Subfractionation of Smooth Microsomes From Rat Liver

Abstract

Total smooth microsomes from rat liver isolated on a Cs+-containing sucrose gradient were concentrated and subsequently fractionated by zone centrifugation on a stabilizing sucrose gradient. The prerequisite for fractionation is to prepare total smooth microsomes in a nonaggregated condition, as well as to utilize a procedure which counteracts enzyme inactivation . The median equilibrium density of the various smooth microsomal vesicles ranges from 1 .10 to 1 .18 . The phospholipid/protein ratio is identical in all subfractions, but cholesterol, on a PLP basis, is enriched in the subfractions with the highest sedimentation velocity . The enzyme distribution pattern reveals a pronounced heterogeneity. A number of NADH-and NADPH-oxidizing enzymes are concentrated in the upper part of the gradient and exhibit a certain degree of separation from G6Pase . Mg++-ATPase and AMPase are enriched in the lower part of the gradient . No specific enrichment of newly synthesized NADPH-cytochrome c reductase activity occurs in any of the subfractions after phenobarbital treatment . These data demonstrate that smooth microsomes, by adequate fractionation procedure, can be separated into subfractious of heterogeneous composition .

Analytical Study of Microsomes and Isolated Subcellular Membranes From Rat Liver

“…These results are doubly meaningful, in that they show that the bulk of the microsomal cholesterol is physically independent from the structures bearing group b and ¢ enzymes, and also that the latter structures do not contain appreciable amounts of cholesterol per unit weight, at least in a form accessible to digitonin binding. Our conclusion, which is at variance with the views of other authors (13,23), will be further supported in the next paper of this series (5). The intracellular localization of cholesterol is examined below.…”

Salt-stimulated Adenosine Triphosphatase from Smooth Microsomes of Turnip

“…The origin of the two smooth membrane fractions in which the ATPase is concentrated (Table I) is not clear. The lighter of the two fractions may consist of plasma-membranes (9) but whether this is plasmalemma, tonoplast, or both and which way they are folded is not clear. However, it is tempting to suggest that the ATPase is located on the outside of the tonoplast membrane, i.e., the cytoplasmic side of the tonoplast, because it (a) showed specificity for anions (21) and was maximally stimulated by organic anions likely to be accumulated in the vacuole; (b) was saturated at high salt concentration (50 mM) in accordance with the (system 2) ion pump proposed on the tonoplast (14).…”

“…The supernatant was then centrifuged at 78,000g (average) for 90 min to sediment the microsomes. Fractionation of the microsomes followed the method of Glauman and Dallner (9). The microsomal pellet was resuspended in 90 ml of 0.25 M sucrose containing 15 mM CsCl, and aliquots of 7 ml were layered over 4.5 ml of 1.3 M sucrose containing 15 mm CsCl.…”

“…An animal microsomal ATPase which requires Mg2+ and is synergistically stimulated by Na+ and K+ has been well characterized (25). However, this activity has now been shown to be concentrated in the plasma membranes which sediment with the endoplasmic reticulum membranes and can be largely separated from them using density gradients (2,9). Plant workers have concentrated on identifying a corresponding microsomal Na+-K+-ATPase activity.…”

Integrated stereological and biochemical studies on hepatocytic membranes. I.V. Heterogeneous distribution of marker enzymes on endoplasmic reticulum membranes in fractions.

“…In recent years, the question as to whether a given amount of marker enzyme activity is associated with a similar amount of ER membrane has become one of increasing importance (15,25,13,44,32,8) . Several papers have suggested that (a) marker enzymes are heterogeneously distributed on the ER and that (b) "ER marker enzymes" may also be attached to membranes other than those of the ER (15,46,2,18,22,29,24,14,41,39,4,38,30) . The existence of such marker enzyme heterogeneities would be expected to have important consequences when one is interpreting bio-J.…”