Immunological and physical comparison of monomeric and dimeric phosphagen kinases: Some evolutionary implications

The antigenic and physical properties of several representative invertebrate phosphagen kinases have been examined in order to further characterize the relationship between taxonomic assignment, quaternary protein structure and evolution of this class of enzymes. Antibodies against dimeric arginine kinase from the sea cucumber cross-reacted with dimeric arginine kinase purified from sea urchin eggs, but failed to react with extracts from any species known to contain monomeric arginine kinase. However, strong immunoreactivity was observed when antibodies against purified dimeric arginine kinase were reacted with pure creatine kinase from the human muscle (CK-MM) and brain (CK-BB) as well as extracts from several species known to contain dimeric creatine kinase. Of particular interest with regard to evolution of the phosphagen kinases, we confirm the presence of creatine kinase activity in the very primitive sponge Tethya aurnatium and detect a reaction with antibodies against dimeric, but not monomeric, arginine kinase. This observation is consistent with recent studies of phosphagen kinase evolution. Substrate utilization was very specific with creatine kinase using only creatine. Arginine kinase catalyzed phosphorylation of arginine but enzymes from several species could also phosphorylate canavanine. No activities were detected with d-arginine. Isoelectric points, evaluated for several pure arginine kinases suggest that generally the monomeric forms are more acidic than the dimeric proteins. Heat inactivation of arginine kinase in several species indicated a wide range of stabilities, which did not appear to be correlated with quaternary structure, but rather distinguished by the organism's environment. On the other hand, homodimeric arginine kinase proteins from species inhabiting disparate environments are sufficiently homologous to form a catalytically active hybrid.     

Purification and characterization of arginine kinase from the American cockroach (Periplaneta americana)

The isolation and characterization of homogeneous arginine kinase from the cockroach is reported. The purification protocol produces 6.6 mg of pure enzyme from 6.8 g of whole cockroach. The purified enzyme cross-reacts with a heterologous antibody and monoclonal antibody against arginine kinase from the shrimp. Both antibody preparations also cross-react with extracts from several species known to contain monomeric arginine kinase, but fail to react with extracts from organisms containing dimeric arginine kinase. Cockroach arginine kinase has a molecular mass of approximately 43,000 determined from measurements by gel filtration and gel electrophoresis. Compared with other arginine kinases, the enzyme from the cockroach is relatively thermostable (50% activity retained at 50 degrees C for 10 min) and has a pH optima of 8.5 and 6.5-7.5, for the forward and reverse reactions, respectively. Treatment with 5,5'dithiobis[2-nitrobenzoic acid] indicates that arginine kinase has a single reactive sulfhydryl group and, interestingly, the reaction is biphasic. The Michaelis constants for the phosphagen substrates, arginine: 0.49 mM, phosphoarginine: 0.94 mM, and nucleotide substrates MgATP: 0.14 mM, MgADP: 0.09 mM, are in the range reported for other arginine kinases. A 1% solution of pure enzyme has an absorbance of 7.0 at 280 nm. Calculations based on circular dichroic spectra indicate that arginine kinase from the cockroach has 12% alpha-helical structure. The intrinsic protein fluorescence emission maximum at 340 nm suggests that tryptophan residues are below the surface of the protein and not exposed to solvent. Arginine kinase from the cockroach and shrimp are known to be deleterious immunogens towards humans. The availability of pure protein, its characterization and potential regulation of activity, will be useful in developing agents to control the cockroach population and its destructive role in agriculture and human health.     

Kinetic analysis of two purified forms of arginine kinase: absence of cooperativity in substrate binding of dimeric phosphagen kinase

Arginine kinase from sea urchin eggs and sea cucumber muscle are dimeric enzymes, unlike the more widely distributed monomeric enzyme found in other invertebrates. Both purified enzymes exhibited features characteristic of the monomeric arginine kinases including pH optima, formation of a catalytic dead-end complex (enzyme-MgADP-arginine) and stabilization of this complex by monovalent anions. A complete analysis of initial velocity data, in both directions for each substrate, indicated that substrate binding cooperativity was either minimal or non-existent. Unlike many other multi-subunit enzymes, the significance of the dimeric state of the phosphagen kinases remains unclear. These present results would suggest that (a) cooperativity, or so-called synergism in substrate binding is not a characteristic of the dimeric state of the protein and (b) the functional significance of the dimeric state is not related to the ability of some of these enzymes to undergo cooperativity in substrate binding. The significance of the dimeric state for the creatine kinases and arginine kinases remains to be established.     

