A 13C nuclear magnetic resonance and circular dichroism study of the collagen-gelatin transformation in enzyme solubilized collagen.

Natural abundance Fourier transform 13C nuclear magnetic resonance (13C NMR) were obtained for enzyme solubilized collagen at 1 degrees intervals through the transition region. The transition of collagen molecules from the rigid triple helical state to single-stranded, random-coil state is accompanied by a change from broadened carbon resonances unobservable under high-resolution conditions to narrow line spectra. Thus distinction can be made between helical and random-coil states of individual residues. The transition is monophasic, as determined by examination of 14 different carbon resonances, and the entire structure is found to melt cooperatively over a temperature interval of 5 +/- 1 degrees. All the residues seem to be involved in the unfolding process concurrently. The transition was also studied by examining the changes in the circular dichroism spectrum brought about by heating. The experiments corroborated the observation that the transition proceeded cooperatively over a temperature interval of 4 degrees. Enzyme soluble collagen is seen to melt less cooperatively than native collagen. The enthalpy change was determined by assuming an equilibrium between three random coil gelatin chains and tropocollogen molecules. From the enthalpy, the average length of the tripeptide sequences (70-85) involved in the transition can be estimated. The shortening of the cooperative unit could arise as a result of some alteration of the native conformation through proctase treatment.     

Conformational analysis and stability of collagen peptides by CD and by 1H- and 13C-NMR spectroscopies

Four small type I collagen CNBr peptides containing complete natural sequences were purified from bovine skin and investigated by CD and 1H- and 13C-nmr spectroscopies to obtain information concerning their conformation and thermal stability. CD showed that a triple helix was formed at 10 degrees C in acidic aqueous solution by peptide alpha l(I) CB2 only, and to lesser extent, by alpha 1(I) CB4, whereas peptides alpha 1(I) CB5 and alpha 2(I) CB2 remained unstructured. Analytical gel filtration confirmed that peptides alpha 1(I) CB2 and alpha 1(I) CB4 only were able to form trimeric species at temperature between 14 and 20 degrees C, and indicated that the monomer = trimer equilibrium was influenced by the chaotropic nature of the salt present in the eluent, by its concentration, and by temperature variations. CD measurements at increasing temperatures showed that alpha 1(I) CB2 was less stable than its synthetic counterpart due to incomplete prolyl hydroxylation of the preparation from the natural source. 1H- and 13C-nmr spectra acquired in the temperature range 0-47 and 0-27 degrees C, respectively, indicated that with decreasing temperature the most abundant from of alpha 1(I) CB2 was in slow exchange with an assembled form, characterized by broad lines, as expected for the triple-helical conformation. A large number of trimer cross peaks was observed both in the proton and carbon spectra, and these were most likely due to the nonequivalence of the environments of the three chains in the triple helix. This nonequivalence may have implications for the aggregation of collagen molecules and for collagen binding to other molecules. The thermal transition from trimer to monomer was also monitored by 1H-nmr following the change in area of the signal belonging to one of the two beta protons of the C-terminal homoserine. The unfolding process was found to be fully reversible with a melting temperature of 13.4 degrees C, in agreement with CD results. The qualitative superposition of the melting curves obtained by CD for the peptide bond characteristics and by nmr for a side chain suggests that triple-helical backbone and side chains constitute a single unit.     

Age-related thermal stability and susceptibility to proteolysis of rat bone collagen.

