High Protein Density Research Articles

Supramolecular photosystem II organization in grana thylakoid membranes: evidence for a structured arrangement.

The distribution of photosystem (PS) II complexes in stacked grana thylakoids derived from electron microscopic images of freeze-fractured chloroplasts are examined for the first time using mathematical methods. These characterize the particle distribution in terms of a nearest neighbor distribution function and a pair correlation function. The data were compared with purely random distributions calculated by a Monte Carlo simulation. The analysis reveals that the PSII distribution in grana thylakoids does not correspond to a random protein mixture but that ordering forces lead to a structured arrangement on a supramolecular level. Neighboring photosystems are significantly more separated than would be the case in a purely random distribution. These results are explained by structural models, in which boundary lipids and light-harvesting complex (LHC) II trimers are arranged between neighboring PSII. Furthermore, the diffusion of PSII was analyzed by a Monte Carlo simulation with a protein density of 80% area occupation (determined for grana membranes). The mobility of the photosystems is severely reduced by the high protein density. From an estimate of the mean migration time of PSII from grana thylakoids to stroma lamellae, it becomes evident that this diffusion contributes significantly to the velocity of the repair cycle of photoinhibited PSII.

Electron-microscopic structure of the V-ATPase from mung bean.

The vacuolar H(+)-ATPase from mung bean (Vigna radiata L. cv. Wilczek) was purified to homogeneity. The purified complex contained all the reported subunits from mung bean, but also included a 40-kDa subunit, corresponding to the membrane-associated subunit d, which has not previously been observed. The structure of the V-ATPase from mung bean was studied by electron microscopy of negatively stained samples. An analysis of over 6,000 single-particle images obtained by electron microscopy of the purified complex revealed that the complex, similar to other V-ATPases, is organized into two major domains V1 and Vo with overall dimensions of 25 nm x 13.7 nm and a stalk region connecting the V1 and Vo domains. Several individual areas of protein density were observed in the stalk region, indicating its complexity. The projections clearly showed that the complex contained one central stalk and at least two peripheral stalks. Subcomplexes containing subunits A, B and E, dissociated from the tonoplast membrane by KI, were purified. The structure of the subcomplex was also studied by electron microscopy followed by single-molecule analysis of 13,000 projections. Our preliminary results reveal an area of high protein density at the bottom of the subcomplex immediately below the cavity formed by the A and B subunits, indicating the position of subunit E.

On the origin of intracellular compartmentation and organized metabolic systems.

The history of the development of the ideas and research of organized metabolic systems during last three decades is shortly reviewed. The cell cytoplasm is crowded with solutes, soluble macromolecules such as enzymes, nucleic acids, structural proteins and membranes. The high protein density within the large compartments of the cells predominantly determines the major characteristics of cellular environment such as viscosity, diffusion and inhomogeneity. The fact that the solvent viscosity of cytoplasm is not substantially different from the water is explained by intracellular structural heterogeneity: the intrinsic macromolecular density is relatively low within the interstitial voids in the cell because many soluble enzymes are apparently integral parts of the insoluble cytomatrix and are not distributed homogeneously. The molecular crowding and sieving restrict the mobility of very large solutes, binding severely restrict the mobility of smaller solutes. One of consequence of molecular crowding and hindered diffusion is the need to compartmentalize metabolic pathway to overcome diffusive barriers. Although the movement of small molecules is slowed down in the cytoplasm, the metabolism can successfully proceed and even be facilitated by metabolite channeling which directly transfers the intermediate from one enzyme to an adjacent enzyme without the need of free aqueous-phase diffusion. The enhanced probability for intermediates to be transferred from one active site to the other by sequential enzymes requires stable or transient interactions of the relevant enzymes, which associate physically in non-dissociable, static multienzyme complexes--metabolones, particles containing enzymes of a part or whole metabolic systems. Therefore, within the living cell the metabolism depends on the structural organization of enzymes forming microcompartments. Since cells contain many compartments and microenvironments, the measurement of the concentration of metabolites in whole cells or tissues gives an average cellular concentration and not that which is actually sensed by the active site of a specific enzyme. Thus, the microcompartmentation could provide a mechanism which can control metabolic pathways. Independently and in parallel to the developments described above, the ideas of compartmentation came into existence from the necessity to explain important physiological phenomena, in particular in heart research and in cardiac electrophysiology. These phenomena demonstrated the physiological importance of the biophysical and biochemical mechanisms described in this review.

