植物及动物钙调素抗体免疫反应特性的比较研究

用酶联免疫吸附测定和胶体金免疫电镜定位技术对三种钙调素抗体与植物和动物钙调素的免疫反应特性进行了比较研究。结果表明,在与小麦钙调素的相对亲和力中,抗小麦钙调素抗体大于抗 DNP 修饰猪脑钙调素抗体,抗牛脑钙调素抗体则很弱。抗小麦钙调素抗体与小麦钙调素的 K_D(解离常数)值为2.50×10~(-9)mol/L;抗 DNP 修饰猪脑钙调素抗体与小麦钙调素的 K_D 值为2.82×10~(-8)mol/L,而它与牛脑钙调素的 K_D 值为1.90×10~(-)mol/L。定位玉米根尖细胞钙调素,抗小麦钙调素抗体比抗 DNP 修饰猪脑钙调素抗体有更高的标记密度.定量小麦钙调素,抗小麦钙调素抗体比抗 DNP 修饰猪脑钙调素抗体有较高的检测灵敏度。     

牛小脑γ-氨基丁酸受体的生化特性

用放射性配基结合实验方法鉴定了牛小脑γ-氨基丁酸(GABA)受体的生化特性。高速离心制备的牛小脑突触膜与~3H-GABA有一饱和结合,K_D值为96nM,B_(max)为1.02pmol/mg蛋白,Hill系数为0.99。这一结合具有药理专一性和立体专一性,动力学实验测得结合速度常数为9.6×10~6M~(-1)min~(-1),解离速度常数为0.115min~(-1)。冻融、洗涤、超声波和TritonX-100处理突触膜,使~3H-GABA结合活性增加114倍。     

钙调神经磷酸酶B亚基及钙调素的EPR研究

根据顺磁离子Mn~(2+)的取代特性,用EPR方法研究了钙调神经磷酸酶B亚基与其4个Ca~(2+)的结合位点,以及它们亲和力的细微差别。并同时进行了钙调素的对比研究。实验和Scatchard作图表明,B亚基有4个Ca~(2+)结合位点,2个高亲和力结合位点,其解离常数为4×10~(-6)mol/L;2个低亲和力结合位点,解离常数为9×10~(-5)mol/L。钙调素也有2个Ca~(2+)高亲和力结合位点,其解离常数为8×10~(-6)mol/L,2个低亲和力结合位点,解离常数为7×10~(-5)mol/L。钙调神经磷酸酶B亚基和钙调素Mn~(2+)结合位点的EPR研究对B亚基和钙调素在共同调节钙调神经磷酸酶中的作用提供了有用的信息。     

人肝癌细胞表皮生长因子受体以及佛波酯对它的调变

利用放射受体结合测定证明了两株人体肝癌细胞株7402和7721细胞均有EGF受体的存在。受体数目分别为每细胞6.2×10~4和2.5×10~4,代表它们亲和力的解离常数K_D值分别为1.2 nmol/L和0.80 nmol/L。PMA处理对7721细胞的EGF受体及其亲和力均无明显影响;对7402细胞EGF受体却显示了调变作用。受体数目虽同样没有变化;但亲和力却随药物处理时间有一个下降、恢复的变化过程。在PMA处理1小时,EGF结合抑制达最大,此时受体的解离常数K_D值为3.0 nmol/L;在处理96小时,受体亲和力恢复并略有增高,此时K_D值为0.95 nmol/L。~3H-TdR参入实验表明,在PMA抑制7402细胞EGF受体亲和力的同时,细胞DNA的合成速率也相应下降。对于佛波酯等因子对EGF受体的调变作用,我们认为是属于生长因子启动细胞DNA合成进行细胞分裂的整个生物学过程中感受性因子对进行性因子的调节作用。     

CDK11 P58调节细胞增殖与凋亡机制

CDK11 P58属于CDK11/PITSLRE蛋白激酶家族成员,由Cdc2L1和Cdc2L2基因编码,通过使用内部核糖体进入位点（IRES）翻译而来.它在有丝分裂、凋亡、纺锤体形成、微管稳定肿瘤发生发展和中枢神经损伤等方面均有重要作用.目前发现,CDK11 P58能与HBO1、细胞周期蛋白D3、β-1,4-半乳糖转移酶、雌激素受体、雄激素受体等相互作用.进一步研究CDK11 P58的功能将为如肿瘤和代谢疾病的医治带来新的思路.     