The shrinkage temperature (Ts) and the pepsin-solubilizability of collagen fibrils in bone matrix obtained from decalcified femur diaphysis from 2-, 5-, 15- and 25-month-old rats were found to decrease with age. Digestion with human fibroblast collagenase dissolved less than half of the collagen, whereas sequential treatment by pepsin followed by collagenase resulted in its complete dissolution. This result shows that collagenase and a telopeptide-cleaving enzyme, when acting in an appropriate sequence, have a great potential for the degradation of bone collagen. The 'melting' profile of the pepsin-solubilized collagen showed a biphasic transition with transition peak at 35.9 degrees C and 40.8 degrees C. With increasing age an increasing proportion of the collagen 'melted' in the transition peak at 35.9 degrees C (pre-transition), and the 'melting' temperature (Tm) of the collagen decreased in parallel with Ts in relation to age. Both Ts and Tm decreased by 3 degrees C in the age span investigated. The age-related change in Ts could therefore be accounted for by the decrease in molecular stability. The collagenase-cleavage products of the bone collagen obtained by the sequential treatment with pepsin and collagenase showed only one peak transition (at 35.1 degrees C), and the Tm for the products was independent of age. The results indicate that the pre-transition for the pepsin-solubilized collagen is due to an age-related decrease in thermal stability may have implications for the mechanical strength and turnover of the bone collagen. In contrast with bone collagen, soft-tissue collagen showed neither the age-dependency of thermal stability nor the characteristic biphasic 'melting' profile.     

Dual mesomorphic assemblage of chitin normal acylates and rapid enthalpy relaxation of their side chains

Chitin derivatives having normalacyl groups (C(n)H(2n-1)O-; n = 4-20) were synthesized with pyridine, p-toluenesulfonyl chloride, and normal alkanoic acid in an N,N-dimethylacetamide-lithium chloride homogeneous system. The products (C(n)-ACs; degree of acyl substitution, DS = 1.7-1.9) showed an n-dependent thermal transition behavior: no evident transition (n = 4-10), a glass transition (n = 12 and 14), and a pseudo-first-order phase transition (n = 16-20), the latter two occurring usually below room temperature when examined by differential scanning calorimetry. Wide-angle X-ray diffractometry (WAXD) at 20 degrees C displayed a sharp diffraction peak (2theta = 2 degrees -7 degrees ) and a diffuse halo (2theta approximately 20 degrees ) for the respective C(n)-ACs. The former d-spacing (1.5-3.6 nm) increased with an increase in n to yield two stages of mutually different increasing rates, which reflects a systematic n-dependence of the period of a layered structure of the main chains. The molecular assembly of C(n)-ACs exhibited "dual mesomorphy"; nematic ordering for the semirigid carbohydrate trunk and smectic one for the flexible side chains. On the other hand, WAXD profiles of C(n)-ACs (n = 14-18) indicated almost no temperature dependence from -150 to +220 degrees C. Therefore, it was reasonably assumed that the pseudo-first-order transition observed in thermograms of C(n)-ACs (n = 16-20) was due to the enthalpy relaxation of the side-chain assemblage. An insight was provided into the kinetics of the characteristic aging behavior as a liquid-crystalline glass, in comparison with the corresponding data for other noncrystalline macromolecules.     

Membrane packing geometry of diphytanoylphosphatidylcholine is highly sensitive to hydration: phospholipid polymorphism induced by molecular rearrangement in the headgroup region.

Diphytanoylphosphatidylcholine (DPhPC) has often been used in the study of protein-lipid interaction and membrane channel activity, because of the general belief that it has high bilayer stability, low ion leakage, and fatty acyl packing comparable to that of phospholipid bilayers in the liquid-crystalline state. In this solid-state 31P and 2H NMR study, we find that the membrane packing geometry and headgroup orientation of DPhPC are highly sensitive to the temperature studied and its water content. The phosphocholine headgroup of DPhPC starts to change its orientation at a water content as high as approximately 16 water molecules per lipid, as evidenced by hydration-dependent 2H NMR study at room temperature. In addition, a temperature-induced structural transition in the headgroup orientation is detected in the temperature range of approximately 20-60 degrees C for lipids with approximately 8-11 water molecules per DPhPC. Dehydration of the lipid by one more water molecule leads to a nonlamellar, presumably cubic, phase formation. The lipid packing becomes a hexagonal phase at approximately 6 water molecules per lipid. A phase diagram of DPhPC in the temperature range of -40 degrees C to 80 degrees C is thus constructed on the basis of NMR results. The newly observed hydration-dependent DPhPC lipid polymorphism emphasizes the importance of molecular packing in the headgroup region in modulating membrane structure and protein-induced pore formation of the DPhPC bilayer.     