Diabetes Mellitus Has an Additional Effect on Coronary Artery Disease. To Decrease Plasma Adiponectin Levels

We investigated whether plasma levels of adiponectin in patients with both coronary artery disease (CAD) and diabetes mellitus (DM) are lower than in patients with CAD alone. We examined plasma adiponectin levels in 113 patients, 82 with CAD (40 of whom had both CAD and type 2 DM) and 31 normal controls. We found differences in plasma adiponectin levels between CAD patients with and without DM (7.8 +/- 4.75 versus 12.1 +/- 6.87 microg/mL, P = 0.002), between patients with CAD and controls (10.0 +/- 6.27 versus 15.3 +/- 5.38 microg/mL, P < 0.0001), and between men and women (10.2 +/- 6.41 versus 13.1 +/- 6.22 microg/mL, P = 0.017). Plasma adiponectin levels were correlated negatively with body mass index, triglyceride, total cholesterol, hemoglobin A1c, and fibrinogen levels (r = -0.456, P < 0.0001; r = -0.355, P < 0.0001; r = -0.286, P = 0.002; r = -0.299, P < 0.0001; r = -0.400, P < 0.0001, respectively), but were not significantly correlated with high sensitivity C-reactive protein or low density lipoprotein levels (r = -0.088, P = 0.352; r = -0.167, P = 0.077, respectively). Plasma adiponectin levels correlated positively with high density lipoprotein levels (r = 0.410, P < 0.0001). Our study demonstrates that plasma adiponectin levels in patients with both CAD and DM are lower than in patients with CAD alone. We speculate that people who have very low plasma adiponectin levels may be at increased risk of developing both CAD and DM.

Flow-Mediated On-Surface Reconstitution of G-Protein Coupled Receptors for Applications in Surface Plasmon Resonance Biosensors

To facilitate biosensor studies of G-protein coupled receptors (GPCR) and other membrane proteins, reliable methods for preparation of sensor surfaces with high protein density are required. We present here a method for the easy and rapid immobilization and reconstitution of GPCR on carboxylated dextran surfaces modified with long alkyl groups. Following amine coupling of the detergent-solubilized receptor, lipid/detergent-mixed micelles were adhered as they were injected over the immobilized surface, taking advantage of the integrated flow cells. The detergent was eluted in the subsequent buffer flow and the remaining lipid formed a bilayer on the chip surface. With this procedure, rhodopsin was functionally reconstituted in a lipid environment in approximately 1 min. This method can also be used for the easy formation of pure supported lipid bilayers for use in model membrane interaction studies.

A novel superoxide dismutase with a high isoelectric point in higher plants. expression, regulation, and protein localization.

Several isoforms of superoxide dismutase (SOD) with a high isoelectric point (pI) have been identified by isoelectric focusing chromatography in protein extracts from Scots pine (Pinus sylvestris) needles. One of these isoforms, a CuZn-SOD with a pI of about 10 and thus denoted hipI-SOD, has been isolated and purified to apparent homogeneity. A cDNA encoding the hipI-SOD protein was cloned and sequenced. Northern hybridization of mRNA isolated from different organs and tissues showed that hipI-SOD has a markedly different pattern of expression compared with chloroplastic and cytosolic SOD. Furthermore, the transcript levels of hipI-SOD and cytosolic SOD were found to respond differently to mechanical wounding, treatment with oxidized glutathione, paraquat, and ozone. Immunogold electron microscopy localized the hipI-SOD in the plasma membrane of sieve cells and the Golgi apparatus of albuminous cells. Moreover, high protein density was also detected in extracellular spaces such as secondary cell wall thickenings of the xylem and sclerenchyma and in intercellular spaces of parenchyma cells.

Origin of multiexponential T(2) relaxation in muscle myowater.

To obtain a further understanding of the nature of the multiexponential T(2) relaxation seen in muscle tissue water (myowater), relaxation measurements were carried out on whole, minced, and homogenized pork of three different qualities with regard to water-holding capacity (normal, red soft exudative, and dark firm dry). Whole, minced, and homogenized pork all resulted in multiexponential T(2) relaxation (three components) independently of the quality, even though microscopic studies on homogenized meat revealed considerable disruption of the macroscopic structure. This states that the relaxation behavior in meat cannot be explained by intra-/extracellular compartmentalization of the water as suggested in earlier studies. Subsequent studies of T(2) relaxation in either whole meat, where the structure integrity was changed by the introduction of dimethyl sulfoxide (membrane disruption) or urea (protein denaturation), or minced meat with added NaCl (inter-/intraprotein interactions) lead to the suggestion that in whole meat (i) the fastest relaxation component reflects water tightly associated with macromolecules, (ii) the intermediate relaxation component reflects water located within highly organized protein structures, for example, water in tertiary and/or quaternary protein structures and spatials with high myofibrillar protein densities including actin and myosin filament structures, and (iii) the slowest relaxation component reflects the extra-myofibrillar water containing the sarcoplasmatic protein fraction. Finally, relaxation patterns in heat-set gels of superprecipitated actomyosin and bovine serum albumin similar to that identified in whole meat support the proposed nature of T(2) relaxation in muscle myowater.

