Expression,purification, and characterization of arginine kinase from the sea cucumber Stichopus japonicus

The arginine kinase gene of sea cucumber Stichopus japonicus was cloned and inserted into the prokaryotic expression plasmid pET-21b. The protein was expressed in a soluble and functional form in Escherichia coli and purified by Blue Sepharose CL-6B, DEAE-32, and Sephadex G-100 chromotography with a final yield of 83 mgL(-1) of LB medium. The specific activity, electrophoretic mobility, and isoelectric focusing were all identical with those of arginine kinase that was purified from sea cucumber muscle. The fluorescence emission spectrum of arginine kinase had a maximum fluorescence at a wavelength of 330 nm upon excitation at 295 nm. These results are the first report of this purified protein.     

Gastropod arginine kinases from Cellana grata and Aplysia kurodai. Isolation and cDNA-derived amino acid sequences

Arginine kinase (AK) was isolated from the radular muscle of the gastropod molluscs Cellana grata (subclass Prosobranchia) and Aplysia kurodai (subclass Opisthobranchia), respectively, by ammonium sulfate fractionation, Sephadex G-75 gel filtration and DEAE-ion exchange chromatography. The denatured relative molecular mass values were estimated to be 40 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isolated enzyme from Aplysia gave a K_m value of 0.6 mM for arginine and a V_max value of 13 μmole Pi min⁻¹ mg protein⁻¹ for the forward reaction. These values are comparable to other molluscan AKs. The cDNAs encoding Cellana and Aplysia AKs were amplified by polymerase chain reaction, and the nucleotide sequences of 1608 and 1239 bp, respectively, were determined. The open reading frame for Cellana AK is 1044 nucleotides in length and encodes a protein with 347 amino acid residues, and that for A. kurodai is 1077 nucleotides and 354 residues. The cDNA-derived amino acid sequences were validated by chemical sequencing of internal lysyl endopeptidase peptides. The amino acid sequences of Cellana and Aplysia AKs showed the highest percent identity (66–73%) with those of the abalone Nordotis and turbanshell Battilus belonging to the same class Gastropoda. These AK sequences still have a strong homology (63–71%) with that of the chiton Liolophura (class Polyplacophora), which is believed to be one of the most primitive molluscs. On the other hand, these AK sequences are less homologous (55–57%) with that of the clam Pseudocardium (class Bivalvia), suggesting that the biological position of the class Polyplacophora should be reconsidered.     

Gonad development of the sea anemone Anthopleura asiatica in clonal populations

Seasonal fluctuations in relative gonad volume and oocyte size of the sea anemone Anthopleura asiatica were examined in 3 unisexual (male) populations and one bisexual population in the Seto Inland Sea of Japan from December 1982 to December 1985. A distinct annual cycle of gonadal maturation with a peak in the summer was found in all of the populations, although they appeared to be sustained only by asexual reproduction. Spawning occured synchronously between the 2 sexes early in the fall in the bisexual population while it was one to one and a half months later in the unisexual populations.     

Phylogeny and biogeography of Anthopleura in the North Atlantic Ocean

Two species of Anthopleura, A. ballii and A. thallia, co-occur in Northern Europe with Bunodosoma biscayensis, a species closely allied to Anthopleura. The historical factors underlying their distribution are here investigated as part of a preliminary phylogenetic analysis of morphological data. Based on this analysis, Bunodosoma is nested within Anthopleura, and B. biscayensis is more closely related to members of Anthopleura than to other members of Bunodosoma. Because B. biscayensis also has adhesive verrucae, rather than non-adhesive, rounded vesicles, it is transferred to Anthopleura.The tree consists of two major clades, each of which includes species from the Atlantic and the Pacific. The tree shows geographic structure: one clade includes species found only in the Northern Hemisphere, and the other includes widespread species and species found only in the Southern Hemisphere. Although all of the European species of Anthopleura are part of the Northern Hemisphere clade, none are one another’s closest relative.     