Thermodynamic characteristics of collagen fibrils reconstructed in vitro at different temperatures and concentrations

The results of a calorimetric study of type I collagen fibrillogenesis were analyzed. The dependence of the half-width of the temperature transition of a collagen solution on the concentration and temperature of collagen formation was studied. It was demonstrated that, by varying temperature and collagen concentration, one can regulate the density of packing and dimensions of cooperative fibril blocks. At temperatures below the physiological level (25 degrees C and 30 degrees C), and a relatively low concentration of collagen (0.3 mg/ml), fibrils with the lowest density of packing are formed. The degree of order does not change as the collagen concentration increases twofold but grows as the concentration increases fourfold. It was shown that, at the physiological temperature (35 degrees C), fibrils with a dense packing of molecules are formed at all collagen concentrations studied. The value of fibril formation enthalpy is minimal at a temperature of 35 degrees C, pH 7.2, an ionic strength of 0.17 M and a concentration of 1.2 mg/ml. Based on the results obtained, a conclusion was made that the packing density of fibrils formed at physiological temperature does not depend on collagen concentration over the concentration range of 0.3 - 1.2 mg/ml.     

Temperature induced denaturation of collagen in acidic solution

The denaturation of collagen solution in acetic acid has been investigated by using ultra-sensitive differential scanning calorimetry (US-DSC), circular dichroism (CD), and laser light scattering (LLS). US-DSC measurements reveal that the collagen exhibits a bimodal transition, i.e., there exists a shoulder transition before the major transition. Such a shoulder transition can recover from a cooling when the collagen is heated to a temperature below 35 degrees C. However, when the heating temperature is above 37 degrees C, both the shoulder and major transitions are irreversible. CD measurements demonstrate the content of triple helix slowly decreases with temperature at a temperature below 35 degrees C, but it drastically decreases at a higher temperature. Our experiments suggest that the shoulder transition and major transition arise from the defibrillation and denaturation of collagen, respectively. LLS measurements show the average hydrodynamic radius R(h), radius of gyration R(g)of the collagen gradually decrease before a sharp decrease at a higher temperature. Meanwhile, the ratio R(g)/R(h) gradually increases at a temperature below approximately 34 degrees C and drastically increases in the range 34-40 degrees C, further indicating the defibrillation of collagen before the denaturation.     

Glucosylation of galactosylhydroxylysyl residues in collagen in vitro by collagen glucosyltransferase. Inhibition by triple-helical conformation of the substrate.

Glucosylation of galactosylhydroxylysyl residues in various collagen polypeptide chains and in small peptides prepared from collagen was studied in vitro using collagen glucosyltransferase purified about 200 to 500-fold from extract prepared from chick embryos. When various denatured polypeptide or peptide chains were compared as substrates for the enzyme, no significant differences were found between citrate-soluble collagens from normal or lathyritic rats and isolated alpha1 and alpha2 chains. In contrast, gelatinized insoluble calf skin collagen, and peptides prepared from collagen and having an average molecular weight of about 500 were clearly less effective substrates as judged from their Km and V values. A marked difference was found between native and heat-denatured citrate-soluble collagen in that no synthesis of glucosylgalactosylhydroxylysine was observed with the native collagen when the reaction was studied at 30 degrees C with different times, enzyme concentrations, and substrate concentrations. When the reaction was studied as a function of temperature, little glucosylation of native collagen was observed below 37 degrees C, but there was a sharp transition in the rate of glucosylation of native collagen at temperatures above 37 degrees C, similar to that observable in the melting curve of collagen. The data suggest that triple-helical conformation of collagen prevents that glucosylation of galactosylhydroxylysyl residues.     