Conformational change and inactivation of arginine kinase from shrimp Feneropenaeus chinensis in oxidized dithiothreitol solutions.

The effect of oxidized dithiothreitol (DTT) on the conformation and function of arginine kinase from shrimp Feneropenaeus chinensis was investigated with the methods of intrinsic fluorescence, ANS fluorescence, size exclusion chromatography (SEC), sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE), and activity assay. The excess molecular oxidized dithiothreitol could result in a loss of activity and conformational change of arginine kinase. The oxidized arginine kinase was characterized by monitoring the changes of fluorescence emission wavelength (excitation wavelength: 295 nm) and the intensity of 1-anilino-8-naphthalenesulfonate (ANS) binding (excitation wavelength: 380 nm) to the protein. The results of fluorescence spectra showed that the presence of oxidized DTT could result in a marked change in the enzyme tertiary structure. The conformational changes of native and oxidized arginine kinase are induced by the presence of the full set of transition state analog (TSA) components. The results of size exclusion chromatography and SDS-PAGE indicated that no disulfide bond was formed among the protein molecules in the oxidized-DTT solution.     

Tabtoxin-resistant protein: overexpression, purification, and characterization

One of the self-protection mechanisms in Pseudomonas syringae pv. tabaci, a pathogen of tobacco wildfire, is thought to be due to its tabtoxin-resistance gene (ttr). In this study, the ttr gene was inserted into an expression vector, pQE30, and successfully expressed in Escherichia coli M15 at high levels. The purified recombinant tabtoxin-resistant protein (TTR) had an apparent molecular mass of about 21 kDa on SDS-PAGE as well as by mass spectroscopy and had a pI of 6.6 on isoelectric focusing-PAGE. Spectral analysis showed that TTR possesses a maximum fluorescence wavelength (lambda(max)) of 325 nm upon excitation at 282 nm and a positive band with a maximum at 195 nm and a broad negative band with a minimum at 215 nm in the far-UV CD spectrum. The spectrophotometric assay demonstrated the strong detoxification activity of TTR. These results are the first report of the characterization of the purified tabtoxin-resistant protein. Its capacity to detoxify tabtoxinine-beta-lactam shows that it must be one of the self-protection mechanisms in pv. tabaci.     

Ultraviolet-Induced Photodegradation of Cucumber (Cucumis sativus L.) Microsomal and Soluble Protein Tryptophanyl Residues in Vitro

The in vitro effects of ultraviolet B (280-320 nm) radiation on microsomal membrane proteins and partially purified ribulose bisphosphate carboxylase (Rubisco) from cucumber (Cucumis sativus L.) was investigated by measuring the direct photolytic reduction of tryptophan fluorescence and the formation of fluorescent photooxidation products. Exposure of microsomes and Rubisco to monochromatic 300-nm radiation resulted in the loss of intrinsic tryptophan fluorescence and the production of blue-emitting fluorophores. The major product of tryptophan photolysis was tentatively identified as N-formylkynurenine (N-FK). Even though the rates of tryptophan photodegradation and N-FK formation were similar, the amount of blue fluorescence produced was significantly higher in the microsomes relative to Rubisco. Studies with various free radical scavengers and other modifiers indicated that tryptophan photodegradation requires oxygen and that the subsequent formation of N-FK may involve reactive oxygen species. The optimum wavelengths for loss of typtophan fluorescence were 290 nm for the microsomes and 280 nm for Rubisco. The temperature dependence of tryptophan fluorescence and rate of tryptophan photodegradation indicated an alteration in the cucumber microsomal membranes at about 24[deg]C, which influenced protein structure and tryptophan photosensitivity.     

