Stabilization of dry phospholipid bilayers and proteins by sugars.
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Animals↗
Biomedical subjects
J F Carpenter
Publications and source records attributed to J F Carpenter.
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↗
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↗
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↗
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↗
Comparison of pH-dependent allostery and dissociation for phosphofructokinases from Artemia embryos and rabbit muscle: nature of the enzymes acylated with diethylpyrocarbonate.
Purified Artemia phosphofructokinase (PFK), unlike the rabbit skeletal muscle enzyme, displays allosteric kinetics at pH 8, a feature that is functionally significant since the intracellular pH of the developing brine shrimp embryo is greater than or equal to 7.9. Catalytic activity of the Artemia enzyme is severely suppressed by acidic pH even when assayed at the adenylate nucleotide concentrations existing in anaerobic embryos, which is consistent with the lack of a Pasteur effect in these organisms. For both PFK homologs, carbethoxylation reduces the sensitivity to ATP and citrate inhibition, the cooperativity as a function of fructose 6-phosphate concentration and the degree of activation in the presence ADP, AMP, and fructose 2,6-bisphosphate. Considering the role of histidine protonation in PFK allosteric control, the capacity for regulatory kinetics seen at pH 8 in the Artemia enzyme could be explained in part by upward shifts in pKa values of ionizable residues. pH-induced dissociation of tetrameric Artemia PFK into inactive subunits does not occur during catalytic inhibition at acidic pH (pH 6.5, 6 degrees C), as judged by 90 degree light scattering. This observation contrasts markedly with the dimerization and inactivation of rabbit PFK, but is shown not to be unique when compared to other selected PFK homologs. Neither the acute pH sensitivity of Artemia PFK nor the pH-induced hysteretic inactivation displayed by the rabbit enzyme are altered by carbethoxylation, suggesting that ionizable residues involved in these two processes are not the same ones involved in allosteric kinetics.
Acylation↗
pH-induced hysteretic properties of phosphofructokinase purified from rat myocardium.
Phosphofructokinase (PFK) purified from the rat myocardium is reversibly inactivated under a pH regime approximating that reported for ischemic hearts. At pH 6.5 and 37 degrees C, the enzyme displays a hysteretic loss of activity during 60-min incubations, declining to 48% of control (pH 7.1, 37 degrees C) values. Citric acid increases the degree of inactivation (28% of control), whereas fructose 1,6-bisphosphate reduces the decline in activity. Simultaneous measurements of 90 decreases light scattering and catalytic activity suggest the inactivation is temporally linked to dissociation of active tetrameric enzyme into an inactive form of lower molecular weight. Fluorescence enhancement of the extrinsic probe sodium mansate, which binds preferentially to dimeric PFK, indicates that the equilibrium dimer concentration (cp1 infinity) increases as pH is lowered. This increase in cp1 infinity exhibits a strong inverse correlation (r = 0.984) with catalytic activity across the pH range of 8.0 to 6.5. Returning solution pH to 7.0 or above promotes a time-dependent reactivation and repolymerization of PFK. The rate of reactivation is increased at higher enzyme concentrations and in the presence of trimethylamine-N-oxide, a nitrogenous osmolyte noted for its ability to promote protein aggregation reactions. Thus these results demonstrate the capacity of rat heart PFK to undergo reversible inactivation and dissociation in vitro and represent the first phase of a two-part study testing the hypothesis that these pH-induced hysteretic processes are operative in the ischemic myocardium. The data are evaluated in terms of the potential roles of hysteretic enzymes in metabolic regulation.
Animals↗
Reversible dissociation and inactivation of phosphofructokinase in the ischemic rat heart.
To determine if shifts in the ratio of active phosphofructokinase (PFK) tetramers to the inactive dimeric form of the enzyme occur in vivo in the ischemic rat heart, we have developed a rocket immunoelectrophoretic (IEP) assay that provides a sensitive means by which to measure relative differences in this ratio among crude heart extracts. In ischemic hearts, in the face of a drop in intracellular pH from 6.95 to 6.25, there is a time-dependent decrease (63%) in the ratio of tetramer to dimer IEP rocket height relative to perfused controls. Concomitant with this hysteretic depolymerization is a 50% loss of PFK catalytic activity. Realkalinizing extracts of ischemic hearts fosters a recovery of 86% of the activity lost during ischemia and a return of the tetramer-to-dinner ratio to near control value. The amount of reactivation is directly dependent on the degree of enzyme dissociation that occurred during ischemia. Importantly, ischemia-induced dimerization is also reversed in vivo by postischemic reperfusion. The data are consistent with those in the previous study [Hand and Carpenter, Am. J. Physiol. 250 (Regulatory Integrative Comp. Physiol. 19): R505-R511, 1986] that characterized the pH-dependent hysteretic dissociation of heart PFK in vitro, and together they represent the first demonstration that this molecular behavior is operative in intact tissue. Other vertebrate muscle systems in which this mechanism might be functioning during pH-dependent glycolytic inhibition are discussed.
Acids↗
Effects of intravenous dantrolene sodium on respiratory and cardiovascular functions.