Temperature and compositional dependence of the structure of hydrated dimyristoyl lecithin.

Differential scanning calorimetry and x-ray diffraction techniques have been used to investigate the structure and phase behavior of hydrated dimyristoyl lecithin (DML) in the hydration range 7.5 to 60 weight % water and the temperature range -10 to +60 degrees C. Four different calorimetric transitions have been observed: T1, a low enthalpy transition (deltaH approximately equal to 1 kcal/mol of DML) at 0 degrees C between lamellar phases (L leads to Lbeta); T2, the low enthalpy "pretransition" at water contents greater than 20 weight % corresponding to the transition Lbeta leads to Pbeta; T3, the hydrocarbon chain order-disorder transition (deltaH = 6 to 7 kcal/mol of DML) representing the transition of the more ordered low temperature phases (Lbeta, Pbeta, or crystal C, depending on the water content) to the lamellar Lalpha phase; T4, a transition occurring at 25--27 degrees C at low water contents representing the transition from the lamellar Lbeta phase to a hydrated crystalline phase C. The structures of the Lbeta, Pbeta, C, and Lalpha phases have been examined as a function of temperature and water content. The Lbeta structure has a lamellar bilayer organization with the hydrocarbon chains fully extended and tilted with respect to the normal to the bilayer plane, but packed in a distorted quasihexagonal lattice. The Pbeta structure consists of lipid bilayer lamellae distorted by a periodic "ripple" in the plane of the lamellae; the hydrocarbon chains are tilted but appear to be packed in a regular hexagonal lattice. The diffraction pattern from the crystalline phase C indexes according to an orthorhombic cell with a = 53.8 A, b = 9.33 A, c = 8.82 A. In the lamellae bilayer Lalpha strucure, the hydrocarbon chains adopt a liquid-like conformation. Analysis of the hydration characteristics and bilayer parameters (lipid thickness, surface area/molecule) of synthetic lecithins permits an evaluation of the generalized hydration and structural behavior of this class of lipids.     

Gel phase polymorphism in ether-linked dihexadecylphosphatidylcholine bilayers

The structure and properties of the ether-linked 1,2-dihexadecylphosphatidylcholine (DHPC) have been examined as a function of hydration. By differential scanning calorimetry, DHPC exhibits an endothermic (chain melting) transition with the transition temperature (limiting value, 44.2 degrees C) and enthalpy (limiting value, delta H = 8.0 kcal/mol) being hydration dependent. For hydration values greater than 30 wt % water, DHPC exhibits a pretransition at approximately 36 degrees C (delta H = 1.1 kcal/mol) and a subtransition at approximately 5 degrees C (delta H = 0.2 kcal/mol). By X-ray diffraction, at 22 degrees C DHPC exhibits a normal bilayer gel structure with the bilayer periodicity increasing from 58.0 to 62.5 A over the hydration range 9.5-25.4% water. At 30-32% water, two coexisting gel phases are observed with d = 63-64 A and d = 44-45 A; at higher hydration, only the latter phase is present, reaching a limiting d = 47.0 A at 37.5% water. Two different gel phases clearly exist at low and high hydrations. Electron density profiles at low hydration (9.5-25.4%) show a bilayer thickness dp-p = 46 A, whereas at greater than 32% water the bilayer thickness is markedly reduced, dp-p = 30 A. These and other structural parameters indicate a hydration-dependent gel----gel structural transition between a normal bilayer (two chains per polar group) and the chain-interdigitated bilayer (four chains per polar group) arrangement described previously for DHPC [Ruocco, M. J., Siminovitch, D. J., & Griffin, R. G. (1985) Biochemistry 24, 2406-2411].(ABSTRACT TRUNCATED AT 250 WORDS)     

Titration of the phase transition of phosphatidylserine bilayer membranes. Effects of pH, surface electrostatics, ion binding, and head-group hydration

