Solubilization, partial purification and photolabeling of the integral membrane protein lysophospholipid:acyl-CoA acyltransferase (LAT).

In the present study, we defined experimental conditions that allowed the extraction of the integral membrane protein lysophospholipid:acyl-CoA acyltransferase (LAT, EC 2.3.1.23) from membranes while maintaining the full enzyme activity using the nonionic detergent n-octyl glucopyranoside (OGP) and solutions of high ionic strength. We found that the optimal OGP concentration depended on the ionic strength of the solubilization buffer. Fluorescence measurements with 1,6-diphenyl-1,3,5-hexatriene indicated that the critical micellar concentration (CMC) of OGP decreased with increasing salt concentrations. Analogous studies revealed that the zwitterionic detergent Chaps was ineffective in extracting LAT from membranes in the absence of salt, whereas its solubilization efficiency increased with increasing salt concentrations. Detailed lipid analysis of the different protein/lipid/detergent mixed micelles showed that the protein/lipid/OGP mixed micelles were relatively enriched with sphingomyelin (SPM) compared to protein/lipid/Chaps mixed micelles, indicating that the differences in the solubilization efficiency may be due to the ability to extract more SPM from membranes. When the protein/lipid/OGP mixed micelles were dissociated into protein/detergent and lipid/detergent complexes by the addition of increasing Chaps concentrations, one-tenth of the LAT enzyme activity was preserved making the enzyme accessible to protein purification. Analysis by native PAGE revealed that in the presence of excess Chaps a high molecular mass protein complex migrated into the gel which could be photolabeled by 125I-labelled-18-(4'-azido-2'-hydroxybenzoylamino)-oleyl-CoA. This fatty acid analogue has been shown to be a competitive inhibitor of LAT enzyme activity in the dark, and an irreversible inhibitor after photolysis. Therefore, this protein complex is assumed to contain the LAT enzyme.

Quantitative measurement of different ceramide species from crude cellular extracts by electrospray ionization tandem mass spectrometry (ESI-MS/MS).

Ceramide (CER) is an important signaling molecule involved in a variety of cellular processes, including differentiation, cell growth, and apoptosis. Currently, different techniques are applied for CER quantitation, some of which are relatively insensitive and/or time consuming. Tandem mass spectrometry with its high selectivity and sensitivity is a very useful technique for detection of low abundant metabolites without prior purification or derivatization. In contrast to existing mass spectrometry methods, the developed electrospray tandem mass spectrometry (ESI-MS/MS) technique is capable of quantifying different CER species from crude cellular lipid extracts. The ESI-MS/MS is performed with a continuous flow injection and the use of an autosampler, resulting in a high throughput capability. The collision-induced fragmentation of CER produced, in addition to others, a characteristic fragment of m/z 264, making a precursor ion scan of 264 well suited for CER quantitation. Quantitation is achieved by use of a constant concentration of a non-naturally occurring internal standard C8-CER, together with a calibration curve established by spiking different concentrations of naturally occurring CER. The calibration curves showed linearity over a wide concentration range and sample volumes equivalent to 10 microg of cell protein corresponding to about 20, 000 fibroblasts were sufficient for CER analysis. Moreover this assay showed a detection limit at the subpicomole level. In summary, this methodology enables accurate and rapid analysis of CER from small samples without prior separation steps, thus providing a useful tool for signal transduction research.

HDL-mediated efflux of intracellular cholesterol is impaired in fibroblasts from Tangier disease patients.

