Cellular localization and biochemical characterization of a novel calcium-dependent protein kinase from tobacco

By screening tobacco cDNA library with MCK1 as a probe, we isolated a cDNA clone NtCPK5 （accession number AY971376）, which encodes a typical calcium-dependent protein kinase. Sequence analyses indicated that NtCPK5 is related to both CPKs and CRKs superfamilies and has all of the three conserved domains of CPKs. The biochemical activity of NtCPK5 was calcium-dependent. NtCPK5 had Vmax and Km of 526nmol/min/mg and 210μg/ml respectively with calf thymus histone （fraction Ⅲ, abbreviated to histone Ⅲs） as substrate. For substrate syntide-2, NtCPK5 showed a higher Vmax of 2008 nmol/min/mg and a lower Km of 30μM. The K0.5 of calcium activation was 0.04μM or 0.06μM for histone Ⅲs or syntide-2 respectively. The putative myristoylation and palmitoylation consensus sequence of NtCPK5 suggests that it could be a membrane-anchoring protein. Indeed, our transient expression experiments with wild type and mutant forms of NtCPK5/GFP fusion proteins showed that NtCPK5 was localized to the plasma membrane of onion epidermal cells and that the localization required the N-terminal acylation sites of NtCPK5/GFP. Taking together, our data have demonstrated the biochemical characteristics of a novel protein NtCPK5 and its subcellular localization as a membrane-anchoring protein.     

ALG-2 interacts with the amino-terminal domain of annexin XI in a Ca(2+)-dependent manner

The apoptosis-linked protein ALG-2 is a Ca(2+)-binding protein that belongs to the penta-EF-hand protein family. ALG-2 forms a homodimer, a heterodimer with another penta-EF-hand protein, peflin, and a complex with its interacting protein, named AIP1 or Alix. By yeast two-hybrid screening using human ALG-2 as bait, we isolated a cDNA of a novel ALG-2-interacting protein, which turned out to be annexin XI. Deletion analysis revealed that ALG-2 interacted with the N-terminal domain of annexin XI (AnxN), which has an amino acid sequence similar to that of the C-terminal region of AIP1/Alix. Using recombinant biotin-tagged ALG-2 and the glutathione S-transferase (GST) fusion protein of AnxN, the direct interaction was analyzed by an ALG-2 overlay assay and by real-time interaction analysis with a surface plasmon resonance (SPR) biosensor. The dissociation constant (K(d)) was estimated to be approximately 70 nM. The Ca(2+)-dependent fluorescence change of ALG-2 in the presence of the hydrophobicity fluorescent probe 2-p-toluidinylnaphthalene-6-sulfonate (TNS) was inhibited by mixing with GST-AnxN, suggesting that the Pro/Gly/Tyr/Ala-rich hydrophobic region in AnxN masked the Ca(2+)-dependently exposed hydrophobic surface of ALG-2.     

Degradation of skeletal muscle plasma membrane proteins by calpain

Summary Observations described here provide the first demonstration that calpain (Ca²⁺-dependent cysteine protease) can degrade proteins of skeletal muscle plasma membranes. Frog muscle plasma membrane vesicles were incubated with calpain preparations and alterations of protein composition were revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Calpain II (activated by millimolar concentrations of Ca²⁺) was isolated from frog skeletal muscle, but the activity of calpain I (activated by micromolar concentrations of Ca²⁺) was lost during attempts at fractionation. Calpain I obtained from skeletal muscle and erythrocytes of rats was tested instead, and exerted effects similar to those of frog muscle calpain on the membrane proteins. All of the calpain preparations caused striking losses of a major membrane protein of molecular mass of approximately 97 kDa, designated band c, and diminution of a thinner band of approximately 200 kDa. There were concomitant increases in 83-and 77-kDa polypeptides. These effects were absolutely dependent on the presence of free Ca²⁺, and were completely blocked by calpastatin, a specific inhibitor of calpain action. Frog muscle calpain differed only in being relatively more active at 0°C than were the calpains from rat tissues. Experimental observations suggest that calpain acts at the cytoplasmic surface of the plasma membrane.     