The dependence of the gel-to-fluid phase transition temperature of dimyristoyl- and dipalmitoylphosphatidylserine bilayers on pH, NaCl concentration, and degree of hydration has been studied with differential scanning calorimetry and with spin-labels. On protonation of the carboxyl group (pK2app = 5.5), the transition temperature increases from 36 to 44 degrees C in the fully hydrated state of dimyristoylphosphatidylserine (from 54 to 62 degrees C for dipalmitoylphosphatidylserine), at ionic strength J = 0.1. In addition, at least two less hydrated states, differing progressively by 1 H2O/PS, are observed at low pH with transition temperatures of 48 and 52 degrees C for dimyristoyl- and 65 and 68.5 degrees C for dipalmitoylphosphatidylserine. On deprotonation of the amino group (pK3app = 11.55) the transition temperature decreases to approximately 15 degrees C for dimyristoyl- and 32 degrees C for dipalmitoylphosphatidylserine, and a pretransition is observed at approximately 6 degrees C (dimyristoylphosphatidylserine) and 21.5 degrees C (dipalmitoylphosphatidylserine), at J = 0.1. No titration of the transition is observed for the fully hydrated phosphate group down to pH less than or equal to 0.5, but it affinity for water binding decreases steeply at pH greater than or equal to 2.6. Increasing the NaCl concentration from 0.1 to 2.0 M increases the transition temperature of dimyristoyphosphatidylserine by approximately 8 degrees C at pH 7, by approximately 5 degrees at pH 13, and by approximately 0 degrees C at pH 1. These increases are attributed to the screening of the electrostatic titration-induced shifts in transition temperature. On a further increase of the NaCl concentration to 5.5 M, the transition temperature increases by an additional 9 degree C at pH 7, 13 degree C at pH 13, approximately 7 degree C in the fully hydrated state at pH 1, and approximately 4 and approximately 0 degree C in the two less hydrated states. These shifts are attributed to displacement of water of hydration by ion binding. From the salt dependence it is deduced that the transition temperature shift at the carboxyl titration can be accounted for completely by the surface charge and change in hydration of approximately 1 H2O/lipid, whereas that of the amino group titration arises mostly from other sources, probably hydrogen bonding. The shifts in pK (delta pK2 = 2.85, delta pK3 = 1.56) are consistent with a reduced polarity in the head-group region, corresponding to an effective dielectric constant epsilon approximately or equal to 30, together with surface potentials of psi congruent to -100 and -150 mV at the carboxyl and amino group pKs, respectively. The transition temperature of dimyristoylphosphatidylserine-water mixtures decreases by approximately 4 degree C each water/lipid molecule added, reaching a limiting value at a water content of approximately 9-10 H2O/lipid molecule.     

Heat-induced secondary structure and conformation change of bovine serum albumin investigated by Fourier transform infrared spectroscopy

Fourier transform infrared (FT-IR) spectra were measured for an aqueous solution (pD = 5.40) of defatted monomer bovine serum albumin (BSA) over a temperature range of 25-90 degrees C to investigate temperature-induced secondary structure and conformation changes. The curve fitting method combined with the Fourier self-deconvolution technique allowed us to explore details of the secondary structure and conformation changes in defatted BSA. Particularly striking in the FT-IR spectra was an observation of the formation of an irreversible intermolecular beta-sheet of BSA on heating above 70 degrees C. A band at 1630 cm(-1) in the spectra was assigned to short-segment chains connecting alpha-helical segments. The transition temperature for the short-segment chains connecting alpha-helical segments is lower by 17-18 degrees C, when compared to those of the alpha-helix, turn, and intermolecular beta-sheet structures of BSA, suggesting that the alpha-helix and turn structures of BSA are cooperatively denatured on heating. Moreover, the results give an important feature in heat-induced denaturation of BSA that the conformation changes occur twice around both 57 and 75 degrees C. The appearance of two peaks is interpreted by the collapse of the N-terminal BSA domain due to the crevice in the vicinity between domains I and II at low-temperature transition and by the change in cooperative unit composed of the other two BSA domains at high-temperature transition.     

