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In two-component phosphatidylcholine bilayers with coexisting liquid and P beta' gel phases, the distribution between phases of low concentrations of glycosphingolipids can be determined by freeze-etch electron microscopy after labeling the glycolipid with a suitable protein. We have found that the distribution depends upon the glycosphingolipid species (Rock, P. et al., (1991) Biochemistry 30, 19-25). Using this technique with cholera toxin as the protein label and bilayers formed from dipalmitoyl- and dielaidoylphosphatidylcholine (1:1) containing < 1 mol% GM1, we have studied the distribution of a family of GM1 homologues differing in the acyl chain and sphingoid base moieties. The GM1 preference for the P beta' ripple phase decreases with decreasing acyl chain length and increasing unsaturation. GM1 with either a C18:1 or C20:1 sphingoid base shows similar distributions in liquid and gel phases. When the molecules are preferentially found in the P beta' phase, they are positioned along unique loci in both A and A/2 forms of the ripple structure. This localization and acyl chain dependence reflect the volume, shape and localization of molecular packing defects in the P beta' phase. The ganglioside inclusions stabilize the P beta' phase and form compositional domains of unique topography.

We demonstrate that supported synthetic phospholipid bilayers, which are stabilized by lateral cross-linking in both leaflets, can be used for specimen preparation for atomic force microscopy of purified membrane proteins with high stability and excellent reproducibility under water or low-salt buffer. A bilayer containing 1,2-dipentacosa-10,12-diynoyl-phosphatidylcholine and 20 mol% ganglioside (GMI) was transferred onto the surface of mica from a Langmuir trough. Cholera toxin, both the B-subunit and the complete molecule randomly bound to the gangliosides, were imaged by atomic force microscopy in solution with a resolution of better than 2 nm. The pentameric structure of the B-subunit oligomers was well resolved. This result indicates that, with this preparation procedure, other membrane proteins may be studied at intermediate to high resolution under physiologically relevant conditions without the need for crystallization.

The development of sarcoplasmic reticulum membranes was studied in vivo and in tissue culture in chicken pectoralis muscle cells. The concentration of the calcium- and magnesium-activated ATPase measured by selective labeling of the enzyme with [32P]ATP in whole muscle homogenates was found to increase in developing chicken pectoralis muscle in vivo from 0.01 nmol/mg of protein in 12-day embryos to 0.3 to 0.4 nmol/mg of protein in 1-month-old chicks, where it constitutes about 3% of the total protein content of muscle. In cultured muscle cells the concentration of calcium-sensitive phosphoprotein increased from 0.015 nmol/mg of protein at 2 days to 0.04 to 0.05 nmol/mg of protein after 5 days of culture. This amount represents about 0.5% of the protein content of the muscle cells. The accumulation of Ca2+ transport ATPase began during fusion and continued with a linear rate during 8 days of culture. The density of 75 A intramembranous particles seen by freeze-etch electron microscopy on fracture faces of sarcoplasmic reticulum membranes is about 4,000/mum2 in adult chick pectoralis muscle but only 400/mum2 in cultured muscle cells in rough proportion to the concentration of Ca2+-sensitive phosphoprotein. The Ca2+, Na+, and K+ concentration of the medium and addition of ouabain, caffeine, or the calcium ionophores A23187 and X537A sharply influence the concentration of calcium transport ATPase in cultured muscle cells, parallel with their effect upon cell fusion and growth. These observations are consistent with the proposition that the gene expression leading to the accumulation of Ca2+ transport ATPase during development in culture may be regulated by intracellular ion concentrations.

A radioactively-labelled glycosphingolipid, asialo-GM1, has been incorporated into phosphatidylcholine multilamellar vesicles. After incubation with ferritin-Ricinus communis agglutinin 60 (RCA 60) conjugate at different temperatures, the vesicles were separated from the conjugate by discontinuous density gradient ultracentrifugation. Measurement of the distribution of the radioactively-labelled asialo-GM1 in the pelleted conjugate fraction and freeze-etch electron microscopy of the vesicle fraction indicate that the decrease in labelling of asialo-G M 1 -containing vesicles by ferritin-RCA 60 conjugate with increasing temperatures (Tillack, T.W., Wong, M., Allietta, M. and Thompson, T.E. (1982) Biochim. Biophys. Acta 691, 261–273) reflects a decrease in apparent binding affinity rather than an ability of the conjugate to extract glycolipid from the phospholipid bilayer after binding.

Molecules of the ganglioside GM1 are randomly distributed in liquid-crystalline 1-palmitoyl-2-oleoyl phosphatidylcholine bilayers. This conclusion is based on a freeze-etch electron microscopic study using ferritin-conjugated cholera toxin and cholera toxin alone as ganglioside labels. The average number of GM1 molecules under a label is calculated by a novel method from the dependence of the fraction of bilayer area covered by the label on the mole fraction of GM1 in the bilayer.

During the initial 5 minutes of T2 bacteriophage infection of Escherichia coli, there is substantial incorporation of labeled guanine into the transfer RNA (tRNA) fraction, strongly suggesting that new tRNA is synthesized early in infection. In this 5-minute period the amount of labeled guanine incorporated into the tRNA fraction of T2-infected bacteria is 23% of that in uninfected bacteria during an equivalent period. The tRNA fractions from T2-infected bacteria and uninfected bacteria were compared by pulse-labeling cultures of each for 5 minutes with 3H and 14C isotopes of guanine and partially resolving a mixture of the doubly labeled tRNA species by reversed phase chromatography. The chromatographic pattern of the labeled tRNA from uninfected cultures of E. coli was very similar to that of nonlabeled carrier tRNA from uninfected cultures. tRNA prepared from pulse-labeled T2-infected cultures of E. coli had a chromatographic pattern that was radically different from that of tRNA prepared from uninfected cultures. These results indicate either that the tRNA synthesized after T2 infection of E. coli represents species of tRNA entirely different from those made in uninfected bacteria, or that relative proportions of the different species made are greatly altered after phage infection, some being made preferentially and others in reduced amounts. Evidence is presented that a substantial amount of the material in the tRNA fraction made during T2 infection can accept amino acids.