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Isolation of plasma membrane from human blood monocytes. Subcellular fractionation and marker distribution
Abstract:
The isolation of plasma membrane from human peripheral blood monocytes is described. Monocytes were isolated by centrifugal elutriation, to eliminate an adherence step, thus minimizing functional and surface antigenic alterations to the cells. Monocytes were surface-labelled with a radiolabelled monoclonal antibody, 125I-WVH-1, and then disrupted by nitrogen cavitation. Membranes were separated according to equilibrium buoyant density by isopycnic centrifugation on a sucrose gradient. The subcellular membranes were localized using marker enzymes for the plasma membrane, 5'-nucleotidase and leucine 2-naphthylamidase (leucine aminopeptidase), and for intracellular membranes: galactosyltransferase (Golgi), arylsulfatase C (endoplasmic reticulum), monoamine oxidase (mitochondria), catalase (peroxisomes), beta-hexosaminidase and beta-glucuronidase (lysosomal vesicles) and lactate dehydrogenase (cytosol). The monoclonal antibody 125I-WVH-1 was shown to label the plasma membrane, as judged by known markers, and represents a highly specific trace label, applicable to the use of plasma membrane as an immunogen for monoclonal antibody production. The NAD-splitting enzyme, NAD+ nucleosidase, was detected and its presence on the plasma membrane was demonstrated. The subcellular localization of non-specific esterase in human mononuclear phagocytes is controversial. No evidence was found for alpha-naphthyl acetate esterase activity on the plasma membrane or in lysosomal vesicles. However, a membrane-bound esterase in fractions with properties similar to the smooth endoplasmic reticulum was detected.
Insights
This study details a method for isolating human peripheral blood monocyte plasma membranes using centrifugal elutriation and nitrogen cavitation. The technique ensures cell integrity and identifies specific membrane-bound enzymes, aiding in understanding monocyte function.
Area of Science:
- Cell Biology
- Immunology
- Biochemistry
Background:
- Plasma membrane isolation is crucial for studying cell surface properties.
- Previous methods often alter monocyte function and surface antigens.
- Accurate isolation techniques are needed for reliable immunogen development.
Purpose of the Study:
- To describe a novel method for isolating plasma membrane from human peripheral blood monocytes.
- To validate the purity and integrity of isolated membranes using specific enzyme markers.
- To investigate the localization of NAD+ nucleosidase and non-specific esterase in monocytes.
Main Methods:
- Monocytes isolated via centrifugal elutriation, avoiding adherence steps.
- Surface labeling with radiolabeled monoclonal antibody (125I-WVH-1).
- Cell disruption by nitrogen cavitation followed by isopycnic centrifugation on sucrose gradients.
Main Results:
- Isolated plasma membranes were identified using 5'-nucleotidase and leucine 2-naphthylamidase.
- 125I-WVH-1 confirmed as a specific plasma membrane label.
- NAD+ nucleosidase was detected on the plasma membrane.
- Non-specific esterase activity was not found on the plasma membrane or in lysosomes but was detected in smooth endoplasmic reticulum-like fractions.
Conclusions:
- Centrifugal elutriation and nitrogen cavitation provide a method for high-purity plasma membrane isolation from human monocytes.
- The radiolabeled antibody is a specific marker for plasma membrane studies and immunogen development.
- The localization of NAD+ nucleosidase and non-specific esterase provides insights into monocyte enzymatic activity and potential functional roles.