Characterization of an activated ribosomal S6 kinase variant from maturing sea star oocytes: association with phosphatase 2A and substrate specificity.

Two ribosomal protein S6 kinases (i.e., pp52(S6K) and pp70(S6K)) of the p70 S6 kinase family were markedly activated during meiotic maturation of Pisaster ochraceus sea star oocytes. A rapid protocol was developed for the purification from the oocyte cytosol of pp52(S6K) by approximately 50,000-fold with a specific enzyme activity of 1.6 micromol per min per mg. The purified enzyme apparently featured the N- and C-terminal regions of pp70(S6K) as it immunoreacted with antibodies directed to peptides patterned after these amino acid sequences in mammalian pp70(S6K). pp52(S6K) was inhibited by fluoride (IC(50) approximately 60 mM), but was relatively insensitive to beta-glycerolphosphate, EGTA, dithiothreitol, spermine, heparin, NaCl, and metal ions such as Mn(2+), Zn(2+), and Ca(2+). The consensus sequence for substrate phosphorylation was determined to be RXXSXR, which was partially distinct from mammalian p70(S6K) in its requirement for an amino-terminal arginine. Phosphorylation of ribosomal protein S6 by p52(S6K) occurred exclusively on serine on at least five tryptic peptides. Inhibition of sea star p52(S6K) phosphotransferase activity after treatment with protein serine/threonine phosphatases confirmed that p52(S6K) was still regulated by phosphorylation. The sea star S6 kinase was purified to near homogeneity with the regulatory and catalytic subunits of protein-serine phosphatase 2A and the heat shock protein 60. The association of an S6 kinase with phosphatase 2A was confirmed by coimmunoprecipitation of S6 kinase activity with phosphatase 2A-specific antibodies. The purified S6 kinase and the sea star oocyte system will be useful for analysis of upstream and downstream signaling events that lead to phosphorylation of the S6 protein and other targets.     

Thermal inactivation and unfolding of a dimeric arginine kinase

Thermal inactivation and unfolding of the dimeric arginine kinase (AK) from sea cucumber Stichopus japonicus was investigated. The activation energy was calculated to be 388 kJ/mol. Based on the analysis of the denaturation course at 58 degrees C, a model is suggested for the thermal unfolding of this dimeric AK. In addition, the effect of free Mg(2+) and the potential biological significance on the thermal unfolding of dimeric AK is discussed.     

Purification and properties of phosphoglycerate kinase from Thermus thermophilus strain HB8.

(1) A glycolytic enzyme, phosphoglycerate kinase [EC 2.7.2.3], was purified from cells of an extreme thermophile, Thermus thermophilus strain HB8. The enzyme was resistant to heat, and no loss of activity was observed after incubation for 10--20 min at 79 degrees C. (2) Catalytic properties such as pH optimum (pH 6--8.5), kinetic parameters (Km=0.28 mM for ATP, 1.79 mM for glycerate 3-phosphate), substrate specificity and inhibitors of the enzyme were investigated and compared with those of phosphoglycerate kinase from other sources. (3) The enzyme protein consists of a single polypeptide chain of molecular weight 44,600. The isoelectric point is 5.0 The amino acid composition of the enzyme was studied. The contents of ordered secondary structures were estimated to be 29% alpha-helix and 11% pleated sheet from the circular dichroic spectrum of the enzyme protein. (4) The fluorescence spectrum of the enzyme protein showed an emission maximum at 320 nm when excited at 280 nm. The quantum yield was 0.19. Tryptophyl fluorescence was not quenched, in contrast to the fluorescence reported for yeast phosphoglycerate kinase.     

Urea Induced Inactivation and Unfolding of Arginine Kinase from the Sea Cucumber Stichopus japonicus

Urea titration was used to study the inactivation and unfolding equilibrium of arginine kinase (AK) from the sea cucumber Stichopus japonicus. Both fluorescence spectral and circular dichroism spectral data indicated that an unfolding intermediate of AK existed in the presence of 1.0 to 2.0 M urea. This was further supported by the results of size exclusion chromatography. The spectral data suggested that this unfolding intermediate shared many structural characteristics with the native form of AK including its secondary structure, tertiary structure, as well as its quaternary structure. Furthermore, according to the residual activity curve, this unfolding intermediate form still retained its catalytic function although its activity was lower than that of native AK. Taken together, the results of our study give direct evidence that an intermediate with partial activity exists in unfolding equilibrium states of AK during titration with urea.     

