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A comparative study of ribonuclease hydrolysis of rat brain-cortex and liver membrane-bound ribosomes.

ABSTRACT: Because it has been proposed that the ribosome-membrane interaction is different in endoplasmic reticulum derived from a non-secretory and secretory cell we undertook a study to determine whether attachment of the ribosome to the membrane involved ribosomal RNA and if the rRNA in ribosomes derived from the two classes of cell possessed an altered susceptibility to RNAase (ribonuclease) hydrolysis. We found that brain ribosomes appeared to possess more regions accessible to nuclease attack, independent of whether a sequence-dependent RNAase (T(1)) or a sterically hindered RNAase bound to Enzite polymer was employed. These results were independent of whether the ribosomes were membrane-bound or detached from the endoplasmic reticulum membranes, but at high RNAase concentration these differences became negligible. No conclusions, however, could be drawn as to whether ribosomal RNA is involved in the attachment of the ribosome to the endoplasmic reticulum membrane, because of the presence of endogeneous membrane-associated RNAases. Analysis of the rRNA fragments by polyacrylamide-gel electrophoresis suggests that the sites available for attack by low concentrations of nuclease in bound-ribosomes derived from brain cortex are different from those of liver.

SUBMITTER: Simpkins H

PROVIDER: S-EPMC1165823 | biostudies-other | 1973 Oct

REPOSITORIES: biostudies-other

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Project description:1. The intrinsic Na(+), K(+), Mg(2+) and Ca(2+) contents of a preparation of membrane fragments from ox brain were determined by emission flame photometry. 2. Centrifugal washing of the preparation with imidazole-buffered EDTA solutions decreased the bound Na(+) from 90+/-20 to 24+/-12, the bound K(+) from 27+/-3 to 7+/-2, the bound Mg(2+) from 20+/-2 to 3+/-1 and the bound calcium from 8+/-1 to <1nmol/mg of protein. 3. The activities of the Na(+)+K(+)+Mg(2+)-stimulated adenosine triphosphatase and the Na(+)-dependent reaction forming bound phosphate were compared in the unwashed and washed preparations at an ATP concentration of 2.5mum (ATP/protein ratio 12.5pmol/mug). 4. The Na(+)-dependent hydrolysis of ATP as well as the plateau concentration of bound phosphate and the rate of dephosphorylation were decreased in the washed preparation. The time-course of formation and decline of bound phosphate was fully restored by the addition of 2.5mum-magnesium chloride and 2mum-potassium chloride. Addition of 2.5mum-magnesium chloride alone fully restored the plateau concentration of bound phosphate, but the rate of dephosphorylation was only slightly increased. Na(+)-dependent ATP hydrolysis was partly restored with 2.5mum-magnesium chloride; addition of K(+) in the range 2-10mum-potassium chloride then further restored hydrolysis but not to the control rate. 5. Pretreatment of the washed preparation at 0 degrees C with 0.5nmol of K(+)/mg of protein so that the final added K(+) in the reaction mixture was 0.1mum restored the Na(+)-dependent hydrolysis of ATP and the time-course of the reaction forming bound phosphate. 6. The binding of [(42)K]potassium chloride by the washed membrane preparation was examined. Binding in a solution containing 10nmol of K(+)/mg of protein was linear over a period of 20min and was inhibited by Na(+). Half-maximal inhibition of (42)K(+)-binding required a 100-fold excess of sodium chloride. 7. It was concluded (a) that a significant fraction of the apparent Na(+)-dependent hydrolysis of ATP observed in the unwashed preparation is due to activation by bound K(+) and Mg(2+) of the Na(+)+K(+)+Mg(2+)-stimulated adenosine triphosphatase system and (b) that the enzyme system is able to bind K(+) from a solution of 0.5mum-potassium chloride.

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A comparative study of ribonuclease hydrolysis of rat brain-cortex and liver membrane-bound ribosomes.

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