Outer membrane of Pseudomonas aeruginosa: heat- 2-mercaptoethanol-modifiable proteins.

A number of polyacrylamide gel systems and solubilization procedures were studied to define the number and nature of "major" polypeptide bands in the outer membrane of Pseudomonas aeruginosa. It was shown that five of the eight major outer membrane proteins were "heat modifiable" in that their mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was determined by the solubilization temperature. Four of these heat-modifiable proteins had characteristics similar to protein II of the Escherichia coli outer membrane. Addition of lipopolysaccharide subsequent to solubilization caused reversal of the heat modification. The other heat-modifiable protein, the porin protein F, was unusually stable to sodium dodecyl sulfate. Long periods of boiling in sodium dodecyl sulfate were required to cause conversion to the heat-modified form. This was demonstrated both with outer membrane-associated and purified lipopolysaccharide-depleted protein F. Furthermore, lipopolysaccharide treatment had no effect on the mobility of heat-modified protein F. Thus it is concluded that protein F represents a new class of heat-modifiable protein. It was further demonstrated that the electrophoretic mobility of protein F was modified by 2-mercaptoethanol and that the 2-mercaptoethanol and heat modification of mobility were independent of one another. The optimal conditions for the examination of the outer membrane proteins of P. aeruginosa by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis are discussed.     

Differential extraction of hydrophobic proteins from chloroplast envelope membranes: a subcellular-specific proteomic approach to identify rare intrinsic membrane proteins

Identification of rare hydrophobic membrane proteins is a major biological problem that is limited by the specific biochemical approaches required to extract these proteins from membranes and purify them. This is especially true for membranes, such as plastid envelope membranes, that have a high lipid content, present a wide variety of specific functions and therefore contain a large number of unique, but minor, proteins. We have optimized a procedure, based on the differential solubilization of membrane proteins in chloroform/methanol mixtures, to extract and concentrate the most hydrophobic proteins from chloroplast envelope membrane preparations, while more hydrophilic proteins were excluded. In addition to previously characterized chloroplast envelope proteins, such as the phosphate/triose phosphate translocator, we have identified new proteins that were shown to contain putative transmembrane α-helices. Moreover, using different chloroform/methanol mixtures, we have obtained differential solubilization of envelope proteins as a function of their hydrophobicity. All the proteins identified were genuine chloroplast envelope proteins, most of them being localized within the inner membrane. Our procedure enables direct mapping (by classical SDS-PAGE) and identification of hydrophobic membrane proteins, whatever their isoelectric point was, that are minor components of specific subcellular compartments. Thus, it complements other techniques that give access to peripheral membrane proteins. If applied to various cell membranes, it is anticipated that it can expedite the identification of hydrophobic proteins involved in transport systems for ions or organic solutes, or it may act as signal receptors or to control metabolic processes and vesicle trafficking.     

Development and application of a two-phase, on-membrane digestion method in the analysis of membrane proteome

Analysis of membrane proteins, particularly integral membrane proteins, still presents a great challenge due to their poor water solubility and low abundance though much effort has been devoted to the solubilization and enrichment of the protein class. In this paper, a two-phase, on-membrane digestion method was developed and applied in the analysis of rat liver membrane proteome. The two-phase system was constituted by mixing n-butanol and 25 mM NH4HCO3. Comparative experiments indicated that the proteins on membranes could be digested in the two-phase system more efficiently than in both 60% methanol and 25 mM NH4HCO3 solutions under the same conditions, thereby improving the identification of the membrane proteins. When the established two-phase system and CapLC-MS/MS was used to analyze rat liver membrane proteome, a total of 411 membrane proteins were identified, more than 80% of which were transmembrane proteins with 1-12 mapped transmembrane domains (TMDs). Because of its extraction and dissolution actions, the two-phase on-membrane digestion system we developed could efficiently improve the digestion and removal of adsorbed nonmembrane proteins, and remarkably increase the number and coverage of identified membrane proteins, particularly the transmembrane proteins. Using our procedure to identify a complementary protein set from all fractions of the two-phase system could achieve a higher coverage of the membrane proteome.     