To further elucidate the cellular mechanisms leading to HDL deficiency in Tangier disease, HDL-mediated cholesterol efflux was studied in cultured skin fibroblasts from Tangier patients. Both Tangier and control fibroblasts show specific saturable binding of HDL3 to the cell membrane (Bmax = 70 and 52 ng/mg protein, respectively; Kd = 8.8 and 10.6 micrograms/mL, respectively). There was no appreciable uptake of HDL3 by Tangier and control fibroblasts, indicating that cholesterol efflux from fibroblasts occurs at the cell membrane. When cellular cholesterol was labeled to equilibrium by [14C]cholesterol incubation for 48 hours at 37 degrees C, HDL3-mediated cholesterol efflux from Tangier fibroblasts was only 50% of control fibroblasts. To define this abnormality in HDL3-mediated cholesterol efflux more precisely, several additional experiments were performed. First, membrane desorption of cholesterol was determined after cell membranes were labeled with [14C]cholesterol for 3 hours at 15 degrees C. With this labeling protocol, there was no difference in HDL3-mediated cholesterol efflux between control and Tangier fibroblasts. Second, efflux of newly synthesized sterols was determined after incorporation of the precursor [14C]mevalonolactone. Under these conditions, specific HDL3-mediated efflux of sterols was almost absent in Tangier fibroblasts. Third, cells were labeled by incubation with reconstituted [3H]cholesteryl-linoleate-LDL. Efflux of LDL-derived cholesterol was only slightly reduced for the first 4 hours of incubation. After 12 hours, there was no difference between control and Tangier cells. The combined data indicate that the reduced efflux of cholesterol from Tangier fibroblasts observed after homogeneous labeling is due to severely reduced efflux of newly synthesized sterol.(ABSTRACT TRUNCATED AT 250 WORDS)

Improved separation of radioactively labelled cellular phospholipids by high-performance liquid chromatography.

An improved high-performance liquid chromatographic method for the separation and determination of radioactively labelled cellular phospholipids is described. The method is based on separation of phospholipids on a 250 x 4 mm I.D. LiChrospher DIOL 100 (5 microns) column, fitted with a 50 x 4 mm I.D. LiChrospher Si 60 (5 microns) precolumn and a gradient of 5% H3PO4 and acetonitrile. It allows the determination of small amounts of labelled phosphatidylcholine and sphingomyelin due to the sharp elution profile in spite of long retention times.

Diamine ligand release from the cisplatin analogue [meso-1,2-bis(2,6-dichloro-4- hydroxyphenyl)ethylenediamine]dichloroplatinum(II) in cell culture medium.

The stability of the five-membered chelate ring of the cisplatin analogue [meso-1,2-bis(2,6-dichloro-4- hydroxyphenyl)ethylenediamine]dichloroplatinum(II) was investigated under typical cell culture conditions (IMEM-Richter's medium with 10% fetal calf serum, 37 degrees C). For this purpose, the platinum compound was radiolabeled with tritium in the meta position of the aromatic ring by an acid-catalyzed tritium-exchange reaction, and a reversed-phase HPLC assay with radiochemical detection was developed to monitor for the presence of the free diamine ligand in the cell culture medium. A gradual increase in radioactivity attributed to the free diamine was found in medium containing the dichloroplatinum(II) complex (ca. 25% after 24 h), indicating that the diamine ligand was being released from the metal atom. When 1 mM glutathione (GSH) was included in the incubation medium, the amount of free diamine nearly doubled after 24 h, while the amount of radioactivity attributed to serum protein-platinum adducts decreased relative to incubations without GSH. On the other hand, the omission of serum from the incubations resulted in a dramatic decrease in the amount of radioactivity eluting under the diamine peak, while the concentrations of the two methionine-Pt adducts, which formed in a 1:1 ratio, rose. Through the use of liquid secondary ion mass spectroscopy, the two methionine-Pt adducts were identified as monomethionine metabolites of the title compound, whereby the two chloride ligands have been replaced by the amino acid. These compounds are probably diastereomers since the sulfur of methionine can coordinate to platinum with equal probability either cis or trans to the R-configured benzylamine carbon. On the basis of the chemical shifts of the MeS groups in the 250-MHz 1H NMR, it is concluded that a S,N-five-membered chelate ring is present in these methionine-Pt adducts.