Cloning, expression, purification, and characterization of arginine kinase from Locusta migratoria manilensis

Arginine kinase (AK) is a phosphotransferase that plays a critical role in energy metabolism in invertebrates. The gene encoding Locusta migratoria manilensis AK was cloned and expressed in Escherichia coli by two prokaryotic expression plasmids, pET-30a and pET-28a. The recombinant protein was expressed as inclusion bodies using pET-30a. After denaturation, the recombinant AK was successfully renatured and confirmed to be enzymatically active. Addition of Tween-20 and SDS to the dilution system led to higher renaturation efficiency. Using another expression plasmid, pET-28a, and changing the expression conditions resulted in a soluble and functional form of AK, which was purified by an improved method using Sephadex G-75 chromotography to a final yield of 358 mg L^− 1 of LB medium. Some parameters for the renatured and soluble forms of AK, including K_m, K_d, specific activity, electrophoretic mobility and isoelectric focusing, were identical with those of AK obtained directly from L. migratoria manilensis leg muscle. Comparison of kinetic constants with those of AKs from other sources indicated that L. migratoria manilensis AKs have the highest k_cat and stronger synergistic substrate binding. The first report of a concise purification method enables the enzyme to be prepared in large quantities. This research should enable further detailed investigations of the enzymatic mechanism by site directed mutagenesis techniques.     

Cytoplasmic and mitochondrial arginine kinases inDrosophila: Evidence for a single gene

Mitochondrial and cytoplasmic isozymes of arginine kinase have been identified inDrosophila melanogaster. On the basis of their immunological similarity, parallel dosage responses, and cosegregation of electrophoretic mobility differences, it is concluded that both isozymes are the product of a single gene. The consequences of this in relation to the regulation and evolution of this unusual gene-enzyme system are discussed. It is inferred that the origin of the phosphagen shuttle must predate the divergence of invertebrates and vertebrates.     

Amino acid residues 62 and 193 play the key role in regulating the synergism of substrate binding in oyster arginine kinase

The purpose of this study is to clarify the amino acid residues responsible for the synergism in substrate binding of arginine kinase (AK), a key enzyme in invertebrate energy metabolism. AKs contain a pair of highly conserved amino acids (D62 and R193) that form an ion pair, and replacement of these residues can cause a pronounced loss of activity. Interestingly, in the oyster Crassostrea AK, these residues are replaced by an N and a K, respectively. Despite this replacement, the enzyme retains high activity and moderate synergism in substrate binding (Kd/Km=2.3). We replaced the N62 by G or D and the K193 by G or R in Crassostrea AK, and also constructed the double mutants of N62G/K193G and N62D/K193R. All of the mutants retained 50-90% of the wild-type activity. In N62G and N62D mutants, the Kmarg for arginine binding was comparable to that of wild-type enzyme, but the Kdarg was increased 2-5-fold, resulting in a strong synergism (Kd/Km=4.9-11.3). On the other hand, in K193G and K193R mutants, the Kmarg was increased 4-fold, and synergism was lost almost completely (Kd/Km=1.0-1.4). The N62G/K193G double mutant showed similar characteristics to the K193G and K193R mutants. Another double mutant, N62D/K193R, similar to the amino acid pair in the wild-type enzyme, had characteristics similar to those of the wild-type enzyme. These results indicate that the amino acid residues 62 and 193 play the key role in mediating the synergism in substrate binding of oyster arginine kinase.     

Hormonally regulated expression of arginine kinase in Drosophila melanogaster

Summary Arginine kinase (AK) is present throughout the life cycle of Drosophila melanogaster, but there is a sharp, transient peak of AK activity during the prepupal period and a second period of elevated activity at the time of eclosion of the adult. Imaginal discs show the greatest increase in AK activity at the prepupal stage of those tissues assayed. The prepupal peak is not seen when the temperature-sensitive ecdysoneless mutant ecd-1 is shifted to 29° C at mid-third instar larval stage. The peak in activity reappears when ecd-1 is either shifted back to 20° C after 60 h at 29° C or is fed 20-hydroxyecdysone. At the restrictive temperature, imaginal discs from ecd-1 larvae progressively lose AK activity, whereas discs from 20-hydroxyecdysone-fed larvae have a marked increase in AK activity at stage P3 of the prepupal period. These data suggest that the prepupal peak is regulated by the hormone 20-hydroxyecdysone.     

