Synthesis and in vitro and in vivo activity of a hybrid composed of ricin B chain-barley ribosome-inactivating protein

Michael Ovadia, Ronald G. Wiley, Cynthia Hager, Chris LaRocca, Thomas N. Oeltmann

Research output: Contribution to journalArticlepeer-review

Abstract

In our continued studies on hybrid proteins for use as cytotoxins and possible suicide transport agents, we have begun to investigate the use of ribosome-inactivating proteins (RIP) isolated from grain. The RIP from barley is a single polypeptide chain (Mr 32,000) and is nontoxic to intact cells. The barley RIP has been purified to homogeneity by modifications of the methods of Roberts and Selitrennikoff and crosslinked to the binding subunit B of the seed toxin ricin (RTB). The resulting hybrid was purified by a combination of gel filtration and affinity chromatography on acid-washed Sepharose 4B. This model suicide transport agent was assayed in vitro against K-562 cells and was found to be cytotoxic in a dose-dependent manner (ID50 = 0.15 μg/ml). Lactose inhibited the toxicity of the hybrid, indicating that cytotoxicity was dependent on the cell binding property of the ricin B moiety. In addition, free RIP and free ricin B, either alone or in combination, were nontoxic over this concentration range. The in vivo effects of the RTB-RIP hybrid were assessed by pressure microinjection into the vagus nerves of rats. Injection of 0.18 to 6.5 μg of conjugate resulted in death of vagal sensory but not motor neurons after 3-17 days. The cytotoxic changes in vagal sensory neurons were identical to those previously observed with a variety of RIP toxins such as ricin.

Original languageEnglish
Pages (from-to)168-175
Number of pages8
JournalArchives of Biochemistry and Biophysics
Volume264
Issue number1
DOIs
StatePublished - Jul 1988

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