电离辐射后大鼠离体胰腺腺泡淀粉酶释放及M受体数量变化

我们曾观察到大鼠经γ-射线照射后胰淀粉酶活性降低和分泌减少[1],为进一步探讨照射后胰酶分泌减少的机制,本研究制备出分散的大鼠胰腺腺泡悬液并以不同浓度的~3H-二苯羟乙酸-3-喹咛环酯(~3Hquinuclidinyll benzilatc,简称~3H-QNB)进行M受体结合测定,同时观察胆碱能介质氨甲酰胆碱刺激腺泡所引起的淀粉酶释放反应。结果表明,γ-射线10Gy照射后3天,大鼠分散的胰腺腺泡在氨甲酰胆碱刺激时淀粉酶释放量减少到对照的50％,腺泡M受体与~3H-QNB最大结合量(Bmax)减少到对照的38％,伋M受体与~3H-QNB结合的解离常数(K_D)无改变,说明胰腺腺泡细胞M受体数量的减少可能是照射后胰腺腺泡分泌淀粉酶减少的原因之一。     

Cdc25B在小鼠受精卵的表达和定位

为阐明细胞分裂周期(Cdc)25B调控小鼠受精卵发育的机制,利用Western印迹检测小鼠受精卵各时期Cdc25B的表达及Cdc2-Tyr15的磷酸化状态。利用间接免疫荧光技术观察Cdc25B在小鼠受精卵的定位。构建pEGFP-Cdc25B融合表达载体并显微注射到受精卵中,观察Cdc25B在受精卵M期的定位变化。结果表明Cdc25B在G1和S期被磷酸化,在G2和M期去磷酸化。Cdc2-Tyr15在G1和S期处于磷酸化状态,G2期只检测到Cdc2-Tyr15轻微的磷酸化信号,M期未检测到任何Cdc2-Tyr15的磷酸化信号。Cdc25B在G1期定位于细胞质和细胞核中,S和G2期定位于细胞质的皮质部分,M期由细胞质转向核区。证明Cdc25B核输出后激活有丝分裂促进因子,从而启动小鼠受精卵的有丝分裂。     

胍变性肌酸激酶复性复活过程中构象与活力的比较研究

本文讨论了经3M盐酸胍变性的兔肌肌酸激酶复性和复活的动力学过程,对二者进行了比较,以期从量的关系上研究酶的构象与催化活力之间的关系。从萤光和紫外差吸收光谱的变化看,复性过程基本上是变性过程的逆转。变性肌酸激酶复性遵循一级反应方程,以萤光强度变化为标志的速度常数k=2.2×10~(-3)秒~(-1)。酶复活过程却表明由两个一级反应所组成,其速度常数分别为k_1=0.97×10~(-3)秒~(-1),k_2=0.17×10~(-3)秒~(-1)。可见构象变化速度与复活过程中较快的反应速度相近。这说明在反映色氨酸及酪氨酸微环境的构象变化基本完成之后,活力恢复的过程还没有终结。可以认为兔肌肌酸激酶的构象与活力密切相关,酶的构象完整是催化活力的基础。     

胍变性肌酸激酶复性复活过程中构象与活力的比较研究

本文讨论了经3M盐酸胍变性的兔肌肌酸激酶复性和复活的动力学过程,对二者进行了比较,以期从量的关系上研究酶的构象与催化活力之间的关系。从萤光和紫外差吸收光谱的变化看,复性过程基本上是变性过程的逆转。变性肌酸激酶复性遵循一级反应方程,以萤光强度变化为标志的速度常数k=2.2×10~(-3)秒~(-1)。酶复活过程却表明由两个一级反应所组成,其速度常数分别为k_1=0.97×10~(-3)秒~(-1),k_2=0.17×10~(-3)秒~(-1)。可见构象变化速度与复活过程中较快的反应速度相近。这说明在反映色氨酸及酪氨酸微环境的构象变化基本完成之后,活力恢复的过程还没有终结。可以认为兔肌肌酸激酶的构象与活力密切相关,酶的构象完整是催化活力的基础。     

Insights into mad2 regulation in the spindle checkpoint revealed by the crystal structure of the symmetric mad2 dimer