Trypanosoma brucei: Two mitogen activated protein kinase kinases are dispensable for growth and virulence of the bloodstream form

Mitogen activated protein kinase cascades function in eukaryotic responses to the environment and stress. Trypanosomatid parasites possess protein kinases with sequences characteristic of kinases in such cascades. In this report we use gene knockouts to demonstrate that two mitogen activated kinase kinase genes, MKK1 (Tb927.3.4860) and MKK5 (Tb927.10.5270), are not essential in the pathogenic bloodstream stage of Trypanosoma brucei, either in vitro or in vivo. Bloodstream forms lacking MKK1 showed decreased growth at 39 °C as compared to the parental line. However, unlike its Leishmania orthologue, T. brucei MKK1 does not appear to play a significant role in flagellar biogenesis.     

小桐子植物特异性酪蛋白激酶PS-CK1-5基因的克隆及原核表达分析

酪蛋白激酶(casein kinase, CK)作为一类普遍存在的Ser/Thr蛋白激酶，通过调节靶标蛋白的活性与稳定性，在植物整个生理过程及信号转导途径中发挥重要作用。基于同源序列比对，该研究对小桐子(Jatropha curcas)酪蛋白激酶基因家族进行鉴定与表达分析。结果表明，小桐子基因组中共鉴定到7个酪蛋白激酶1基因(CK1)、5个植物特异性酪蛋白激酶1基因(PS-CK1)、3个酪蛋白激酶2α亚基基因(CK2-α)、2个酪蛋白激酶2β亚基基因(CK2-β)，4个亚家族成员在氨基酸长度、等电点及外显子数目等都有其家族特异性。蛋白的氨基酸序列比对表明，小桐子酪蛋白激酶1都包含N端保守激酶结构域，同时其内部都鉴定到典型的激酶活性环基序、ATP结合核心基序、核定位信号肽。qRT-PCR表达分析表明，小桐子JcPS-CK1-5基因在叶片与根中都属于低温诱导基因，可能参与小桐子抗冷性过程。构建其原核表达载体pET-32a-JcPS-CK1-5，并在BL21(DE3)中诱导表达，得到81.6 kD的条带，与理论融合蛋白的分子量一致。这可为小桐子CK基因的功能鉴定及逆境信号转导机制研究提供参考。     

Identification and characterization of two arginine kinases from the parasitic insect Ctenocephalides felis

Arginine kinase (ATP:l-arginine ω-N-phosphotransferase, EC2.7.3.3.; AK) is an enzyme crucial for the energy metabolism of insects and other invertebrates, that has known allergenic potential in humans and that has been proposed as a pesticidal drug target. Here we report the identification, cDNA cloning, genomic gene structure and functional expression of AK genes from Ctenocephalides (C.) felis (cat flea). In contrast to other insect species investigated so far, C. felis possesses two AK genes, cfak1 and cfak2, encoding the functional enzymes CfAK1 and CfAK2 that can be distinguished by their guanidino substrate specificity and the kinetic parameters for their natural substrates. Molecular modelling on CfAK1 and CfAK2 based on the Limulus polyphemus AK X-ray structure (Zhou et al., 1998) and substrate docking studies provide a potential rational for the observed specificities. Evidence is provided that adult fleas express predominantly CfAK1 as an abundant soluble protein, and that in vivo in C. felis, the AK metabolites are present in concentration ranges relevant for this enzyme.     

Enzymes of arginine utilization and their formation in Aeromonas formicans NCIB 9232

In Aeromonas formicans two inducible catabolic pathways of L-arginine have been characterized. The arginine decarboxylase is induced by arginine which also induces the three enzymes of the arginine deiminase pathway but only in stress conditions such as a shift from aerobic growth conditions to very low oxygen tension. Addition of glucose to medium containing arginine leads to repression of the enzymes involved in the arginine deiminase pathway while exogenous cAMP prevents that repression of enzyme synthesis by glucose. This suggests that the induction of arginine deiminase pathway is regulated by carbon catabolite repression and the energetic state of the cell.     

