Step Density Gradient Research Articles

Studies on smooth muscle plasma membrane: I. Isolation and characterization of plasma membrane from rat myometrium

1. 1. The plasma membrane has been isolated from rat myometrium using a single step density gradient centrifugation without any salt extraction. A new kind of continuous sucrose density gradient was prepared using an Instrumentation Specialities Co. (ISCO) density gradient former. Other subcellular fractions were also obtained by this technique. 2. 2. Electron microscopic studies revealed the homogeneity of the plasma membrane fraction. Also the cytoplasmic membranes and mitochondria were relatively free of detectable contamination by other particles. The plasma membrane fraction was mainly vesicular in nature and a unit membrane structure was evident in some sections. 3. 3. The cholesterol:phospholipid ratio of plasma membrane was 0.82. A small amount of RNA-P was found to be associated with the plasma membrane. DNA-P was mainly associated with the nuclear fraction. 4. 4. The following enzymes were found to be concentrated in the plasma membrane: 5′-nucleotidase (EC 3.1.3.5), l-leucyl-β-naphthylamidase (EC 3.4.1.1), p-nitrophenylphosphatase [acid phosphatase (EC 3.1.3.1) and alkaline phosphatase (EC 3.1.3.2)]; phosphodiesterase (EC 3.1.4.1) and ATPase (EC 3.6.1.4). Cytochrome c oxidase (EC 1.9.3.1) and NADH-cytochrome c reductase (EC 1.6.2.1) were concentrated in a fraction which contained mitochondria and cytoplasmic membranes (not separated in this case). 5. 5. 14–27% of the total protein and 59% of the total 5′-nucleotidase activity were found to be present in the plasma membrane fraction. 6. 6. We conclude that high purity and good yield of the plasma membrane from smooth muscle and recovery of other fractions at the same time was achieved by the method presented in this article. This material should be suitable for a variety of pharmacological studies.

Studies on smooth muscle plasma membrane: I. Isolation and characterization of plasma membrane from rat myometrium

1. 1. The plasma membrane has been isolated from rat myometrium using a single step density gradient centrifugation without any salt extraction. A new kind of continuous sucrose density gradient was prepared using an Instrumentation Specialities Co. (ISCO) density gradient former. Other subcellular fractions were also obtained by this technique. 2. 2. Electron microscopic studies revealed the homogeneity of the plasma membrane fraction. Also the cytoplasmic membranes and mitochondria were relatively free of detectable contamination by other particles. The plasma membrane fraction was mainly vesicular in nature and a unit membrane structure was evident in some sections. 3. 3. The cholesterol: phospholipid ratio of plasma membrane was 0.82. A small amount of RNA-P was found to be associated with the plasma membrane. DNA-P was mainly associated with the nuclear fraction. 4. 4. The following enzymes were found to be concentrated in the plasma membrane: 5′-nucleotidase (EC 3.1.3.5), l-leucyl-ß-naphthylamidase (EC 3.4.1.1.), p-nitrophenylphosphatase, [acid phosphatase (EC 3.1.3.1) and alkaline phosphatase (EC 3.1.3.2)]; phosphodiesterase (EC 3.1.4.1) and ATPase (EC 3.6.1.4). Cytochrome c oxidase (EC 1.9.3.1) and NADH-cytochrome c reductase (EC 1.6.2.1) were concentrated in a fraction which contained mitochondria and cytoplasmic membranes (not separated in this case). 5. 5. 14–27% of the total protein and 59% of the total 5′-nucleotidase activity were found to be present in the plasma membrane fraction. 6. 6. We conclude that high purity and good yield of the plasma membrane from smooth muscle and recovery of other fractions at the same time was achieved by the method presented in this article. This material should be suitable for a variety of pharmacological studies.

The lipid components of the smooth-surfaced membrane-bounded vesicles of the columnar cells of the rat intestinal epithelium during fat absorption

Homogenates of rat intestinal epithelium fragments and cells have been fractionated by step density gradient centrifugation. The isolated fractions have been defined by electron microscopy and the lipid content analyzed when labeled fat had been fed to the animals one-half and one hour prior to sacrifice. The apical vesicles rich in fat contained the majority of labeled fed oleic acid, mainly in the form of fatty acids and triglyceride. Cholesterol fed together with the triglyceride fat was mainly localized to the smooth-surfaced membranes of the vesicles. The results agree with the concept that the apical vesicles of the columnar cells are the site of triglyceride resynthesis.