Thermal denaturation of UV-irradiated wet rat tail tendon collagen

The thermal helix-coil transition of UV irradiated collagen in rat tail tendon has been investigated by differential scanning calorimetry. During UVB irradiation the tendons were immersed in water to keep the collagen fibers in a fully hydrated condition at all times. UV irradiation induced changes in collagen which caused both stabilization and destabilization of the triple helix in fibers. The helix-coil transition for non-irradiated collagen occurred near 64 degrees C, for irradiated 1 and 3 h at 66 and 67 degrees C, respectively. After irradiating for longer times (20-66 h) the helix-coil transition peak occurred at much lower temperatures. The peak was very broad and suggested that collagen was reduced by UV to different polypeptides of different molecular weight and different lower thermal stabilities. It was caused by the disruption of a network of hydrogen-bonded water molecules surrounding the collagen macromolecule.     

Isolation and characterization of pepsin-treated type III collagen from calf skin.

Calf skin collagen was solubilized by incubating acid-extracted calf skin with pepsin at pH 2.0 and 25 degrees C, conditions that did not cause degradation of the triple helical region of collagen. Type III collagen was separated from type I collagen by differential salt precipitation at pH 7.5. The isolated type III collagen contained mainly gamma and higher molecular weight components cross-linked by reducible and/or non-reducible bonds. The isolated alpha1 (III) chains had an amino acid composition characteristic of type III collagen. Denatured but unreduced type III collagen, chromatographed on carboxymethyl-cellulose, eluted in the alpha 2 region, while after reduction and alkylation the alpha1 (III) chains eluted between the positions of alpha1 (I) and alpha2. The mid-point melting temperature temperature (tm) of type III collagen (35.1 degrees C) in a citrate buffer at pH 3.7 was somewhat lower than that of type I collagen (35.9 degrees C). Renaturation experiments at 25 degrees C showed that denatured type III collagen molecules with intact intramolecular disulfide bridges (gamma components) reform the triple helical structure of collagen much faster than reduced and carboxymethylated alpha1 (III) chains.     

Discrete reduction of type I collagen thermal stability upon oxidation

The oxidation of acid-soluble calf skin collagen type I caused by metal-dependent free radical generating systems, Fe(II)/H2O2 and Cu(II)/H2O2, was found to bring down in a specific, discrete way the collagen thermal stability, as determined by microcalorimetry and scanning densitometry. Initial oxidation results in splitting of the collagen denaturational transition into two components. Along with the endotherm at 41 degrees C typical for non-oxidized collagen, a second, similarly cooperative endotherm appears at 35 degrees C and increases in enthalpy with the oxidant concentration and exposure time, while the first peak correspondingly decreases. The two transitions at 35 and 41 degrees C were registered by densitometry as stepwise increases of the collagen-specific volume. Further oxidation results in massive collagen destruction manifested as abolishment of both denaturational transitions. The two oxidative systems used produce identical effects on the collagen stability but at higher concentrations of Cu(II) in comparison to Fe(II). The discrete reduction of the protein thermal stability is accompanied by a decrease of the free amino groups, suggestive of an oxidation attack of the side chains of lysine residues. Since the denaturation temperature of collagen shifts from above to below body temperature (41 degrees C-35 degrees C) upon oxidation, it appears important to account for this effect in a context of the possible physiological implications of collagen oxidation.     