Genetic structure of endangered Rhodiola sachalinensis

Rh. sachalinensis is a perennial,medicinal herb. The species is rare with apatchy distribution. Due to heavy disturbanceby human activities, it is now endangered.Allozyme analysis was employed to estimate itsgenetic diversity. Nineteen loci encoding 12enzyme systems were used. Rh.sachalinensis has a lower genetic variationwithin patches and higher differentiationbetween patches than other endangered, endemicspecies with limited distributions. The geneticdifferentiation between patches withingeographic regions is lower than that amonggeographic regions. Genetic drift is likely adominant factor contributing to the observedpattern of genetic structure. Because a higherdegree of genetic differentiation existsbetween patches in Rh. sachalinensis,more populations should be protected insitu, and samples from more populations shouldbe collected for ex situ conservation.     

The role of Arg-96 in Danio rerio creatine kinase in substrate recognition and active center configuration

In creatine kinases (CKs), the amino acid residue-96 is a strictly conserved arginine. This residue is not directly associated with substrate binding, but it is located close to the binding site of the substrate creatine. On the other hand, the residue-96 is known to be involved in expression in the substrate specificity of various other phosphagen (guanidino) kinases, since each enzyme has a specific residue at this position: arginine kinase (Tyr), glycocyamine kinase (Ile), taurocyamine kinase (His) and lombricine kinase (Lys). To gain a greater understanding of the role of residue-96 in CKs, we replaced this residue in zebra fish Danio rerio cytoplasmic CK with other 19 amino acids, and expressed these constructs in Escherichia coli. All the twenty recombinant enzymes, including the wild-type, were obtained as soluble form, and their activities were determined in the forward direction. Compared with the activity of wild-type, the R96K mutant showed significant activity (8.3% to the wild-type), but 10 mutants (R96Y, A, S, E, H, T, F, C, V and N) showed a weak activity (0.056–1.0%). In the remaining mutants (R96Q, G, M, P, L, W, D and I), the activity was less than 0.05%. Our mutagenesis studies indicated that Arg-96 in Danio CK can be substituted for partially by Lys, but other replacements caused remarkable loss of activity. From careful inspection of the crystal structures (transition state analog complex (TSAC) and open state) of Torpedo cytoplasmic CK, we found that the side chain of R96 forms hydrogen bonds with A339 and D340 only in the TSAC structure. Based on the assumption that CKs consist of four dynamic domains (domains 1–3, and fixed domain), the above hydrogen bonds act to link putative domains 1 and 3 in TSAC structure. We suggest that residue-96 in CK and equivalent residues in other phosphagen kinases, which are structurally similar, have dual roles: (1) one involves in distinguishing guanidino substrates, and (2) the other plays a key role in organizing the hydrogen-bond network around residue-96 which offers an appropriate active center for the high catalytic turnover. The mode of development of the network appears to be unique each phosphagen kinase, reflecting evolution of each enzyme.     

Functional expression and characterization of four novel neurotoxins from sea anemone Anthopleura sp

The genes of four novel neurotoxins, named Hk2a, Hk7a, Hk8a, and Hk16a, were obtained from sea anemone Anthopleura sp. All four neurotoxins were composed of 47 amino acid residues and the variable residues among them were found in positions 14, 22, 25, and 37. To study their activities, the four toxins fused to the Escherichia coli thioredoxin were overexpressed by BL21 (DE3), cleaved off from the fusion partner, purified, and characterized with MALDI-TOF and CD assays. Contractile force studies of isolated SD atria indicated that rHk2a had the strongest and rHk7a the longest heart stimulation effect. Consequently, the Arg14, a highly conserved residue in various sea anemone neurotoxins, can be inferred to contribute to the duration but not the intensity of contraction-stimulating activity. Our work renders useful information to studies of sea anemone neurotoxins, especially to the clarification of the function of the disputative Arg14.     

Gene expression in Plasmodium: from gametocytes to sporozoites

Completion of the complex developmental program of Plasmodium in the mosquito is essential for parasite transmission, yet this part of its life cycle is still poorly understood. In recent years, considerable progress has been made in the identification and characterization of genes expressed during parasite development in the mosquito. This line of investigation was greatly facilitated by the availability of the genome sequence of several Plasmodium, and by the application of approaches such as proteomics, microarrays, gene disruption by homologous recombination (gene knockout) and by use of subtraction libraries. Here, we review what is presently known about genes expressed in gametocytes and during the Plasmodium life cycle in the mosquito.     