1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholines: influence on aggregation and [3H]serotonin release of human thrombocytes

1-O-Alkyl-2-O-acetyl-sn-glycero-3-phosphocholines (platelet activating factor, PAF) aggregate human thrombocytes in a concentration dependent fashion. After a short lag-phase, maximum aggregation is reached within 2 min. PAF releases serotonin from human thrombocytes within 1 min. Indomethacin and creatine phosphate (CP)/creatine phosphokinase (CPK) are able to inhibit the second phase of the aggregation by PAF, while xylocain reduces both the first and second phase of aggregation of human thrombocytes. Hirudine neither influences the first nor the second phase of aggregation by PAF.     

Measurement of phosphate esters in extract of fetal rat heart by automated high pressure liquid chromatography

Creatine phosphate, nucleotides and glycolytic phosphate esters were estimated in extract of beating, in situ freeze clamped, $\text{131}\text{2}$ to $\text{191}\text{2}$ day fetal rat hearts by automated phosphate ester chromatography. Creatine phosphate increased more than 4-fold to almost 9 n moles per mg. protein at $\text{191}\text{2}$ days, while ATP remained relatively constant at about 19 to 21 n moles per mg. protein. Most other nucleotides decreased as gestation advanced. ATP rather than creatine phosphate appears to be the major energy source of fetal rat heart. Except for glucose-6-phosphate, which increased, the glycolytic phosphate esters decreased only very slightly with advancing gestational age, suggesting a relatively stable basal glycolytic activity. Methodology includes correction for phosphate esters of whole blood trapped in extracts of in situ freeze clamped tissues.     

Analytical techniques for cell fractions. XXIV. Isoelectric point stadnards for two-dimensional electrophoresis.

We have investigated the two-dimensional electrophoretic behavior of a number of proteins throughout the course of carbamylation induced by heating in urea solution. Stepwise charge changes occur which are apparently commensurate with the number of free amino groups in the protein. Characteristic changes also occur in the sodium dodecyl sulfate (SDS) electrophoretic mobility (SDS molecular weight) of some heavily carbamylated proteins, implying altered structure or SDS binding. A mixture of all carbamylation intermediates of a protein provides a simple solution to the problem of internal isoelectric point standardization in the two-dimensional electrophoretic technique of P. H. O'Farrell (1975, J. Biol. Chem.250, 4007–4021).     

Schistosoma mansoni: One- and two-dimensional electrophoresis of proteins synthesized in vitro by males,females, and juveniles

Polyacrylamide gel electrophoresis coupled with fluorography is a sensitive method for visualizing individual gene products synthesized in vitro by Schistosoma mansoni (K. Atkinson and B. G. Atkinson 1980, Nature (London)283, 478–479). In vitro labelling with radioactive amino acids ensures that the proteins are of parasite origin and fluorography permits detection of minute amounts of newly synthesized, electrophoretically separated gene products. One-dimensional electrophoretic separation in polyacrylamide gels with sodium dodecyl sulphate and fluorography of juvenile and adult proteins reveal that juveniles produce most adult proteins. Although similar studies with proteins from sexed adults imply that analogous gene products are elaborated by both sexes, a number of sex-specific gene products are resolvable by more rigorous two-dimensional electrophoretic separations. The homogametic male produces 5 polypeptides not produced by the heterogametic female. Three outstanding male-specific gene products include a polypeptide with a molecular weight (MW) of 88 kilodaltons (kd) and an isoelectric point (pI) of 5.65, one with an MW of 66 kd and a pI of 5.25, and one with an MW of 58 kd and a pI of 5.25. Other, readily detectable male-specific polypeptides include one which coelectrophoreses with β-actin and one which coelectrophoreses with β-tropomyosin. The female synthesizes 4 specific polypeptides which have isoelectric points between 4.3 and 4.7, are of low molecular weight, and are resolvable only with 12% acrylamide gels. Two-dimensional electrophoresis resolves 74 major polypeptides synthesized by adult worms, and a code is presented which identifies each polypeptide by sex specificity, isoelectric point, and molecular weight.     

