Purification and Characterization of Chick Embryo Interferon

Abstract

468PURIFICATION AND CHARACTERIZAT'ION OF INTERFERON sorption of HC03-ions per se, but rather is the secretion of H+ ions which react with filtered HC03-to generate H2C03. Second, the accumulation of excess H2C03 in tubular fluid disproves the previously suggested hypothesis (3,7) that endogenous carbonic anhydrase might be located in the luminal membrane of renal tubular cells, thereby exerting a catalytic action on dehydration of excess H2C03 in tubular fluid.Finally, the calculated pH gradient (pHB-pHTE') between blood and tubular fluid was 1.07. For a pH gradient of this magnitude to result from passive distribution of H+ ions along an electrochemical gradient (8,9), it can be calculated from the Nernst equation [ET = -61.5 (pHB-pHTF)] that a transtubular potential difference ( ET) greater than 66 mV, lumen negative to blood, would be required. Previous measurement of proximal ET under conditions of NaHC03 diuresis gave an average value of only -20 mV( 10). Therefore, it is concluded that H+ ions are secreted into the lumen against an electrochemical gradient by an active transport process.Various virus-host animal or virus-cell culture systems have been shown to elaborate a "soluble" substance called interferon ( 1 ) , which interferes with the propagation of viruses in related host or host cell systems. Interferon appears to be an active principle in the virus interference phenomenon. Interferon, as first described by Isaacs and Lindenmann( l ) ? was prepared in fragments of chick chorioallantoic membrane incubated with hea t-inac tivated influenza virus. Wagner ( 2 ) demonstrated the presence of an interferon in the allantoic fluid of embryonated eggs infected with influenza virus and other workers have demonstrated interferons in other systems. Several groups of workers (2-6) have described the biological and biophysical properties of crude or semi-purified interferons. The present report describes the biological and biophysical qualities of a highly purified interferon derived from the allantoic fluid of embryonated hens' eggs infected with the WS strain of influenza virus.Materials and methods. Preparation of crude interferon. Nine-or 10-day-old embryonated eggs were inoculated by the allantoic route with lW5 ID50 of WS influenza strain RIE 5372 virus obtained from Dr. I. Tamm. The eggs were incubated at 36°C for 84-96 hours after which they were chilled a t 4OC and the allantoic fluid containing the interferon was harvested. The crude fluids were centrifuged in the cold for 20 minutes at 1500 rpm and the supernates were stored at 4°C for periods up to 14 days prior to processing for purification. In vitro assay for interferon activity. Serial 2-fold dilutions of the interferon sample were diluted in mixture 199 containing 1% calf serum and antibiotics, and one ml amounts were added in by guest on August 11, 2015 ebm.sagepub.com Downloaded from

Purification and Characterization of Mouse L Cell Interferon

“…Although considerable progress has been made toward purification of interferon, especially of chick [2,6,8], mouse [8,9] and rabbit [13] origins, even the best preparations so far obtained appear to be far from being pure. The difficulties in the purification of interferon may reside, among other things, in ( 1 ) obtaining large amounts of starting material with high titers, preferably of high specific activity, (2) difficulty in preventing inactivation during purification and characterization processes, and (3) laborious assay of activity.…”

“…(1) Zinc acetate precipitation [6]. To crude interferon samples was added 1/50 volume of 1 M zinc acetate in the cold, and the pH was adjusted to 7 by adding N NaOH.…”

“…(2) Carboxymethyl Sephadex (CMS) chromatography [6,8]. CMS, C-25 (Pharmacia), equilibrated in 0.1 M phosphate buffer at pH 5.9 and boiled to sterilize, was packed in a column and the concentrated interferon preparation was charged after adjusting its pH to about 5.9 with N HC1.…”

Studies on the Mechanism of Interferon Action

“…Such work has failed to confirm results obtained in earlier experiments in which crude interferon was used. Thus, reports on an inhibition of cellular RNA synthesis were not confirmed when highly purified interferon was used (1), and effects on glucose metabolism (2) were not observed with purified material (3). Similarly, purified interferon had no effect on cell division (4), an action which had been reported earlier.…”

Interferon induction by influenza type C

“…The crude interferon was partially purified by the method of Lampson, Tytell, Nemes & Hilleman (1963), omitting the polumn chromatography and all subsequent steps. This partially purified material contained 1280 units of interferon/ml.…”