Ultracentrifugation, sucrose density-gradient

The properties of the crystalline enzyme from calf liver have been studied by Levine et al. (1969). Despite several recrystallizations the enzyme preparations showed up to three minor components on analytical ultracentrifugation, sucrose density gradient centrifugation, or polyacrylamide gel electrophoresis. This was not due to the presence of impurities, but rather to the occurence of multimers of the enzyme, i.e., monomer, dimer, trimer, and tetramer. Evidence to this effect was obtained from the close correspondence between protein content and enzyme activity in the fractions isolated by density gradient centrifugation and gel electrophoresis. Furthermore, the separated fractions slowly redistributed to yield analytical patterns similar to that of the native enzyme, indicating that interconversion between the various molecular species was taking place. 

Several investigators have presented evidence for low molecular weight forms exhibiting urease activity. Hand (33), in 1939, obtained diffusion data indicating particles of 17,000 daltons or less that retained enzymic activity. More recently, sucrose density gradient ultracentrifugation (34)... 

The molecular size and molecular homogeneity of antibodies may be determined by a sucrose density-gradient ultracentrifugation method.31 32 Samples of 0.2 mL of 0.4% solutions of each antibody preparation are placed carefully on top of separate sucrose density-gradient tubes prepared from 5,10,20,30, and 40% sucrose. The tubes are centrifuged in a swinging... 

Fig. 4.—Ultracentrifugation in sucrose-density gradients Se = immune serum Ab = antibody Glc-Ox = D-glucose oxidase. (Reprinted from Carbohydrate Research, Volume 124, J. H. Pazur, M. S. Erikson, M. Tay, and P. Z. Allen, pp. 253-263, copyright 1983, with permission from Elsevier Science Ltd., The Boulevard, Langford Lane, Kidlington 0X5 1GB, UK.)...

D-isomer. It has been possible to separate and identify the different variants of alkaline phosphatases in human placenta by Sephadex-gel filtration, sucrose density-gradient centrifugation, and ultracentrifugation, and in these studies starch-gel electrophoresis has proved to be unique in characterization of different isoenzyme fractions. Figure 17... 

Human a-2-macroglobulin was prepared from freshly drawn blood essentially as described by Harpel (V7) except that, rather than a KBr gradient, a sucrose density gradient (minimum 0.2 M maximum 1.0 M sucrose) was employed. After ultracentrifugation (Beckman L8-55 SW 28.1 rotor at 28,000 rpm for 16 hr), the a-2-macroglobulin activity was present largely in the hypophase. Immunodiffusion... 

Such complexes are visible with the electron microscope and also may be detected by sucrose density gradient ultracentrifugation (Bytne et al., 1964). It has also been shown that the addition of ribosomes in the in vitro RNA polymerase system enhances RNA synthesis and the liberation of RNA from its complex with the template and the enzyme (Shin and Moldave, 1966 Jones et al., 1968). Thus in the bacterial cell the processes of transcription and translation usually are coupled (see Martin, 1969). 

During the incubation, construct sucrose density gradients. Pour 1 ml of 60% sucrose solution into an autoclaved ultracentrifuge tube. Then, add 1 ml of 40% sucrose solution, and overlay with 8 ml of 30% sucrose solution. 

Figure 1. Effects of preincubation with Mg2+ and MgATP, singly or in coinbination, on the time course of PRPP synthesis catalyzed by the tetrameric form of PRPP synthetase. Highly purified PRPP synthetase was subjected to sucrose density gradient ultracentrifugation in 1 mM Pi, 1 itiM dithiothreitol (pH 7.4) as previously described. Enzyme sedimenting at 7.1s (corresponding to the 4 subunit aggregated form) was preincubated for 30 minutes at 37 in 1 mM Pi, 1 mM dithiothreitol (pH 7.4) with the additions indicated below. PRPP synthesis was then measured at 26° with final concentrations of 32 mM Pi, 1 mM MgATP,...

A 280-fold purified enzyme preparation was mixed with o -chymo-trypsinogen and bovine serum albumin and subjected to sucrose density gradient ultracentrifugation (Fig. 3). Comparison of the sedimentation distance for APRT with that for the protein standards assigned the enzyme an 3.35. 

Fig. 3. Sucrose density gradient ultracentrifugation of human adenine phosphoribosyltransferase (From Thomas, Arnold and Kelley, 1973).

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