Ultrastructural Localization of Antigens in the Mitotic Spindle of Budding Yeast Using High Presure Freezing and Freeze Substitution

Abstract The preservation of S. cerevisiae ultrastructure is not a trivial task, because the cell wall serves as a diffusion barrier during all steps of preparation. In addition, a high density of proteins in the cytoplasm and in the nucleus renders the visualization of cellular details difficult. High pressure freezing in combination with freeze substitution has proven to be an advantageous technique for the preservation of the yeast mitotic spindle (1). However, the composition of the freeze substitution “cocktail” is crucial, when immunolabeling of antigens in the sample is intended. A high concentration of crosslinking agents might improve ultrastructural preservation of the sample, but will possibly destroy the antigenicity of the target proteins. In contrast, under conditions of high labeling efficiency, the ultrastructural detail may be lost. We were interested in a method, which gives a good ultrastructural preservation of the mitotic spindle and maintains the antigenicity of proteins associated with the spindle.

Two-Dimensional Crystallization on Lipid Layer: A Successful Approach for Membrane Proteins

A considerable interest exists currently in designing innovative strategies to produce two-dimensional crystals of membrane proteins that are amenable to structural analysis by electron crystallography. We have developed a protocol for crystallizing membrane protein that is derived from the classical lipid-layer two-dimensional crystallization at the air/water interface used so far for soluble proteins. Lipid derivatized with a Ni2+-chelating head group provided a general approach to crystallizing histidine-tagged transmembrane proteins. The processes of protein binding and two-dimensional crystallization were analyzed by electron microscopy, using two prototypic membrane proteins: FhuA, a high-affinity receptor from the outer membrane of Escherichia coli, and the F0F1-ATP synthase from thermophilic Bacillus PS3. Conditions were found to avoid solubilization of the lipid layer by the detergent present with the purified membrane proteins and thus to allow binding of micellar proteins to the functionalized lipid head groups. After detergent removal using polystyrene beads, membrane sheets of several hundreds of square micrometers were reconstituted at the interface. High protein density in these membrane sheets allowed further formation of planar two-dimensional crystals. We believe that this strategy represents a new promising alternative to conventional dialysis methods for membrane protein 2D crystallization, with the additional advantage of necessitating little purified protein.

Phase-feeding to optimize performance and quality of Belgian Blue double-muscled bulls

Abstract To optimize performance and carcass quality of Belgian Blue double-muscled bulls four phased feeding regimens were evaluated. All diets were offered ad libitum and consisted of 650 g concentrates and 350 g maize silage per kg dry matter (DM). Total period was divided into three phases (ca. 360 to 460, 460 to 570 and 570 to 680 kg). All four groups received the same maize silage but the different energy and protein densities were applied using different concentrates. During the three phases, the negative control (NC) group constantly received a diet with a low protein and a moderate energy density; the second group (DP) received with each phase diets with decreasing protein density while the energy density remained moderate; the third group (IE) received diets with increasing energy density at a constantly high protein density and the last group (DPIE) received diets which decreased in protein and increased in energy density. The NC group grew significantly slower during the first interval (1·37 v. 1·62 kg/day for the other groups), resulting in a significantly longer total finishing period. During the third period IE had the slowest growth. The NC group needed 21 and 20 days more than the DP and DPIE groups, respectively, to reach the same slaughter weight. The NC group had the lowest DM intake during the first interval, the IE group during the third interval. No significant differences (P &gt; 0·05) were found for food conversion, when expressed as DM or NEF (net energy for fattening). IE had the worst protein conversion while NC had the best, with the two other groups being intermediate. The only important significant difference concerning carcass quality was the improved dressing proportion of the IE and DP groups. Although significant, differences in the fat content of the m. longissimus thoracis are of minor importance. These results proved that for Belgian Blue double-muscled bulls, protein density of the diet can be decreased with increasing live weight, while energy should be increased to give optimal performance.

