Centrin is a member of the EF-hand super family that plays critical role in the centrosome duplication and separation. To investigate the role of Asp37 in the process of metal-binding and conformational change of cili...Centrin is a member of the EF-hand super family that plays critical role in the centrosome duplication and separation. To investigate the role of Asp37 in the process of metal-binding and conformational change of ciliate Euplotes octocarinatus centrin (EoCen), the mutant D37K, in which aspartic acid 37 had been replaced by lysine, was first obtained by the site-directed mutagenesis. Then 2-p-toluidinylnaphthalene-6- sulfonate (TNS) was used as a fluorescence probe to detect the conformational change of the protein. The results show that the metal- binding capability of the site I of EoCen was lost by the mutation of Asp37→Lys. In comparison the Tb3+-saturated EoCen, the hydrophobic surface of D37K, which is exposed by the binding of Tb3+, has shrunk sharply, suggesting that Asp37 plays an important role in maintaining the proper conformation of EoCen in the presence of Tb3+. Meanwhile, the conditional binding constants of TNS with Tb3+-loaded EoCen and D37K were obtained, K(Tb3+-EoCen-TNS)=(7.38 ±0.25)×105 M-1 and K(Tb3+-D37K-TNS)=(1.16±0.05)×106 M-1.展开更多
Centrins are members of the centrosome protein family, which is highly conserved during revolution. The homologous genes of centrin in many organisms had been cloned, but the sequences of the rat centrin genes were no...Centrins are members of the centrosome protein family, which is highly conserved during revolution. The homologous genes of centrin in many organisms had been cloned, but the sequences of the rat centrin genes were not reported yet in GenBank. We cloned the cDNA fragments of centrin-1, -2 and -3 from the rat testis by RT-PCR, and analyzed the homology of the deduced amino acid sequences. The expression characterization of centrin genes in rat spermatogenesis was carried out by semi-quantitative RT-PCR. The results show that the homology of the corresponding centrin proteins in human, mouse and rat is high. The expression of centrin-1 is testis-specific, spermatogenic cell-specific and developmental stage-related. Centrin-1 begins to be transcribed when the meiosis occurs, and its mRNA level reaches the peak in round spermatids. Centrin-2 and centrin-3 are highly expressed in spermatogonia and their mRNA level decreases markedly when meiosis occurs. These results suggest that centrin-1 may play roles in meiosis and spermiogenesis, and centrin-2 and centrin-3 may be related to mitosis.展开更多
To study the relations between male infertility and centrosome protein (centrin) and the functions of centrin in spermatogenesis, the matured spermatozoa of 10 normal male people and 18 male infertility patients were ...To study the relations between male infertility and centrosome protein (centrin) and the functions of centrin in spermatogenesis, the matured spermatozoa of 10 normal male people and 18 male infertility patients were stained by immunofluorescence labeling antibody against centrin. The results showed that two fluorescence signal dots appeared in the normal male spermatozoa and were located at the base of flagellum. They are proximal centriole and distal centriole. However, in some spermatozoa of the male infertility, centrin protein was located abnormally at the base of flagellum and its staining signals were spread, the normal proximal and distal centrioles were confused and could not be recognized separately. These results suggest that abnormality of centrosome protein may be related to male infertility. This discovery may be used as a marker of abnormal sperm and male infertility.展开更多
To understand the unfolding of ciliate Euplotes octocarinatus centrin（EoCen）, the glycine positioned at 115, the sixth residue of the loop of the protein＇s third EF-hand, was mutated into tryptophan（Trp）.Intrinsi...To understand the unfolding of ciliate Euplotes octocarinatus centrin（EoCen）, the glycine positioned at 115, the sixth residue of the loop of the protein＇s third EF-hand, was mutated into tryptophan（Trp）.Intrinsic fluorescence and Tb（Ⅲ） binding properties of wild type EoCen and G115W mutant were monitored by fluorescence spectra in 10 mmol/L Hepes. The emission maximum of EoCen was 306 nm and mutation had no impact on the Tb（Ⅲ） binding properties. The properties of G115W were investigated by fluorescence, far-UV circular dichroism（CD） spectra and fluorescence decays in the absence or in the presence of 6 mol/L guanidine hydrochloride（GdnHCl）. For the increase in polarity of microenvironment around Trp residue, the emission maximum of apoG115 W at 343 nm is shifted to 359 nm in 6 mol/L GdnHCI. Also the secondary structure is lost nearly and fluorescence lifetime decreases in 6 mol/L GdnHCI. The unfolding of G115W induced by GdnHCI was assessed by using the model of structural element The unfolding of proteins is a sequential reaction, namely two-transition. three-state process. The first transition belongs to the unfolding of the C-terminal domain, and the second transition is assigned to the unfolding of the N-terminal domain. The ⊿〈△G_（total）~0（H_2O）〉 was used to determine the effect of Tb（Ⅲ） on the stability of apoprotein. The 〈△G_（total）~0（H_2 O）〉for Tb_2-G115 W has a less increase of0.68 kJ/mol compared with apoG115W, proving Tb（Ⅲ） situated at C-terminal has negligible impact on the stability of protein. Whereas the 〈△G_（total）~0（H_2 O）〉 for Tb_4-G115W has a rise of 1.29 kJ/mol compared with Tb_2-G115W, manifesting Tb（Ⅲ） lcocated at low affinity sites has considerable influence on protein stability. mainly stabilizing the N-terminal domain.展开更多
