Lipid composition of squirrel monkey (Saimiri sciureus) saliva

The content and composition of lipids in saliva of healthy caries-free squirrel monkeys were investigated. The dialyzed and lyophilized saliva on extraction with chloroform/methanol yielded 8.0 +/- 0.9 mg of lipids/100 ml of saliva. Following fractionation on silicic acid column, 30.9% of lipids were found in the neutral lipid fraction, 58.8% in the glycolipid fraction, and 10.3% in the phospholipid fraction. The neutral lipids exhibited high content of free fatty acids (58.8%) and triglycerides (23.3%), the glycolipids consisted mainly of neutral and sulfated glyceroglucolipids (95%), while the phospholipids were rich in sphingomyelin and phosphatidylcholine. The results show that squirrel monkey saliva, while displaying lipid content similar to that of caries-susceptible humans, contains 50% less lipids than saliva of periodontal disease-prone marmoset.     

Comparison of the Lipid Composition of Oat Root and Coleoptile Plasma Membranes : Lack of Short-Term Change in Response to Auxin

The total lipid composition of plasma membranes (PM), isolated by the phase partitioning method from two different oat (Avena sativa L.) tissues, the root and coleoptile, was compared. In general, the PM lipid composition was not conserved between these two organs of the oat seedling. Oat roots contained 50 mole percent phospholipid, 25 mole percent glycolipid, and 25 mole percent free sterol, whereas comparable amounts in the coleoptile were 42, 39, and 19 mole percent, respectively. Individual lipid components within each lipid class also showed large variations between the two tissues. Maximum specific ATPase activity in the root PM was more than double the activity in the coleoptile. Treatment of coleoptile with auxin for 1 hour resulted in no detectable changes in PM lipids or extractable ATPase activity. Differences in the PM lipid composition between the two tissues that may define the limits of ATPase activity are discussed.     

A novel glycolipid and phospholipid in the purple membrane

A novel glycolipid of mass 1935 and a phospholipid of mass 1522 are the main residual lipids (along with traces of PGP-Me, S-TGD-1, and PG) specifically associated with "delipidated" bacteriorhodopsin fractions BR I and BR II, prepared by Triton X-100 treatment of purple membrane (PM), from a genetically engineered strain (L33) of Halobacterium salinarum, and chromatography on phenyl-Sepharose CL-4B. The novel glycolipid and phospholipid are components of the PM matrix not previously described. The TLC isolated and purified novel glycolipid and phospholipid were shown, by chemical degradation, mass spectrometry, and NMR analyses, to have the structure, respectively, of a phosphosulfoglycolipid, 3-HSO(3)-Galp-beta1,6Manp-alpha1,2Glcp-alpha1,1-[sn-2, 3-di-O-phytanylglycerol]-6-[phospho-sn-2,3-di-O-phytanylglycero l], and of a glycerol diether analogue of bisphosphatidylglycerol (cardiolipin), sn-2,3-di-O-phytanyl-1-phosphoglycerol-3-phospho-sn-2, 3-di-O-phytanylglycerol.     

A Monte Carlo study of the self-assembly of bacteriorhodopsin

Bacteriorhodopsin (BR) and specific lipid molecules self-assemble into a quasi two-dimensional lattice structure known as the purple membrane (PM). In the PM, BR molecules exist in a trimeric form with lipid molecules present in the space enclosed by each trimeric unit and in the inter-trimer space. These trimeric units, which have a roughly circular cross-section, are arranged in hexagonal patterns with long-ranged crystalline order. In this work, we investigate the self-assembly of BR in the PM via Monte Carlo simulations of a two-dimensional model of the membrane and proteins. The protein molecules are modeled as 120 degrees sectors of a circle and the lipid molecules enter into the model through effective protein-protein interactions. The sectors cannot overlap with each other, and in addition to this excluded volume interaction there are site-site attractive interactions between specific points of the proteins to mimic interactions between helices on the proteins and lipid-induced interactions. At low values of the attractive well depth, the proteins are found in the monomeric form at all concentrations. At moderate and high values of the attractive well depth, trimers are formed as the concentration increases, and with a further increase in concentration the trimers organize into a hexagonal lattice. The interactions between the proteins and those induced by the intra-trimer lipids play an equally important role in the formation of trimers and the lattice. The lipids in the inter-trimer space cause the trimers to orient in a specific direction in the hexagonal crystal lattice.     

