Purification of a 60‐kDa Protein from Chicken Liver Associated with the Internal Nuclear Matrix and Closely Related to Carboxylesterases

Abstract

A 60-kDa protein was purified from chicken liver internal nuclear matrix and its nuclear localization was confirmed by immunofluorescence analysis. Structural information acquired from sequence analysis of the intact protein and of fragments obtained from enzymatic and chemical cleavages strongly suggests that it belongs to the carboxylesterases family, even if with some very peculiar features. The N-terminal sequence of the 60-kDa protein is completely different from the other carboxylesterases, but is similar to a region that is normally internal to all mammalian esterase sequences and localized after the serine residue at the active site. This suggests that the protein may be derived from a gene duplication and/or rearrangement. Since the 60-kDa protein shows a low esterase activity of about 0.2 pmol . min-' . mg-' using either p-nitrophenyl acetate or p-nitrophenyl butyrate as substrates, it is not possible to rule out that the protein shares only a sequence similarity with carboxylesterases and is not a true esterase. Otherwise it could be an esterase which has developed different properties, i.e. a special substrate specificity, the requirement of additional factors or a different stability in solution. In the latter case, this protein could be related to the physiological control of hydrolysis of exogenous and endogenous esters which can act on nuclear functions and/or metabolism.Keywords: nuclear matrix proteins ; carboxylesterase; protein purification.The understanding of many important functions concerning the expression and regulation of the eukaryotic genome needs a better knowledge of the arrangement of nuclear components and the way in which they interact to form the complex architecture of the cell nucleus. The nuclear matrix is a residual structure that has been isolated from a variety of eukaryotic cells and consists mainly of the nuclear lamina, residual nucleoli and a proteinaceus network that is in contact with segments of nuclear DNA [l -41. Considerable experimental evidence suggests that the nuclear matrix plays an important role in the organization and function of the nucleus. Processes like replication, transcription, RNA splicing and DNA loop attachment are associated with the nuclear matrix (4-61. Moreover, hormone receptor function, transcription factors and various enzymatic activities are localized at the nuclear matrix level (4, 6-81, Nuclear matrix proteins have been widely investigated and it has been observed that their composition is tissue-specific and varies during cell differentiation and between normal and transformed cell types [9-121. Several proteins associated with the nuclear matrix have been identified and characterized [ Abbreviufzons PhMeSO,F, phenylmethylsulfonyl fluoride; NaWP,, phosphate-buffered saline; M60k, a 60-kDa protein purified from chicken liver internal nuclear mairix. make contacts with both cytoskeletal proteins and chrmiatin [18, 191. Considering the complexity and the importance of the nuclear matrix, we have developed procedures to purify sing...

Immunoprecipitation of DNA–Protein Complexes Cross-Linked by cis-Diamminedichloroplatinum

“…S.A), polyclonal anti-actin antibody (Sigma), and anti-chicken nuclear carboxylesterase rabbit serum (19). The last protein has been previously shown to be one of the nuclear matrix proteins that are unbound to DNA (F. Altieri and A. Ferraro, unpublished results).…”

“…Figure 3 shows the results obtained with three different antibodies against nuclear proteins, anti-carboxylesterase (19), anti-actin, antiERp57. The three proteins are present in the nuclear fraction but only the actin and ERp57 are cross-linked to DNA, confirming the validity of our procedure.…”

Nuclear halo from Bradysia hygida (Diptera:Sciaridae) salivary gland polytene cells

“…Observations using electronic microscopy and biochemical studies of chromosomes and nuclei lacking histones supported the hypothesis that eukaryotic DNA was organized in loops associated with the chromosomal skeleton (scaffold) or the nuclear matrix (Cook and Brazell, 1976;Paulson and Laemmli, 1977). Additional analyses demonstrated that these loops, roughly ranging from 5 to 100 kb or more, were anchored in the nuclear matrix through specific sequences called Scaffold Attached Regions (SARs) or Matrix Attached Regions (MARs) (Altieri et al, 1996;Berrios et al, 1985;Phi-Van and Strätling, 1990). The nuclear matrix forms a fibrogranular structure, resulting from the extraction of a high saline concentration (2M NaCl) or the use of detergents such as LIS (3.5 lithium diiodosalicilate).…”

“…However, it also depends on cell type, as well as the isolation procedure of the nuclear matrix, the internal network and/or the remaining nucleolar fraction, which may be absent, while the external lamina is present in all the extractions (Neri et al, 1997). A large number of proteins with enzymatic functions such as oxidoredutases, transferases, hidrolases and glycoproteins are found associated with the nuclear matrix (Altieri et al, 1996;Ferraro et al, 1994;S'iakste and S'iakste, 1994), as well as enzymes such as DNA and RNA polymerases, and DNA metilases and ligases, which are involved in the synthesis processes (processing and repairing) (Berrios et al, 1985;Stein et al, 1991;Berezney, 1987a, Vemuri et al, 1993). The presence of a minimum group of constituent proteins from the nuclear matrix such as topoisomerases, histone H1 and lamina, in plant and animal cells, suggests a conserved structure (Bouliskas, 1995).…”

“…The presence of a minimum group of constituent proteins from the nuclear matrix such as topoisomerases, histone H1 and lamina, in plant and animal cells, suggests a conserved structure (Bouliskas, 1995). Some of the internal network proteins from the nuclear matrix are specific and some changes in this protein composition have been related with tumor growth, viral infection, mitosis, cell differentiation, embryo development and sign transduction (Altieri et al, 1996;Brancolini and Schneider, 1991;Stuurman et al, 1990;Zbarskii, 1990). The characterization of specific nuclear matrix proteins has as a goal the possible use of these proteins as markers of malignant phenotypes in the early diagnosis of bladder, prostate, breast and larynx cancers, among others (Barboro et al, 1996;Donat et al, 1996;Cupo, 1991;Filatova and Zbarskii, 1993;Getzenberg et al, 1996).…”