Characterization of the calcium-binding contractile protein centrin from Tetraselmis striata (Pleurastrophyceae).

Centrin is a major protein of the contractile striated flagellar roots of the green alga Tetraselmis striata. We present a newly modified procedure for the preparation of centrin in sufficient quantity and purity to allow for detailed biochemical characterization. We establish that centrin purified by differential solubility, followed by phenyl-Sepharose and DEAE-Sephacel chromatography is identical with the protein extracted directly from striated flagellar roots with regard to molecular weight, isoelectric point, and calcium-dependent behavior in SDS-PAGE. We also compare the biochemical properties of purified centrin with calmodulin isolated from Tetraselmis and calmodulin isolated from mammalian brain. Centrin can be fully distinguished from either algal or mammalian calmodulin on the basis of molecular weight, isoelectric point, calcium-dependent behavior in SDS-PAGE, proteolytic peptide maps, amino acid composition, ability to activate bovine brain phosphodiesterase, and reactivity with specific antibodies.     

Flagellar membrane proteins of Tetraselmis striata butcher (Chlorophyta)

Highly purified flagella of the green alga Tetraselmis striata (Chlorophyta) were extracted by Triton X-114 phase partitioning. SDS-PAGE analysis revealed that most proteins were present in the aqueous phase, only two prominent flagellar membrane proteins (fmp) of apparent molecular weight 145 and 57 kDa (fmp145 and fmp57) were enriched in the detergent phase. Fmp145 was purified by gel permeation chromatography. Glycosidase treatment in combination with lectin blot analysis showed that fmp145 is a glycoprotein containing 3-5 N-glycans of the high mannose and/or hybrid type. A polyclonal antibody (anti-fmp136) was raised against the deglycosylated form of fmp145 and used to localize fmp145 by immunofluorescence and immunoelectron microscopy. Immunogold labeling showed fmp145 to be present between the scale layers and the flagellar membrane. During flagellar regeneration fmp145 is incorporated evenly and rapidly into the newly developing flagella. Anti-fmp136 specifically cross-reacted with flagella of only a subgroup of Tetraselmis strains characterized by a specific flagellar hair type (type II according to Marin et al. 1993) and thus could be a useful immunomarker for the identification of Tetraselmis strains by fluorescence microscopy.     

The Assimilation of Nitrate-nitrogen by Nitrogen-starved Cells of Platymonas (Tetraselmis) striata (Prasinophyceae)

Nitrate refeeding of nitrogen-starved cells of Platymonas striataresulted in approximately a doubling of average cellular nitrogenwithin 24 h. All the nitrate-nitrogen removed from the culturemedium could be accounted for as non-nitrate nitrogen withincells. Thus no significantly sized nitrate pool existed in Platymonasstriata and no assimilated nitrogen was lost from the cellsto the medium over the 48 h period studied. The slight fallin average cellular nitrogen which occurred from 24 to 36 hcould be attributed to cell division. The majority (70–80per cent) of the assimilated nitrate was recovered in the trichloroaceticacid (TCA)-insoluble fraction. There was some increase in thepercentage of nitrogen found in the TCA-soluble fraction duringthe period of most rapid nitrate assimilation (0–24 h).This presumably reflects an inability of the cells to assimilatelow-molecular-weight metabolic intermediates into macromoleculesat the same rate at which they were being formed. The majorityof the TCA-soluble fraction could be accounted for in termsof amino acids, purine and pyrimidine bases and ammonia. Cellswith adequate nitrogen nutrition seemed to maintain amino acidand purine + pyrimidine base nitrogen pools of about 0.8–0.9and 0.3–0.4 pg per average cell respectively. Algal amino acids, algal purine and pyrimidine bases, algal ammonia     

Antibodies against the Calcium-Binding Protein: Calsequestrin from Streptanthus tortuosus (Brassicaceae)