DNA Binding and Aggregation Properties of the Vaccinia Virus I3L Gene Product

The vaccinia virus I3L gene encodes a single-stranded DNA-binding protein which may play a role in viral replication and genetic recombination. We have purified native and recombinant forms of gpI3L and characterized both the DNA-binding reaction and the structural properties of DNA-protein complexes. The purified proteins displayed anomalous electrophoretic properties in the presence of sodium dodecyl sulfate, behaving as if they were 4-kDa larger than the true mass. Agarose gel shift analysis was used to monitor the formation of complexes composed of single-stranded DNA plus gpI3L protein. These experiments detected two different DNA binding modes whose formation was dependent upon the protein density. The transition between the two binding modes occurred at a nucleotide to protein ratio of about 31 nucleotides per gpI3L monomer. S1 nuclease protection assay revealed that at saturating protein densities, each gpI3L monomer occludes 9.5 +/- 2.5 nucleotides. In the presence of magnesium, gpI3L promoted the formation of large DNA aggregates from which double-stranded DNA was excluded. Electron microscopy showed that, in the absence of magnesium and at low protein densities, gpI3L forms beaded structures on DNA. At high protein density the complexes display a smoother and less compacted morphology. In the presence of magnesium the complexes contained long fibrous and tangled arrays. These results suggest that gpI3L can form octameric complexes on DNA much like those formed by Escherichia coli single-stranded DNA protein. Moreover, the capacity to aggregate DNA may provide an environment in which hybrid DNA formation could occur during DNA replication.

Spin-label studies of lipid-protein interactions with reconstituted band 3, the human erythrocyte chloride-bicarbonate exchanger

Lipid-protein interactions in reconstituted band 3 preparations were investigated by using spin-labeled lipids in conjunction with electron paramagnetic resonance (EPR) spectroscopy. Purified erythrocyte band 3 was reconstituted into egg phosphatidylcholine liposomes at high protein density with preservation predominantly of the dimeric state. Lipid-protein associations were revealed by the presence of a component in the EPR spectra that, when compared to spectra obtained from protein-free bilayers, indicated that lipid chain motions are restricted by interactions with the protein. From the fraction of the motionally restricted component obtained from the phosphatidylcholine spin-label, a value of 64 ± 14 annular lipids per band 3 dimer was obtained. This agrees with a value of 62 for the number of lipids that may be accommodated around the electron density map of a band 3 dimer. Selectivity of various spin-labeled lipids for the protein revealed that androstanol had a lower affinity for the band 3 interface, whereas a distinct preference was observed for the negatively charged lipids phosphatidylglycerol and stearic acid over phosphatidylcholine. This preference for negatively charged lipids could not be screened by 1-M salt, indicating that electrostatic lipid-protein interactions are not dominant. Estimates of annular lipid exchange rates from measured acyl chain segmental motions suggested that the rate of exchange between bilayer and boundary lipids was ~106 s-1, at least an order of magnitude slower than the rate of lipid lateral diffusion in protein-free bilayers.Key words: band 3, reconstitution, electron paramagnetic resonance, lipid-protein interactions.

Rapid diffusion of green fluorescent protein in the mitochondrial matrix.

It is thought that the high protein density in the mitochondrial matrix results in severely restricted solute diffusion and metabolite channeling from one enzyme to another without free aqueous-phase diffusion. To test this hypothesis, we measured the diffusion of green fluorescent protein (GFP) expressed in the mitochondrial matrix of fibroblast, liver, skeletal muscle, and epithelial cell lines. Spot photobleaching of GFP with a 100x objective (0.8-micron spot diam) gave half-times for fluorescence recovery of 15-19 ms with >90% of the GFP mobile. As predicted for aqueous-phase diffusion in a confined compartment, fluorescence recovery was slowed or abolished by increased laser spot size or bleach time, and by paraformaldehyde fixation. Quantitative analysis of bleach data using a mathematical model of matrix diffusion gave GFP diffusion coefficients of 2-3 x 10(-7) cm2/s, only three to fourfold less than that for GFP diffusion in water. In contrast, little recovery was found for bleaching of GFP in fusion with subunits of the fatty acid beta-oxidation multienzyme complex that are normally present in the matrix. Measurement of the rotation of unconjugated GFP by time-resolved anisotropy gave a rotational correlation time of 23.3 +/- 1 ns, similar to that of 20 ns for GFP rotation in water. A rapid rotational correlation time of 325 ps was also found for a small fluorescent probe (BCECF, approximately 0.5 kD) in the matrix of isolated liver mitochondria. The rapid and unrestricted diffusion of solutes in the mitochondrial matrix suggests that metabolite channeling may not be required to overcome diffusive barriers. We propose that the clustering of matrix enzymes in membrane-associated complexes might serve to establish a relatively uncrowded aqueous space in which solutes can freely diffuse.