In response to misaligned sister chromatids during mitosis, the spindle checkpoint protein Mad2 inhibits the anaphase-promoting complex or cyclosome (APC/C) through binding to its mitotic activator Cdc20, thus delaying anaphase onset. Mad1, an upstream regulator of Mad2, forms a tight core complex with Mad2 and facilitates Mad2 binding to Cdc20. In the absence of its binding proteins, free Mad2 has two natively folded conformers, termed N1-Mad2/open-Mad2 (O-Mad2) and N2-Mad2/closed Mad2 (C-Mad2), with C-Mad2 being more active in APC/C^Cdc20 inhibition. Here, we show that whereas O-Mad2 is monomeric, C-Mad2 forms either symmetric C-Mad2–C-Mad2 (C–C) or asymmetric O-Mad2–C-Mad2 (O–C) dimers. We also report the crystal structure of the symmetric C–C Mad2 dimer, revealing the basis for the ability of unliganded C-Mad2, but not O-Mad2 or liganded C-Mad2, to form symmetric dimers. A Mad2 mutant that predominantly forms the C–C dimer is functional in vitro and in living cells. Finally, the Mad1–Mad2 core complex facilitates the conversion of O-Mad2 to C-Mad2 in vitro. Collectively, our results establish the existence of a symmetric Mad2 dimer and provide insights into Mad1-assisted conformational activation of Mad2 in the spindle checkpoint.     

Accumulation of Mad2-Cdc20 complex during spindle checkpoint activation requires binding of open and closed conformers of Mad2 in Saccharomyces cerevisiae

The spindle assembly checkpoint (SAC) coordinates mitotic progression with sister chromatid alignment. In mitosis, the checkpoint machinery accumulates at kinetochores, which are scaffolds devoted to microtubule capture. The checkpoint protein Mad2 (mitotic arrest deficient 2) adopts two conformations: open (O-Mad2) and closed (C-Mad2). C-Mad2 forms when Mad2 binds its checkpoint target Cdc20 or its kinetochore receptor Mad1. When unbound to these ligands, Mad2 folds as O-Mad2. In HeLa cells, an essential interaction between C- and O-Mad2 conformers allows Mad1-bound C-Mad2 to recruit cytosolic O-Mad2 to kinetochores. In this study, we show that the interaction of the O and C conformers of Mad2 is conserved in Saccharomyces cerevisiae. MAD2 mutant alleles impaired in this interaction fail to restore the SAC in a mad2 deletion strain. The corresponding mutant proteins bind Mad1 normally, but their ability to bind Cdc20 is dramatically impaired in vivo. Our biochemical and genetic evidence shows that the interaction of O- and C-Mad2 is essential for the SAC and is conserved in evolution.     

The Mad2 conformational dimer: structure and implications for the spindle assembly checkpoint

The 25 kDa Mad2 protein is a key player in the spindle assembly checkpoint, a safeguard against chromosome segregation errors in mitosis. Mad2 combines three unusual properties. First, Mad2 adopts two conformations with distinct topologies, open (O) and closed (C) Mad2. Second, C-Mad2 forms topological links with its two best-characterized protein ligands, Mad1 and Cdc20. Third, O-Mad2 and C-Mad2 engage in a "conformational" dimer that is essential for spindle checkpoint function in different organisms. The crystal structure of the O-Mad2-C-Mad2 conformational dimer, reported here, reveals an asymmetric interface that explains the selective dimerization of the O-Mad2 and C-Mad2 conformers. The structure also identifies several buried hydrophobic residues whose rearrangement correlates with the Mad2 topological change. The structure of the O-Mad2-C-Mad2 conformational dimer is consistent with a catalytic model in which a C-Mad2 template facilitates the binding of O-Mad2 to Cdc20, the target of Mad2 in the spindle checkpoint.     

Determinants of conformational dimerization of Mad2 and its inhibition by p31comet

The spindle assembly checkpoint (SAC) monitors chromosome attachment to spindle microtubules. SAC proteins operate at kinetochores, scaffolds mediating chromosome-microtubule attachment. The ubiquitous SAC constituents Mad1 and Mad2 are recruited to kinetochores in prometaphase. Mad2 sequesters Cdc20 to prevent its ability to mediate anaphase onset. Its function is counteracted by p31comet (formerly CMT2). Upon binding Cdc20, Mad2 changes its conformation from O-Mad2 (Open) to C-Mad2 (Closed). A Mad1-bound C-Mad2 template, to which O-Mad2 binds prior to being converted into Cdc20-bound C-Mad2, assists this process. A molecular understanding of this prion-like property of Mad2 is missing. We characterized the molecular determinants of the O-Mad2:C-Mad2 conformational dimer and derived a rationalization of the binding interface in terms of symmetric and asymmetric components. Mutation of individual interface residues abrogates the SAC in Saccharomyces cerevisiae. NMR chemical shift perturbations indicate that O-Mad2 undergoes a major conformational rearrangement upon binding C-Mad2, suggesting that dimerization facilitates the structural conversion of O-Mad2 required to bind Cdc20. We also show that the negative effects of p31comet on the SAC are based on its competition with O-Mad2 for C-Mad2 binding.     

