Abstract
Plasma membranes from 6 spontaneously metastasizing and 4 non-metastasizing rat mammary carcinomata were isolated by discontinuous sucrose density gradient centrifugation of microsomal pellets. The starting microsomal fraction contained 40-50% plasma membranes as determined by the levels of 5'-nucleotidase activity, with a negligible amount of nuclear (1%), mitochondrial (5%) and lysomal (7%) contamination. Five distinct fractions (F1-F5) were banded at densities 1 X 09, 1 X 13, 1 X 15, 1 X 17 and 1 X 21 at 25 degrees C, in addition to a pellet (F6) obtained by centrifuging at 76,000 g for 17 h. The fractions F1 through F5, all contained various concentrations of membranous structures, while the pellet (F6) contained only amorphous materials as evidenced by electron microscopy. The F3 fraction at the gradient 1 X 15 had the highest specific as well as total activity of the plasma membrane marker enzyme, with aggregates of the least contaminated plasma membranes in vesicular forms. This fraction also had the lowest specific activity for glucose-6-phosphatase (smooth ER marker) and for beta-D-glucuronidase (lysomal marker), and therefore was considered to be the "cleanest" plasma membrane fraction. When the activity of 4 additional plasma membrane marker enzymes, i.e., alkaline phosphatase, phosphodiesterase I, nucleotide pyrophosphatase and alkaline ribonuclease was determined in the same F3 fraction, their levels were significantly lower in every metastasizing tumour than in the non-metastasizing ones, with the enzyme activity decreasing in direct proportion to the metastasizing capacity. On the other hand, the marker enzymes were high in all non-metastasizing tumours, with the activity seemingly increasing with the immunogenicity of tumour cells. There was no significant difference between the 2 groups of mammary tumours in the levels of sialic acid, hexosamine, phospholipid or cholesterol in the plasma membranes. Thus, the level of plasma membrane marker enzymes is considered an accurate indicator for metastasizing capacity in the rat mammary tumour system.
Highlights
Summary.-Plasma membranes from 6 spontaneously metastasizing and 4 nonmetastasizing rat mammary carcinomata were isolated by discontinuous sucrose density gradient centrifugation of microsomal pellets
Spontaneously meta- phate and p-nitrophenyl-thymidine-5'-phosstasizing rat mammary carcinomata
Five distinct bands designated as Fl, F2, F3, F4 and F5, in addition to the containing 0-25 mol/l sucrose and 0-1 mmol/l pellet (F6), were obtained and carefully CaCl2, with its pH adjusted to 8-0 with collected with pasteur pipettes and their tris-HCl, was added for each g of pressed sucrose concentration adjusted to 0-25 mol/l tumour
Summary
Phenyl tetrazolium chloride (INT), p-nitrophenyl fl-D-glueuronide, p-nitrophenyl phos-. The supernatant was spun at 78,000 g for 90 min., and the microsomal of lymph node and other secondary organ pellet for the sucrose gradient centrifugation involvements. The pellet was suspended in to the lung whereas others involved primarily a solution containing 100 mmol/l tris-HCl lymph nodes. Five distinct bands designated as Fl, F2, F3, F4 and F5, in addition to the containing 0-25 mol/l sucrose and 0-1 mmol/l pellet (F6), were obtained and carefully CaCl2, with its pH adjusted to 8-0 with collected with pasteur pipettes and their tris-HCl, was added for each g of pressed sucrose concentration adjusted to 0-25 mol/l tumour. The homogenate was centrifuged at 200 g for 1-10 min depending on the tumour bring it down in a pellet form and its protein concentration was adjusted to 1-2 mg/ml strain. After the second homo- Enzyme assays-.Succinic dehydrogenase genization, more than 90%o of the cells (EC 1.3.99.1) was chosen for the mitowere disrupted, as verified by the standard chondrial marker and was assayed according trypan blue dye exclusion test
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