Spin-label studies of lipid-protein interactions with reconstituted band 3, the human erythrocyte chloride-bicarbonate exchanger.

Lipid-protein interactions in reconstituted band 3 preparations were investigated by using spin-labeled lipids in conjunction with electron paramagnetic resonance (EPR) spectroscopy. Purified erythrocyte band 3 was reconstituted into egg phosphatidylcholine liposomes at high protein density with preservation predominantly of the dimeric state. Lipid-protein associations were revealed by the presence of a component in the EPR spectra that, when compared to spectra obtained from protein-free bilayers, indicated that lipid chain motions are restricted by interactions with the protein. From the fraction of the motionally restricted component obtained from the phosphatidylcholine spin-label, a value of 64 +/- 14 annular lipids per band 3 dimer was obtained. This agrees with a value of 62 for the number of lipids that may be accommodated around the electron density map of a band 3 dimer. Selectivity of various spin-labeled lipids for the protein revealed that androstanol had a lower affinity for the band 3 interface, whereas a distinct preference was observed for the negatively charged lipids phosphatidylglycerol and stearic acid over phosphatidylcholine. This preference for negatively charged lipids could not be screened by 1-M salt, indicating that electrostatic lipid-protein interactions are not dominant. Estimates of annular lipid exchange rates from measured acyl chain segmental motions suggested that the rate of exchange between bilayer and boundary lipids was approximately 10(6) s(-1), at least an order of magnitude slower than the rate of lipid lateral diffusion in protein-free bilayers.

Preparaciôn y caracterización química y microestructural de surimi de merluza (Merluccius merluccius) y de jurel (Trachurus trachurus) / Preparation and chemical and microstructural characterization of surimi from hake (Merluccius merluccius) and horse mackerel (Trachurus trachurus)

Surimi and kamaboko prepared from hake ( Merluccius merluccius) and horse mackerel ( Trachurus trachurus) were characterized chemically and microstructurally. During the process, protein and lipid values decreased while ash, chloride and free nitrogen extract values increased. Hake surimi and kamaboko had less humidity and a higher value of protein than horse mackerel surimi and kamaboko. In both gels water activity decreased and whiteness increased. Horse mackerel kamaboko was more elastic while hake kamaboko was harder and more brittle. The microstructural study showed that the typical structure of fish muscle was lost, giving rise to a porous protein matrix with regions of high and low protein density, as well as some fragments of muscle fibres.

NATURAL FREEZING SURVIVAL IN ANIMALS

▪ Abstract Natural freeze-tolerance supports the winter survival of many animals including numerous terrestrial insects, many intertidal marine invertebrates, and selected species of terrestrially hibernating amphibians and reptiles. Freeze-tolerant animals typically endure the conversion of 50% or more of total body water into extracellular ice and employ a suite of adaptations that counter the negative consequences of freezing. Specific adaptations control the sites and rate of ice formation to prevent physical damage by ice. Other adaptations regulate cell-volume change: Colligative cryoprotectants minimize cell shrinkage during extracellular ice formation; other protectants stabilize membrane structure; and a high density of membrane transporter proteins ensure rapid cryoprotectant distribution. Cell survival during freezing is also potentiated by anoxia tolerance, mechanisms of metabolic rate depression, and antioxidant defenses. The net result of these protective mechanisms is the ability to reactivate vital functions after days or weeks of continuous freezing. Magnetic resonance imaging has allowed visual examinations of the mode of ice penetration through the body of freeze-tolerant frogs and turtles, and cryomicroscopy has illustrated the effects of freezing on the cellular and microvasculature structure of tissues. Various metabolic adaptations for freezing survival appear to have evolved out of pre-existing physiological capacities of animals, including desiccation-resistance and anoxia-tolerance.

Matrix accumulation in diabetic nephropathy: Studies by confocal microscopy

Summary: Confocal scanning laser microscopy (CSLM) has the potential to provide new insights into matrix deposition in renal pathology. For example laser stimulated light emission from Kimmelstiel Wilson (KW) nodules emphasizes a difference between peripheral and central nodule contents, an observation matching immunoperoxidase studies, which fails to demonstrate high density of renal matrix proteins in the core.