Ciliate Euplotes octocarinatus centrin (EoCen) is an EF-hand calcium-binding protein closely related to the prototypical calcium sensor protein calmodulin.The first amino acid of the Ca2+-binding loops found in the EF...Ciliate Euplotes octocarinatus centrin (EoCen) is an EF-hand calcium-binding protein closely related to the prototypical calcium sensor protein calmodulin.The first amino acid of the Ca2+-binding loops found in the EF-hand calcium-binding proteins is a highly conserved aspartic acid residue.The D37K mutant was produced to elucidate the metal binding role of the first aspartic acid of the EF-loop I of EoCen.Aromatic-sensitized Tb3+fluorescence results indicated that the metal binding ability of loop I was lost due to the D37K mutation.Based on fluorescence titration curves of Lu2-D37K,the conditional binding constants of the EoCen loop II were quantitatively found to be KII=(1.61±0.04)×105 L mol-1 and KII=(3.52±0.08)×102 L mol-1 with Tb3+ and Ca2+,respectively.Using 2-p-toluidinylnaphthalene-6-sulfonate as a hydrophobic probe,exposure of the hydrophobic surface upon metal binding was found to be significantly reduced for the metal ion-saturated EoCen D37K mutant.展开更多
Interactions between model target peptide melittin (ME) and Euplotes octocarinatus centrin (EoCen) were investigated by fluorescence spectra, circular dichroism (CD) spectra and native polyacrylamide gel electrophores...Interactions between model target peptide melittin (ME) and Euplotes octocarinatus centrin (EoCen) were investigated by fluorescence spectra, circular dichroism (CD) spectra and native polyacrylamide gel electrophoresis (PAGE). In 0.1 mol/L N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (Hepes) and 150 mmol/L NaCl at pH 7.4, EoCen and isolated short C-terminal domain of EoCen (SC-EoCen) form 1:1 peptide:protein complexes. However, no detectable signal changes can be observed while isolated N-terminal domain of EoCen (N-EoCen) or isolated long C-terminal domain of EoCen (LC-EoCen) was added into solution of ME. The interaction between EoCen and ME is specified exclusively for the short C-terminal domain of EoCen. On the basis of fluorescence titration curves, the conditional binding constants of ME with EoCen and SC-EoCen were calculated to be logKME-EoCen = 6.81±0.33 and log- KME-SC-EoCen = 6.51±0.45, respectively.展开更多
文摘Centrin is a member of the EF-hand super family that plays critical role in the centrosome duplication and separation. To investigate the role of Asp37 in the process of metal-binding and conformational change of ciliate Euplotes octocarinatus centrin (EoCen), the mutant D37K, in which aspartic acid 37 had been replaced by lysine, was first obtained by the site-directed mutagenesis. Then 2-p-toluidinylnaphthalene-6- sulfonate (TNS) was used as a fluorescence probe to detect the conformational change of the protein. The results show that the metal- binding capability of the site I of EoCen was lost by the mutation of Asp37→Lys. In comparison the Tb3+-saturated EoCen, the hydrophobic surface of D37K, which is exposed by the binding of Tb3+, has shrunk sharply, suggesting that Asp37 plays an important role in maintaining the proper conformation of EoCen in the presence of Tb3+. Meanwhile, the conditional binding constants of TNS with Tb3+-loaded EoCen and D37K were obtained, K(Tb3+-EoCen-TNS)=(7.38 ±0.25)×105 M-1 and K(Tb3+-D37K-TNS)=(1.16±0.05)×106 M-1.
基金This work was supported by the Special Funds for Major State Basic Research Project (Grant No. G1999055901)
文摘Centrins are members of the centrosome protein family, which is highly conserved during revolution. The homologous genes of centrin in many organisms had been cloned, but the sequences of the rat centrin genes were not reported yet in GenBank. We cloned the cDNA fragments of centrin-1, -2 and -3 from the rat testis by RT-PCR, and analyzed the homology of the deduced amino acid sequences. The expression characterization of centrin genes in rat spermatogenesis was carried out by semi-quantitative RT-PCR. The results show that the homology of the corresponding centrin proteins in human, mouse and rat is high. The expression of centrin-1 is testis-specific, spermatogenic cell-specific and developmental stage-related. Centrin-1 begins to be transcribed when the meiosis occurs, and its mRNA level reaches the peak in round spermatids. Centrin-2 and centrin-3 are highly expressed in spermatogonia and their mRNA level decreases markedly when meiosis occurs. These results suggest that centrin-1 may play roles in meiosis and spermiogenesis, and centrin-2 and centrin-3 may be related to mitosis.
基金This work was supported by the National "973" Program of China (Grant No. G19990559-01).