刺参海带饲料原料褐藻胶降解菌的分离与鉴定

从海带及刺参养殖环境中筛选有效降解褐藻胶,且对刺参无致病性的微生物,对海带饲料原料进行降解处理,以降低海带饲料中刺参难以消化的褐藻胶成分,显著提高饲料利用率,增加海带原料价值。以褐藻胶为唯一碳源选择培养基初筛;DNS法测定褐藻胶裂解酶酶活;16S r DNA测序及生理生化试验对菌种进行鉴定;高浓度腹腔攻毒试验考察筛选所得菌株对刺参的潜在致病性;分子排阻色谱及高效凝胶色谱法对微生物酶解褐藻胶的终产物进行分析。系统发育树分析表明,菌株WB1与Bacillus amyloliquifaciens有最高同源性,对刺参无潜在致病性;其褐藻胶裂解酶酶解褐藻胶的终产物主要为二糖和三糖,相对含量分别为74.1%和25.9%,平均分子量为516 Da。解淀粉芽胞杆菌WB1可作为一种安全的有益微生物用于刺参海带饲料原料中褐藻胶成分的降解。     

Intermediates in the inactivation and unfolding of dimeric arginine kinase induced by GdnHCl

Equilibrium studies of guanidine hydrochloride (GdnHCl)-induced unfolding of dimeric arginine kinase (AK) from sea cucumber have been performed by monitoring by enzyme activity, intrinsic protein fluorescence, circular dichroism (CD), 1-anilinonaphthalene-8sulfonate (ANS) binding, size-exclusion chromatography and glutaraldehyde cross-linking. The unfolding is a multiphasic process involving at least two dimeric intermediates. The first intermediate, I1, which exists at 0-0.4 M GdnHCl, is a compact inactive dimer lacking partial global structure, while the second dimeric intermediate, I2, formed at 0.5-2.0 M GdnHCl, possesses characteristics similar to the globular folding intermediates described in the literature. The whole unfolding process can be described as follows: (1) inactivation and the appearance of the dimeric intermediate I1; (2) sudden unwinding of I1 to another dimeric intermediate, I2; (3) dissociation of dimeric intermediate I2 to monomers U. The refolding processes initiated by rapid dilution in renaturation buffers indicate that denaturation at low GdnHCl concentrations (below 0.4 M GdnHCl) is reversible and that there seems to be an energy barrier between the two intermediates (0.4-0.5 M GdnHCl), which makes it difficult for AK denatured at high GdnHCl concentrations (above 0.5 M) to reconstitute and regain its catalytic activity completely.     

Anomalous fluorescence of yeast 3-phosphoglucerate kinase.

The 3-phosphoglycerate kinase (EC 2.7.2.3) of yeast which contains two tryptophyl and eight tyrosyl residues per molecule, displayed an unusualy fluorescence emission spectrum with a maximum at 308 nm when excited at 280 nm. The emission peak shifted to 329 nm when excited at 295 nm. We could confirm that it was due to the efficient quenching of tryptophyl fluorescence as well as to the incomplete energy transfer from tyrosyl to tryptophyl residues. The average fluorescence quantum yield of this protein was 0.076 (excitation at 280 nm) and that of tryptophyl residues was 0.046 (excitation at 295 nm). As the pH of the solution was lowered, the fluorescence intensity of phosphoglycerate kinase at 329 nm dramatically increased between pH 5 and 4, while the position of the peak remained unchanged. When denatured in 4 M guanidine hydrochloride, the protein showed two emission peaks, one at 343 nm and the other at 303 nm.     

Properties of adenylate kinase after modification of Arg-97 by phenylglyoxal.

Adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) isolated from porcine skeletal and heart muscle and from rabbit muscle are inactivated when a single arginine residue is modified. In adenylate kinase from pig the modified residue was identified as Arg-97 by peptide-mapping. In native adenylate kinase Arg-97 is located at the bottom of the active site cleft. The protein fluorescence of modified adenylate kinase is reduced. Whereas the addition of AMP, ADP and MgATP quench the fluorescence of native adenylate kinase, the fluorescence of phenylglyoxal-modified adenylate kinase is only affected by ADP and MgATP. This finding is discussed in connection with the structural isomerization observed in native adenylate kinase by X-ray diffraction analysis.     

饲料中添加甘草酸对刺参生长、免疫及抗病力的影响

以初始体重为(6.80±0.00)g左右的刺参(Apostichopus japonicus Selenka)为研究对象,在室内循环水系统中进行8周饲喂实验,研究饲料中添加不同梯度的甘草酸对刺参生长、免疫及其抗病力的影响。以基础饲料为对照组,在基础饲料中分别添加50、100和200mg/kg的甘草酸,共配制4种实验饲料。结果表明:饲料中添加甘草酸对刺参的成活率没有影响,各处理组均为100%。饲料中添加200mg/kg甘草酸可显著提高刺参的特定生长率(SGR)(P0.05)。养殖实验结束后,通过注射刺参腐皮综合症致病菌灿烂弧菌(Vibriosplendidus)进行刺参攻毒实验,攻毒后14d内对照组与50mg/kg添加组的累计发病率(分别为38.3%和36.7%)显著高于100和200mg/kg添加组(分别为30.0%和26.7%)(P<0.05)。实验可得到以下结论:(1)饲料中添加200mg/kg甘草酸可以提高刺参养殖的产量,同时可以提高刺参的非特异性免疫力和抗病力;(2)在研究中,全周期养殖期间投喂甘草酸不会产生免疫疲劳或其他副作用。       

Ultrastructural,fluorescence microscopic and microfluorimetric study of the innervation of the axial complex in the sea urchin,Sphaerechinus granularis (Lam.)

Summary In juxtaposition with the contractile epithelia of the axial complex of the sea urchin, Sphaerechinus granularis, several types of nerve fibers with different vesicle populations were determined. Nerve terminals, filled with clear vesicles and dense core vesicles, form synaptoid neuromuscular junctions. Close to the somatocoelic epithelia of the axial and terminal sinus septa, numerous axon profiles form a nerve plexus. Among the epithelial cells covering the plexus, two types of nerve cells can be distinguished which presumably produce neurosecretory and aminergic granules, respectively. Monoamine fluorescence (formaldehyde-induced fluorescence, Falck-Hillarp technique) was analyzed microspectrofluorimetrically. The emission spectrum of the fluorophores occurring in the present material shows a maximum at 475 nm and is characteristic of catecholamines; the excitation maximum at 380 nm after formaldehyde treatment is typical of catecholamines at low pH only. Since the peak ratio (370:320 nm) does not change after HCl-vapor treatment, the fluorophores are likely to be indicative of dopamine.     

Simultaneous purification of three mitochondrial enzymes. Acetylglutamate kinase, acetylglutamyl-phosphate reductase and carbamoyl-phosphate synthetase from Neurospora crassa

The early enzymes of arginine biosynthesis in Neurospora crassa are localized in the mitochondrion and catalyze the conversion of glutamate to citrulline. The final conversion of citrulline to arginine occurs via two enzymatic steps in the cytoplasm. We have devised a method for the isolation and purification of three of the mitochondrial arginine biosynthetic enzymes from a single extract. Acetylglutamate kinase and acetylglutamyl-phosphate reductase (both products of the complex arg-6 locus) were purified to homogeneity and near homogeneity, respectively. The large catalytic subunit of carbamoyl-phosphate synthetase was also purified to homogeneity. The three enzymes were resolved into separate fractions by chromatography on three dye-ligand affinity resins, which are specific for nucleotide binding enzymes and have a high protein binding capacity. High performance liquid chromatography was employed in the final stages of purification and was extremely effective in fractionating both acetylglutamate kinase and acetylglutamyl-phosphate reductase from proteins with very similar properties, which were not removed by other techniques. The purified proteins were used to raise specific antisera against these proteins. Acetylglutamate kinase and acetylglutamyl-phosphate reductase were shown to be immunologically unrelated. This finding suggests that the arg-6 locus encompasses two nonoverlapping cistrons. The antisera raised against carbamoyl-phosphate synthetase has been shown to cross-react with related enzymes in Saccharomyces cerevisiae, Escherichia coli, and rat liver (Ness, S. A., and Weiss, R. L. (1985) J. Biol. Chem. 260, 14355-14362). Acetylglutamate kinase is a regulatory enzyme and has been shown to be feedback-inhibited by arginine. We have determined the submitochondrial localization of acetylglutamate kinase and the second arg-6 product, acetylglutamyl-phosphate reductase. Both enzymes were shown to be soluble matrix enzymes. We discuss the relevance of this finding with respect to possible mechanisms for end product inhibition of a mitochondrial enzyme by a cytoplasmic effector.     

