Incorporation of Histamine Ribonucleoside into Soluble Proteins

Abstract

ISOTOPICALLY labelled histamine ribonucleoside can be detected in the urine of experimental animals after intraperitoneal administration of either 2(ring)⁻¹⁴C-labelled histamine¹, or similarly labelled histamine-adenine-dinucleotides². However, in such experiments the quantities of histamine ribonucleoside recovered from the urine represent only a small percentage of the expected amount. This apparent disappearance of histamine ribonucleoside in vivo suggested the possibility of its combination with proteins. The incorporation of amines into proteins, probably through transamidation, was studied by Waelsch et al.^3,4. A similar system, that is, a 100,000g supernatant of a guinea-pig liver homogenate³, was used in our investigations. Histamine ribonucleoside was prepared from histamine adenine dinucleotide phosphate⁵, as already described². The degree of purity of this material was checked ionophoretically^1,2, and only a single spot was observed in either tris-hydrochloric acid (pH 8.0) or sodium borate (pH 9.1). As shown in Table 1, there was appreciable incorporation of radioactivity into the acid insoluble fraction of the incubation mixtures when either histamine, or histamine ribonucleoside, or histamine-adenine-dinucleotide-phosphate was used. This incorporation was Ca²⁺-dependent. Thus, in controls from which Ca²⁺ was omitted, very few scintillations were counted, and radioactivity approached background values (Table 1). When tested under conditions similar to those in Table 1, the guinea-pig liver supernatant used in our investigations was found free of activities capable of liberating inorganic phosphate from the dinucleotide. This may be partly due to the lack of the proper enzymes (that is, pyrophosphatases and phosphatases) and partly because of the lack of magnesium ions. In any event, this finding led us to the conclusion that the dinucleotide was probably attached as such on the protein molecule.