Subject ultracentrifugation

The most important distinction between the two structures is that only the helical model is compatible with all of the published structural data, including results obtained from limited proteolysis [81,83], chemical cross-linking [83], analytical ultracentrifugation [81], and mutant complementation [84,85]. Although the double helix provides a useful model with good predictive power, it will no doubt be subjected to further investigation by, for example, atomic force microscopy or X-ray crystallography. Such experiments should refine the structural information on PKSs, and point the way toward the productive modification and immobilization of the synthases. 

The PSI-200 preparation can be subjected to further fractionation by solubilisation in the detergent dodecyl maltoside, followed by sucrose gradient ultracentrifugation (Bassi and Simpson 1987c). This... 

If a further purification is desired, then the supernatant (above) can be subjected to ammonium sulfate fractionation, gel filtration on Sephadex G-200, and finally on a-aminopropane-agarose affinity column. On polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate, the final product migrated as a single band with an estimated molecular weight of 113,000. Upon sedimentation equilibrium velocity ultracentrifugation, an estimated molecular weight value of near 117,000 was obtained. 

Note that electrophoresis separates proteins on the basis of their net charge. If the electrophoretic picture shows only one protein component, and this is observed at several pH values, it may be suspected that the protein is homogeneous. To establish homogeneity more firmly, the protein should also be subjected to a procedure that analyzes it according to size, for example ultracentrifugation and/or electrophoresis in a polyacrylamide gel in the presence of urea and sodium dodecyl sulfate. 

Figure 4. Distribution of [1 ]C—DDT in larval M. sexta hemolymph. 19 h after topical application, hemoTymph was subjected to density gradient ultracentrifugation as shown in Figure 3. Following centrifugation the tube was fractionated and the radioactivity in each fraction determined. Most of the labeled pesticide was found in the lipophorin fraction.

Structural Aspects of Microemulsions. Several investigators have studied the structure of microemulsions using various techniques such as ultracentrifugation, high resolution NMR, spin-spin relaxation time, ultrasonic absorption, p-jump, T-jump, stopped-flow, electrical resistance and viscosity measurements (56-58). The useful compilation of different studies on this subject is found in the books by Robb (68) and Shah and Schechter (69). Several structural models of microemulsions have been proposed and we will discuss only a few important studies here. 

The CDC has also defined a reference method for LDL cholesterol based on the same techniques described above for HDL cholesterol. After ultracentrifugation to remove the VLDL and any chylomicrons present, the bottom fraction (d > 1.006) is subjected to precipitation by heparin and manganese as described previously. After measurement of cholesterol in the d > 1.006 fraction and in the heparin-Mn supernatant solution, LDL cholesterol is calculated by difference. It should be noted that the LDL fraction as measured by this reference method is a so-called broad-cut fraction including any IDL and Lp(a). 

In addition, P-VLDL can also be observed directly by subjecting the VLDL fraction to agarose gel electrophoresis, where it migrates electrophoretically with LDL rather than VLDL (see Figure 26-26). The combination of a VLDL cholesterol/plasma triglyceride ratio of 0.3 or higher and the observation of p-VLDL in the ultracentrifugal supernatant establishes the type III lipoprotein pattern. 

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