Relationship between the tyrosination state of tubulin and the activities of tubulin: tyrosine ligase and tubulin carboxypeptidase in rat muscle during development

Abstract

Tubulin can be post-translationally modified by the incorporation or the release of a tyrosine residue at the COOH-terminus of the ct subunit. The present study demonstrates that rat muscle soluble preparations contain tubulin carboxypeptidase besides tubulin : tyrosine ligase.The state of tyrosination of tubulin and the activities of both the ligase and the carboxypeptidase were examined in rat muscle during development. The proportion of tyrosinated tubulin with respect to tyrosinable tubulin (tyrosinated plus detyrosinated tubulin) decreased from 83% (new-born rats) to 28% (adult rats) with the corresponding increase in detyrosinated tubulin. The activities of the enzymes decreased continuously and in a near parallel fashion during development. These results indicate that the changes in the tyrosination state of tubulin can not be explained merely by changes in the enzyme activities.We also compared the ability of rat muscle and brain [14C]tyrosinated tubulin to act as substrate of the carboxypeptidase. Muscle tubulin was found to be a less efficient substrate than brain tubulin.Tubulin is subjected to a post-translational modification by which a tyrosine residue is removed or added back at the COOH-terminus of the ct chain [l -51. Two enzymes are involved in this tyrosination-detyrosination reaction, tubulin : tyrosine ligase and tubulin carboxypeptidase, respectively. The ligase was first found in brain tissue but soon after was shown to be present in other tissues [6] and also in lower eukaryotic organisms [7, 81. On the other hand, the biochemical characterization of the carboxypeptidase was only done in brain. In this paper we show that rat skeletal muscle contains tubulin carboxypeptidase.In vitro, the ligase was shown to act on non-assembled tubulin but not on microtubules [9]. Using brain slices, we were unable. to determine unequivocally the in vivo specificity of this enzyme [lo]. Studies with chicken erythrocytes [ l l ] and cultured cells [12] showed the preference of the ligase for nonassembled tubulin so that this pool in living cells is apparently maintained fully tyrosinated. On the other hand, the carboxypeptidase acts preferentially on microtubules [9, 13, 141 in such a way that the proportions of tyrosinated and detyrosinated tubulin (referred to as the tyrosination state) in these structures vary with time [15]. lmmunofluorescence with specific peptide antibodies has previously shown that tyrosinated and detyrosinated tubulin are present in distinct, but overlapping, subsets of microtubules in cultured cells [16, 171. Total soluble tubulin obtained under depolymerizing conditions is composed by a mixture of the tyrosinated and detyrosinated forms whose relative proportions have been shown to change with development [18 -201. Among the possible mechanisms that could determine the tyrosination state of tubulin we considered the two most likely to be: (a) the

Reference genes for proximal femoral epiphysiolysis expression studies in broilers cartilage

“…However, recent studies evaluating the stability of reference genes have reported outstanding stabilities of ribosomal proteins in different cell lines and tissues of mammals [ 61 – 63 ], fish [ 64 ], shellfish [ 65 ] and plants [ 66 ]. The gene RPLP1 was studied as a reference gene in several animal species, such as Homo sapiens [ 58 ], Mus musculus [ 67 ], Rattus norvegicus [ 68 , 69 ], Gallus gallus [ 43 , 70 ], Bos Taurus [ 71 ] and Anopheles gambiae [ 72 ], having a role in the elongation step of protein synthesis [ 73 ]. This gene had a divergent stability ranking between neonatal and cardiosphere-derived adult cells, indicating that its gene expression was age-dependent [ 50 ].…”

cDNA Cloning and Overexpression of Acidic Ribosomal Phosphoprotein P1 Gene (RPLP1) from the Giant Panda

“…RPLP1 gene was cloned and studied from many animals including Homo sapiens 17-19, Mus musculus 20, Rattus norvegicus 21, 22, Gallus gallus 23, Danio rerio 24, Bos Taurus 25, Anopheles gambiae 26, Drosophila melanogaster 27, Bombyx mori 28 etc. However, RPLP1 gene from the Giant Panda ( Ailuropoda melanoleuca ) has not been reported yet.…”