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Here we used electrospray ionization mass spectrometry for quantitative determination of lipid molecular species in human fibroblasts and their plasma membrane incorporated into enveloped viruses. Both influenza virus selecting ordered domains and vesicular stomatitis virus (VSV) depleted of such domains [Scheiffele, P., et al. (1999) J. Biol. Chem. 274, 2038-2044] were analyzed. The major difference between influenza and VSV was found to be a marked enrichment of glycosphingolipids in the former. The effect of chronic cholesterol loading on viral lipid composition was studied in Niemann-Pick type C (NPC) fibroblasts. Both NPC-derived influenza and VSV virions contained increased amounts of cholesterol. Furthermore, polyunsaturated phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine were enriched in NPC-derived virions at the expense of the monounsaturated ones. When normal fibroblasts were acutely loaded with cholesterol using cyclodextrin complexes, an adjustment toward increasingly unsaturated phospholipid species was observed, most clearly for phosphatidylcholine and sphingomyelin. Our results provide evidence that (1) glycosphingolipids are enriched in domains through which influenza virus buds, (2) chronic cholesterol accumulation increases the cholesterol content of both glycosphingolipid-enriched and intervening plasma membrane domains, and (3) an increase in membrane cholesterol content is accompanied by an increased level of polyunsaturated species of the major membrane phospholipids. We suggest that remodeling of phospholipids toward higher unsaturation may serve as both an acute and a long-term adaptive mechanism in human cellular membranes against cholesterol excess.

Tissue-type plasminogen activator (t-PA), the serine protease responsible for catalyzing the production of plasmin from plasminogen at the site of blood clots, is synthesized as a single-chain polypeptide precursor. Proteolytic cleavage at the C-terminal side of Arg275 generates a two-chain form of the enzyme whose subunits are held together by a single disulfide bond. We have measured the activities of both forms of the wild-type enzyme, as well as that of a mutant enzyme (Arg275----Gly), created by oligonucleotide-directed mutagenesis, that cannot be cleaved into a two-chain form. Both types of single-chain t-PAs are enzymatically active and exhibit identical Vmax and Km values when assayed with synthetic peptide substrates, indicating that the single amino acid change had no effect on the amidolytic activity of the enzyme. However, cleavage of wild-type t-PA into the two-chain form results in increased activity both on a peptide substrate and on the natural substrates Lys- and Glu-plasminogen in the absence or presence of stimulation by soluble fibrin. The enhanced activity is due to a 3-5-fold increase in the Vmax of the cleaved enzyme, rather than to any change in the Km values for the various substrates. During incubation with plasminogen, the single-chain form of wild-type t-PA is converted to the two-chain form by plasmin generated during the reaction. This conversion, from the less active form of the enzyme, results in a reaction that displays biphasic kinetics.(ABSTRACT TRUNCATED AT 250 WORDS)