The abnormal phosphorylation of spectrin in human hereditary spherocytosis

The phosphorylation of the proteins of the erythrocyte membrane of patients suffering from hereditary spherocytosis is investigated in intact erythrocytes by their incubation in the presence of radioactive inorganic phosphate. Examination of the phosphorylated components by high-resolution two-dimensional gel electrophoresis reveals only one defect in the pathological membranes, a depressed phosphorylation of the smaller polypeptide of spectrin; band 2. The phosphorylation of band 2 is measured with reference to the phosphorylation of syndein ($\text{2.1 + 2.2 + 2.3}$). In patients showing overt clinical symptoms and for whom splenectomy is advocated the phosphorylation of band 2 is depressed by approx. 70%. After splenectomy the phosphorylation of membrane proteins is restored to normal levels.     

X-ray diffraction and time-resolved fluorescence analyses of Aequorea green fluorescent protein crystals

The energy transfer protein, green fluorescent protein, from the hydromedusan jellyfish Aequorea victoria has been crystallized in two morphologies suitable for x-ray diffraction analysis. Hexagonal plates have been obtained in the P6122 or P6522 space group with a = b = 77.5, c = 370 A, and no more than three molecules per asymmetric unit. Monoclinic parallel-epipeds have been obtained in the C2 space group with a = 93.3, b = 66.5, c = 45.5 A, beta = 108 degrees, and one molecule per asymmetric unit. The monoclinic form is better suited for use in a structure determination, and a data set was collected from the native crystal. Time-resolved fluorescence measurements of large single crystals are possible due to the unique, covalently bound chromophore present in this molecule. Fluorescence emission spectra of Aequorea green fluorescent protein in solution and from either the hexagonal or monoclinic single crystal show similar profiles suggesting that the conformations of protein in solution and in the crystal are similar. Multifrequency phase fluorimetric data obtained from a single crystal were best fit by a single fluorescence lifetime very close to that exhibited by the protein in solution. The complementary structural data obtained from fluorescence spectroscopy and x-ray diffraction crystallography will aid in the elucidation of this novel protein's structure-function relationship.     

A calorimetric study of the thermotropic behavior of aqueous dispersions of natural and synthetic sphingomyelins.

A recently developed differential scanning calorimeter has been used to characterize the thermotropic behavior of aqueous dispersions of liposomes containing sphingomyelin. Liposomes derived from sheep brain sphingomyelin exhibit a broad gel-liquid crystalline phase transition in the temperature range of 20-45 degrees C. The transition is characterized by maxima in the heat capacity function at 31.2 and 37.1 degrees C and a total enthalpy change of 7.2 +/-0.4 kcal/mol. Beef brain sphingomyelin liposomes behave similarly but exhibit heat capacity maxima at 30, 32, and 38 degrees C and a total enthalpy change of 6.9 kcal/mol. The thermotropic behavior of four pure synthetic sphingomyelins is reminiscent of multilamellar lecithin liposomes in that a single, sharp, main transition is observed. Results obtained for liposomes containing mixtures of different sphingomyelins are complex. A colyophilized mixture of N-palmitoylsphingosinephosphorylcholine, N-stearoylsphingosinephosphorylcholine, and N-lignocerylsphingosinephosphorylcholine in a 1 : 1 : 1 mol ratio exhibits a single transition with a Tm below that observed for the individual components. On the other hand a 1 : 1 mixture of N-stearoylsphingosinephosphorylcholine and 1-palmitoyl-2-oleylphosphatidylcholine exhibits three maxima in the heat capacity function. It is clear from these results that the thermotropic behavior of sphingomyelin-containing liposomes is a complex function of the exact composition. Furthermore, it appears that the behavior of the liposomes derived from natural sphingomyelins cannot be explained in terms of phase separation of the individual components.