Abstract
The 70–90,000 molecular weight (MW) alpha (α) component of the human lymphotoxin (LT) system has been purified to electrophoretic homogeneity. The α LT containing supernatants were obtained from a phorbol myristate (PMA) stimulated cloned continuous human B lymphoblastoid cell line IR 3.4. Supernatants were subjected to a biochemical separation scheme that consisted of chromatography on control pore glass beads, DEAE ion-exchange chromatography, lentil-lectin affinity chromatography, and electrophoresis on 7% native preparative polyacrylamide gels. The specific activity of the α LT in the final fractions was from 10 7 to 5 × 10 7 units of LT activity/mg protein. Approximately 3 to 5% of the initial α LT was recovered in the final fractions and a purification factor of 25,000 to 30,000 fold was required to achieve homogeneity. The α LT preparation from preparative PAGE exhibited concident migration of bioactivity and radioactivity on 5 and 7% native PAGE tube gels. Only a single protein peak was observed when the radiolabeled α LT was subjected to a two-dimensional SDS-reducing slab gel.
Highlights
Supernatants from phorbol myristate (PMA) activated IR 3.4 cells were first subjected to chromatography on CPG-240 columns
The results of this study indicate that the lentil-lectin samole contained multiole components but a good separation of lytic material (Rf 0.32) and noniytic material (Rf b.84) was achieved so the final separation of the lentil-lectin material could be achieved on a high capacity preparative native polyacrylamide gels (PAGE) column
Additional studies revealed the material obtained from the preparative PAGE column elutes from molecular sieving on Ultrogel AcA 44 as a 70-90,000 molecular weight (MW) a LT class
Summary
Phorbol myristate acetate (PMA) mitoymcin-C, ethylene glycol, and a-methyl-D-mannoside (C&M) Washed cells were resuspended in RPMI-1640 supplemented with LAH at 0.1% as a serum substitute and cultured in the presence of 20 ng/ml PMA for three days (Yamamoto et al, 1983). One hundred mls of the CPG-240 desorbed sample was loaded on a 2.5 X 4.5 cm DEAE column equilibrated in Tris buffer. The LT sample was eluted with a linear gradient of NaCl from 0.05 to 0.3 M Tris buffer and protein, conductivity, and lytic activity were measured as previously described (Granger et al, 1978). Twenty drop fractions were collected as material ran off the gel at a flow rate of 120 drops/hr and was irrmediately assayed for radioactivity and lytic activity. The fractions containing lytic activity were pooled, concentrated, reiodinated, and subjected to 5 and 7% native PAGE tube gels by the technique of Davis (1964). Gels were dried and autoradiographed by XRP-5 X-OMAT film (Eastman Kodak, Rochester, NY) with Cronex Lighting Plus intensifying screens (DuPont, Claremont, CA)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
