Myxococcus xanthus spore coat protein S may have a similar structure to vertebrate lens beta gamma-crystallins

The Gram-negative bacterium Myxococcus xanthus has a complex life cycle during which large amounts of a protein of relative molecular mass (Mr) 19,000, known as protein S, are assembled into a spore surface coat by a process that specifically requires calcium ions. The gene for protein S has been cloned and the DNA sequence shows that the gene product is composed of four internally repeated homologous sequences, each 40 amino acids long. Although protein S resembles calmodulin both in its internally duplicated structure and its ability to bind calcium, it apparently has a beta-sheet secondary structure rather than the helix-loop-helix motifs that characterize the calmodulin family. We now show that protein S has a striking homology with the beta- and gamma-crystallins of the vertebrate eye lens which are beta-sheet proteins with internally duplicated structures. This implies that the beta- and gamma-crystallins evolved from already existing proteins, whose ancestors occurred in the prokaryotes. The biological function of protein S, as a closely packed, stable protein in a relatively dehydrated environment, has implications for the functions of crystallins, which are found closely packed in the lens fibre cells, where their stability is essential for maintenance of transparency.     

Recruitment of cytosolic proteins to a secretory granule membrane depends on Ca2+-calmodulin

An increase in free calcium triggers catecholamine secretion from chromaffin cells and calmodulin is strongly implicated as the intracellular Ca2+ receptor. In our recent studies of calmodulin action in the chromaffin cell, micromolar Ca2+ concentrations resulted in calmodulin and cytosolic proteins becoming bound to the chromaffin granule membranes. We now report that calmodulin is bound with high affinity to granule membrane proteins of molecular weights (Mrs) 25,000 and 22,000 (25K and 22K) at low Ca2+ (less than 10(-8) M) and to proteins with Mrs 69K and 50K at high Ca2+ (greater than 1 microM). Other cytosolic components (Mrs 70K, 36K, 34K and 32K) require calmodulin for their interfraction with membrane. These proteins separately bound to calmodulin-Sepharose at high Ca2+ concentrations. Although the functions of these adrenal proteins have not been established, the 34K and 32K Mr components co-migrate with clathrin light chains isolated from medullary coated vesicles and the Mr 34K components from both sources share the same one-dimensional peptide map. These interactions were observed at micromolar Ca2+ levels at 'intracellular' conditions of pH and ionic strength and would be expected to occur during secretion from the chromaffin cell.     

动物体液中的胞外钙调素结合蛋白（快报）

利用以生物素标记钙调素为探针的凝胶覆盖技术检测动物体液中胞外钙调素结合蛋白（ＣａＭＢＰ）。结果，人的唾液中检测到至少３种分子量分别为１４ｋＤ，２４ｋＤ和５２ｋＤ的ＣａＭＢＰｓ。其中，５２ｋＤ蛋白与ＣａＭ的结合依赖于Ｃａ２＋的存在，而２４ｋＤ和１４ｋＤ蛋白则不依赖于Ｃａ２＋。在鸡血清里，检测到以９４ｋＤ，４４／４５ｋＤ蛋白为主的４～５种胞外ＣａＭＢＰｓ，所有的这些蛋白与ＣａＭ的结合都依赖于Ｃａ２－。此外，在牛奶中也检出胞外ＣａＭＢＰｓ。以上结果，证明了在动物中普遍存在胞外ＣａＭＢＰｓ，为胞外钙调素的作用机理提供了新线索。     

A bacterial calcium-binding protein homologous to calmodulin

Many of the effects of calcium ions in eukaryotic cells are mediated by calcium-binding regulatory proteins such as calmodulin, in which each calcium-binding site has a distinctive helix-loop-helix conformation termed the EF hand. Protein S from the spore coat of the Gram-negative bacterium Myxococcus xanthus has been shown to resemble calmodulin in its internally-duplicated structure and ability to bind calcium. However, it has a beta-sheet secondary structure rather than the helix-loop-helix arrangement of the eukaryotic proteins. We have determined the complete amino-acid sequence of a calcium-binding protein from the Gram-positive bacterium "Streptomyces erythraeus" by cloning and sequencing the corresponding gene. It contains four EF-hand motifs bearing remarkable sequence similarity to the calcium-binding sites in calmodulin. This implies that the EF-hand super-family may have evolved from ancient proteins present in prokaryotes.     