Lipid-protein stoichiometries in a crystalline biological membrane: NMR quantitative analysis of the lipid extract of the purple membrane.

The lipid/protein stoichiometries of a naturally crystalline biological membrane, the purple membrane (PM) of Halobacterium salinarum, have been obtained by a combination of (31)P- and (1)H-NMR analyses of the lipid extract. In total, 10 lipid molecules per retinal were found to be present in the PM lipid extract: 2-3 molecules of phosphatidylglycerophosphate methyl ester (PGP-Me), 3 of glycolipid sulfate, 1 of phosphatidylglycerol, 1 of archaeal glycocardiolipin (GlyC), 2 of squalene plus minor amounts of phosphatidylglycerosulfate (PGS) and bisphosphatidylglycerol (archaeal cardiolipin) (BPG) and a negligible amount of vitamin MK8.The novel data of the present study are necessary to identify the lipids in the electron density map, and to shed light on the structural relationships of the lipid and protein components of the PM.     

Lipid-Induced Modulation of the Protein Packing in Two-Dimensional Crystals of Bacteriorhodopsin

The mechanism by which bacteriorhodopsin (BR), the light-driven proton pump from the purple membrane (PM) of Halobacterium halobium, arranges in a 2D hexagonal array has been studied by reconstitution of BR in complexes of two types of bilayer made either with PM-derived lipids or with PM lipids and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). The unit cell dimensions of the 2D protein crystals, determined by correlation averaging analysis of freeze-fracture electron micrographs, were compared with the lattice constant of the PM. In complexes made with delipidated BR and with the polar lipids extracted from H. halobium cells (HHPL), BR trimers are arranged in a hexagonal lattice with the same lattice constant of 5.9 ± 0.2 nm as found in the PM. In BR-containing complexes made with PM-derived lipids and DMPC at several protein:lipid mole ratios, BR trimers are also arranged in a hexagonal lattice, but with a unit cell dimension of 9.2 ± 0.2 nm, which is about one-third larger compared to that measured in PM (Michel et al. , 1980). In a subclass of this type of complexes, orthogonal BR arrays were observed with a lattice constant of 5.9 × 9.9 ± 0.2 nm. It appears that insertion of DMPC into the BP/PM-derived lipid complexes increases the center-to-center distances in both array types by a discrete amount.     

Endosomes Derived from Clathrin-Independent Endocytosis Serve as Precursors for Endothelial Lumen Formation

Clathrin-independent endocytosis (CIE) is a form of bulk plasma membrane (PM) endocytosis that allows cells to sample and evaluate PM composition. Once in endosomes, the internalized proteins and lipids can be recycled back to the PM or delivered to lysosomes for degradation. Endosomes arising from CIE contain lipid and signaling molecules suggesting that they might be involved in important biological processes. During vasculogenesis, new blood vessels are formed from precursor cells in a process involving internalization and accumulation of endocytic vesicles. Here, we found that CIE has a role in endothelial lumen formation. Specifically, we found that human vascular endothelial cells (HUVECs) utilize CIE for internalization of distinct cargo molecules and that in three-dimensional cultures CIE membranes are delivered to the newly formed lumen.     

The chromophore retinal hinders passive proton/hydroxide ion translocation through bacteriorhodopsin

Experiments have been performed to examine any influence of the chromophore retinal in bacteriorhodopsin (BR) on the passive proton/hydroxide ion flux through this integral membrane protein. BR was reconstituted into dimyristoylphosphatidylcholine (DMPC)-phosphatidylserine or DMPC-dimyristoylphosphatidylglycerol unilamellar vesicles with molar lipid to protein ratios ranging from 30 to 150. The entrapped fluorescence dye pyranine served as a reliable indicator of the internal proton concentration. Transmembrane pH-gradients were quickly established across the vesicular membrane and the kinetics of the induced fluorescence changes were compared for vesicles with incorporated native BR, BR bleached to the chromophore-free protein bacterioopsin, and BR regenerated from bacterioopsin with all-trans-retinal, respectively. For aggregated protein molecules, the H+/OH- diffusion across bacterioopsin was always considerably faster than that through the protein containing covalently bound retinal. The decay rate of the imposed pH-gradient was 4.4-9.1 and 2.0-5.1 times slower for native and regenerated BR, respectively, as compared to bacterioopsin. Stepwise regeneration of bacterioopsin with all-trans-retinal revealed a linear dependence of the predominant delta pH-decay time on the degree of regeneration. Essentially the same observations were made with monomeric protein molecules in vesicular lipid membranes. The results demonstrate that the chromophore retinal itself blocks the H+/OH- conducting pathway across the transmembrane protein BR or indirectly controls this path by inducing conformational changes in the protein upon binding.     