Plant microsomes contain a protein clearly related to a calcium-binding protein, calsequestrin, originally found in the sarcoplasmic reticulum of muscle cells, responsible for the rapid release and uptake of Ca²⁺ within the cells. The location and role of calsequestrin in plant cells is unknown. To generate monoclonal antibodies specific to plant calsequestrin, mice were immunized with a microsomal fraction from cultured cells of Streptanthus tortuosus (Brassicaceae). Two clones cross-reacted with one protein band with a molecular weight equal to that of calsequestrin (57 kilodaltons) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. This band is able to bind ⁴⁵Ca²⁺ and can be recognized by a polyclonal antibody against the canine cardiac muscle calsequestrin. Rabbit skeletal muscle calsequestrin cross-reacted with the plant monoclonal antibodies. The plant monoclonal antibodies generated here are specific to calsequestrin protein.     

Calcium-Binding Capacity of Centrin2 Is Required for Linear POC5 Assembly but Not for Nucleotide Excision Repair

Centrosomes, the principal microtubule-organising centres in animal cells, contain centrins, small, conserved calcium-binding proteins unique to eukaryotes. Centrin2 binds to xeroderma pigmentosum group C protein (XPC), stabilising it, and its presence slightly increases nucleotide excision repair (NER) activity in vitro. In previous work, we deleted all three centrin isoforms present in chicken DT40 cells and observed delayed repair of UV-induced DNA lesions, but no centrosome abnormalities. Here, we explore how centrin2 controls NER. In the centrin null cells, we expressed centrin2 mutants that cannot bind calcium or that lack sites for phosphorylation by regulatory kinases. Expression of any of these mutants restored the UV sensitivity of centrin null cells to normal as effectively as expression of wild-type centrin. However, calcium-binding-deficient and T118A mutants showed greatly compromised localisation to centrosomes. XPC recruitment to laser-induced UV-like lesions was only slightly slower in centrin-deficient cells than in controls, and levels of XPC and its partner HRAD23B were unaffected by centrin deficiency. Interestingly, we found that overexpression of the centrin interactor POC5 leads to the assembly of linear, centrin-dependent structures that recruit other centrosomal proteins such as PCM-1 and NEDD1. Together, these observations suggest that assembly of centrins into complex structures requires calcium binding capacity, but that such assembly is not required for centrin activity in NER.     

Cloning of cDNA Sequences Encoding the Calcium-Binding Protein, Calmodulin, from Barley (Hordeum vulgare L.)

Full- and partial-length cDNAs encoding calmodulin mRNA have been cloned and sequenced from barley (Hordeum vulgare L.). Barley leaf mRNA, size-fractionated in sucrose density gradients, was used to synthesize double-stranded cDNA. The cDNA was cloned in λgt10 and screened with a synthetic, 14-nucleotide oligonucleotide probe, which was designed using the predicted coding sequences of the carboxy termini of spinach and wheat calmodulin proteins. The primary structure of barley calmodulin, predicted from DNA sequencing experiments, consists of 148 amino acids and differs from that of wheat calmodulin in only three positions. In two of the three positions, the amino acid changes are conservative, while the third change consists of an apparent deletion/insertion. The overall nucleotide sequence similarity between the amino acid coding regions of barley and vertebrate calmodulin mRNAs is approximately 77%. However, a region encoding 11 amino acids of the second Ca²⁺-binding domain is very highly conserved at the nucleotide level compared with the rest of the coding sequences (94% sequence identity between barley and chicken calmodulin mRNAs). Genomic Southern blots reveal that barley calmodulin is encoded by a single copy gene. This gene is expressed as a single size class of mRNA in all tissues of 7-day-old barley seedlings. In addition, these analyses indicate that a barley calmodulin cDNA coding region subclone is suitable as a probe for isolating calmodulin genes from other plants.     

