Mouse fibroblast interferon

In this communication we extend our earlier observations, which primarily dealt with the antiviral action of mouse fibroblast interferon, to its antigrowth activity and to antiviral and antigrowth activities of mouse T-cell interferon. We will show that inhibition by common gangiiosides Is restricted to both activities of fibroblast interferon alone. T-cell interferon, although its biological activities are analogous to those of fibroblast Interferon, neither binds to nor Is inhibited by these glycol ipids. Furthermore we demonstrate that mouse leukemia L—1210 cells that were selected for resistance to fibroblast interferon (6), respond equally well to T-cell Interferon as the parent cells which are responsive to both Interferons. 

Column Chromatography. Sepharose beads containing covalently linked gangliosides (0.2 ml packed volume) were placed into a pasteur pipette containing a small amount of glass wool. Columns were washed with HEM containing 50 ug/ml bovine serum albumin (3 ml). Interferon solutions in MEM-albumin (1 ml) were placed on the columns, which were eluted with MEM-albumin at a flow rate of no more than one drop per minute. Fractions of 1 ml were collected and interferon titers determined in each fraction after serial two-fold dilution. Columns onto which mouse fibroblast interferon had been loaded, were eluted with MEM-albumin first, then with 0.07 M N-acetylneuraminyl lactose at pH 2. 

Since interferons appear to be species-specific, we investigated whether the ganglioside fraction from mouse brain was more potent in inhibiting antiviral activity of mouse fibroblast interferon than that obtained from heterologous brain extracts. As seen in Figure 1 bovine brain gangliosides were almost as potent... 

Figure 1. Effects of mouse ( ) and bovine (Q) brain gangliosides on antiviral activity of mouse fibroblast interferon...

Since the ceramide portions of more and less inhibitory glycoliplds are very similar, differential Inhibition of antiviral activity of mouse fibroblast interferon must be related to their carbohydrate side chains. We therefore assayed antiviral activity in the presence of various saccharides contained in gangiiosides. As seen in Figure 3, both N-acetylneuraminyl lactose and N-acetyl-neuraminic acid inhibited antiviral activity, requiring approximately equal concentrations to obtain complete inhibition (60 mM). However, in comparison to GM3, 600-fold higher concentrations of these sugars had to be employed to yield complete inhibition of antiviral activity. N-glycolyineuraminic acid and the 6-methyi-... 

Figure 4. Binding of mouse fibroblast interferon to Sepharose-ganglioside col-ums and elution with N-acetylneuraminyl lactose...

Figure 5. Effect of bovine brain ganglio-sides on growth inhibition of L-1210S cells by mouse fibroblast interferon...

Cells were grown in RPMI medium plus 5 % fetal bovine serum in 1 mL total volume as described in Materials and Methods. At 24-hr intervals the cells were counted in a Coulter counter. Control cells 0, cells from cultures containing 1000 lU/mL mouse fibroblast interferon , cells from cultures containing bovine brain gangliosides at a concentration corresponding to 35 p.M sialic acid , cells from cultures containing both interferon (1000 IU/mL) and gangliosides (35 p.M sialic acid). 

Figure 6. Effects of mouse fibroblast interferon L-1210S and L-1210R cells in the presence of increasing ganglioside concentrations...

Under conditions where over 90% of mouse fibroblast interferon was retained (as shown in Figure k) T-cell interferon quantitatively eluted in the breakthrough of the column (Figure 8). T-cell interferon, after passage through the affinity column, was still insensitive to ganglioside inhibition, excluding the possi-... 

Experiment was carried out as described in Figure 1, using 20 lU/mL of both interferons. , Mouse fibroblast interferon , T-cell interferon , T-cell interferon after passage through a Sepharose—ganglioside column (see Figure 8). T EMC titer in the absence of interferon (19). 

Hood, " which incorporates substantial modifications in the design of the commercial Beckman instrument coupled with the computer assisted identification of phenylthiohydantoin derivatives by reversed-phase HPLC. These methods have enabled HunkapUler and Hood to determine 24 residues of the mouse fibroblast interferon amino-acid sequence starting with only 0-6 fig of material. This remarkable achievement is now being succeeded by developments in the same laboratory. A machine of even greater sensitivity, based on coupling and cleavage in the gas phase, has been constructed. The sensitivity of the automatic amino-acid sequencer is apparently beginning to surpass most of the analytical techniques commonly used to detect proteins. 

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