Retinal fatty acid binding protein reduce lipid peroxidation stimulated by long-chain fatty acid hydroperoxides on rod outer segments

In the present study we have investigated the effect of partially purified retinal fatty acid binding protein (FABP) against nonenzymatic lipid peroxidation stimulated by hydroperoxides derived from fatty acids on rod outer segment (ROS) membranes. Linoleic acid hydroperoxide (LHP), arachidonic acid hydroperoxide (AHP) and docosahexaenoic acid hydroperoxide (DHP) were prepared from linoleic acid, arachidonic acid and docosahexaenoic acid, respectively, by means of lipoxidase. ROS membranes were peroxidized using an ascorbate-Fe(+2) experimental system. The effect on the peroxidation of ROS containing different amounts of lipid hydroperoxides (LOOH) was studied; ROS deprived of exogenously added LOOH was utilized as control. The degradative process was measured simultaneously by determining chemiluminescence and fatty acid composition of total lipids isolated from ROS. The addition of hydroperoxides to ROS produced a marked increase in light emission. This increase was hydroperoxide concentration-dependent. The highest value of activation was produced by DHP. The decrease percentage of the more polyunsaturated fatty acids (PUFAs) (20:4 n6 and 22:6 n3) was used to evaluate the fatty acid alterations observed during the process. We have compared the fatty acid composition of total lipids isolated from native ROS and peroxidized ROS that were incubated with and without hydroperoxides. The major difference in the fatty acid composition was found in the docosahexaenoic acid content, which decreased by 45.51+/-1.07% in the peroxidized group compared to native ROS; the decrease was even higher, 81.38+/-1.11%, when the lipid peroxidation was stimulated by DHP. Retinal FABP was partially purified from retinal cytosol. Afterwards, we measured its effect on the reaction of lipid peroxidation induced by LOOH. As a result, we observed a decrease of chemiluminescence (inhibition of lipid peroxidation) when adding increasing amounts (0.2 to 0.6 mg) of retinal FABP to ROS. The inhibitory effect reaches its highest value in the presence of DHP (41.81+/-10.18%). Under these conditions, bovine serum albumin (BSA) produces a smaller inhibitory effect (20.2+/-7.06%) than FABP.     

Chlorosome lipids from Chlorobium tepidum: characterization and quantification of polar lipids and wax esters

We have extracted polar lipids and waxes from isolated chlorosomes from the green sulfur bacterium Chlorobium tepidum and determined the fatty acid composition of each lipid class. Polar lipids amounted to 4.8 mol per 100 mol bacteriochlorophyll in the chlorosomes, while non-polar lipids (waxes) were present at a ratio of 5.9 mol per 100 mol bacteriochlorophyll. Glycolipids constitute 60 % of the polar lipids while phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, and an aminoglycosphingolipid make up respectively 15, 3, 8 and 12 %. A novel glycolipid was identified as a rhamnose derivative of monogalactosyldiacylglycerol, while the other major glycolipid was monogalactosyldiacylglycerol. Tetradecanoic acid was the major fatty acid in the aminoglycosphingolipid, while the other polar lipids contained predominantly hexandecanoic acid. The chlorosome waxes are esters of unbranched fatty acids and fatty alcohols with 14 or 16 carbon atoms, joined to form molecules with between 28 and 32 carbon atoms. The stoichiometry between lipids and bacteriochlorophyll suggests that much of the chlorosome surface is covered by protein.     

Evidence for a Composite State of an F′his,gnd Element and a Cryptic Plasmid in a Derivative of Salmonella typhimurium LT2