A comparison of techniques for isolation of the outer membrane proteins of Haemophilus influenzae type b

We compared several rapid techniques used for extraction of outer membrane proteins from gram-negative enteric bacteria to Haemophilus influenzae type b. After lysis of cells with a French press, the inner and outer membranes were separated by isopycnic centrifugation. Each membrane was identified by density, morphology, enzymatic activity, and susceptibility to solid-phase iodination of intact cells. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we identified 10 polypeptides which were enriched in the outer membrane band compared to the inner membrane band. Using these proteins, we compared the polypeptide pattern of outer membranes with that obtained by (1) selective solubilization with sodium dodecyl-beta-D-maltoside, octyl-beta-D-glucopyranoside, Triton X-100, sodium, or cholamidopropyl dimethylaminopropanesulfonate; (2) extraction with chaotropic agents and heat; and (3) differential centrifugation of vesicles shed during transition from log growth phase to stationary growth phase. There were definable differences between the polypeptide pattern of membranes obtained with each rapid technique compared to the polypeptide pattern of isolated outer membranes. The polypeptide pattern of lithium extracts and the Triton X-100 insoluble fractions of total membranes most closely approximated the polypeptide pattern of isopycnically isolated outer membranes. Depending on the outer membrane protein sought, one of these rapid techniques can be utilized when a rapid method of outer membrane protein isolation is required.     

Purification of a Synaptic Membrane Na+/Ca2+ Antiporter and Immunoextraction with Antibodies to a 36-kDa Protein

The conditions for optimal solubilization and reconstitution of bovine brain synaptic plasma membrane Na+/Ca2+ exchange activity were examined and a series of chromatographic procedures were used for the isolation of a protein involved in this transport activity. The zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate in the presence of 20% (vol/vol) glycerol led to optimal solubilization, and soybean phospholipids in low-pH medium were found to produce optimal reconstitution of activity after dialysis to remove the detergent. Sequential chromatography steps involving the use of gel filtration on Sephacryl S-400 HR, ion exchange on diethylaminoethyl-Sephacel, and metal chelate chromatography on tris-(carboxymethyl)ethylenediamine loaded with LaCl3 led to the isolation of a fraction highly enriched in both Na+/Ca2+ exchange activity and two protein bands identified by denaturing electrophoresis. The estimated molecular masses of the two proteins were 50 and 36 kDa. Development of polyclonal antibodies to the 36-kDa protein permitted immunoextraction of greater than 95% of the antiporter activity from solubilized synaptic plasma membranes. These antibodies cross-reacted with the electroeluted 50-kDa protein on enzyme-linked immunosorbent assays, suggesting a close relationship between the two proteins. These results indicate that the 36-kDa protein is at least a component of the brain membrane Na+/Ca2+ antiporter.     

Enrichment of integral membrane proteins for proteomic analysis using liquid chromatography-tandem mass spectrometry

An increasing number of proteomic strategies rely on liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect and identify constituent peptides of enzymatically digested proteins obtained from various organisms and cell types. However, sample preparation methods for isolating membrane proteins typically involve the use of detergents and chaotropes that often interfere with chromatographic separation and/or electrospray ionization. To address this problem, a sample preparation method combining carbonate extraction, surfactant-free organic solvent-assisted solubilization, and proteolysis was developed and demonstrated to target the membrane subproteome of Deinococcus radiodurans. Out of 503 proteins identified, 135 were recognized as hydrophobic on the basis of their calculated hydropathy values (GRAVY index), corresponding to coverage of 15% of the predicted hydrophobic proteome. Using the PSORT algorithm, 53 of the proteins identified were classified as integral outer membrane proteins and 215 were classified as integral cytoplasmic membrane proteins. All identified integral cytoplasmic membrane proteins had from 1 to 16 mapped transmembrane domains (TMDs), and 65% of those containing four or more mapped TMDs were identified by at least one hydrophobic membrane spanning peptide. The extensive coverage of the membrane subproteome (24%) by identification of highly hydrophobic proteins containing multiple TMDs validates the efficacy of the described sample preparation technique to isolate and solubilize hydrophobic integral membrane proteins from complex protein mixtures.     

