase in the activity recovered, and the presence of cadmium or manganese leads to a minor increase. Magnesium and calcium are ineffective in this respect. During slow drying, the presence of cadmium or calcium leads to increased preservation, magnesium and manganese have no influence on stabilization, and copper and nickel inactive the enzyme.

Air↗
PubMed1987

Preservation of dry liposomes does not require retention of residual water.

Certain sugars, particularly trehalose, dramatically alter physical properties of dry phospholipids in ways that mimic the presence of water. As a result, these sugars are capable of preserving the integrity of dry liposomes and membranes. Since these effects could conceivably be due to the presence of small amounts of water in the dry preparations of sugar and lipid, we have done careful measurements of the residual water contents in the dry samples and report the results here. Lyophilized liposomes composed of palmitoyloleoylphosphatidylcholine and phosphatidylserine (9:1) contain at most 0.2 mol of H2O per mol of lipid. When the trehalose concentration in the dry mixtures is increased, there is no increase in the apparent water content of the samples over a wide range of sugar concentrations. Over the same range of trehalose contents the maximal effect of trehalose on physical properties of the lipids and on stabilization of liposomes is achieved. We conclude that the stabilization does not require retention of residual amounts of water in the dry trehalose-phospholipid preparations. Similar studies with other sugars show a relationship between the amount of sugar interacting with the lipid and the ability of the same sugar to stabilize dry liposomes.

Carbohydrates↗
PubMed1986

Cryoprotection of phosphofructokinase with organic solutes: characterization of enhanced protection in the presence of divalent cations.

Phosphofructokinase (PFK) purified from rabbit skeletal muscle is fully inactivated after being frozen in liquid nitrogen for 30 s and thawed. The addition of 500 mM trehalose, sucrose, or proline to the enzyme solution prior to freezing results in a recovery of over 70% of the original activity after thawing. Slightly less stabilization is imparted by maltose and 4-hydroxyproline whereas glucose, glycine, inositol, and glycerol at concentrations up to 500 mM are relatively ineffective at protecting PFK. With 50 mM trimethylamine-N-oxide, almost 50% of the prefreeze activity is recovered, and this same level of cryoprotection is noted at concentrations up to 500 mM. The addition of ionic zinc to enzyme-organic solute mixtures prior to freezing greatly enhances the cryoprotection imparted by all of the solutes tested. This effect is not simply due to the summation of the individual cryoprotective capacities of zinc and the organic solute because in many instances a great degree of cryoprotection is noted when each component is present at a concentration at which, by itself, it is totally ineffective. In the presence of a constant 50 mM organic solute concentration, freeze-thaw stabilization of PFK is increased as the concentration of zinc is increased. When the zinc concentration is held constant (0.6 mM) and organic solute concentration varied, the maximum cryoprotection, in most cases, is noted with less than 50 mM organic solute. At higher solute concentrations the degree of enhancement decreases such that with 500 mM organic solute the addition of zinc results in only a slight increase in protection. The addition of ionic copper, cadmium, nickel, and cobalt to trehalose-PFK solutions prior to freezing also increases the percentage of activity recovered after thawing. Magnesium, manganese, and calcium are ineffective in this respect.

Amino Acids↗
PubMed1986

Effects of three stabilizing agents--proline, betaine, and trehalose--on membrane phospholipids.

We have studied the interaction between three compounds which accumulate in organisms under hydration stress--proline, betaine, and trehalose--and the membrane phospholipids dimyristoylphosphatidylcholine (DMPC), palmitoyloleoylphosphatidylcholine (POPC), and dimyristoylphosphatidylethanolamine in bulk solution. Film balance studies reveal that these compounds increase the area/molecule of these lipids. Differential scanning calorimetry has been employed to investigate the effect these agents have on the gel-to-liquid crystalline phase transition of multilamellar and small unilamellar vesicles of DMPC, dipalmitoylphosphatidylcholine, and POPC:phosphatidylserine (90:10 mole ratio) in bulk solution. In the presence of 1 M proline, trehalose, or betaine, the midtransition temperature in small unilamellar vesicles is reduced (up to 7 degrees C in 1 M trehalose), and the transition broadened. In contrast, multilamellar vesicles of similar lipid composition show an increased transition temperature in the presence of the same concentration of these compounds. This result suggests that the inner lamellae in multilamellar vesicles may be dehydrated with only a few outer lamellae exposed to the protective compound. Finally, we have used stereomodels of phosphatidylcholine to investigate the mechanism of action of these agents. Hydrogen bonding of trehalose to the head group region results in an increase in the distance between head groups of 6.9 A. This amount of spreading compares well with data from the monolayer experiments which indicate that maximal spreading of DMPC monolayers by trehalose is 6.5 A. Molecular models of proline and betaine have also been constructed, and these models suggest potential interactions between these compounds and phosphatidylcholines. For the amphipath proline, this interaction may involve intercalation between phospholipid head groups.