Kunitz-type protease inhibitors from acrorhagi of three species of sea anemones

Sea anemones are rich in biologically active polypeptides such as toxins and protease inhibitors. These polypeptides have so far been isolated from whole bodies, tentacles or secreted mucus. Recently, two novel peptide toxins with crab lethality have been isolated from acrorhagi (specialized aggressive organs elaborated by only certain species of sea anemones belonging to the family Actiniidae) of Actinia equina. This prompted us to survey biologically active polypeptides in the acrorhagi of two species of sea anemones, Anthopleura aff. xanthogrammica and Anthopleura fuscoviridis. No potent crab lethality was displayed by the acrorhagial extracts of both species. However, significantly high protease inhibitory activity was instead detected in the acrorhagial extracts of the two species and also in that of A. equina. From the acrorhagi of A. equina, A. aff. xanthogrammica and A. fuscoviridis, one (AEAPI), one (AXAPI) and two (AFAPI-I and AFAPI-III) protease inhibitors were isolated, respectively. The complete amino acid sequences of the four inhibitors were elucidated by N-terminal sequencing and sequencing of the C-terminal peptide fragment produced upon asparaginylendopeptidase digestion. The determined amino acid sequences revealed that all the four inhibitors are new members of the Kunitz-type protease inhibitor family.     

毛竹PeSCL3基因表达特征及其启动子活性研究

为探讨毛竹(Phyllostachys edulis)SCL3基因的表达特征及其启动子活性,采用同源克隆的方法从毛竹中分离到SCL3同源基因Pe SCL3的编码区(ORF)和上游启动子序列(Pe SCL3p)。序列分析表明,Pe SCL3的ORF为1335 bp,推测编码含444氨基酸的蛋白,该蛋白与水稻(Oryza sativa)的SCL3同源性高达93.9%。Pe SCL3p长度为1358 bp,含有脱落酸(ABA)应答元件ABRE、赤霉素(GA3)应答元件GARE-motif和P-box、干旱诱导MYB结合位点等多种作用元件。实时定量PCR分析结果表明,Pe SCL3在毛竹叶中的表达丰度最高,其次是茎和根,而鞘中的最低;Pe SCL3的表达受GA3的抑制,受ABA、Na Cl和干旱的诱导。转Pe SCL3p∷GUS拟南芥(Arabidposis thaliana)的GUS染色结果表明,根尖、顶端生长点和子叶叶柄均被染成蓝色,尤其根尖的染色最深。这表明Pe SCL3对毛竹的生长发育,尤其是根系,可能起着重要的调控作用。     

黄河裸裂尻鱼肌酸激酶M-CK cDNA的克隆及组织表达分析

肌酸激酶(CK)能够催化磷酸基在二磷酸腺苷(ADP)和磷酸肌酸之间的可逆性转移,在细胞能量代谢过程中发挥重要作用。本研究利用RT-PCR和RACE方法克隆了黄河裸裂尻鱼(Schizopygopsis pylzovi)肌酸激酶基因全长c DNA序列,并进行了生物信息学分析和系统发育关系研究。黄河裸裂尻鱼肌酸激酶c DNA全长1 599 bp,开放阅读框(ORF)1 143 bp,编码380个氨基酸,具有典型的N-端结构域和C-端催化结构域。氨基酸序列同源性分析表明,黄河裸裂尻鱼肌酸激酶与斑马鱼(Danio rerio)和鲤(Cyprinus carpio)M3-CK有较高的同源性,其序列一致度达94%以上。系统发育分析显示,黄河裸裂尻鱼肌酸激酶与斑马鱼和鲤的M3-CK聚在一支,形成一个单系群,初步判定克隆获得的黄河裸裂尻鱼肌酸激酶基因可能与鲤和斑马鱼M3-CK是直系同源基因。利用Real-time PCR方法检测和分析了黄河裸裂尻鱼主要组织肌酸激酶m RNA表达水平,肌肉、肠、眼、心中肌酸激酶转录本表达较高,在肝胰脏和脑中表达微弱。肌酸激酶在肌肉、肠、和心中高表达与其能量代谢功能相适应,而在眼组织中的高表达是否与肌酸激酶的其他功能相关,有待于进一步研究。