甜菊钙调蛋白基因的克隆及结构分析

钙调蛋白（Calmodulin）是生物细胞内一种重要的调控蛋白，通过其与靶酶的相互作用控制细胞正常的生长发育及细胞对外界环境变化的反应，我们从甜菊顶芽和花芽中提取总RNA，逆转录合成cDNA第一链，以此为模板，参考GenBank上已发表植物的钙调蛋白基因序列合成5′端和3′端引物，利用多聚酶链式反应（PCR）扩增并克隆得到了甜菊钙调蛋白基因的两个异型基因，序列分析表明，它们均由450个核苷酸组成，编码148个氨基酸，在核苷酸序列上与迄今已知的多种植物钙调蛋白均有很高的同源性，同源率在83％以上，编码的氨基酸序列同源性更同，同源率高达95％以上，这两个基因之间存在差异，其核苷酸序列同源率为85％，编码区的氨基酸序列的同源率为99％，仅在第122个氨基酸由ALA代替了VAL。     

Involvement of heterotrimeric G protein in signal transduction of extracellular calmodulin in regulatingrbcS expression

The role of heterotrimeric G protein in signal transduction pathway of extracellular calmodulin in regulating rbcS expression was examined in suspension-cultured cells of transgenic tobacco. Pharmalogical experiments indicated that G protein agonist cholera toxin enhanced rbcS expression and heterotrimeric G protein antagonist pertussis toxin inhibited rbcS expression in transgenic tobacco cells. Pertussis toxin also inhibited the enhancement effect caused by exogenous purified calmodulin on rbcS expression, whereas cholera toxin completely reversed the inhibitory effects caused by anti-calmodulin serum on rbcS expression. The right side-out vesicles from tobacco cell membrane were purified, which contained all of substrates for fluometric assay of GTPase activity. Exogenous purified calmodulin, when adding directly to the medium of plasma membrane vesicles, significantly activated GTPase activity in the right side-out plasma membrane vesicles, and this increase in GTPase activity was completely inhibited both by heterotrimeric G proteins antagonist pertussis toxin and nonhy-drolyzable GTP analogs GMP-PCP. These results provided the evidence that heterotrimeric G proteins may be involved in signal transduction pathways of extracellular calmodulin to regulate rbcS gene expression.     

植物细胞钙靶蛋白的研究进展

简述了植物细胞的钙离子调控过程，对钙调蛋白、钙调磷酸酶B类蛋白和钙离子依赖型蛋白质激酶3类钙靶蛋白的最新研究进展进行了综述。     

Three-dimensional structure of the ATPase fragment of a 70K heat-shock cognate protein

The three-dimensional structure of the amino-terminal 44K ATPase fragment of the 70K bovine heat-shock cognate protein has been solved to a resolution of 2.2 A. The ATPase fragment has two structural lobes with a deep cleft between them; ATP binds at the base of the cleft. Surprisingly, the nucleotide-binding 'core' of the ATPase fragment has a tertiary structure similar to that of hexokinase, although the remainder of the structures of the two proteins are completely dissimilar, suggesting that both the phosphotransferase mechanism and the substrate-induced conformational change intrinsic to the hexokinases may be used by the 70K heat shock-related proteins.     

Penicillopepsin from Penicillium janthinellum crystal structure at 2.8 A and sequence homology with porcine pepsin.

The polypeptide chain of the acid protease penicillo pepsin folds via an 18-stranded mixed beta-sheet into two distinct lobes separated by a 30-A long groove which is the extended substrate binding site. The catalytic residues Asp-32 and Asp-215 are located in this groove and their carboxyl groups are in intimate contact. Alignment of the amino acid sequence with that of pepsin shows regions of high homology.     