Calcium-Binding Proteins in Pretectum of Turtles (Immunohistochemical Study)

An immunohistochemical study was carried out to reveal distribution of immunoreactivity (IR) against Ca-binding proteins (CaBPr): calbindin (Calb), calretinin (Calr), and parvalbumin (Parv) in pretectum of the Central Asian terrestrial turtles (Testudo horsfieldi) and the pond turtles (Emys orbicularis). A wide distribution of CaBPr-IR neurons and terminals with a significant overlapping was established in most of pretectal nuclei (Lm, Pd, Pe, Po, and ndCP) with some differences in density of IR elements for various proteins. The alternative distribution of Parv-IR (a high density of IR neurons) and Calb/Calr-IR (a most low content of IR neurons and terminals) was found in the non-retinorecipient nucleus Ptv. Comparison of data obtained in turtles with literature data obtained in other species of reptiles and mammals has revealed a significant variability in the CaBPr distribution in some, presumably homologous, pretectal nuclei among various representatives of amniotes. The conclusion is made that in the process of evolution of amniotes, apart from conservatism of CaBPr-IR characteristics, there occurred their plastic rearrangements due to morpho-functional specialization of pretectal nuclei.     

The Heparin-Binding Activity of Secreted Modular Calcium-Binding Protein 1 (SMOC-1) Modulates Its Cell Adhesion Properties

Secreted modular calcium-binding proteins 1 and 2 (SMOC-1 and SMOC-1) are extracellular calcium- binding proteins belonging to the BM-40 family of proteins. In this work we have identified a highly basic region in the extracellular calcium-binding (EC) domain of the SMOC-1 similar to other known glycosaminoglycan-binding motifs. Size-exclusion chromatography shows that full length SMOC-1 as well as its C-terminal EC domain alone bind heparin and heparan sulfate, but not the related chondroitin sulfate or dermatan sulfate glycosaminoglycans. Intrinsic tryptophan fluorescence measurements were used to quantify the binding of heparin to full length SMOC-1 and the EC domain alone. The calculated equilibrium dissociation constants were in the lower micromolar range. The binding site consists of two antiparallel alpha helices and mutagenesis experiments have shown that heparin-binding residues in both helices must be replaced in order to abolish heparin binding. Furthermore, we show that the SMOC-1 EC domain, like the SMOC-2 EC domain, supports the adhesion of epithelial HaCaT cells. Heparin-binding impaired mutants failed to support S1EC-mediated cell adhesion and together with the observation that S1EC in complex with soluble heparin attenuated cell adhesion we conclude that a functional and accessible S1EC heparin-binding site mediates adhesion of epithelial cells to SMOC-1.     

Characterization of the Structure and Intermolecular Interactions between the Connexin 32 Carboxyl-terminal Domain and the Protein Partners Synapse-associated Protein 97 and Calmodulin

In Schwann cells, connexin 32 (Cx32) can oligomerize to form intracellular gap junction channels facilitating a shorter pathway for metabolite diffusion across the layers of the myelin sheath. The mechanisms of Cx32 intracellular channel regulation have not been clearly defined. However, Ca(2+), pH, and the phosphorylation state can regulate Cx32 gap junction channels, in addition to the direct interaction of protein partners with the carboxyl-terminal (CT) domain. In this study, we used different biophysical methods to determine the structure and characterize the interaction of the Cx32CT domain with the protein partners synapse-associated protein 97 (SAP97) and calmodulin (CaM). Our results revealed that the Cx32CT is an intrinsically disordered protein that becomes α-helical upon binding CaM. We identified the GUK domain as the minimal SAP97 region necessary for the Cx32CT interaction. The Cx32CT residues affected by the binding of CaM and the SAP97 GUK domain were determined as well as the dissociation constants for these interactions. We characterized three Cx32CT Charcot-Marie-Tooth disease mutants (R219H, R230C, and F235C) and identified that whereas they all formed functional channels, they all showed reduced binding affinity for SAP97 and CaM. Additionally, we report that in RT4-D6P2T rat schwannoma cells, Cx32 is differentially phosphorylated and exists in a complex with SAP97 and CaM. Our studies support the importance of protein-protein interactions in the regulation of Cx32 gap junction channels and myelin homeostasis.     