Explaining the oligomerization properties of the spindle assembly checkpoint protein Mad2

Mad2 is an essential component of the spindle assembly checkpoint (SAC), a molecular device designed to coordinate anaphase onset with the completion of chromosome attachment to the spindle. Capture of chromosome by microtubules occur on protein scaffolds known as kinetochores. The SAC proteins are recruited to kinetochores in prometaphase where they generate a signal that halts anaphase until all sister chromatid pairs are bipolarly oriented. Mad2 is a subunit of the mitotic checkpoint complex, which is regarded as the effector of the spindle checkpoint. Its function is the sequestration of Cdc20, a protein required for progression into anaphase. The function of Mad2 in the checkpoint correlates with a dramatic conformational rearrangement of the Mad2 protein. Mad2 adopts a closed conformation (C-Mad2) when bound to Cdc20, and an open conformation (O-Mad2) when unbound to this ligand. Checkpoint activation promotes the conversion of O-Mad2 to Cdc20-bound C-Mad2. We show that this conversion requires a C-Mad2 template and we identify this in Mad1-bound Mad2. In our proposition, Mad1-bound C-Mad2 recruits O-Mad2 to kinetochores, stimulating Cdc20 capture, implying that O-Mad2 and C-Mad2 form dimers. We discuss Mad2 oligomerization and link our discoveries to previous observations related to Mad2 oligomerization.     

Conformation-specific anti-Mad2 monoclonal antibodies for the dissection of checkpoint signaling

The spindle assembly checkpoint (SAC) ensures accurate chromosome segregation during mitosis by delaying the activation of the anaphase-promoting complex/cyclosome (APC/C) in response to unattached kinetochores. The Mad2 protein is essential for a functional checkpoint because it binds directly to Cdc20, the mitotic co-activator of the APC/C, thereby inhibiting progression into anaphase. Mad2 exists in at least 2 different conformations, open-Mad2 (O-Mad2) and closed-Mad2 (C-Mad2), with the latter representing the active form that is able to bind Cdc20. Our ability to dissect Mad2 biology in vivo is limited by the absence of monoclonal antibodies (mAbs) useful for recognizing the different conformations of Mad2. Here, we describe and extensively characterize mAbs specific for either O-Mad2 or C-Mad2, as well as a pan-Mad2 antibody, and use these to investigate the different Mad2 complexes present in mitotic cells. Our antibodies validate current Mad2 models but also suggest that O-Mad2 can associate with checkpoint complexes, most likely through dimerization with C-Mad2. Furthermore, we investigate the makeup of checkpoint complexes bound to the APC/C, which indicate the presence of both Cdc20-BubR1-Bub3 and Mad2-Cdc20-BubR1-Bub3 complexes, with Cdc20 being ubiquitinated in both. Thus, our defined mAbs provide insight into checkpoint signaling and provide useful tools for future research on Mad2 function and regulation.     

The Influence of Catalysis on Mad2 Activation Dynamics

Mad2 is a key component of the spindle assembly checkpoint, a safety device ensuring faithful sister chromatid separation in mitosis. The target of Mad2 is Cdc20, an activator of the anaphase-promoting complex/cyclosome (APC/C). Mad2 binding to Cdc20 is a complex reaction that entails the conformational conversion of Mad2 from an open (O-Mad2) to a closed (C-Mad2) conformer. Previously, it has been hypothesized that the conversion of O-Mad2 is accelerated by its conformational dimerization with C-Mad2. This hypothesis, known as the Mad2-template hypothesis, is based on the unproven assumption that the natural conversion of O-Mad2 required to bind Cdc20 is slow. Here, we provide evidence for this fundamental assumption and demonstrate that conformational dimerization of Mad2 accelerates the rate of Mad2 binding to Cdc20. On the basis of our measurements, we developed a set of rate equations that deliver excellent predictions of experimental binding curves under a variety of different conditions. Our results strongly suggest that the interaction of Mad2 with Cdc20 is rate limiting for activation of the spindle checkpoint. Conformational dimerization of Mad2 is essential to accelerate Cdc20 binding, but it does not modify the equilibrium of the Mad2:Cdc20 interaction, i.e., it is purely catalytic. These results surpass previously formulated objections to the Mad2-template model and predict that the release of Mad2 from Cdc20 is an energy-driven process.     

