Enzymatic composition of mitogen-induced lymphoblasts and untreated lymphocytes fractionated by rate-zonal centrifugation

• Published in: Biochimica et biophysica acta Vol. 676; no. 3; pp. 321 - 328
• Main Authors: Harrison, Earl H., Zbuzek, Vratislav, Labadie, Jon H., Bowers, William E.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 04.09.1981
• Subjects: Animals; Cell Separation; Centrifugation, Zonal; Enzyme composition; Galactose Oxidase - metabolism; Lymph Nodes - cytology; Lymphoblast; Lymphocyte; Lymphocyte Activation; Lymphocytes - enzymology; Mitogen; Neuraminidase - metabolism; Rate-zonal centrifugation; Rats; Lymphoblast; Mitogen; Rate-zonal centrifugation; Lymphocyte; Enzyme composition
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/0304-4165(81)90166-5

Treatment of rat lymph node cells with neuraminidase plus galactose oxidase leads to blast transformation of T lymphocytes. Rate-zonal centrifugation was used to separate both untreated lymph node cells and neuraminidase plus galactose oxidase-treated lymph node cells cultured for 2 days. The majority of untreated lymph node cells were small lymphocytes with a mean size of 130 μm 3 as determined by electronic sizing. Only 3% of the cells sedimented rapidly enough to reach fractions distal from the axis of rotation. In contrast, 20% of the cells in cultures of the neuraminidase plus galactose oxidase-treated lymph node cells sedimented rapidly and these were almost exclusively large, transformed lymphoblasts (mean size 300 μm 3). The most slowly sedimenting cells in these cultures were small, untransformed lymphocytes. Rate-zonal centrifugation can by used to separate and recover in high yield mitogen-induced lymphoblasts from lymphocyte cultures and thus allows the direct comparison of their biochemical properties with those of the small precursor cells. After neuraminidase plus galactose oxidase-induced blastogenesis, the amount per cell of DNA, cytochrome oxidase, β-galactosidase. cathepsin D, and arylsulfatase in whole cultures were similar to those of untreated lymph node cells. In contrast, the levels of protein, RNA, 5′-nucleotidase, γ-glutamyltranspeptidase, alkaline phosphatase, N-acetyl- β-glucosaminidase and N-acetyl- β- galactosaminidase increased 2–3-fold. Moreover, separation of these cells revealed that for each of these latter constituents there was a progressive increase in the amount per cells as the mean size increased. Thus, three plasma membrane-associated enzymes and two lysosomal acid hydrolases increased markedly in mitogen-induced blast cells as compared to the small precursor cells, but these increases were quntitatively similar to the increases in mean cellular volume and protein content.