Calcium binding and conformational properties of calmodulin complexed with peptides derived from myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP)

The myristoylated alanine-rich C kinase substrate (MARCKS) and the MARCKS-related protein (MRP) are members of a distinct family of protein ki-nase C (PKC) substrates that bind calmodulin (CaM) in a manner regulated by Ca²⁺ and phosphorylation by PKC. The CaM binding region overlaps with the PKC phosphorylation sites, suggesting a potential coupling between Ca²⁺-CaM signalling and PKC-mediated phosphorylation cascades. We have studied Ca²⁺ binding of CaM complexed with CaM binding peptides from MARCKS and MRP using flow dialysis, NMR and circular dichroism (CD) spectroscopy. The wild-type MARCKS and MRP peptides induced significant increases in the Ca²⁺ affinity of CaM (pCa 6.1 and 5.8, respectively, compared to 5.2, for CaM in the absence of bound peptides), whereas a modified MARCKS peptide, in which the four serine residues susceptible to phosphorylation in the wild-type sequence have been replaced with aspartate residues to mimic phosphorylation, had smaller effect (pCa 5.6). These results are consistent with the notions that phosphorylation of MARCKS reduces its binding affinity for CaM and that the CaM binding affinity of the peptides is coupled to the Ca²⁺ affinity of CaM. All three MARCKS/MRP peptides perturbed the backbone NMR resonances of residues in both the N- and C-terminal domains of CaM and, in addition, the wild-type MARCKS and the MRP peptides induced strong positive cooperativity in Ca²⁺ binding by CaM, suggesting that the peptides interact with the amino- and carboxy-terminal domains of CaM simultaneously. NMR analysis of the Ca²⁺-CaM-MRP peptide complex, as well as CD measurements of Ca²⁺-CaM in the presence and absence of MARCKS/MRP peptides suggest that the peptide bound to CaM is non-helical, in contrast to the α-helical conformation found in the CaM binding regions of myosin light-chain kinase and CaM-dependent protein kinase II. The adaptation of the CaM molecule for binding the peptide requires disruption of its central helical linker between residues Lys-75 and Glu-82. Received: 26 September 1996 / 22 October 1996     

Purification and Characterization of a Cryoprotective Protein (Cryoprotectin) from the Leaves of Cold-Acclimated Cabbage

We have purified a protein (cryoprotectin) from the leaves of cold-acclimated cabbage (Brassica oleracea L.) that protects thylakoids from nonacclimated spinach (Spinacia oleracea L.) against freeze-thaw damage. The procedure involves precipitations by heat, ammonium sulfate, and the glycosaminoglycan heparin and column chromatography on Polyamide 6 and a C18 reverse-phase matrix. After reverse-phase chromatography we obtained a single band of an apparent molecular mass of 7 kD when fractions that showed cryoprotective activity were analyzed by sodium dodecyl sulfate gel electrophoresis and silver staining. Gel-filtration experiments confirmed that the active protein is a monomer of 7 kD native molecular mass. This 7-kD protein could be purified only from cold-acclimated cabbage, but not from plants grown under nonacclimating conditions. Using peroxidase-labeled lectins, we show that cryoprotectin is a glycoprotein and that the saccharide moiety contains [alpha]1-3-linked fucose.     

Biochemical and Bioinformatic Characterization of Type II Metacaspase Protein (TaeMCAII) from Wheat

Plant Molecular Biology Reporter - The biochemical analysis and homology modeling of a tertiary structure of a cereal type II metacaspase protein from wheat (Triticum aestivum), TaeMCAII, are...     

