The Association of Cholesterol, 5'-nucleotidase, and Alkaline Phosphodiesterase I with a Distinct Group of Microsomal I Particles

When a microsome fraction is prepared from rat liver by the method of de Duve et al . (11), it contains about 20% of the proteins of the homogenate, 75% of characteristic microsomal hydrolases (such as glucose 6-phosphatase, nucleoside diphosphatase, esterase), almost the same proportion of typical microsomal oxidoreductases (NADPH and NADH: cytochrome c reductases, cytochrome b 5 , aminopyrine demethylase, cytochrome P 450), and 40-50% of the homogenate content in cholesterol, 5'-nucleotidase, and alkaline phosphodiesterase I (acting on p-nitrophenyl 5'-thymidylate) . The latter three components exhibit a "nucleo-microsomal distribution" in the sense that the amount recovered in the nuclear fraction is much higher than that expected for particles of microsome size or observed for typical microsomal constituents . Since the high activity of 5'-nucleotidase and the abundance of cholesterol are characteristic properties of purified plasma membrane preparations isolated from low speed sediments (for a review see ref. 5), the obvious interpretation for the presence of these components in the nuclear fraction is that they belong to plasma membrane fragments of large size that sediment together with the nuclei and large cell debris . In line with this interpretation, which is widely accepted, it is tempting to assume that small fragments of plasma membrane sedimenting together with the fragments of the endoplasmic reticulum are responsible for the microsomal localization of most of the remaining cholesterol, 5'-nucleotidase, and alkaline phosphodiesterase I . Another possible interpretation is that the microsomal plasma membrane constituents belong to membranous entities that do not originate directly from the plasma membrane, but are related to it either as biosynthetic precursors or as derivatives (pinocytic vesicles, for instance). Finally, it is also possible that they are entirely unrelated to the plasma membrane and are associated with the main components of the microsomal fraction, i .e ., with true elements of the endoplasmic reticulum. Owing to lack of definitive experimental data, most workers have kept a non-ommittal attitude on this subject, but there is a tendency to believe that at least the cholesterol of the microsomal fraction belongs largely to the endoplasmic reticulum proper (10) . The results described in this note argue against this view and add further support to our earlier contention that the plasma membrane constituents found in the microsomal fraction, including most, if not all, of its cholesterol content, are associated with a trace component different from those that bear the typical microsomal hydrolases and oxidoreductases (1, 2) . We have previously reported how various enzymes are distributed after equilibration of the microsome fraction in a number of different density gradients. (Some examples of the distribution patterns observed in a sucrose-H20 gradient are given in Fig . I) . These results may be summarized as follows : All components equilibrate over a wide range of density and their distribution patterns overlap to a large extent. There are, however, significant differences which allow the assayed components to be classified into four distinct groups of increasing median density : (a) Monoamine oxidase, 5'-nucleotidase and cholesterol ; (b) microsomal oxidoreductases ; (c) microsomal hydrolases; (d) RNA and some enzymes presumably adsorbed on ribosomes . The distribution of the proteins is almost superposable to that of the hydrolases, while that of the phospholipids is very close to that of the oxidoreductases . When the composition of the density gradient is changed, the last three groups retain their identity, but 5'-nucleotidase and monoamine oxidase do not behave in the same manner and are thus presumably linked to different particles . More recent (unpublished) observations have shown that 5'-nucleotidase and monoamine oxidase can also be distinguished from each other and from the bulk of the microsome fraction by their rate of sedimentation in shallow stabilizing sucrose density gradients : 5'-nucleotidase sediments faster, and monoamine oxidase sediments more slowly, than do most other microsomal enzymes. We have