钙调素依赖性的环核苷酸磷酸二酯酶的制备

通过两次离子交换柱层析, 从牛脑中制备钙调素依赖性的环核苷酸磷酸二酯酶． 所得酶不含内源性钙调素, 能被足量外加的钙调素激活 １４ 倍, 可用于测定细胞中的钙调素含量     

Structural basis for the activation of anthrax adenylyl cyclase exotoxin by calmodulin.

Oedema factor, a calmodulin-activated adenylyl cyclase, is important in the pathogenesis of anthrax. Here we report the X-ray structures of oedema factor with and without bound calmodulin. Oedema factor shares no significant structural homology with mammalian adenylyl cyclases or other proteins. In the active site, 3'-deoxy-ATP and a single metal ion are well positioned for catalysis with histidine 351 as the catalytic base. This mechanism differs from the mechanism of two-metal-ion catalysis proposed for mammalian adenylyl cyclases. Four discrete regions of oedema factor form a surface that recognizes an extended conformation of calmodulin, which is very different from the collapsed conformation observed in other structures of calmodulin bound to effector peptides. On calmodulin binding, an oedema factor helical domain of relative molecular mass 15,000 undergoes a 15 A translation and a 30 degrees rotation away from the oedema factor catalytic core, which stabilizes a disordered loop and leads to enzyme activation. These allosteric changes provide the first molecular details of how calmodulin modulates one of its targets.     

Clathrin light chains and secretory vesicle binding proteins are distinct

Recently, several groups have initiated studies on cytosolic proteins that bind to isolated secretory vesicle membranes in the presence of Ca2+ in order to identify proteins that may regulate exocytosis. Two major chromaffin granule binding proteins, of molecular weights 32,000 (32K) and 34,000 (34K), were reported to have the same mobility on one-dimensional SDS gels as clathrin-associated light chains from the adrenal medulla, and the 34K granule binding protein the same one-dimensional peptide map as the 34K clathrin light chain. These observations support the hypothesis that Ca2+-dependent recruitment of soluble light chains to the vesicle membrane may nucleate the assembly of a clathrin coat and initiate endocytosis. Here we report that two-dimensional peptide maps of the clathrin light chains and of all chromaffin granule membrane binding proteins in the 30K range are distinct, and therefore fail to support this hypothesis. It has also been suggested that some or all of the vesicle binding proteins require calmodulin for their interaction with the membrane. However, we find that antagonism of calmodulin by trifluoperazine does not prevent the association of the other cytosolic proteins with the chromaffin granule membrane.     

Identification of a secretory granule-binding protein as caldesmon

Stimulation of adrenal chromaffin cells results in a rise in the concentration of intracellular free calcium which initiates catecholamine secretion by exocytosis. An understanding of the molecular basis of exocytosis will require knowledge of the sites of action of calcium. A role for calmodulin has been implicated in secretion from chromaffin cells, and isolated granule membranes bind both calmodulin and a series of cytosolic proteins in a calcium-dependent fashion. Here, we demonstrate that one of the cytosolic granule-binding proteins with a relative molecular mass (Mr) of 70,000 (70K) is a form of the calmodulin-regulated actin-binding protein caldesmon, first isolated from smooth muscle. Cytoplasmic gels assembled from an adrenal medullary extract in the absence of Ca2+ contained actin and the 70K protein. The association of both of these proteins with the cytoplasmic gel was inhibited by a micromolar concentration of Ca2+. In addition, we have demonstrated that the 70K protein is localized at the periphery of chromaffin cells. These results are consistent with the notion that 70K protein (caldesmon) has a role in regulating the organization of actin filaments of the cell periphery during the secretory process.     

