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Lipid-free apolipoprotein E (apo E) and canine apo E HDLc, a cholesterol-rich lipoprotein containing apo E as the only apolipoprotein, show very different circular dichroism (CD) spectra. To determine the cause of the spectral difference, we estimated the CD contribution of phospholipid, cholesterol, and cholesteryl ester in liposomes and microemulsions. We prepared microemulsions, containing nearly equal amounts of egg phosphatidylcholine (PC) and cholesteryl oleate (mean diameter 320 A), by an injection technique. Both microemulsions and cholesterol-containing liposomes exhibit intense negative CD bands in the far-ultraviolet region. Lipids contribute about 20% of the spectral difference between apo E and apo E HDLc at 222 nm, and about 60% of the spectral difference at 208 nm. The remainder of the spectral difference is attributable to lipid-protein interaction corresponding to a 15-30% increase in helicity of apo E. CD analysis indicates that the helical content of apo E in apo E HDLc resembles that in the ternary complex apo E-PC-cholesterol (or apo E-PC-cholesteryl ester) more than that in the binary complex apo E-PC, suggesting that cholesterol affects the conformation of apo E. Our data indicate that in going from a lipid-free state to a lipid environment, apo E undergoes a random to helix transition, assuming the maximal helicity predicted from its primary structure.

Some physicochemical properties of a homogeneous preparation of a bifunctional enzyme, fructose-6-phosphate 2-kinase:fructose-2,6-bisphosphatase, were determined. The molecular weight of the enzyme is 101 000 as determined by high-speed sedimentation equilibrium. The molecular weight of dissociated enzyme is 55 000 in 6 M guanidinium chloride by sedimentation equilibrium and in sodium dodecyl sulfate by polyacrylamide gel electrophoresis. A value of 4.7 was observed for the isoelectric point. Tryptic peptide maps and high-performance liquid chromatography of the trypsin-digested enzyme revealed approximately 60 peptides. Amino acid analysis of the enzyme shows that it contains 27 lysine and 36 arginine residues per 55 000 daltons. No free N-terminal amino acid residue was detectable, suggesting that it is blocked. Hydrolysis of the enzyme by carboxypeptidases A and B releases tyrosine followed by histidine and arginine, indicating that the amino acid sequence at the carboxyl terminus is probably -Arg-His-Tyr. Tryptic digestion of [32P]phosphofructose-6-phosphate 2-kinase:fructose-2,6-bisphosphatase yields a 32P-labeled peptide detected by tryptic peptide mapping and high-performance liquid chromatography. Thermolysin digestion of CNBr-cleaved 32P-enzyme also yields a single 32P-peptide. These results indicate that fructose-6-phosphate 2-kinase:fructose-2,6-bisphosphatase is a dimer of 55 000 daltons and the subunits are very similar, if not identical.