Membrane proteomic analysis of Arabidopsis thaliana using alternative solubilization techniques

This study presents a comparative proteomic analysis of the membrane subproteome of whole Arabidopsis seedlings using 2% Brij-58 or 60% methanol to enrich and solubilize membrane proteins for strong cation exchange fractionation and reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 441 proteins were identified by our Brij-58 method, and 300 proteins were detected by our methanol-based solubilization approach. Although the total number of proteins obtained using the nonionic detergent was higher than the total obtained by organic solvent, the ratio of predicted membrane proteins to total proteins identified indicates up to an 18.6% greater enrichment efficiency using methanol. Using two different bioinformatics approaches, between 31.0 and 40.0% of the total proteins identified by the methanol-based method were classified as containing at least one putative transmembrane domain as compared to 22.0-23.4% for Brij-58. In terms of protein hydrophobicity as determined by the GRAVY index, it was revealed that methanol was more effective than Brij-58 for solubilizing membrane proteins ranging from -0.4 (hydrophilic) to +0.4 (hydrophobic). Methanol was also approximately 3-fold more effective than Brij-58 in identifying leucine-rich repeat receptor-like kinases. The ability of methanol to effectively solubilize and denature both hydrophobic and hydrophilic proteins was demonstrated using bacteriorhodopsin and cytochrome c, respectively, where both proteins were identified with at least 82% sequence coverage from a single reversed-phase LC-MS/MS analysis. Overall, our data show that methanol is a better alternative for identifying a wider range of membrane proteins than the nonionic detergent Brij-58.     

Evaluation of the application of sodium deoxycholate to proteomic analysis of rat hippocampal plasma membrane

Detergents have been widely used for the solubilization of membrane proteins and the improvement of their digestion. In this paper, we have evaluated the application of sodium deoxycholate (SDC) to the solubilization and digestion of rat hippocampal plasma membrane (PM) proteins. For in-solution digestion, rat hippocampal PM fraction from sucrose-density gradient centrifugation was solubilized by boiling in 1.0% SDC, and directly digested without dilution. During the in-gel digestion of the hippocampal PM proteins separated by SDS-PAGE, 0.1% SDC was added. Before analysis of peptide mixture by liquid chromatography and electrospray mass spectrometry, SDC in the tryptic digests was removed by centrifugation following acidification. Use of 1.0% SDC in solubilization and in-solution digestion of rat PM proteins had led to 77 PM or membrane-associated proteins identified, a more than 2-fold increase over that by use of SDS. The addition of 0.1% SDC to the in-gel digestion of SDS-PAGE-resolved membrane proteins remarkably enhanced the coverage of tryptic peptides and the number of hydrophobic membrane proteins identified. Being a cheaper and more tractable acid-insoluble detergent, SDC could be used at higher concentration in the solubilization and tryptic digestion of proteins including PM proteins with the purpose of enhancing the protein solubility and at the same time making no interference with trypsin activity and subsequent analyses.     

Calmodulin-binding proteins in granule and plasma membranes from bovine chromaffin cells

Calmodulin-binding proteins in chromaffin granule membrane and chromaffin cell plasma membranes have been investigated and compared. Chromaffin granules were purified by centrifugation over a 1.7 M sucrose layer. Plasma membranes were obtained in a highly purified form by differential and isopycnic centrifugation. Enzymatic determinations of 5'-nucleotidase, a generally accepted plasma membrane marker, showed a 40-50-fold enrichment as compared to the cell homogenate. Marker enzyme studies demonstrated only minimal contamination by other subcellular organelles. After solubilization with Triton X-100, calmodulin-binding proteins were isolated from chromaffin granule membranes and plasma membranes by affinity chromatography on a calmodulin/Sepharose 4B column. On two-dimensional polyacrylamide gelelectrophoresis a prominent protein (Mr = 65,000, pI ranging from 5.1 to 6) consisting of multiple spots, was present in the calmodulin-binding fraction from chromaffin granule membranes as well as from plasma membranes. Besides this 65 kDa protein both fractions had at least four groups of proteins in common. Also, proteins typical for either preparation were observed. In the calmodulin-binding protein preparations from chromaffin granule membranes a prominent spot with Mr = 80,000 and a pH ranging from 5.0 to 5.7 was present. This protein was enzymatically and immunologically identified as dopamine-beta-monooxygenase.     