文摘To study the relations between male infertility and centrosome protein (centrin) and the functions of centrin in spermatogenesis, the matured spermatozoa of 10 normal male people and 18 male infertility patients were stained by immunofluorescence labeling antibody against centrin. The results showed that two fluorescence signal dots appeared in the normal male spermatozoa and were located at the base of flagellum. They are proximal centriole and distal centriole. However, in some spermatozoa of the male infertility, centrin protein was located abnormally at the base of flagellum and its staining signals were spread, the normal proximal and distal centrioles were confused and could not be recognized separately. These results suggest that abnormality of centrosome protein may be related to male infertility. This discovery may be used as a marker of abnormal sperm and male infertility.
基金Project supported by the National Natural Science Foundation of China(21571117)the PhD Programs Foundation of the Ministry of Education of China(20131401110011)
文摘To understand the unfolding of ciliate Euplotes octocarinatus centrin（EoCen）, the glycine positioned at 115, the sixth residue of the loop of the protein＇s third EF-hand, was mutated into tryptophan（Trp）.Intrinsic fluorescence and Tb（Ⅲ） binding properties of wild type EoCen and G115W mutant were monitored by fluorescence spectra in 10 mmol/L Hepes. The emission maximum of EoCen was 306 nm and mutation had no impact on the Tb（Ⅲ） binding properties. The properties of G115W were investigated by fluorescence, far-UV circular dichroism（CD） spectra and fluorescence decays in the absence or in the presence of 6 mol/L guanidine hydrochloride（GdnHCl）. For the increase in polarity of microenvironment around Trp residue, the emission maximum of apoG115 W at 343 nm is shifted to 359 nm in 6 mol/L GdnHCI. Also the secondary structure is lost nearly and fluorescence lifetime decreases in 6 mol/L GdnHCI. The unfolding of G115W induced by GdnHCI was assessed by using the model of structural element The unfolding of proteins is a sequential reaction, namely two-transition. three-state process. The first transition belongs to the unfolding of the C-terminal domain, and the second transition is assigned to the unfolding of the N-terminal domain. The ⊿〈△G_（total）~0（H_2O）〉 was used to determine the effect of Tb（Ⅲ） on the stability of apoprotein. The 〈△G_（total）~0（H_2 O）〉for Tb_2-G115 W has a less increase of0.68 kJ/mol compared with apoG115W, proving Tb（Ⅲ） situated at C-terminal has negligible impact on the stability of protein. Whereas the 〈△G_（total）~0（H_2 O）〉 for Tb_4-G115W has a rise of 1.29 kJ/mol compared with Tb_2-G115W, manifesting Tb（Ⅲ） lcocated at low affinity sites has considerable influence on protein stability. mainly stabilizing the N-terminal domain.
基金supported by the National Natural Science Foundation of China(20771068,20901048)the Ph.D Programs Foundation of Ministry of Education of the People’s Republic of China(20091401110007)the Natural Science Foundation of Shanxi Province(2007011024)
文摘Ciliate Euplotes octocarinatus centrin (EoCen) is an EF-hand calcium-binding protein closely related to the prototypical calcium sensor protein calmodulin.The first amino acid of the Ca2+-binding loops found in the EF-hand calcium-binding proteins is a highly conserved aspartic acid residue.The D37K mutant was produced to elucidate the metal binding role of the first aspartic acid of the EF-loop I of EoCen.Aromatic-sensitized Tb3+fluorescence results indicated that the metal binding ability of loop I was lost due to the D37K mutation.Based on fluorescence titration curves of Lu2-D37K,the conditional binding constants of the EoCen loop II were quantitatively found to be KII=(1.61±0.04)×105 L mol-1 and KII=(3.52±0.08)×102 L mol-1 with Tb3+ and Ca2+,respectively.Using 2-p-toluidinylnaphthalene-6-sulfonate as a hydrophobic probe,exposure of the hydrophobic surface upon metal binding was found to be significantly reduced for the metal ion-saturated EoCen D37K mutant.
基金Supported by the National Natural Science Foundation of China (Grant Nos. 20371031 and 20771068)the Natural Science Foundation of Shanxi Province (Grant No. 2007011024) Young Science Foundation of Shanxi University
文摘Interactions between model target peptide melittin (ME) and Euplotes octocarinatus centrin (EoCen) were investigated by fluorescence spectra, circular dichroism (CD) spectra and native polyacrylamide gel electrophoresis (PAGE). In 0.1 mol/L N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (Hepes) and 150 mmol/L NaCl at pH 7.4, EoCen and isolated short C-terminal domain of EoCen (SC-EoCen) form 1:1 peptide:protein complexes. However, no detectable signal changes can be observed while isolated N-terminal domain of EoCen (N-EoCen) or isolated long C-terminal domain of EoCen (LC-EoCen) was added into solution of ME. The interaction between EoCen and ME is specified exclusively for the short C-terminal domain of EoCen. On the basis of fluorescence titration curves, the conditional binding constants of ME with EoCen and SC-EoCen were calculated to be logKME-EoCen = 6.81±0.33 and log- KME-SC-EoCen = 6.51±0.45, respectively.