The closed form of Mad2 is bound to Mad1 and Cdc20 at unattached kinetochores

The spindle assembly checkpoint (SAC) ensures accurate chromosome segregation by delaying anaphase onset in response to unattached kinetochores. Anaphase is delayed by the generation of the mitotic checkpoint complex (MCC) composed of the checkpoint proteins Mad2 and BubR1/Bub3 bound to the protein Cdc20. Current models assume that MCC production is catalyzed at unattached kinetochores and that the Mad1/Mad2 complex is instrumental in the conversion of Mad2 from an open form (O-Mad2) to a closed form (C-Mad2) that can bind to Cdc20. Importantly the levels of Mad2 at kinetochores correlate with SAC activity but whether C-Mad2 at kinetochores exclusively represents its complex with Mad1 is not fully established. Here we use a recently established C-Mad2 specific monoclonal antibody to show that Cdc20 and C-Mad2 levels correlate at kinetochores and that depletion of Cdc20 reduces Mad2 but not Mad1 kinetochore levels. Importantly reintroducing wild type Cdc20 but not Cdc20 R132A, a mutant form that cannot bind Mad2, restores Mad2 levels. In agreement with this live cell imaging of fluorescent tagged Mad2 reveals that Cdc20 depletion strongly reduces Mad2 localization to kinetochores. These results support the presence of Mad2-Cdc20 complexes at kinetochores in agreement with current models of the SAC but also argue that Mad2 levels at kinetochores cannot be used as a direct readout of Mad1 levels.     

Sustained Mps1 activity is required in mitosis to recruit O-Mad2 to the Mad1–C-Mad2 core complex

Mps1 is an essential component of the spindle assembly checkpoint. In this study, we describe a novel Mps1 inhibitor, AZ3146, and use it to probe the role of Mps1’s catalytic activity during mitosis. When Mps1 is inhibited before mitotic entry, subsequent recruitment of Mad1 and Mad2 to kinetochores is abolished. However, if Mps1 is inhibited after mitotic entry, the Mad1–C-Mad2 core complex remains kinetochore bound, but O-Mad2 is not recruited to the core. Although inhibiting Mps1 also interferes with chromosome alignment, we see no obvious effect on aurora B activity. In contrast, kinetochore recruitment of centromere protein E (CENP-E), a kinesin-related motor protein, is severely impaired. Strikingly, inhibition of Mps1 significantly increases its own abundance at kinetochores. Furthermore, we show that Mps1 can dimerize and transphosphorylate in cells. We propose a model whereby Mps1 transphosphorylation results in its release from kinetochores, thus facilitating recruitment of O-Mad2 and CENP-E and thereby simultaneously promoting checkpoint signaling and chromosome congression.     

Two complexes of spindle checkpoint proteins containing Cdc20 and Mad2 assemble during mitosis independently of the kinetochore in Saccharomyces cerevisiae

Favored models of spindle checkpoint signaling propose that two inhibitory complexes (Mad2-Cdc20 and Mad2-Mad3-Bub3-Cdc20) must be assembled at kinetochores in order to inhibit mitosis. We have directly tested this model in the budding yeast Saccharomyces cerevisiae. The proteins Mad2, Mad3, Bub3, Cdc20, and Cdc27 in yeast were quantified, and there are sufficient amounts to form stoichiometric inhibitors of Cdc20 and the anaphase-promoting complex. Mad2 is present in two separate complexes in cells arrested in mitosis with nocodazole. There is a small amount of Mad2-Mad3-Bub3-Cdc20 and a much larger amount of a complex that contains Mad2-Cdc20. We use conditional mutants to show that both Mad2 and Mad3 are essential for establishment and maintenance of the spindle checkpoint. Both spindle checkpoint complexes containing Mad2 form in mitosis, not in response to checkpoint activation. The kinetochore is not required to form either complex. We propose that the conversion of Mad1-Mad2 to Cdc20-Mad2, a key step in generating inhibitory checkpoint complexes, is limited to mitosis by the availability of Cdc20 and is kinetochore independent.