THE EFFECTS OF SOLUBILIZATION PROCEDURES ON THE RELEASE AND MOLECULAR STATE OF ACETYLCHOLINESTERASE FROM ELECTRIC ORGAN TISSUE

Abstract— A study was made of the effect of various solubilization procedures on the release of AChE from electric organ tissue of the electric eel and on the molecular state of the enzyme. The procedures employed included homogenization in different ionic media or in the presence of detergents, etuymic treatment and chemical modification. Studies were performed on intact electroplax, tissue homogenates and membrane fractions. The apparent AChE activity of intact cells, homogenates and membrane fractions was shown to be governed by diffusion-controlled substrate and hydrogen ion gradients, generated by AChE-catalyscd hydrolysis, leading to a lower substrate concentration and a lower pH in the vicinity of the particulate enzyme.
Treatment of homogenates with NaCl solutions or with NaCl solutions containing the nonionic detergent Triton X-100 causes release of the native'molecular forms of the enzyme (primarily the 18 S species) which aggregate at low ionic strength. For optimal extraction both high ionic strength (e.g. 1 M-NaCl) and the detergent are needed AChE is also solubilized by treatment of tissue homogenates with trypsin, bacterial protease or collagenase. The first two enzymes caused its release as an 11 S non-aggregating form, while collagenase also produces a minor non-aggregating - 16 S component. Treatment of tissue homogenates with maleic anhydride causes release of AChE as a non-aggregating 18 S species. On the basis of the solubilization experiments it is concluded that the interaction of AChE with the excitable membrane is primarily electrostatic. The possible orientation of the enzyme within the synaptic gap is discussed.     

Comparison of SDS- and methanol-assisted protein solubilization and digestion methods for Escherichia coli membrane proteome analysis by 2-D LC-MS/MS

Both organic solvent and surfactant have been used for dissolving membrane proteins for shotgun proteomics. In this work, two methods of protein solubilization, namely using 60% methanol or 1% SDS, to dissolve and analyze the inner membrane fraction of an Escherichia coli K12 cell lysate were compared. A total of 358 proteins (1417 unique peptides) from the methanol-solubilized protein mixture and 299 proteins (892 peptides) from the SDS-solubilized sample-were identified by using trypsin digestion and 2-D LC-ESI MS/MS. It was found that the methanol method detected more hydrophobic peptides, resulting in a greater number of proteins identified, than the SDS method. We found that 159 out of 358 proteins (44%) and 120 out of 299 proteins (40%) detected from the methanol- and SDS-solubilized samples, respectively, are integral membrane proteins. Among the 190 integral membrane proteins 70 were identified exclusively in the methanol-solubilized sample, 89 were identified by both methods, and only 31 proteins were exclusively identified by the SDS method. It is shown that the integral membrane proteins reflected the theoretical proteome for number of transmembrane helices, length, functional class, and topology, indicating there was no bias in the proteins identified.     

Application of high-pressure to subfractionate membrane protein-lipid complexes: a case study of protein kinase C

Current procedures for solubilization of membrane proteins involve the use of detergents. A procedure using high hydrostatic pressures without detergent has been applied in this study to subfractionate membrane proteins and their endogenously associated lipids. Rat brain membrane preparations were suspended in hypotonic buffer containing the membrane fluidizer benzyl alcohol in a sealed pressure cell and subjected to hydrostatic pressures of up to 1500 atmospheres (approx 22,000 psi) in a French press. Under these conditions, specific membrane proteins including protein kinase C, phospholipase A2, calmodulin-binding proteins, G-proteins, and microtubule-associated proteins all coextracted and were associated to lipid particles, suggesting inherent physical contact. Two populations of membrane-associated protein kinase C were identified according to molecular weight estimations. The first coeluted with the lipid particles composed predominantly of phospholipids, while the second contained much less lipid and was similar to the soluble monomer, i.e., cytosolic protein kinase C. This procedure provides an important technique for selective subfractionation of membrane proteins in their native lipid environment which could be used for structure-function studies.     

Smooth muscle cell sarcolemma. Purification and properties of plasma membranes from the rat uterus.