The zoospores of Blastocladiella emersonii, when derived from cultures grown on solid media, contain about 11% total lipid. This lipid was separated chromatographically on silicic acid into neutral lipid (46.6%), glycolipid (15.8%), and phospholipid (37.6%). Each class was fractionated further on columns of silicic acid, Florisil, or diethylaminoethyl-cellulose, and monitored by thin-layer chromatography. Triglycerides were the major neutral lipids, mono- and diglycosyldiglycerides were the major glycolipids, and phosphatidylcholine and phosphatidylethanolamine were the major phospholipids. Other neutral lipids and phospholipids detected were: hydrocarbons, free fatty acids, free sterols, sterol esters, diglycerides, monoglycerides, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidic acid, phosphatidylserine, and phosphatidylinositol. Palmitic, palmitoleic, stearic, oleic, gamma-linolenic, and arachidonic acids were the most frequently occurring fatty acids. When B. emersonii was grown in (14)C-labeled liquid media, lipid again accounted for 11% of both mature plants and zoospores released from them. The composition of the lipid extracted from such plants and spores was also the same; however, it differed markedly from that of the lipid in spores harvested from solid media, consisting of 28.3% neutral lipid, 12.0% glycolipid, and 59.7% phospholipid. The major lipids were again triglycerides for neutral lipids, mono- and diglycosyldiglycerides for glycolipids, and phosphatidyl choline and phosphatidylethanolamine for phospholipids.     

Naturally derived commercial surfactants differ in composition of surfactant lipids and in surface viscosity

Pulmonary surfactant biophysical properties are best described by surface tension and surface viscosity. Besides lecithin, surfactant contains a variety of minor lipids, such as plasmalogens, polyunsaturated fatty acid-containing phospholipids (PUFA-PL), and cholesterol. Plasmalogens and cholesterol improve surface properties of lipid mixtures significantly. High PUFA-PL and plasmalogen content in tracheal aspirate of preterm infants reduces the risk of developing chronic lung disease. Different preparations are available for exogenous surfactant substitution; however, little is known about lipid composition and surface viscosity. Thus lipid composition and surface properties (measured by oscillating drop surfactometer) of three commercial surfactant preparations (Alveofact, Curosurf, Survanta) were compared. Lipid composition exhibited strong differences: Survanta had the highest proportion of disaturated PL and total neutral lipids and the lowest proportion of PUFA-PL. Highest plasmalogen and PUFA-PL concentrations were found in Curosurf (3.8 +/- 0.1 vs. 26 +/- 1 mol%) compared with Alveofact (0.9 +/- 0.3 vs. 11 +/- 1) and Survanta (1.5 +/- 0.2 vs. 6 +/- 1). In Survanta samples, viscosity increased >8 x 10(-6) kg/s at surface tension of 30 mN/m. Curosurf showed only slightly increased surface viscosity below surface tensions of 25 mN/m, and viscosity did not reach 5 x 10(-6) kg/s. By adding defined PL to Survanta, we obtained a Curosurf-like lipid mixture (without plasmalogens) that exhibited biophysical properties like Curosurf. Different lipid compositions could explain some of the differences in surface viscosity. Therefore, PL pattern and minor surfactant lipids are important for biophysical activity and should be considered when designing synthetic surfactant preparations.     

Specific interactions of tryptophan with phosphatidylcholine and digalactosyldiacylglycerol in pure and mixed bilayers in the dry and hydrated state

Amphiphilic solutes play an important role in the desiccation tolerance of plant cells, because they can reversibly partition into cellular membranes during dehydration. Their effects on membrane stability depend on their chemical structure, but also on the lipid composition of the host membrane. We have shown recently that tryptophan destabilizes liposomes during freezing. The degree of destabilization depends on the presence of glycolipids in the membranes, but not on the phase preference (bilayer or non-bilayer) of the lipids in mixtures with the bilayer lipid phosphatidylcholine. Here, we have investigated the influence of tryptophan on the phase behavior and intermolecular interactions in dry and hydrated bilayers made from the phospholipid egg phosphatidylcholine and the plant chloroplast glycolipid digalactosyldiacylglycerol, or from a mixture (1:1) of these lipids, using Fourier-transform infrared spectroscopy. To distinguish effects of the hydrophobic ring structure of tryptophan from those of the amino acid moiety, we also performed experiments with the hydrophilic amino acid glycine. Our data show that there are specific interactions between tryptophan and either phospholipid or glycolipid in the dry state, as well as H-bonding interactions between the lipids and both solutes. In the rehydrated state, the H-bonding interactions between amino acids and lipids are mostly replaced by interactions between water and lipids, while the hydrophobic interactions between lipids and tryptophan mostly persist.     

Kinetics of phospholipid exchange between bilayer membranes.