Dantrolene sodium, a peripherally acting skeletal muscle relaxant, at doses up to 30 mg/kg iv had no effect on respiratory volume, respiratory rate, blood pressure, or heart rate in anesthetized dogs. The ED50 for inhibition of skeletal muscle contractions was 4.5 mg/kg in anesthetized dogs. In anesthetized sheep, the ED50 for skeletal muscle relaxation was 3.2 mg/kg under methoxyflurane anesthesia and 1.7 mg/kg under pentobarbital anesthesia. Unanesthetized sheep administered doses up to 30 mg/kg iv evidenced no dose-related cardiovascular effects. Respiratory volume decreased and respiratory rate increased, with the net result that the respiratory minute volume was not affected by dantrolene sodium. The results indicate that dantrolene sodium has no effect on the cardiovascular or respiratory systems that would preclude its use intravenously in acute conditions where direct relaxation of skeletal muscle is required, as in the management of malignant hyperthermia.
Animals↗
A comparison of skeletal, cardiac, and smooth muscle actions of dantrolene sodium--a skeletal muscle relaxant.
Dantroline sodium, a skeletal muscle relaxant, has been proposed to inhibit the relase of Ca++ from the sarcoplasmic reticulum. Such a mechanism suggests that other muscle contractile systems operating with the same Ca++ denominator should also be affected by the drug. In the present studies dantrolene sodium effects on smooth, cardiac and skeletal muscle have been evaluated with in situ and in vitro techniques. In anesthetized dogs in situ skeletal muscle contractions were inhibitied in a dose-dependent manner, but cardiac muscle contractions were not. Effects on intestinal smooth muscle responses were highly variable and of short duration. In vitro, ED50 values were developed with dantrolene sodium (0.15-120 mg/l) in DMSO for each tissue. Skeletal muscle was the most sensitive (ED50 = 4.1 mg/l), cardiac muscle contractions were not inhibited by the drug and intestinal smooth muscle (ED50 = 59.0 mg/l) was approximately 1/14 as sensitive as skeletal muscle. It is concluded that skeletal muscle is uniquely sensitive to dantrolene sodium. A hypothesis for this specific action is offered.
Animals↗
A comparative study of dantrolene sodium and other skeletal muscle relaxants with the Straub tail mouse.
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Anesthetics, Local↗
Mechanism of control of skeletal-muscle contraction by dantrolene sodium.
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Action Potentials↗
Studies on the mechanism of action of dantrolene sodium. A skeletal muscle relaxant.
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Acetylcholine↗
Stability of subtilisin and lysozyme under high hydrostatic pressure.
The stabilities of subtilisin and lysozyme under hydrostatic pressures up to 200 MPa were investigated for up to 7 days at 25 degrees C. Methods were chosen to assess changes in tertiary and secondary protein structure as well as aggregation state. Tertiary structure was monitored in situ with second derivative UV spectroscopy and after pressure treatment by dynamic light scattering and second derivative UV spectroscopy. Secondary structure and potential secondary structural changes were characterized by second derivative FTIR spectroscopy. Changes in aggregation state were assessed using dynamic light scattering. Additionally, protein concentration balances were carried out to detect any loss of protein as a function of pressure. For the conditions tested, neither protein shows measurable changes in tertiary or secondary structure or signs of aggregation. Lysozyme concentration balances show no dependence on pressure. Subtilisin concentration balances at high protein concentration (4 mg/mL and higher) do not show pressure dependence. However, the concentration balances carried out at 0.4 mg/mL show a clear sign of pressure dependence. These results may be explained by protein interaction with the vial surface and appear to be rate limited by the equilibrium between active and inactive protein on the surface. Pressure increases protein loss, and the estimated partial molar volume change between the two states is estimated to be -20 +/- 10 mL/mol.
Enzyme Stability↗
Manipulation of lyophilization-induced phase separation: implications for pharmaceutical proteins.
Lyophilization, or freeze-drying, of pharmaceutical proteins is often the only processing method that provides requisite long-term product stability. Freezing and drying, however, can cause acute damage to proteins. To alleviate damage, formulations frequently include protein stabilizers (often polymers and/or sugars), as well as buffering salts and "inert" bulking agents. While great efforts are placed on developing a formulation and suitable lyophilization cycle, incompatibilities among components through freezing and drying have been almost completely ignored. We demonstrate that solutions of poly(ethylene glycol) (PEG) and dextran, initially below critical concentrations for phase separation, do indeed experience a liquid-liquid phase separation induced by freeze concentration during the lyophilization cycle. The separation is shown to evolve with annealing at -7 degrees C and can be effectively inhibited simply by replacing NaCl with KCl in the formulation buffer. In addition, we show that phase separation causes unfolding of a model protein, recombinant hemoglobin, when freeze-dried in the PEG/dextran system. When the phase separation is averted by switching to KCl, the protein structural damage is also avoided. Measurements of pH in the frozen solutions show that the structural damage is not a result of pH changes. We suggest that KCl forms a glass with rapid cooling which kinetically prevents the phase separation and thus the protein structural damage.
Chemical Phenomena↗