Although different detergents can give rise to detergent-resistant membranes of different composition, it is unclear whether this represents domain heterogeneity in the original membrane. We compared the mechanism of action of five detergents on supported lipid bilayers composed of equimolar sphingomyelin, cholesterol, and dioleoylphosphatidylcholine imaged by atomic force microscopy, and on raft and nonraft marker proteins in live cells imaged by confocal microscopy. There was a marked correlation between the detergent solubilization of the cell membrane and that of the supported lipid bilayers. In both systems Triton X-100 and CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) distinguished between the nonraft liquiddisordered (l d) and raft liquid ordered (l o) lipid phases by selectively solubilizing the l d phase. A higher concentration of Lubrol was required, and not all the l d phase was solubilized. The solubilization by Brij 96 occurred by a two-stage mechanism that initially resulted in the solubilization of some l d phase and then progressed to the solubilization of both l d and l o phases simultaneously. Octyl glucoside simultaneously solubilized both l o and l d phases. These data show that the mechanism of membrane solubilization is unique to an individual detergent. Our observations have significant implications for using different detergents to isolate membrane rafts from biological systems.
Glycosyl-phosphatidylinositol (GPI)-anchored proteins are enriched in cholesterol- and sphingolipid-rich lipid rafts within the membrane. Rafts are known to have roles in cellular organization and function, but little is understood about the factors controlling the distribution of proteins in rafts. We have used atomic force microscopy to directly visualize proteins in supported lipid bilayers composed of equimolar sphingomyelin, dioleoyl-sn-glycero-3-phosphocholine and cholesterol. The transmembrane anchored angiotensin converting enzyme (TM-ACE) was excluded from the liquid ordered raft domains. Replacement of the transmembrane and cytoplasmic domains of TM-ACE with a GPI anchor (GPI-ACE) promoted the association of the protein with rafts in the bilayers formed with brain sphingomyelin (mainly C18:0). Association with the rafts did not occur if the shorter chain egg sphingomyelin (mainly C16:0) was used. The distribution of GPI-anchored proteins in supported lipid bilayers was investigated further using membrane dipeptidase (MDP) whose GPI anchor contains distearoyl phosphatidylinositol. MDP was also excluded from rafts when egg sphingomyelin was used but associated with raft domains formed using brain sphingomyelin. The effect of sphingomyelin chain length on the distribution of GPI-anchored proteins in rafts was verified using synthetic palmitoyl or stearoyl sphingomyelin. Both GPI-ACE and MDP only associated with the longer chain stearoyl sphingomyelin rafts. These data obtained using supported lipid bilayers provide the first direct evidence that the nature of the membrane-anchoring domain influences the association of a protein with lipid rafts and that acyl chain length hydrophobic mismatch influences the distribution of GPI-anchored proteins in rafts.
Background: Timely detection of acute kidney injury (AKI) in hospital patients has been hampered by the multiple definitions of AKI and difficulties applying their criteria. A laboratory delta check may provide an effective means of detecting patients developing AKI. This study compared three of the proposed AKI definitions and a delta check to detect AKI using serum creatinine results of hospital inpatients. Methods: Serum creatinine results for 2822 inpatients were gathered retrospectively from the clinical biochemistry database. All serum creatinine results within 30 d of admission were included for each patient and assessed for AKI according to four criteria: Risk, Injury, Failure (RIFLE), Acute Kidney Injury Network (AKIN), Waikar & Bonventre or a delta check (increase of .26 mmol/L between two successive values). Results: A total of 149 (11.3%) patients were defined as having AKI by at least one of the four criteria. Different populations of patients were identified by each criterion. The number of patients identified and the incidence of AKI were as follows: RIFLE 94 (7.1%), AKIN 125 (9.5%), Waikar & Bonventre 100 (7.6%) and delta check 146 (11.1%). The delta check detected 132 (98%) of all 135 cases detected by the other three criteria. A further 14 patients were detected solely by the delta check. Conclusions: The different definitions proposed for AKI detect different populations of patients. A laboratory delta check detected 98% of all the patients identified by AKIN, RIFLE and Waikar & Bonventre combined and could therefore provide a practical way of detecting AKI patients.
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