A membrane fraction with sarcolemmal properties was purified from the smooth muscle layers (myometrium) of rat uterus by successive differential and equilibrium centrifugation in sucrose. The putative sarcolemmal fraction was identified by iodination with [125I]iodosulfanilic acid, had an equilibrium density of 1.15, and was enriched in enzyme activities usually associated with the plasma membrane including 5'-nucleotidase (EC 3.1.3.5) and (Na+ + K+) ATPase (EC 3.6.1.3). These membranes were free of mitochondrial or nuclear membrane contamination, suggesting the relative enrichment of sarcolemmal membranes in the fraction. Proteins of the membranes were heterogeneous with respect to molecular weight, but only a few were labelled when intact muscle was radioiodinated. Uniform resistance of sarcolemmal proteins to trypsin digestion and salt extraction suggested many are tightly bound or intrinsic membrane proteins and was a further indication of the homogeneity of membranes in this fraction.     

Optimization of protein solubilization for the analysis of the CD14 human monocyte membrane proteome using LC-MS/MS

Proteomic profiling of membrane proteins is of vital importance in the search for disease biomarkers and drug development. However, the slow pace in this field has resulted mainly from the difficulty to analyze membrane proteins by mass spectrometry (MS). The objective of this investigation was to explore and optimize solubilization of membrane proteins for shotgun membrane proteomics of the CD14 human monocytes by examining different systems that rely on: i) an organic solvent (methanol) ii) an acid-labile detergent 3-[3-(1,1-bisalkyloxyethyl)pyridin-1-yl]propane-1-sulfonate (PPS), iii) a combination of both agents (methanol + PPS). Solubilization efficiency of different buffers was first compared using bacteriorhodopsin as a model membrane protein. Selected approaches were then applied on a membrane subproteome isolated from a highly enriched human monocyte population that was ~ 98% positive for CD14 expression as determined by FACS analysis. A methanol-based buffer yielded 194 proteins of which 93 (48%) were mapped as integral membrane proteins. The combination of methanol and acid-cleavable detergent gave similar results; 203 identified proteins of which 93 (46%) were mapped integral membrane proteins. However, employing PPS 216 proteins were identified of which 75 (35%) were mapped as integral membrane proteins. These results indicate that methanol alone or in combination with PPS yielded significantly higher membrane protein identification/enrichment than the PPS alone.     

Extraction of membrane antigens from Brucella ovis and an assessment of their serological activity by immunoblotting

The efficacy and selectivity of chaotropic and phase-partitioning procedures for the extraction of membrane proteins from Brucella ovis were compared with a standard Sarkosyl method. Major group 1, 2 and 3 outer-membrane proteins (OMPs) of B. ovis stained by Coomassie blue in SDS-PAGE gels had, respectively, apparent molecular masses of 81/82 kDa, 39-41 kDa and 30-32 kDa. The presence of these bands in the Sarkosyl extract of total membrane vesicles (TMVs) indicate that the procedure failed to selectively solubilize only inner-membrane proteins (IMPs). SDS-PAGE analyses also revealed the presence of OMPs and other additional bands following extraction of B. ovis TMVs by butanol phase-partitioning or with extraction solutions based on the chaotropic reagents potassium thiocyanate (KSCN), sodium salicylate (SSC) and lithium acetate (LAE). OMPs are therefore not selectively extracted by any one of these procedures. Based on the number and staining intensity of extracted membrane-associated polypeptides, the efficacy of different extraction procedures could be graded in decreasing order as follows: KSCN, SSC, butanol and LAE. Both butanol and SSC were particularly effective in extracting group 3 OMPs. Sera from chronic excretor rams were used to identify zones of seroreactivity in immunoblots. Essentially, two reactivity patterns were seen: strong antibody binding against polypeptides in zones A (46-85 kDa), C (28-32 kDa) and D (18-22 kDa) in one, and additional reactivity against zones B (34-44 kDa) and E (13-18 kDa) polypeptides in the other.(ABSTRACT TRUNCATED AT 250 WORDS)     

Optimization of protein solubilization for the analysis of the CD14 human monocyte membrane proteome using LC-MS/MS

