Sucrose density gradients, effect

Sphaeroplasts were prepared by slight modifications to published methods [12,13]. Lysis of sphaeroplasts was effected by a combination of osmotic lysis and gentle mechanical disruption [14]. Discontinuous sucrose-density gradients were constructed and fractions were then assayed for protein, PG and marker enzymes for different organelles. 

Hydroperoxide lyase was found first in the 12,000 g supernatant of watermelon seedling extracts (14). On the other hand, Galliard et al reported that cucumber fruit hydroperoxide lyase was associated with a particulate fraction and was solubilized with Triton X-100 ( ,22). Galliard et al also used Triton X-100 to extract hydroperoxide lyase effectively from cucumber fruit (24), tomato fruit (32) and kidney bean leaf (27). Wardale et al attempted to determine the subcellular localization of hydroperoxide lyase in cucumber fruit by sucrose density gradient centrifugation (30). 

In several tissues, like gastric mucosa [6,8,46.47], pancreas [19,20], submandibular gland [22] and intestinal mucosa [36], the anion-sensitive ATPase can be solubilized by Triton X-100, a detergent/protein ratio of 3 1 being most effective [6,8,47]. The solubihzed enzyme from gastric mucosa [46] and pancreas [20] could be partially purified by gel filtration and sucrose density gradient centrifugation. The properties of the solubilized enzyme do not differ very much from those of the particulate enzyme. 

Figure 5. Two sucrose density gradient electrofocusing runs identical except for the polarity of the electrodes. The first, A, has been focused with the anode in the dense solution, i.e., at the bottom of the column, and the second, B, with the cathode in the dense solution. The pH-gradient is hardly affected by the superimposed sucrose density gradient. In the conductivity, however, there is a difference in the two experiments. With the cathode in the bottom, the conductivity lowering effect from the sucrose density gradient will add to the relatively low conductivity of the carrier ampholytes isoelectric near pH 9. In both curves one can observe the low conductivity of the carrier ampholytes around pH 7.

The method we have developed for the isolation of PS II core complexes - a combination of sucrose density-gradient centrifugation and HPLC - is also very effective for the isolation of pure PS I and cytochrome bef complexes. 

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,...

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. 

Figure 4. The effect of or Zn " addition to the CPIa conplex, isolated by SDS-sucrose density gradient centrifugation, on the 77 K emmission spectra.

Fig. 8. The effect of illumination of etiolated maize leaves for 45 minutes on the specific activity [cpm ( P)/O.D.2m] profile obtained by centrifuging an extract of twice-washed plastids in a 10-34% sucrose density gradient (upper figure). The lower figure shows the absorption profile for identification of the position of plastid ribosomes (see Fig. 6).

In a series of investigations in which the incorporation of into plastid RNA was studied by sucrose density gradient analyses of RNA extracted from the organelles, it was found that the effect of light was inductive in the sense that the specific activity of the RNA obtained from plastids of etiolated leaves illuminated for 30 minutes and then returned to darkness for 90 minutes was approximately the same as that of plastids from leaves illuminated continuously for 2 hours. 

Rabbit reticulocytes were incubated with isotopically labeled amino acids in the presence and absence of added hemin after incubation sucrose density gradient analysis was performed to determine the patterns of ribosomes and the radioactivity of the nascent, and completed polypeptide chains (Fig. 6) (Grayzel et al., 1966). The effects... 

Another factor influencing the conductance is the density gradient. The conductance of the solution will be lower the more sucrose it contains. In order to compensate for this effect, the part of the pH-gradient having the best conductivity, that is to say either the most acidic or the most alkaline part, is placed in the most dense region. 

In a second study by the same group phagocytic vacuoles were prepared from PMNs which had been exposed to opsonized oil particles. The phagocytic vesicles could be collected because of the distinctive buoyant density in a discontinuous gradient of sucrose of the oil within phagocytic vacuole. The effects of CN", of chelators, and of EDTA on the appearance of activity in the phagocytic vacuoles... 

Figure 9-29. Effect of a previous injection of Triton WR-1339 on the equilibrium densities of particulate enzymes. Density equilibration of mitochondrial fractions from rat liver in an aqueous sucrose gradient. Upper panel control. Lower panel animal injected intravenously with 170 mg of Triton WR-1339 four days prior to sacrifice. [From Wattiaux et al.. Arch. Intern. Physiol. Biochem., 71 140 (1963) Ciba Foundation Symposium, Lysosomes, Little, Brown, Boston, 1963, p. 176.]...

The affinity-purified connexin-32 was incorporated into unilamellar liposomes as before. The connexin-32 induced a sucrose permeability in liposomes, as assayed by the density-shift technique, and gave results essentially identical to those in Figure 2. Liposomes that were sucrose-permeable did not retain the dye Lucifer Yellow (retained by the sucrose-impermeable liposomes), which is near the upper size-permeability limit for gap junction channels (19, 108, 109). The fraction of the liposomes that were permeable to sucrose decreased by a factor of 4 when the pH in the gradients was changed from 7.5 to 6.0. This effect was partially reversible. 

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