In an accompanying publication by Duckwitz-Peterlein, Eilenberger and Overath ((1977) Biochim. Biophys. Acta 469,311--325) it is shown that the exchange of lipid molecules between negatively charged vesicles consisting of total phospholipid extracts from Escherichia coli occurs by the transfer of single lipid monomers or small micelles through the water. Here a kinetic interpretation is presented in terms of a rate constant, k--, for the escape of lipid molecules from the vesicle bilayer into the water. The evaluated rate constants are kP- = (0.86 +/- 0.05) - 10(-5) S-1 and ke- = (1.09 +/- 0.13) - 10(-6) s-1 for phospholipid molecules with trans-delta 9-hexadecenoate and trans-delta 9-octadecenoate, respectively, as the predominant acyl chain component. The rate constants are discussed in terms of the acyl chain and polar head group composition of the lipids.     

Cell-free synthesis of glycosyl-phosphatidylinositol precursors for the glycolipid membrane anchor of Trypanosoma brucei variant surface glycoproteins. Structural characterization of putative biosynthetic intermediates.

Trypanosome variant surface glycoproteins exemplify a class of eukaryotic cell surface glycoproteins that rely on a carboxyl-terminal covalently-attached inositol-containing glycophospholipid for membrane attachment. The glycolipid anchor is acquired soon after translation of the polypeptide, apparently by replacement of a short carboxyl-terminal peptide sequence with a prefabricated glycolipid. A candidate glycolipid precursor (referred to as P2), and a related glycolipid (P3) have been identified recently in polar lipid extracts from trypanosomes. In this paper we describe the synthesis of P2 and P3 by trypanosome membranes. Analyses of organic solvent extracts from membranes incubated with radioactive sugar nucleotides (GDP-[3H]mannose or UDP-[3H]GlcNAc) showed a spectrum of labelled lipids, ranging from partially glycosylated species to the final products, P2 and P3. Structural analyses of these putative biosynthetic intermediates suggest that glycolipid assembly occurs via the sequential glycosylation of phosphatidylinositol.     

Mechanism of proton permeation through chloroplast lipid membranes.

Electrical measurements were carried out to investigate the contribution of chloroplast lipids to the passive proton permeability of both the thylakoid and inner-envelope membranes. Permeability coefficient and conductance to protons were measured for solvent-free bilayers made from monogalactosyldiglyceride:digalactosyldiglycerid: sulfoquinovosyldiglyceride:phosphatidylglycerol (2:1:0.5:0.5, w/w) in the presence of a pH gradient of 7.4/8.1. The permeability coefficient for protons in glycolipids was 5.5 +/- 1.1 x 10(-4) cm s-1 (n = 14). To determine whether this high H+ permeability could be explained by the presence of lipid contaminants such as weak acids, we investigated the effects of (a) bovine serum albumin, which can remove some amphiphilic molecules such as free fatty acids, (b) 6-ketocholestanol, which increases the membrane dipole potential, (c) oleic acid, and (d) chlorodecane, which increases the dielectric constant of the lipid bilayer. Our results show that free fatty acids are inefficient protonophores, as compared with carbonylcyanide-m-chlorphenythydrazone, and that the hypothesis of a weak acid mechanism is not valid with glycolipid bilayers. In the presence of deuterium oxide the H+ conductane was reduced significantly, indicating that proton transport through the glycolipid matrix could occur directly by a hydrogen bond process. The passive transport of H+ through the glycolipid matrix is discussed with regard to the activity of the thylakoid ATP synthase and the inner-envelope H(+)-ATPase.     

Clinical variation in X-linked adrenoleukodystrophy: fatty acid and lipid metabolism in cultured fibroblasts

To determine whether the clinical phenotype of ALD correlates with the extent of metabolic abnormality, we investigated VLFA metabolism in cultured fibroblasts from patients with the clinically severe childhood from of ALD and the milder AMN variant. No differences were seen in the content of neutral lipids or phospholipids, in incorporation of [1-14C]lignocerate into cellular lipids, or in the fatty acid composition of fibroblasts from patients with childhood ALD or AMN. [1-14C]Lignocerate oxidation was deficient to a similar extent (35-40% of normal) in both intact fibroblasts and cell homogenates from patients with childhood ALD and AMN. With the use of fibroblast homogenates, oxidation of lignocerate was partially inhibited by various long-chain fatty acids, and residual activity in ALD homogenates was more susceptible to inhibition by palmitate than normal. In the presence of competing palmitate, residual lignocerate oxidative activity in fibroblast homogenates was reduced to 20 +/- 4% of normal in childhood ALD and 24 +/- 2% of normal in AMN. These results indicate that residual VLFA oxidative activity, fatty acid composition, VLFA metabolism, and lipid content of cultured fibroblasts do not correlate with the clinical expression of the ALD gene.     