Electron spin resonance study of phospholipid membranes employing a comprehensive line-shape model

The electron spin resonance spectra of the 1-myristoyl-2-[6-(4,4-dimethyloxazolidine-N-oxyl)myristoyl]-sn-glycero- 3-phosphocholine spin-label in highly oriented, fully hydrated bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine have been studied as a function of temperature and magnetic field orientation. The oriented spectra show clear indications of slow motional components (rotational correlation times greater than 3 ns) even in the fluid phase (T greater than 23 degrees C), indicating that motional narrowing theory is not applicable to the spectral analysis. The spectra have been simulated by a comprehensive line-shape model that incorporates trans-gauche isomerization in addition to restricted anisotropic motion of the lipid long molecular axis and that is valid in all motional regimes. In the gel (L beta') phase the spin-label chains are found to be tilted at 28 degrees with respect to the normal of the orienting plane. In the intermediate (P beta') phase there is a continuous distribution of tilt angles between 0 degrees and 25 degrees. In fluid (L alpha) phase there is no net tilt of the lipid chains. The chains rotate at an intermediate rate about their long axis in the fluid phase (tau R,parallel = 1.4-6.6 ns for T = 50-25 degrees C), but the reorientation of the chain axis is much slower (tau R, perpendicular= 13-61 ns for T = 50-25 degrees C), whereas trans-gauche isomerization (at the C-6 position) is rapid (tau J less than or equal to 0.2 ns). Below the chain melting transition both chain reorientation and chain rotation are at the ESR rigid limit (tau R greater than or equal to 100 ns), and trans-gauche isomerization is in the slow-motion regime (tau J = 3.7-9.5 ns for T = 22-2 degrees C). The chain order parameter increases continuously with decreasing temperature in the fluid phase (SZZ = 0.47-0.61 for T = 50-25 degrees C), increases abruptly on going below the chain melting transition, and then increases continuously in the intermediate phase (SZZ = 0.79-0.85 for T = 22-14 degrees C) to an approximately constant value in the gel phase (SZZ congruent to 0.86 for T = 10-2 degrees C).(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of replacement of a double bond by a cyclopropane ring in phosphatidylethanolamines: a 2H NMR study of phase transitions and molecular organization

The thermotropic behavior and molecular properties of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-dihydrosterculoyl-sn-glycero-3-phosphoethanolamine (PDSPE) have been investigated by 2H NMR spectroscopy using samples selectively labeled at the 5'-, 9'-, 10'-, and 16'-positions of the sn-2 chains. Comparison with the corresponding phosphocholine analogues (POPC and PDSPC), obtained as intermediate synthetic products, was used to monitor the role of the polar head group. Replacement of the choline moiety by ethanolamine increased the gel to liquid-crystal transition temperature by 10-32 degrees C and led to a significantly higher ordering of the fatty acyl chains in the liquid-crystalline bilayer state. The lateral compression effect, due to the smaller area per polar head group in PE, results in a bilayer to hexagonal phase transition at elevated temperatures. The effects on both PC and PE due to replacement of the olefinic group by a cyclopropane unit are similar. A decrease in the temperature of the gel to liquid-crystal phase transition, Tc, is observed upon introduction of a cyclopropane ring; it goes from 26 degrees C in POPE to approximately 10 degrees C in PDSPE. In addition, a very significant broadening of the transition profile is observed. These observations are consistent with the poor packing ability of mixed saturated and cyclopropane-containing chains due to the bulky substituent effect. The temperature of the bilayer-hexagonal phase transition of PE samples was decreased by 15-20 degrees C on replacement of oleoyl chains by dihydrosterculoyl chains at the sn-2 position.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inactivation of calcium uptake by EGTA is due to an irreversible thermotropic conformational change in the calcium binding domain of the Ca(2+)-ATPase.