INTRACTABLE MASSIVE ASCITES FOLLOWING RADICAL GASTRECTOMY FOR AN EARLY GASTRIC CANCER -TWO CASE REPORTS-

Two cases of intractable massive ascites following radical gastrectmy for an early gastric cancer are reported. One case was a 79-year-old male and the other was a 57-year-old female. Both cases had been infected hepatitis C virus. Ascites was confirmed hepatic lymphorrhea, because it had appeared clearly yellowish fluid indicating high specific gravity and high protein density and containing much lymphocytes without chyle. Intraperitoneal infusion therapy with OK-432 was performed for both cases. Six times of the infusion procedure (total 75 KE) were done for the former case, and 3 times (total 25 KE) for the other case. After this therapy, ascites disappered. Both patients were discharged on the postoperative 89th and 75th day, respectively. In conclusion, we considered that the intraperiteal infusion regimen with OK-432 may be efficient therapy for intractable ascites due to postoperative hepatic lymphorrhea.

A General Method to Recondition and Reuse Biacore Sensor Chips Fouled with Covalently Immobilized Protein/Peptide

Of significance in the routine use of BIAcore is the cost of the sensor chips. This is particularly evident during the phase of method development of an assay where it is not unusual to expend several chips in a day in attempts to optimize immobilization conditions for a novel peptide or protein. In addition, it is accepted practice to discard a chip once its ligand binding capacity has diminished to an unacceptable level. While the high cost of sensor chips has been addressed to some degree through the recent introduction of research-grade sensor chips, we were interested in assessing the possibility of regenerating or reconditioning sensor chips in order to allow them to be reused. In particular, we concerned ourselves with regenerating sensor chips onto which peptide or protein had been immobilized. Our aim was to develop a general procedure that would allow reuse of such chips but would not decrease ligand immobilization capacity or increase nonspecific ligand adsorption properties. We present a method which employs a combination of enzymatic (Pronase E) and chemical (bromoacetic acid) treatments of used sensor chips. Regeneration requires an overnight incubation of the sensor chip ex situ so that one can continue to perform BIAcore experiments. The data demonstrate that this simple two-step procedure substantially removes immobilized proteins such as IgG, Protein G, an HIV-1 envelope glycoprotein (gp120) and a neoglycoprotein based on bovine serum albumin, as determined by reflectance measurements and X-ray photoelectron spectroscopy. In addition, a high density of proteins can be immobilized onto the reconditioned surfaces, since reactive carboxylic acids are regenerated by the procedure.

Direct enthalpy measurements of the calcium-dependent interaction of annexins V and VI with phospholipid vesicles.

Annexins V and VI are two members of the annexin protein family, each of which associate with phospholipid vesicles in a calcium-dependent manner. They may be important intracellular calcium response elements. Titration calorimetry and spectroscopic techniques showed striking features of these interactions. For annexin V and VI, the total heat release from calcium-dependent binding to bilayer vesicles composed of phosphatidylserine/phosphatidylcholine was -25 and -38 kcal/mol, respectively. The enthalpy of association (delta Hassoc) for the respective protein-calcium interactions was about -11 and -5 kcal/mol. The delta Hassoc for the annexin VI-vesicle interaction was largely independent of the headgroup of the anionic phospholipid and of the pH from 7.1 to 8.4. Use of phosphatidylethanolamine as the neutral phospholipid resulted in a slightly more negative delta Hassoc. Enthalpy was either independent of vesicle size (annexin V) or showed a slightly more negative value for large vesicles (annexin VI). The delta Hassoc for annexin VI-membrane interaction was not constant during protein titration but became more exothermic with higher protein density on the membrane. This behavior was surprising because the equilibrium constant showed negative cooperativity with respect to protein density [Bazzi, M. D., & Nelsestuen, G. L. (1991) Biochemistry 30, 7970-7977]. Apparently, entropy changes occurred which were large and negative, thereby compensating for the increasingly negative enthalpy but decreasing affinity as protein density was increased. In fact, the exothermic process coincided more closely with a change in the intrinsic tryptophan fluorescence of annexin VI than with actual protein-membrane binding. Circular dichroism detected very small changes in protein secondary structure during these events. The observed delta Hassoc for annexin-membrane interaction appeared to involve contributions from the membrane as well as from the protein. Annexin-membrane binding may exert large effects on the membrane that could serve a regulatory capacity in the cell.