Enzyme analysis and subcellular fractionation of human peripheral blood lymphocytes with special reference to the localization of putative plasma membrane enzymes.
Human lymphocytes were isolated from defibrinated blood by Ficoll-Hypaque centrifugation with erythrocyte hypotonic lysis. Homogenates of mixed lymphocytes were subjected to analytical subcellular fractionation by sucrose gradient centrifugation in a Beaufay automatic zonal rotor. The principal organelles were characterized by their marker enzymes: cytosol (lactate dehydrogenase), plasma membrane (5'-nucleotidase), endoplasmic reticulum (neutral alpha-glucosidase), mitochondria (malate dehydrogenase), lysosomes (N-acetyl-beta-glucosaminidase), peroxisomes (catalase). gamma-Glutamyl transferase was exclusively localized to the plasma membrane. Leucine amino-peptidase, especially when assayed in the presence of Co2+, was also partially localized to the plasma membrane. Experiments with diazotized sulphanilic acid, a non-permeant enzyme inhibitor, showed that these plasma membrane enzymes are present on the cell surface. No detectable alkaline phosphatase was found in the lymphocytes. Acid phosphatase and beta-glucuronidase were localized to lysosomes and there was some evidence for lysosomal heterogeneity. Leucine amino peptidase, optimal at pH 8.0, showed a partial localization to intracellular vesicles, possibly lysosomes, especially when assayed in the presence of EDTA. These studies provide a technique for determining the intracellular distribution of hitherto unassigned lymphocyte constituents and serve as a basis for investigating the cell pathology of lymphocytic disorders.
D000469:Alkaline Phosphatase / Q000097:blood; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology*; D002467:Cell Nucleus / Q000201:enzymology; D002499:Centrifugation, Density Gradient; D004247:DNA / Q000097:blood; D004072:Digitonin / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000201:enzymology; D006801:Humans; D006867:Hydrolases / Q000097:blood*; D007931:Leucyl Aminopeptidase / Q000097:blood*; D008214:Lymphocytes / Q000201:enzymology* / Q000648:ultrastructure; D008247:Lysosomes / Q000201:enzymology; D008830:Microbodies / Q000201:enzymology; D008928:Mitochondria / Q000201:enzymology; D010088:Oxidoreductases / Q000097:blood*; D013347:Subcellular Fractions / Q000201:enzymology; D005723:gamma-Glutamyltransferase / Q000097:blood*
D004247:DNA; D010088:Oxidoreductases; D005723:gamma-Glutamyltransferase; D006867:Hydrolases; D000469:Alkaline Phosphatase; D007931:Leucyl Aminopeptidase; D004072:Digitonin