Protein synthesis related to epidermal differentiation.

Abstract

DNA is synthesized in the epidermis of the newborn rat only in the cells of the basal or germinative layer,'-3 while RNA is produced in the spinous and granular layers as well.1' 4'' Radioautographic studies have shown that although amino acids are incorporated into the cells of all three layers6-8, there is a difference in the initial localization of certain amino acids.6Whereas tritiated leucine, phenylalanine, and methionine are first incorporated in the cells of the basal and lower spinous layers, tritiated glycine first appears more concentrated in the cells of the upper spinous and granular layers, and tritiated histidine first localizes in the cells of the upper layer.Methionine-S35 and cystine-S3 seem to be first incorporated in the inner layers.7' 8 This difference in the initial incorporation of amino acids and the histochemically demonstrated9 higher concentration of histidine in the granular relative to the other layers suggest that as epidermal cells differentiate during their migration from the basal layer toward the cornified layer, the synthesis of new and unusual proteins is initiated.In support of this hypothesis, fractions of epidermal protein have been isolated which are soluble in 8 M urea and dilute HC104, have high concentrations of glycine and histidine but little or no methionine, leucine, phenylalanine, or cyst(e)ine, and account for a major portion of the tritiated glycine and histidine initially incorporated in the epidermis under conditions of the previous radio- autographic experiments.Materials and Methods.-Glycine-a-H3 (1mc/ml H20, 200 mc/mmole), D,Lphenylalanine- ring-H3 (0.5 mc/ml H20, 20 mc/mmole), and ILhistidine-HW (0.25 mc/ml 25% ethanol, 1.1 c/ mmole), respectively, were injected intraperitoneally (0.01 ml/animal) to 4-5-day-old rats of the Sprague-Dawley or CFN strain.One to 1.5 hr later, the skin was excised, cooled, and soaked for 15 min at 00 in 0.24 M NH4C1, pH 9.5, to separate the epidermis from the dermis.10The epi- dermis was immediately frozen in liquid N2 and pulverized with a mortar and pestle.The pow- dered tissue was incubated for 1 hr at room temperature in freshly prepared 8 M urea-0.2M Tris acetate, pH 8.5 (2 ml/epidermis), followed by-homogenization in a Ten Broeck tissue grinder.The suspension was centrifuged at 40,000 X g for 10 min at 15°, the residue was again homogenized in 1/2 of the original volume of urea-Tris, and again centrifuged as above.The RNA in the urea extract was degraded at pH 7 by stirring with RNase (5-times recrystallized, protease-free, Sigma Chemical Company, 0.5 mg enzyme/ml) at room temperature for 90 min.1After the pH was adjusted to 10, the extract was dialyzed for 36 hr at 2-4' once against 20 vol of 0.1 M NH40H and then twice against 20 vol of 0.01 M NH40H.The dialyzed solution was lyophilized and the residue was kept overnight under high vacuum in the presence of anhydrous CaCl2.The dry powder (in batches of 500 mg) was stirred with 0.11 N HC104 (1 ml/10 mg) at room temperature for 30 min.The mixture was then filtered through glass wool and the filtrate was neutralized to pH 4.5-5.5 with 2 M Na2CO3 and cooled to 0°.The resulting gummy precipitate was collected by centrifugation at 3,000 X g for 5 min at 00 and then extracted with 0.02 M Na2CO3 (1 mI/10 mg original dried powder) at room temperature for several hours.The suspension was centri- fuged at 6,000 X g for 10 min at 150 and the protein in the supernatant solution was called the "0.1 N HCl04-soluble" fraction.