Rice calcium-dependent protein kinase isoforms OsCDPK2 and OsCDPK11 show different responses to light and different expression patterns during seed development

We investigated the spatial and temporal expression patterns of two rice calcium-dependent protein kinases (CDPKs), OsCDPK2 and OSCDPK11, using isoform-specific antisera. Bands of the expected molecular sizes for OsCDPK2 (59 kDa) and OsCDPK11 (61 kDa) were detected on western blots. OsCDPK2 and OsCDPK11 mRNA and protein levels increased in unison during flower development. However, at the onset of seed development, the protein expression profiles diverged significantly. OsCDPK2 protein was expressed at low levels during early seed development, but increased to high levels that were maintained in later stages (20 days after fertilisation, DAF). Conversely, OsCDPK11 protein levels were high at the beginning of seed development, but fell rapidly from 10 DAF onwards. This decrease in the level of OsCDPK11 protein was associated with the abundant synthesis of a truncated mRNA species. OsCDPK2 expression was also closely associated with light perception. OsCDPK2 protein was barely detectable in green leaves exposed to light, but levels increased sharply when plants were shifted to darkness. Initially, this increase reflected a rapid elevation in the levels of OsCDPK2 mRNA, which was normally located in the mesophyll. Conversely, OsCDPK11 mRNA and protein levels were unaffected by light. These data strongly indicate that two rice CDPK isoforms have different functions in seed development and in response to light in leaves.     

Effects of subacute treatment of ethylene glycol on serum marker enzymes and erythrocyte and tissue antioxidant defense systems and lipid peroxidation in rats

The present study was aimed to investigate the effects of ethylene glycol (EG) on serum marker enzymes, antioxidant defense systems and lipid peroxidation concentration (malondialdehyde=MDA) in various tissues of rats exposed to ethylene glycol. EG (1.25% or 2.5%) in drinking water was administered orally to rats (Sprague-Dawley albino) ad libitum for 21 days continuously. EG treatments caused different effects on the serum marker enzymes, antioxidant defense system and MDA content in various tissues of the treatment groups as compared with the controls. EG also caused a significant increase in the serum marker enzyme activities with 2.5% dosage whereas, no changes were not observed with 1.25% dosage of EG treatment. Lipid peroxidation significantly increased in all the tissues except for in the heart and stomach of rats treated with both dosages of EG. Also, the antioxidative systems were also seriously affected by EG. For example, SOD significantly decreased in the liver treated with both dosages whereas, SOD activity in the erythrocytes, kidney, heart and stomach were significantly increased and not changed in the brain with two dosages of EG. Also, while CAT activity significantly decreased in the erythrocytes, liver and kidney, the activity in the stomach significantly increased, but did not change in the brain and heart with two doses of EG. GR activity significantly decreased in the erythrocytes treated with both dosages of EG whereas GR was not affected in other tissues by EG treatment. GST activity significantly elevated in the heart and brain but did not change in the other tissues of rats treated with both dosages of EG. Meanwhile, GSH depletion in the erythrocytes of rats treated with 2.5% dosage of EG was found to be significant whereas, the level of GSH in the brain was significantly increased treated with both the dosages of EG. The observations presented led us to conclude that the administration of subacute EG promotes lipid peroxidatin content, elevates tissue damage serum marker enzymes and changes in the antioxidative systems in rats. These data, along with the determined changes suggest that EG produced substantial systemic organ toxicity in the erythrocyte, liver, brain, heart kidney and stomach during the period of a 21-day subacute exposure.