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The physico-chemical properties of lipid components isolated from zoospores of the aquatic phycomycete, Blastocladiella emersonii, were investigated with electron spin resonance (ESR) spectroscopy using the spin label, 5-nitroxystearate. Lipid dispersions were made from zoospore phospholipids and glycolipids, both singly and in combination with each other and with isolated neutral lipid components. Plots of the hyperfine splitting parameter (2T parallel) vs. temperature indicate that it is the zoospore glycolipids rather than the phospholipids which are responsible for the phase transformations previously observed in aqueous dispersions of the total lipids extracted from zoospores and in zoospores in vivo. The discontinuities observed in the glycolipid dispersions seem to represent the onset and completion of a gel-to-liquid-crystalline phase transition. Over the temperature range tested, Ca2+ increased the rigidity of the glycolipid dispersions, the major component of which is probably a diglucosyldiglyceride, but had no effect on the phospholipid dispersions. The increase in 2T parallel was not affected by inclusion of neutral lipids into the glycolipid dispersion but was eliminated at high (5 : 1, w/w) phospholipid-to-glycolipid ratios. The Ca2+ effect was relatively independent of both the absolute rigidity of the dispersion and its phase (gel or liquid-crystalline), suggesting an interaction with the glycolipid head group rather than the hydrocarbon core. The Ca2+-induced increase in 2T was neither prevented nor reversed by the presence of K+. The presence of two spin label populations co-existing in a dynamic equilibrium was found in glycolipid/neutral lipid dispersions. Plots of the percentage ([HA/(HA + HB)] X 100 of the spin label population, as measured by the peak height of the low-field peaks, corresponding to the more immobilized component (HA) vs. temperature indicated two break points. The temperatures at which these break points occurred are similar to those obtained for the glycolipid dispersions, and match the break points (TL and TH) found in ESR experiments using zoospores in vivo. The importance of the glycolipids in the development of this organism is discussed.

The physical-chemical properties of the isolated plasma membranes from zoospores of the chytridiomycete Blastocladiella emersonii were investigated, with electron spin resonance (ESR) spectroscopy, using the spin-label 5-nitroxystearate (5-NS). Both isolated plasma membranes and aqueous dispersion of the lipids extracted from the plasma membranes were spin-labeled and analyzed. Plots of the hyperfine splitting parameter (2T) vs. temperature indicated that the middle break point, TM, initially observed in experiments with spin-labeled zoospores in vivo [Leonards, K. S., & Haug, A. (1980) Biochim. Biophys. Acta 600, 805-816], was the result of a lipid-lipid interaction (glycolipid-glycolipid or glycolipid-neutral lipid) rather than a lipid-protein interaction. This interaction was markedly affected by Ca2+ ions, which interacted directly with the lipid components, increasing TM from 11 +/- 1 (Ca2+ removed by EDTA) to 21 +/- 1 degree C (10 mM Ca2+) in the lipid dispersions and from 12 +/- 1 to 23 +/- 1 degree C in the plasma membrane preparations. The initial ESR studies on spin-labeled zoospores in vivo had also demonstrated that the addition of K+ ions could reverse the Ca2+ ion effect, downshifting TM from 22 +/- 1 to 10 +/- 1 degree C. The addition of of K+ ions to the isolated plasma membrane had no affect on TM, indicating that K+ ions do not simply replace Ca2+ ions but exert their effect indirectly on the membrane. However, after the inclusion of ATP, K+ ions could reverse the Ca2+ ion effect. it was determined that the ATp generated an "energized membrane" state which permitted the K+ ions to reverse the Ca2+ effect. Since K+ ions have been shown to depolarize the membrane potential in both zoospores and isolated zoospore plasma membrane preparations (generated by ATP), were suggest that the K+ ion induced reversal of the Ca2+ ion effect, and therefore the change in the lipid-lipid interactions responsible for TM, is a consequence of the K+ ion induced depolarization of the membrane potential.

The roles of lipids and proteins in Ca2+-PO4-induced membrane aggregation were investigated. Cytoskeleton-free vesicles derived from intact human and rabbit erythrocytes (HEves and REves, respectively) were employed as a model system. The HEves and REves have a simplified membrane protein composition [band 3 proteins and glycoproteins PAS-1, -2, and -3 (HEves)] and normal lipid composition. Optimal experimental conditions for pH, [PO4], and [CaCl2] were determined for quantitatively examining the dynamics and extent of HEves and REves aggregation, measured turbidimetrically. The aggregation process was found to be quite sensitive to small changes in pH and [PO4] and much less sensitive to [CaCl2]. The roles of membrane proteins in vesicle aggregation were examined by selectively modifying the proteins enzymatically. The roles of lipids were studied by using sonicated lipid vesicles [small unilamellar vesicles (SUVs)] made from Dodge ghost lipid extracts. Enzymatic treatment with trypsin, chymotrypsin, or Pronase had no effect on either the rates or the extent of vesicle aggregation (2-min incubation period). Neuraminidase treatment reduced both factors by approximately 20%. SUVs aggregated with Ca2+-PO4 in a way which depended on the PO4/lipid ratio. Together the results suggest the following: (1) PO4 is associated with the vesicle surface, involving the membrane lipids; (2) the vesicle + PO4 incubation time component of the PO4 effect is eliminated by enzymatically modifying the vesicle membrane proteins; (3) qualitative, rather than quantitative, properties of sialic acid containing molecules affect vesicle aggregation; and (4) with the exception of the incubation time effect, membrane proteins seem neither to promote nor to inhibit Ca2+-PO4-induced HEves or REves aggregation.