Functional Characterization of Osmotically Inducible Protein C (MG_427) from Mycoplasma genitalium

Mycoplasma genitalium is the smallest self-replicating bacterium and an important human pathogen responsible for a range of urogenital infections and pathologies. Due to its limited genome size, many genes conserved in other bacteria are missing in M. genitalium. Genes encoding catalase and superoxide dismutase are absent, and how this pathogen overcomes oxidative stress remains poorly understood. In this study, we characterized MG_427, a homolog of the conserved osmC, which encodes hydroperoxide peroxidase, shown to protect bacteria against oxidative stress. We found that recombinant MG_427 protein reduced organic and inorganic peroxide substrates. Also, we showed that a deletion mutant of MG_427 was highly sensitive to killing by tert-butyl hydroperoxide and H₂O₂ compared to the sensitivity of the wild type. Further, the fully complemented mutant strain reversed its oxidative sensitivity. Examination of the expression pattern of MG_427 during osmotic shock, oxidative stress, and other stress conditions revealed its lack of induction, distinguishing MG_427 from other previously characterized osmC genes.     

Effect of Zinc on Calmodulin-Stimulated Protein Kinase II and Protein Phosphorylation in Rat Cerebral Cortex

The effect of increasing concentrations of Zn2+ (1 microM-5 mM) on protein phosphorylation was investigated in cytosol (S3) and crude synaptic plasma membrane (P2-M) fractions from rat cerebral cortex and purified calmodulin-stimulated protein kinase II (CMK II). Zn2+ was found to be a potent inhibitor of both protein kinase and protein phosphatase activities, with highly specific effects on CMK II. Only one phosphoprotein band (40 kDa in P2-M phosphorylated under basal conditions) was unaffected by addition of Zn2+. The vast majority of phosphoprotein bands in both basal and calcium/calmodulin-stimulated conditions showed a dose-dependent inhibition of phosphorylation, which varied with individual phosphoproteins. Two basal phosphoprotein bands (58 and 66 kDa in S3) showed a significant stimulation of phosphorylation at 100 microM Zn2+ with decreased stimulation at higher concentrations, which was absent by 5 mM Zn2+. A few Ca2+/calmodulin-stimulated phosphoproteins in P2-M and S3 showed biphasic behavior; inhibition at less than 100 microM Zn2+ and stimulation by millimolar concentrations of Zn2+ in the presence or absence of added Ca2+/calmodulin. The two major phosphoproteins in this group were identified as the alpha and beta subunits of CMK II. Using purified enzyme, Zn2+ was shown to have two direct effects on CMK II: an inhibition of Ca2+/calmodulin-stimulated autophosphorylation and substrate phosphorylation activity at low concentrations and the creation of a new Zn(2+)-stimulated, Ca2+/calmodulin-independent activity at concentrations of greater than 100 microM that produces a redistribution of activity biased toward autophosphorylation and an alpha subunit with an altered mobility on sodium dodecyl sulfate-containing gels.     

Physical map and gene organization of the mitochondrial genome from the unicellular green alga Platymonas (Tetraselmis) subcordiformis (Prasinophyceae)

the entire mitochondrial genome (mt genome) of the unicellular green alga Platymonas subcordiformis (synonym Tetraselmis subcordiformis; Prasinophyceae) was cloned and a physical map for the four restriction enzymes Hind III, Eco RI, Bgl II and Xba I was constructed. The mt genome of P. subcordiformis is a 42.8 kb circular molecule, coding for at least 23 genes. Hybridization and sequence analysis revealed the presence of a ca. 1.5 kb inverted repeat on the mt genome of P. subcordiformis. Phylogenetic analyses based on sequences of several coxI genes were carried out. Our data indicate that mitochondria from P. subcordiformis and from land plants form a natural, monophyletic group.     

Comparison of the 24 kDa Flagellar Calcium-Binding Protein cDNA of Two Strains of Trypanosoma cruzi

ABSTRACT. DNA sequences encoding the 24 kDa flagellar calcium binding protein (FCaBP) of two strains of Trypanosoma cruzi were found to differ at fourteen positions, six of which result in amino acid differences. Four of the amino acid differences are located within the calcium-binding domains of FCaBP; however, none is predicted to affect the calcium-binding ability of the protein. Chromosomes harboring the FCaBP gene clusters differ in size among T. cruzi strains.     

