Enzyme analysis and subcellular fractionation of human peripheral blood lymphocytes with special reference to the localization of putative plasma membrane enzymes

Abstract

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.

Inhibitory effect of anti-aminopeptidase N/CD13 antibodies on fibroblast migration

“…Aminopeptidase N (APN, also known as APN/CD13; EC.3.4.11.2) is a heavily glycosylated transmembrane peptidase [6] that also serves as a receptor for coronaviruses such as the transmissible gastroenteritis virus and human coronavirus 229E [7,8]. It is ubiquitously expressed in endothelial cells, monocytes, macrophages, dendritic cells, neutrophils, fibroblasts, fibroblast-like cells such as synoviocytes, epithelial cells, and synaptic membranes and pericytes [9][10][11][12][13][14]. With dual roles as a peptide-cleaving enzyme and a cell surface receptor, APN/CD13 performs a myriad of functions in tumor cell invasion, proliferation/ apoptosis, differentiation, antigen presentation, and viral infection [15].…”