A comparison of five lipid extraction solvent systems for lipidomic studies of human LDL

Lipidome profile of fluids and tissues is a growing field as the role of lipids as signaling molecules is increasingly understood, relying on an effective and representative extraction of the lipids present. A number of solvent systems suitable for lipid extraction are commonly in use, though no comprehensive investigation of their effectiveness across multiple lipid classes has been carried out. To address this, human LDL from normolipidemic volunteers was used to evaluate five different solvent extraction protocols [Folch, Bligh and Dyer, acidified Bligh and Dyer, methanol (MeOH)-tert-butyl methyl ether (TBME), and hexane-isopropanol] and the extracted lipids were analyzed by LC-MS in a high-resolution instrument equipped with polarity switching. Overall, more than 350 different lipid species from 19 lipid subclasses were identified. Solvent composition had a small effect on the extraction of predominant lipid classes (triacylglycerides, cholesterol esters, and phosphatidylcholines). In contrast, extraction of less abundant lipids (phosphatidylinositols, lyso-lipids, ceramides, and cholesterol sulfates) was greatly influenced by the solvent system used. Overall, the Folch method was most effective for the extraction of a broad range of lipid classes in LDL, although the hexane-isopropanol method was best for apolar lipids and the MeOH-TBME method was suitable for lactosyl ceramides.     

Lipid composition of the isolated rat intestinal microvillus membrane

1. Rat intestinal microvillus plasma membranes were prepared from previously isolated brush borders and the lipid composition was analysed. 2. The molar ratio of cholesterol to phospholipid was greatest in the membranes and closely resembled that reported for myelin. 3. Unesterified cholesterol was the major neutral lipid. However, 30% of the neutral lipid fraction was accounted for by glycerides and fatty acid. 4. Five phospholipid components were identified and measured, including phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, sphingomyelin and lysophosphatidylcholine. Though phosphatidylethanolamine was the chief phospholipid, no plasmalogen was detected. 5. In contrast with other plasma membranes in the rat, the polar lipids of the microvillus membrane were rich in glycolipid. The cholesterol:polar lipid (phospholipid+glycolipid) ratio was about 1:3 for the microvillus membrane. Published data suggest that this ratio resembles that of the liver plasma membrane more closely than myelin or the erythrocyte membrane. 6. The fatty acid composition of membrane lipids was altered markedly by a single feeding of safflower oil. Membrane polar lipids did not contain significantly more saturated fatty acids than cellular polar lipids. Differences in the proportion of some fatty acids in membrane and cellular glycerides were noted. These differences may reflect the presence of specific membrane glycerides.     

Volume and enthalpy changes in the early steps of bacteriorhodopsin photocycle studied by time-resolved photoacoustics.

We have studied the photoinduced volume changes, energetics, and kinetics in the early steps of the bacteriorhodopsin (BR) photocycle with pulsed, time-resolved photoacoustics. Our data show that there are two volume changes. The fast volume change ( < or = 200 ns) is an expansion (2.5 +/- 0.3 A3/molecule) and is observed exclusively in the purple membrane (PM), vanishing in the 3-[(3-cholamidopropyl)-dimethylammonio] -1-propane-sulfonate-sulfonate-solubilized BR sample; the slow change (approximately 1 micros) is a volume contraction (-3.7 +/- 0.3 A3/molecule). The fast expansion is assigned to the restructuring of the aggregated BR in the PM, and the 1-micros contraction to the change in hydrogen bonding of water at Asp 212 (Kandori et al. 1995. J. Am. Chem. Soc. 117:2118-2119). The formation of the K intermediate releases most of the absorbed energy as heat, with delta Hk = -36 +/- 8 kJ/mol. The activation energy of the K --> L step is 49 +/- 6 kJ/mol, but the enthalpy change is small, -4 +/- 10 kJ/mol. On the time scale we studied, the primary photochemical kinetics, enthalpy, and volume changes are not affected by substituting the solvent D2O for H2O. Comparing data on monomeric and aggregated BR, we conclude that the functional unit for the photocycle is the BR monomer, because both the kinetics (rate constant and activation energy) and the enthalpy changes are independent of its aggregation state.