Isolation of rat liver plasma membrane fragments in isotonic sucrose.

Rat liver microsomal fractions have been equilibrated in various types of linear density gradients. 15 fractions were collected and assayed for 27 constituents. As a result of this analysis microsomal constituents have been classified, in the order of increasing median density, into four groups labeled a, b, c, and d. Group a includes: monoamine oxidase, galactosyltransferase, 5'-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase, and cholesterol; group b: NADH cytochrome c reductase, NADPH cytochrome c reductase, aminopyrine demethylase, cytochrome b(5), and cytochrome P 450; group c: glucose 6-phosphatase, nucleoside diphosphatase, esterase, beta-glucuronidase, and glucuronyltransferase; group d: RNA, membrane-bound ribosomes, and some enzymes probably adsorbed on ribosomes: fumarase, aldolase, and glutamine synthetase. Analysis of the microsomal fraction by differential centrifugation in density gradient has further dissociated group a into constituents which sediment more slowly (monoamine oxidase and galactosyltransferase) than those of groups b and c, and 5'-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase, and the bulk of cholesterol which sediment more rapidly (group a2). The microsomal monoamine oxidase is attributed, at least partially, to detached fragments of external mitochondrial membrane. Galactosyltransferase belongs to the Golgi complex. Group a2 constituents are related to plasma membranes. Constituents of groups b and c and RNA belong to microsomal vesicles derived from the endoplasmic reticulum. These latter exhibit a noticeable biochemical heterogeneity and represent at the most 80% of microsomal protein, the rest being accounted for by particles bearing the constituents of groups a and some contaminating mitochondria, lysosomes, and peroxisomes. Attention is called to the operational meaning of microsomal subfractions and to their cytological complexity.

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ANALYTICAL STUDY OF MICROSOMES AND ISOLATED SUBCELLULAR MEMBRANES FROM RAT LIVER III. Subfractionation of the Microsomal Fraction by Isopycnic and Differential Centrifugation in Density Gradients

Purification and properties of the light-activated cyclic nucleotide phosphodiesterase of rod outer segments.

An effect of insulin on cyclic adenosine 3':5'-monophosphate phosphodiesterase activity in fat cells.

Separate Phosphodiesterases for the Hydrolysis of Cyclic Adenosine 3',5'-Monophosphate and Cyclic Guanosine 3',5'-Monophosphate in Rat Liver

A procedure for the isolation and separation of three different subfractions of rat liver plasma membranes is described. The subfractions have different enzyme activities and sucrose buoyant densities of 1.18, 1.16 and 1.13 g/ml respectively. It was found that the low-density subfraction had the following properties:



1
It appeared as smooth membrane vesicles approximately 0.1–0.2 μm in diameter when visualized under positive and negative staining with the electron microscope.

2
Its contamination by microsomes on a protein basis was less than 3% as measured by glucose-6-phosphatase activity, and no succinic dehydrogenase activity or DNA were detected.

3
The low-density subfraction contained high relative specific activities of 5′-nucleotidase, (Na+, K+)-Mg2+-stimulated ATPase, phosphodiesterase, and a glucagon and adrenalin-sensitive adenylate cyclase. This fraction yielded over 30% of the total 5′-nucleotidase, adenylate cyclase and phosphodiesterase activities of the homogenate.

4
A specific 3′: 5′-AMP phosphodiesterase activity of this subfraction was stimulated by insulin. The half-maximal concentration required to produce this stimulation was 2.3 nM. The stimulation was already observed at the shortest time interval tested (30 sec).





The localization in situ of the low-density membrane subfraction on the surface of the hepatic cell is discussed, and it is suggested that it is derived mainly from the microvillar surface of hepatic parenchymal cells facing the perisinusoidal space.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1972.tb19703.x

Paul D. R. House

Papers

Isolation of a plasma-membrane subfraction from rat liver containing an insulin-sensitive cyclic-AMP phosphodiesterase.