The conformational features of dihydrosphingomyelin (DHSM), the major phospholipid of human lens membranes, were investigated by 1H and 31P nuclear magnetic resonance spectroscopy. Several postulates emerge from the observed trends: (a) in partially hydrated samples of DHSM in CDCl3 above 13 mM, at which lipid-lipid interactions prevail, the amide proton is mostly involved in intermolecular H-bonds that link neighboring phospholipids through bridging water molecules. In the absence of water, the NH group is involved in an intramolecular H-bond that restricts the mobility of the phosphate group. (b) In the monomeric form of the lipid molecule, the amide proton of the major conformer is bound intramolecularly with one of the anionic and/or ester oxygens of the phosphate group. A minor conformer may also be present in which the NH proton participates in an intramolecular H-bond linking to the OH group of the sphingoid base. (c) Complete hydration leads to an extension of the head group as water molecules bind to the phosphate and NH groups via H-bonds, thus disrupting the intramolecular H-bonds prevalent at low concentrations.

Sphingolipid long chain bases (LCBs) in Saccharomyces cerevisiae, dihydrosphingosine (DHS) and phytosphingosine (PHS) and their phosphates (DHS-P and PHS-P) are thought to play roles in heat stress. However, quantitative studies of LCBs and LCBPs have been limited by analytical methods. A new analytical procedure allowed us to measure changes in all known LCBPs and LCBs in wild-type and mutant cells during heat shock and to correlate the changes with heat stress resistance. All five molecular species of LCBPs increased rapidly but transiently when log and stationary phase cells were heatstressed and when log-phase cells were induced for thermotolerance, suggesting that LCBPs play a role in heat stress. In support of this hypothesis, cells lacking the minor LCB kinase, Lcb5p, but not the major kinase, Lcb4p, were two-fold less resistant to killing when log-phase cells were induced for thermotolerance. Thus, LCBPs seem to play a minor role in heat-stress resistance. However, their role may be masked by LCBs, which are elevated in mutant strains, such as one lacking Lcb4p. This elevation demonstrates that one function of Lcb4p is to regulate LCB levels. Two new compounds, C 16 DHS and C 16 DHS-P, were identified, with the latter being degraded by the Dpl1p lyase. Our data provide a basis for determining how the basal and heat-induced levels of individual species of LCBs and LCBPs are governed by the Lcb4p and Lcb5p kinases, the Dpl1p lyase and the Lcb3p phosphatase.

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