Calcium uptake by rabbit skeletal sarcoplasmic reticulum (SR) is inhibited with an effective inactivation temperature (TI) of 37 degrees C in EGTA with no effect on ATPase activity. Since the Ca-ATPase denatures at a much higher temperature (49 degrees C) in EGTA, this suggests that a small or localized conformational change of the Ca-ATPase at 37 degrees C results in inability to accumulate calcium by the SR. Using a fluorescent analogue of dicyclohexylcarbodiimide, N-cyclohexyl-N'-[4-(dimethylamino)-alpha-naphthyl]-carbodiimide (NCD-4), the region of the calcium binding sites of the SR Ca-ATPase was labeled. Steady-state and frequency-resolved fluorescence measurements were subsequently performed on the NCD-4-labeled Ca-ATPase. Site-specific information pertaining to the hydrophobicity and segmental flexibility of the region of the calcium binding sites was derived from the steady-state fluorescence intensity, lifetime, and rotational rate of the covalently bound NCD-4 label as a function of temperature (0-50 degrees C). A reversible transition at approximately 15 degrees C and an irreversible transition at approximately 35 degrees C were deduced from the measured fluorescence parameters. The low-temperature transition agrees with the previously observed break in the Arrhenius plot of ATPase activity of the native Ca-ATPase at 15-20 degrees C. The high-temperature transition conforms well with the conformational transition, resulting in uncoupling of Ca translocation from ATP hydrolysis as predicted from the irreversible inactivation of Ca uptake at 31-37 degrees C in 1 mM EGTA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temperature and pH dependent changes of immunoglobulin G structure.

1. The temperature and pH functions of the myeloma IgG(K) conformation were studied by optical rotatory dispersion, circular dichroism, thermal perturbation difference spectroscopy, solvent perturbation difference spectroscopy, electrochemical iodination and difference adiabatic scanning microcalorimetry. 2. The IgG studied was found to be capable of a fully reversible structural change between pH 6.5 and 6.0. A transition occurring at low pH is accompanied by an increase of exposure of the chromophores to the solvent. 3. The "alkaline state" was found to be capable of a fully reversible S-like transition at temperatures between 25 and 35 degrees C. The changes occurring at the higher temperature are accompanied by the screening of 14-15 tyrosine residues and probably by a small increase in the helicity of the protein. These changes are not accompanied by an appreciable heat effect. The thermal denaturation of the "alkaline state" occurs only at 64 degrees C in the narrow temperature interval (3-4 degrees C). 4. The "acid state" is not accompanied by S-like transition at 25-35 degrees C. The thermal denaturation of the "acid state" occurs at 54 degrees C in the wide temperature interval (8-9 degrees C). 5. It was proposed that the ionisation of the invariant histidine residues situated in the "cavity" between the constant and variable domains causes the pH transition studied. The temperature changes in the interval 25-35 degrees C are explained by the alteration of the domains interposition. Similar alterations were investigated as a result of antigen-antibody reaction.     

The inactivation by concanavalin A of the ecto-ATPase and ectoacetylcholinesterase of intact skeletal muscles

The (Ca2+ or Mg2+)-activated ectophosphohydrolase of intact frog muscle liberates, in situ, about 37 mumol inorganic phosphate/g muscle in 20 min at 20 degrees C with 10 mM ATP. Pretreatment with concanavalin A (ConA) at 4 degrees C for 18 h caused ectoenzyme inactivation which plateaued at 35-40% of the control rate. The inhibition was concentration dependent, being maximal at about 500 micrograms ConA/mL Ringer's solution. The lectin mediated its effect via the membrane glycoproteins since the inhibition was specifically prevented by alpha-methyl D-mannopyranoside. As the temperature increased from 10 to 40 degrees C, the ectoenzyme activity of untreated muscles increased linearly between 10 and 35 degrees C, with a "break point" and a clear change in slope at 35 degrees C. When treated with ConA the activity increased linearly from 10 to 40 degrees C, eliminating the transition temperature. The findings suggested that a phase transition toward fluidity in the lipid bilayer may have occurred at 35 degrees C and that this was abolished by the lectin binding. Hence we perturbed the surface membrane phospholipids of muscle pretreated with the lectin. Phospholipase C increased the activation by the lectin; phospholipase D had no effect, but phospholipase A2 completely prevented it. The lectin may require the more fluid fatty acyl chains of membrane lipids to achieve inhibition of this ecto-ATPase. Ectoacetylcholinesterase, in situ, and its inactivation by ConA were measured directly on whole, intact skeletal muscles.