Effect of β-3-Thienylalanine on Antibody Synthesis III. Inhibition of RNA and Protein Synthesis in Immunized Rat Spleen Cells ∗

Previous work from our laboratory (1-3) has indicated that β-3-thienylalanine (β-3-TA), an analog of the essential amino acid phenylalanine, when administered to rats by stomach tube induces marked depression of the production of specific antibody to sheep erythrocytes. This depression is seen when the analog is administered during the induction period and is not noted if β-3-TA is given beginning on the third day after antigen injection. The results of our previous experiments were interpreted as meaning that β-3-TA, like other compounds (4), has an effect on some events occurring during the induction period and will not act on antibody synthesis per se when given during the production phase. The purpose of the work reported here was to investigate further the mechanism by which B-3-TA depresses antibody production and to identify the step or steps which are affected by this analog.

This investigation has pointed out that one of the inhibitory effects is at the level of the synthesis of ribonucleic acid.

Materials and Methods. Sprague-Dawley, young adult, male, albino rats were used in all experiments. They were fed the diet described by Hruban and Wissler (5), prepared without addition of water, and with phenylalanin omitted when B-3-TA was administered. β-3-TA (Nutritional Biochemicals) was suspended in normal saline and heated to the boiling point until dissolved. The solubility of this compound in saline is poor, but by using this method, up to 70 mg/ml can be dissolved, being careful to restore the solution to the original volume after boiling. The preparation was always made up fresh as β-3-TA is rapidly decomposed in solution. Rats were immunized with 1 ml of 2% sheep erythrocytes intravenously on Day 0 and serial bleedings were done from the tail vein.

Sera from one experiment were titriated together to avoid variations in technique. They were stored at —20° until titration was performed. Methods for collection of sera and antibody titration have been previously described (6).

The incorporation of 2 H uridine of ¹⁴C amino acid mixture into spleen cells was performed as follows: Rat spleens were removed and minced onto a piece of 80‐mesh stainless‐steel screen, cells were pushed through gently with a syringe plunger and washed three times in Hanks'balanced salt solution (BSS) prior to incubation. The first wash contained 0.05% EDTA. Two ml of cells (2 × 10⁷/ml) were incubated for 45 min in the amino acid solution from Eagle's medium at 1/10 concentration supplemented with 5 mg/ml glucose.