Purification and partial characterization of cellular retinoic acid-binding protein from human placenta

Cellular retinoic acid-binding protein (CRABP) has been purified to homogeneity from human placenta by a series of procedures, including acetone powder extraction, gel filtration on Sephadex G-50, and ion-exchange chromatography on DEAE-cellulose and on SP-Sephadex. Cellular retinol-binding protein (CRBP) was isolated concurrently. CRABP was purified 75,400-fold, based on total soluble acetone powder extract of placenta. The protein is a single polypeptide chain with a molecular mass of 14,600 Da, estimated by sodium dodecyl sulfate (SDS) gel electrophoresis or gel filtration, and has an isoelectric point of 4.78 (apo-CRABP, 4.82). On analysis of absorption and fluorescence spectra, the protein was seen to exhibit an absorption peak at 350 nm, fluorescence excitation maxima at 350 and 370 nm, and a fluorescence emission maximum at 475 nm. Human CRABP was immunologically distinct from human CRBP and serum retinol-binding protein.     

Neuronal peptides induce oocyte maturation and gamete spawning of sea cucumber, Apostichopus japonicus

Extracts prepared from tissues containing buccal ring nerve or longitudinal radial nerve of sea cucumber induce oocyte maturation and ovulation from ovarian tissues. We purified two small peptides, a pentapeptide and a heptapeptide, from the buccal tissues of Japanese common sea cucumber, Apostichopus japonicas. Both peptides induced oocyte maturation and gamete spawning. The pentapeptide was identified as NGIWYamide. This peptide induced in vitro germinal vesicle breakdown and ovulation of fully-grown oocytes at less than 1 pM and in vivo spawning at 10 nM. A synthetic derivative of the pentapeptide, NGLWYamide, was 10-100 times more potent compared to the natural NGIWYamide. The heptapeptide was less potent, inducing ovulation at 1 μM. NGIWYamide and NGLWYamide induced a characteristic spawning behavior when injected into sexually matured individuals. Mature eggs artificially spawned were fertilized, and developed normally and metamorphosed into young sea cucumbers. The details of the production and the mechanism of action of NGIWYamide are still unclear, but the high biopotency of the peptide will aid understanding of the neuronal and hormonal control of reproduction of sea cucumber.     

Interaction of sea cucumber saponins with multilamellar liposomes

The effect of three sea cucumber saponins, echinoside A, bivittoside D and holothurin A, on multilamellar liposomes was investigated. An ideal osmotic behavior of liposomes was described as a linear relationship between the reciprocal $\text{3}\text{2}\text{s}$ power of absorbance at 450 nm and the osmotic gradient across the membrane. Sea cucumber saponins at concentrations below critical micelle concentration (CMC) disturbed this linear relationship in liposomes composed of egg phosphatidylcholine, phosphatidic acid and cholesterol. Cholesterol-free liposomes were not susceptible to these saponins. Results of optical measurements were consistent with those of transmission electron microscopy, which showed saponin-induced changes in liposomal structure. The lytic activity of sea cucumber saponins on liposomes depended on their chemical structure.These results suggest that sea cucumber saponins as monomers can interact with liposomes and that cholesterol serve as a principal binding site for the sea cucumber saponins.