Betaine↗
PubMed1986

Inhibition of dehydration-induced fusion between liposomal membranes by carbohydrates as measured by fluorescence energy transfer.

The relative abilities of a number of naturally occurring carbohydrates to inhibit dehydration-induced fusion between palmitoyloleoylphosphatidylcholine:phosphatidylserine (85:15) large unilamellar vesicles have been studied. Fusion events were quantified using a fluorescence resonance energy transfer technique. Trehalose was most effective at inhibiting fusion (0.4 g/trehalose/g lipid showed 30% probe intermixing), followed by maltose (60% intermixing), fructose (60%), sucrose (70%), glucose (80%), cellobiose, glycerol, raffinose, and myo-inositol (90%). The relative abilities of these carbohydrates to inhibit fusion correlate directly with their abilities to interact with phospholipids, maintain bilayer fluidity, and preserve biological membranes. The results are discussed in relation to the crystalline structure of the carbohydrates and their possible influence on level of interaction with phosphate head groups.

Energy Transfer↗
PubMed1986

A calorimetric and infrared spectroscopic study of the stabilizing solute proline.

We have studied the calorimetric and infrared spectroscopic properties of the amino acid proline which has been implicated in the stabilization of biomacromolecules during reduced water states. It has been suggested that the ability of this molecule to protect biomacromolecules during these stress states may be related to the formation of polymeric aggregates of proline monomers in solution. The structure of this aggregate is thought to be an alternates stack, forming a hydrophilic colloid-like polymer which is thought to interact with hydrophobic moieties of biomacromolecules, reducing the exposed hydrophobic area during reduced water conditions. Calorimetric data presented in this work show that in increasing concentration of proline in solution the enthalpy associated with the melting of bulk water is greatly reduced, indicating strong hydrogen bonding character of proline in aqueous solution. Proline shows two eutectic phase separations at moderate concentrations and one of these eutectics may be the proposed intermolecular state. A partial phase diagram for proline is presented. Fourier-transform infrared spectroscopic data indicate that the COO- asymmetric stretch of proline shows marked splitting with increasing proline concentration. This suggests that the carboxylate is in different environments, with the high energy vibrations representing COO- groups which are participating in the hydrogen bonding pattern associated with the formation of the intermolecular stack. Changes in the CH2 asymmetric and symmetric stretches of the pyrrolidine rings of proline are consistent with the proposed stack structure. We also suggest a possible mechanism by which these intermolecular associations may be important in the protection of biomacromolecules during reduced water states.

Calorimetry↗
PubMed1985

Preservation of freeze-dried liposomes by trehalose.

One of the practical difficulties with the frequently proposed use of liposomes for delivery of water-soluble substances to cells in whole organisms is that liposomes are relatively unstable during storage. We have studied the ability of trehalose, a carbohydrate commonly found at high concentrations in organisms capable of surviving dehydration, to stabilize dry liposomes. With trehalose both inside and outside the bilayer, almost 100% of trapped solute was retained in rehydrated vesicles previously freeze-dried with 1.8 g trehalose/g dry phospholipid. Trehalose is very effective at inhibiting fusion between liposomes during drying, as assessed by freeze-fracture and resonance energy transfer between fluorescent probes incorporated into the bilayer. However, inhibition of fusion alone does not account for the preservation of the dry liposomes, since the concentration of trehalose required to prevent leakage is more than 10-fold that required to prevent fusion. We provide evidence that stabilization of the dry liposomes requires depression of transition temperature and consequent maintenance of the constituent lipids in the dry liposomes in a liquid crystalline phase.

Chromatography, Gel↗
PubMed1985

Interaction of carbohydrates with dry dipalmitoylphosphatidylcholine.