Purification and Characterization of a 70-Kilodalton Polyadenylate-Binding Protein from Pea (Pisum sativum)

A polyadenylate-binding protein (PABP) was purified from cell-free extracts prepared from pea seedlings (Pisum sativum) by ammonium sulfate precipitation and Affi-Gel Blue and polyadenylate-Sepharose 4B affinity chromatography. The final preparation from polyadenylate-Sepharose 4B columns contained a single 70-kilodalton polypeptide with high polyadenylate-binding activity. The purified protein was active over a broad range of ionic strengths and showed temperature and pH optima of 37°C and pH 6.5, respectively. Specificity studies indicated that the pea PABP was most active with polyadenylic acids, showed some activity with polyguanylic acid, and did not bind to polycytidylic acid. Moreover, longer polyadenylate molecules were bound more effectively than shorter ones. Because these properties are similar to PABPs isolated from other sources, we conclude that we have identified, purified, and characterized a plant PABP analogous to those described in yeast and animal systems.     

High yield mixotrophic cultures of the marine microalga Tetraselmis suecica (Kylin) Butcher (Prasinophyceae)

The effects of three organic compounds were tested on one of the most used marine micro-algae in the aquaculture of molluscs and crustaceans, Tetraselmis suecica. Studies were made in axenic conditions with yeast extract, peptone and glucose added to the culture medium, each alone, in combinations of two or all together. Medium without any organic compound was used for the control. Cultures containing yeast extract grew best, reaching maximum cell density of 3.79 × 10⁶ and 3.84 × 10⁶ cells ml⁻¹. The organic carbon source affected the biochemical composition. The components most affected were the carbohydrates, with values between 6.5 pg cell⁻¹ in control cultures and 48.5 pg cell⁻¹ in glucose cultures. Protein content ranged between 27.5 pg cell⁻¹ in control cultures and 88.6 pg cell⁻¹ in yeast + glucose + peptone cultures. The lipid content changed little. Maximum protein yields were reached in cultures with yeast + glucose and with yeast - glucose - peptone, with values of 24.6 and 28.2 mg 1⁻¹ d⁻¹, respectively. These values are 22 and 25 times those in control cultures. A maximum carbohydrate yield of 7.9 mg carbohydrate per litre per day was obtained in yeast + glucose + peptone cultures, 27 times that in the control cultures. The maximum lipid yield was obtained with yeast + glucose + peptone and yeast + glucose. Maximum energy values were 308 kcal 1⁻ in yeast extract - glucose - peptone cultures and 279 kcal 1⁻¹ in yeast extract + glucose cultures. Gross energy values in control cultures were 24.5 kcal 1⁻¹, but peptone cultures presented the minimum energy value, 22 kcal 1⁻¹. The yeast extract: glucose ratio in the culture medium was optimized. A ratio 2:1 produced the best yields in cells, protein, carbohydrate and gross energy.     

Ergopeptine-Sensitive Calcium-Dependent Protein Phosphorylation System in the Brain

We studied a protein phosphorylation system that is regulated by the dopamine-mimetic ergot bromocriptine. Bromocriptine was found to inhibit selectively the endogenous phosphorylation of a threonine residue(s) in 50,000- and 60,000-dalton proteins in a synaptosome fraction. The bromocriptine-sensitive phosphorylation is stimulated by calcium and by calmodulin, and occurs predominantly in the brain. The inhibitory effect of bromocriptine was not mimicked by 3,4-dihydroxyphenylethylamine or by any of the neurotransmitters and related agents tested, but was mimicked, although less effectively, by other ergots that contain peptide moieties. In the hippocampus, the brain region with the highest content of the 50,000- and 60,000-dalton proteins, the ergopeptine-sensitive protein phosphorylation appears to be localized to interneurons or cell bodies whose axons synapse outside the hippocampus. The results raise the possibility that some of the bromocriptine- and ergopeptine-induced pharmacological effects in the CNS may be mediated by the inhibition of the calcium/calmodulin-dependent phosphorylation of these specific proteins.