Proteomic profiling of membrane proteins is of vital importance in the search for disease biomarkers and drug development. However, the slow pace in this field has resulted mainly from the difficulty to analyze membrane proteins by mass spectrometry (MS). The objective of this investigation was to explore and optimize solubilization of membrane proteins for shotgun membrane proteomics of the CD14 human monocytes by examining different systems that rely on: i) an organic solvent (methanol) ii) an acid-labile detergent 3-[3-(1,1-bisalkyloxyethyl)pyridin-1-yl]propane-1-sulfonate (PPS), iii) a combination of both agents (methanol + PPS). Solubilization efficiency of different buffers was first compared using bacteriorhodopsin as a model membrane protein. Selected approaches were then applied on a membrane subproteome isolated from a highly enriched human monocyte population that was ~ 98% positive for CD14 expression as determined by FACS analysis. A methanol-based buffer yielded 194 proteins of which 93 (48%) were mapped as integral membrane proteins. The combination of methanol and acid-cleavable detergent gave similar results; 203 identified proteins of which 93 (46%) were mapped integral membrane proteins. However, employing PPS 216 proteins were identified of which 75 (35%) were mapped as integral membrane proteins. These results indicate that methanol alone or in combination with PPS yielded significantly higher membrane protein identification/enrichment than the PPS alone.     

Effects of L-arginine on solubilization and purification of plant membrane proteins

Biochemical analysis of membrane proteins is problematic at the level of solubilization and/or purification because of their hydrophobic nature. Here, we developed methods for efficient solubilization and purification of membrane proteins using L-arginine. The addition of 100 mM of basic amino acids (L-arginine, L-lysine, and L-ornithine) to a detergent-containing solubilization buffer enhanced solubilization (by 2.6-4.3 fold) of a model membrane protein-polygalacturonic acid synthase. Of all the amino acids, arginine was the most effective additive for solubilization of this membrane protein. Arginine addition also resulted in the best solubilization of other plant membrane proteins. Next, we examined the effects of arginine on purification of a model membrane protein. In anion-exchange chromatography, the addition of arginine to the loading and elution buffers resulted in a greater recovery of a membrane protein. In ultrafiltration, the addition of arginine to a protein solution significantly improved the recovery of a membrane protein. These results were thought to be due to the properties of arginine that prevent aggregation of hydrophobic proteins. Taken together, the results of our study showed that arginine is useful for solubilization and purification of aggregate-prone membrane proteins.     

Molecular Dynamics Simulation of WSK-3, a Computationally Designed, Water-Soluble Variant of the Integral Membrane Protein KcsA

Poor solubility and low expression levels often make membrane proteins difficult to study. An alternative to the use of detergents to solubilize these aggregation-prone proteins is the partial redesign of the sequence so as to confer water solubility. Recently, computationally assisted membrane protein solubilization (CAMPS) has been reported, where exposed hydrophobic residues on a protein's surface are computationally redesigned. Herein, the structure and fluctuations of a designed, water-soluble variant of KcsA (WSK-3) were studied using molecular dynamics simulations. The root mean square deviation of the protein from its starting structure, where the backbone coordinates are those of KcsA, was 1.8 angstroms. The structure of salt bridges involved in structural specificity and solubility were examined. The preferred configuration of ions and water in the selectivity filter of WSK-3 was consistent with the reported preferences for KcsA. The structure of the selectivity filter was maintained, which is consistent with WSK-3 having an affinity for agitoxin2 comparable to that of wild-type KcsA. In contrast to KcsA, the central cavity's side chains were observed to reorient, allowing water diffusion through the side of the cavity wall. These simulations provide an atomistic analysis of the CAMPS strategy and its implications for further investigations of membrane proteins.     

Identification of two proteins associated with mammalian ATP synthase

Bovine mitochondrial ATP synthase commonly is isolated as a monomeric complex that contains 16 protein subunits and the natural IF(1) inhibitor protein in substoichiometric amounts. Alternatively ATP synthase can be isolated in dimeric and higher oligomeric states using digitonin for membrane solubilization and blue native or clear native electrophoresis for separation of the native mitochondrial complexes. Using blue native electrophoresis we could identify two ATP synthase-associated membrane proteins with masses smaller than 7 kDa and isoelectric points close to 10 that previously had been removed during purification. We show that in the mitochondrial membrane both proteins are almost quantitatively bound to ATP synthase. Both proteins had been identified earlier in a different context, but their association with ATP synthase was unknown. The first one had been named 6.8-kDa mitochondrial proteolipid because it can be isolated by chloroform/methanol extraction from mitochondrial membranes. The second one had been denoted as diabetes-associated protein in insulin-sensitive tissue (DAPIT), which may provide a clue for further functional and clinical investigations.