Effect of saturated phosphatidylcholines on the functional properties of reconstituted cytochrome oxidase

1986 ◽  
Vol 64 (2) ◽  
pp. 91-98 ◽  
Author(s):  
Michael A. Singer ◽  
Maria Dinda ◽  
Marlene Young ◽  
Leonard Finegold
Keyword(s):  
Cytochrome Oxidase ◽  
Freeze Fracture ◽  
Respiratory Control ◽  
Scanning Calorimetry ◽  
Lipid Ratios ◽  
Low Protein ◽  
Two Temperatures ◽  
Freeze Fracture Electron Microscopy ◽  
Fracture Electron

Cytochrome oxidase was incorporated into liposomes, at various protein/lipid ratios, composed of either a phosphatidylcholine of varying chain length and symmetry or asolectin. Catalytic activity and respiratory control were assayed at two temperatures. All preparations showed higher activity at low protein/lipid ratios, but only asolectin showed respiratory control. A spectroscopic determination of the vectorial orientation of oxidase molecules showed that, for proteoliposomes with saturated lipids, 100% of oxidase molecules could be reduced by external substrate as compared with 75% for asolectin proteoliposomes. Freeze-fracture electron microscopy confirmed that oxidase was incorporated into these proteoliposomes and differential scanning calorimetry indicated that the protein induces significant disruption in the long range packing of the saturated phospholipids. We propose that the oxidase molecules in proteoliposomes formed from saturated phosphatidylcholines do not display respiratory control because they are unable to assume the transmembrane orientation necessary for full vectorial activity.

Freeze-fracture electron microscopy and enzyme activity of reconstituted cytochrome oxidase

1992 ◽  
Vol 50 (1) ◽  
pp. 710-711
Author(s):  
M. Tihova ◽  
B. Tattrie ◽  
P. Nicholls
Keyword(s):  
Electron Microscopy ◽  
Enzyme Activity ◽  
Cytochrome Oxidase ◽  
Freeze Fracture ◽  
Crystal Form ◽  
Dimensional Structure ◽  
Inner Mitochondrial Membrane ◽  
3 Dimensional ◽  
Freeze Fracture Electron Microscopy ◽  
Fracture Electron

Eukaryotic cytochrome oxidase (CO) is a multisubunit enzyme spanning the inner mitochondrial membrane. As the terminal component in the respiratory assembly it catalyzes the reduction of oxygen to water. The size, shape and other gross features of the 3-dimensional structure of this protein have been obtained only for its 2-dimensional crystal form. The enzyme can be incorporated into liposomes of a defined lipid composition. Freeze-fracture electron microscopy of cytochrome oxidase vesicles (COV) has been combined with kinetic and spectroscopic measurements to study the phospholipid requirements for enzyme activity and protein orientation within the bilayers.

Image Analysis of Intramembrane Particles in Freeze-Fractured Biomembranes Using Computer Simulation Techniques

1975 ◽  
Vol 33 ◽  
pp. 214-215
Author(s):  
D.J. Benefiel ◽  
R.S. Weinstein
Keyword(s):  
Membrane Proteins ◽  
Freeze Fracture ◽  
Integral Membrane Proteins ◽  
Systematic Evaluation ◽  
Intramembrane Particles ◽  
Modeling Methods ◽  
Simulation Techniques ◽  
Freeze Fracture Electron Microscopy ◽  
Fracture Electron ◽  
Computer Simulation Techniques

Intramembrane particles (IMP or MAP) are components of most biomembranes. They are visualized by freeze-fracture electron microscopy, and they probably represent replicas of integral membrane proteins. The presence of MAP in biomembranes has been extensively investigated but their detailed ultrastructure has been largely ignored. In this study, we have attempted to lay groundwork for a systematic evaluation of MAP ultrastructure. Using mathematical modeling methods, we have simulated the electron optical appearances of idealized globular proteins as they might be expected to appear in replicas under defined conditions. By comparing these images with the apearances of MAPs in replicas, we have attempted to evaluate dimensional and shape distortions that may be introduced by the freeze-fracture technique and further to deduce the actual shapes of integral membrane proteins from their freezefracture images.

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