Interactions of six carbohydrates (trehalose, sucrose, glucose, raffinose, inositol, and glycerol) with dry dipalmitoylphosphatidylcholine (DPPC) were studied using differential scanning calorimetry (DSC) and infrared spectroscopy (ir) in order to elucidate the mechanism by which some of these carbohydrates preserve structural and functional integrity of dry membranes. Results with DSC showed that trehalose depressed the main transition temperature (Tmid) of dry DPPC below that of fully hydrated DPPC, and raised the enthalpy of that transition more than did addition of water. Results obtained with ir spectroscopy suggested a potential mechanism for this interaction. In the presence of most of the carbohydrates the ir spectrum for DPPC showed changes similar to those seen when water was added to dry DPPC, and the asymmetric P = O stretching band was diminished in intensity. The degree to which the carbohydrates tested affected the integrated intensity of this band and the Tmid was correlated with the ability of those carbohydrates to preserve dry membranes. Also, bands assigned to -OH deformations in the trehalose and other carbohydrates were depressed in the presence of DPPC. Based on these observations, it is suggested that the mechanism of interaction between the carbohydrate and lipid involves hydrogen bonding between -OH groups on the carbohydrate and the phosphate head group of the phospholipid. The only exceptions to this pattern are glycerol, which depresses Tmid of dry DPPC, and myo-inositol, which has no effect on Tmid or the ir spectrum of DPPC; neither carbohydrate can preserve dry membranes. It is suggested, based on ir spectroscopy and previous results with monolayer preparations, that glycerol interacts with phospholipids by a mechanism different from that shown by the other carbohydrates.

Calorimetry, Differential Scanning↗
PubMed1985

Degradation of functional integrity during long-term storage of a freeze-dried biological membrane.

Trehalose, and to some extent a few other carbohydrates, is capable of stabilizing the structure and function of isolated biological membranes during lyophilization. In this paper the results of investigations into the long-term stability of the lyophilized membrane-carbohydrate mixtures were reported. The effects of varying water content, oxygen level, and light on the rates of oxidation, browning, and degradation of biological activity were reported. The efficiency with which three carbohydrates stabilized membrane structure was also reported, with glucose shown to be less efficient than maltose or trehalose. Increased water content accelerated loss of biological activity, possibly because, under the same conditions, nonenzymatic browning and photooxidation were accelerated also. Glucose-containing samples were especially unstable at elevated humidities. Efficiency of preservation could be maximized by storage under conditions of low oxygen, low humidity, and dark, and by the inclusion of high levels of trehalose.

Animals↗
PubMed1985

Membrane stabilization during freezing: the role of two natural cryoprotectants, trehalose and proline.

The relative effectiveness of two natural cryoprotectants, proline and trehalose, in preserving membrane structure and function during freezing was studied. Isolated vesicles of sarcoplasmic reticulum (SR) from lobster muscle (Homarus americanus) were employed to study changes in structure and function during rapid freeze-thaw conditions. Both proline and trehalose were shown to effectively preserve the structure (assessed with freeze fracture) and function (assessed by the ability of the membranes to transport calcium) in the frozen vesicles. As a first step toward determining the mechanism of cryoprotection by these compounds, we have investigated their effectiveness in inhibiting freezing induced fusion between phospholipid vesicles. Pamiltoyloleoyl-phosphatidylcholine: phosphatidylserine (85:15 mole ratio) small unilamellar vesicles (SUVs) were made incorporating one of the following fluorescent probes, and energy donor, cholesteryl anthracene-9-carboxylate, or an energy acceptor, nitrobenzo-2-oxa-1,3-diazole phosphatidylethanolamine to investigate the amount of membrane mixing during rapid freeze-thaw cycles, and storage at -20 degrees C. Membrane mixing was measured as an energy transfer from donor to acceptor when donor vesicles and acceptor vesicles were mixed before a particular freezing treatment. Membrane mixing was correlated with structural changes in these membranes by freeze-fracture analysis. Both trehalose and proline were found to be more effective in preventing membrane mixing between SUVs than the standard protectants, glycerol and dimethylsulfoxide.

Animals↗
PubMed1984

Preservation of functional integrity during long term storage of a biological membrane.

Sarcoplasmic reticulum vesicles freeze-dried in the presence of trehalose retain most of their original biological activity for short periods. When the dry vesicles are stored for longer periods in air, Ca2+-transport becomes uncoupled from ATPase activity within a few days. However, when they are stored under vacuum, ATPase activity, Ca2+ transport, and coupling between Ca2+ transport and ATP utilization are maintained essentially intact for at least 110 days.

Adenosine Triphosphatases↗
PubMed1984

Effects of carbohydrates on membrane stability at low water activities.

The relative effectiveness of a variety of carbohydrates in preserving the structural and functional integrity of membranes at low water activities was studied, using Ca-transporting microsomes from muscle as a model membrane. The order of effectiveness (greatest to lowest) was: trehalose, lactose, maltose, cellobiose, sucrose, glucose, fructose, sorbitol, raffinose, myo-inositol, glycerol. At the highest concentrations of the most effective sugars tested, microsomes were obtained upon rehydration that were similar structurally and functionally to fresh membranes. The least effective carbohydrates, alcohol sugars, all appear to be fusogenic. A structural explanation for relative effectiveness of the sugars was sought, but no clear relationship was found, except that effectiveness does not appear to be related to the number of position of hydroxyl groups available for hydrogen bonding.

Animals↗