ABSTRACT
Among a number of tissues and peripheral blood cells in chicken, leukocytes, bone marrow cells, liver and spleen showed high ADP-ribosyltransferase activity, with leukocytes having the highest. Density gradient centrifugation of the leukocytes revealed that the leukocyte ADP-ribosyltransferase originates in the polymorphonuclear cells, so called heterophils. Subcellular distribution of the cells showed the localization of the enzyme in the granule fraction. Based on the obtained amino acid sequences of arginine-specific ADP-ribosyltransferase purified from chicken peripheral heterophils, two arginine-specific ADP-ribosyltransferase cDNAs (designated AT1 and AT2) were obtained from chicken bone marrow cells. Each cDNA encodes a different peptide of 312 amino acid residues. Homology of the deduced amino acid sequences between AT1 and AT2 was 78.3%. Arginine-specific ADP-ribosyltransferase activity was detected in culture medium of COS 7 cells transiently transfected with AT1 cDNA, while activity from the cells transfected with AT2 cDNA was found in both culture medium and cell lysate. AT1 transferase required 2-mercaptoethanol (MSH) for the activity and in the presence of NaCl, the activity was inhibited while the AT2 enzyme was activated by either agent. Highly conserved regions were observed among the deduced amino acid sequences of AT1, AT2, chicken erythroblast and rabbit and human skeletal muscle ADP-ribosyltransferases, and rodent T-cell surface antigen RT6. Two forms of the transferase with much the same properties as AT1 and AT2 proteins, regarding the effect of NaCl and MSH, were detected in bone marrow cells. Based on these results it seems that AT1 and AT2 cDNAs encode the two forms of arginine-specific ADP-ribosyltransferase detected in chicken bone marrow cells.
Subject(s)
ADP Ribose Transferases/genetics , Bone Marrow/enzymology , ADP Ribose Transferases/chemistry , ADP Ribose Transferases/metabolism , Amino Acid Sequence , Animals , Base Sequence , Bone Marrow Cells , Chickens , Cloning, Molecular , DNA, Complementary , Molecular Sequence Data , Tissue DistributionABSTRACT
Two arginine-specific ADP-ribosyltransferase cDNAs (designated AT1 and AT2) were cloned from chicken bone marrow cells. Each cDNA encodes a different peptide of 312 amino acid residues. Homology of deduced amino acid sequences between AT1 and AT2 was 78.3%. We found all six combined peptide sequences of 222 amino acid residues derived from purified chicken heterophil ADP-ribosyltransferase (Mishima, K., Terashima, M., Obara, S., Yamada, K., Imai, K., and Shimoyama, M. (1991) J. Biochem. (Tokyo) 110, 388-394) in the deduced amino acid sequence of AT1, with two amino acid mismatches. Arginine-specific ADP-ribosyltransferase activity was detected in culture medium of COS 7 cells transiently transfected with AT1 cDNA, while activity from the cells transfected with AT2 cDNA was found in both culture medium and cell lysate. AT1 transferase required 2-mercaptoethanol for the activity. The activity was inhibited in the presence of NaCl while AT2 enzyme was activated by either agent. On zymographic in situ gel analysis, estimated molecular masses of the AT1, AT2 and purified chicken heterophil transferases were 32, 34, and 27.5 kDa, respectively. Northern blot analysis with specific probes to AT1 or AT2 cDNAs revealed about a 1.5-kilobase message in chicken bone marrow cells but no signals were observed in heterophils, spleen, and liver of chicken or human HL-60 cells. Highly conserved regions were observed among the deduced amino acid sequences of AT1, AT2, rabbit skeletal muscle transferase, and rodent T-cell surface antigen RT6s.