动脉硬化性脑梗死继发性癫痫

动脉硬化性脑梗死继发性癫痫217例,梗死灶主要位于脑叶,提示痫性活动与皮层梗死密切相关,早发性痫性发作（发生于脑梗死2周内）为短暂性痫性发作,可能为脑代谢异常所致,一般仅临时用安定,预后好,晚发性痫性发作（发生于脑梗死2周后）可能为癫痫灶所致,半数病例需服抗癫痫药。     

皮肤特异表达钙调素类蛋白研究进展

钙调素是广泛存在于各种动植物细胞的信号转导分子,近年在皮肤中发现了一些钙调素类蛋白,其分布、特征、功能都与钙调素不同,在表皮的分化发育中发挥重要作用。本文就此作一简单介绍。     

Discription of a New Species of Pseudocrangonyx from AnHui Lava Tubes, China (Amphipoda: Gammaridae )

本文记述了采自安徽含山熔洞的伪克钩虾属一新种,含山伪克钩虾Pseudocrangonyx hanshansis sp, nov.,新种与近缘种亚洲伪克钩虾p, asiaticu。之不同在于。第一尾足内肢短于柄部,内缘具2枚长粗利,第三尾足外肢第1节较柄部长3倍,端节很小,约为第1节的1/6;尾节板左右两叶,每叶具2枚端刺,外侧稍长;第二触角柄部之末节长于次末节,约与鞭节等长,腹部侧板2之下缘具1利,偶2利,侧板3之下缘具2利。     

金标银染技术在表达文库筛选中的应用

以胶体多标记钙调素作为探针再辅以银染法（金标银染技术）,自玉米ｃＣＮＡ表达文库中筛迁钙调素结合蛋白。该探针保持钙调素的活性,特异性地与钙调素结合蛋白相结合,在胶体金标记的基础上进行银染,灵敏度获得显著提高（达ｎｇ级）。应用这一方法对玉米ＣＮＤＡ表达文库进行筛选获得了一个与探针特异结合的阳性克隆。以生物素标记的钙调素对该克隆表达的融合蛋白进行结合实验,结果表明该克隆编码的蛋白为一个钙调素结合蛋白。本     

Calmodulin bifurcates the local Ca2+ signal that modulates P/Q-type Ca2+ channels

Acute modulation of P/Q-type (alpha1A) calcium channels by neuronal activity-dependent changes in intracellular Ca2+ concentration may contribute to short-term synaptic plasticity, potentially enriching the neurocomputational capabilities of the brain. An unconventional mechanism for such channel modulation has been proposed in which calmodulin (CaM) may exert two opposing effects on individual channels, initially promoting ('facilitation') and then inhibiting ('inactivation') channel opening. Here we report that such dual regulation arises from surprising Ca2+-transduction capabilities of CaM. First, although facilitation and inactivation are two competing processes, both require Ca2+-CaM binding to a single 'IQ-like' domain on the carboxy tail of alpha1A; a previously identified 'CBD' CaM-binding site has no detectable role. Second, expression of a CaM mutant with impairment of all four of its Ca2+-binding sites (CaM1234) eliminates both forms of modulation. This result confirms that CaM is the Ca2+ sensor for channel regulation, and indicates that CaM may associate with the channel even before local Ca2+ concentration rises. Finally, the bifunctional capability of CaM arises from bifurcation of Ca2+ signalling by the lobes of CaM: Ca2+ binding to the amino-terminal lobe selectively initiates channel inactivation, whereas Ca2+ sensing by the carboxy-terminal lobe induces facilitation. Such lobe-specific detection provides a compact means to decode local Ca2+ signals in two ways, and to separately initiate distinct actions on a single molecular complex.     

The structure of the myosin VI motor reveals the mechanism of directionality reversal

Here we solve a 2.4-A structure of a truncated version of the reverse-direction myosin motor, myosin VI, that contains the motor domain and binding sites for two calmodulin molecules. The structure reveals only minor differences in the motor domain from that in plus-end directed myosins, with the exception of two unique inserts. The first is near the nucleotide-binding pocket and alters the rates of nucleotide association and dissociation. The second unique insert forms an integral part of the myosin VI converter domain along with a calmodulin bound to a novel target motif within the insert. This serves to redirect the effective 'lever arm' of myosin VI, which includes a second calmodulin bound to an 'IQ motif', towards the pointed (minus) end of the actin filament. This repositioning largely accounts for the reverse directionality of this class of myosin motors. We propose a model incorporating a kinesin-like uncoupling/docking mechanism to provide a full explanation of the movements of myosin VI.