Symbiotic Association Between Symbiodinium and the Gastropod Strombus gigas: Larval Acquisition of Symbionts

The importance of the dinoflagellate Symbiodinium sp. was studied in the early life stages of the gastropod Strombus gigas. This dinoflagellate was not found in the eggs or the gelatinous mass surrounding the eggs of the mollusk; therefore, Symbiodinium is not inherited directly. To determine whether the planktonic veligers can acquire these algae from the environment, they were exposed to freshly isolated Symbiodinium from adult S. gigas (homologous). The optimal stage for Symbiodinium inoculation was found at 48 h post-hatching. Survival and growth rates of veligers and juveniles were higher when inoculated with freshly isolated Symbiodinium in conjunction with daily feeding of Isochrysis spp. Veligers inoculated with Symbiodinium freshly isolated from three host species elicited distinct responses: (1) veligers did not take up Symbiodinium isolated from the hydrozoan Millepora alcicornis suggesting that there is discrimination on contact prior to ingestion, (2) veligers did take up Symbiodinium isolated from the anemone Bartholomea annulata, but the algae did not persist in the host tissue suggesting that selection against this type took place after ingestion or that the algae did not divide in the host, and (3) veligers did take up Symbiodinium isolated from Pterogorgia anceps where it persisted and was associated with metamorphosis of the larvae. In contrast, the Symbiodinium freshly isolated from S. gigas were not associated with metamorphosis and required an inducer such as the red alga Laurencia poitei. These data present a significant advancement for the establishment of a new approach in the aquaculture of this important but declining Caribbean species.     

Organospecific responses of lupin seedlings to lead I. localization of lead ions and stress proteins

A tissue-printing technique was used to follow distribution of lead ions in different organs of lupin seedling with the histological localization of pathogenesis-related proteins designated as PR-L1 to PR-L6, which were found to be induced in lupin roots by heavy metals (Przymusiński and Gwóźdź 1999). Lead nitrate solution was supplied to the root tips and the histological distribution of the metal in lupin organs was visualized by staining with 0.6 % (w/v) of sodium rhodizonate. As the distance from the site of lead application increased, the amount of free lead ions decreased and in the petioles the metal was not detected at all. Lead ions were localized mostly in vascular bundles, which suggests that it was transported into the upper parts of seedlings with the transpiration stream. Immunohistochemical analysis of the tissue prints showed that as compared to the control lead visibly increased the accumulation of the PR proteins in roots, hypocotyls, stems and leaf petioles of the lupin seedling. The histological distribution of the PR protein differs from that of lead, and was localized in parenchymatic cells of root cortex, hypocotyl and stem. It is worth noticing that the stress protein was also observed in the leaf petioles where lead was not detected. This fact as well as marked enhancement of PR (L1–L6) proteins accumulation in lead treated seedlings and our earlier studies (Przymusiński and Gwóźdź 1994, 1999, Przymusiński et al. 1995) suggests that these proteins could be elements of plant’s defence system against both biotic and abiotic stressing factors.     

De-regulation of D-3-phosphoglycerate dehydrogenase by domain removal.

Escherichia coli 3-phosphoglycerate dehydrogenase (PGDH) catalyzes the first step in serine biosynthesis, and is allosterically inhibited by serine. Structural studies revealed a homotetramer in which the quaternary arrangement of subunits formed an elongated ellipsoid. Each subunit consisted of three domains: nucleotide, substrate and regulatory. In PGDH, extensive interactions are formed between nucleotide binding domains. A second subunit-subunit interaction occurs between regulatory domains creating an extended beta sheet. The serine-binding sites overlap this interface. In these studies, the nucleotide and substrate domains (NSDs) were subcloned to identify changes in both catalytic and physical properties upon removal of a subunit-subunit interface. The NSDs did not vary significantly from PGDH with respect to kinetic parameters with the exception that serine no longer had an effect on catalysis. Temperature dependent dynamic light scattering (DLS) revealed the NSDs aggregated > 5 degrees C before PGDH, indicating decreased stability. DLS and gel filtration studies showed that the truncated enzyme formed a tetramer. This result negated the hypothesis that the removal of the regulatory domain would create an enzyme mimic of the unregulated, closely related dimeric enzymes. Expression of the regulatory domain, to study conformational changes induced by serine binding, yielded a product that by CD spectra contained stable secondary structure. DLS and pulsed field gradient NMR studies of the regulatory domain showed the presence of higher oligomers instead of the predicted dimer. We have concluded that the removal of the regulatory domain is sufficient to eliminate serine inhibition but does not have the expected effect on the quaternary structure.     

Sequence of lamprey vitellogenin. Implications for the lipovitellin crystal structure.

The amino acid sequence of lamprey vitellogenin has been predicted from the nucleotide sequence of cloned cDNA. The sites of proteolytic cleavage that produce the lipovitellin complex from the vitellogenin have been located by comparing the N-terminal sequences of two lamprey lipovitellin polypeptides with the predicted sequence. These results also confirm that the vitellogenin sequence derived here corresponds to the lipovitellin complex for which the crystal structure has been solved previously. Predictions of secondary structure indicate that the region most likely to correspond to the large alpha-helical domain of the crystallographic model consists of vitellogenin residues 300 to 600. Similar to the lipovitellins of Xenopus laevis, lamprey lipovitellin appears to lack approximately 200 C-terminal residues that are present in vitellogenin. However, the lamprey lipovitellin differs from those of Xenopus and chicken in two respects. First, most of the serine-rich domain that is present as the phosvitin polypeptide in the lipovitellins of the higher vertebrates appears to be lost in the maturation of lamprey vitellogenin to lipovitellin. Second, the domains that constitute the large lipovitellin-1 polypeptide in Xenopus and chicken are present in two polypeptides in lamprey, owing to an additional proteolytic processing event.     

Letter: L-3-hydroxyacyl coenzyme A dehydrogenase: crystallographic properties of the pig heart enzyme

Crystals of l-3-hydroxyacyl coenzyme A dehydrogenase from pig heart that are suitable for X-ray analysis have been prepared and characterized. The enzyme is a mitochondrial protein important in the beta oxidation of fatty acids. It is of special interest because it requires coenzyme A-related molecules for its catalytic activity. For one of the crystalline forms of the enzyme, the X-ray data tend to confirm chemical observations that the molecule is a dimer of identical subunits.     

Evidence for multiple sites of ferricyanide reduction in chloroplasts.

Various sites of ferricyanide reduction were studied in spinach chloroplasts. It was found that in the presence of dibromothymoquinone a fraction of ferricyanide reduction was dibromothymoquinone sensitive, implying that ferricyanide can be reduced by photosystem I as well as photosystem II. To separate ferricyanide reduction sites in photosystem II, orthophenanthroline and dichlorophenyl dimethylurea inhibitions were compared at various pHs. It was noted that at low pH ferricyanide reduction was not completely inhibited by orothophenanthroline. At high pH's, however, inhibition of ferricyanide reduction by orthophenanthroline was complete. It was found that varying concentration of orthophenanthroline at a constant pH showed different degrees of inhibition. In the study of ferricyanide reduction by photosystem II various treatments affecting plastocyanin were performed. It was found that Tween-20 or KCN treatments which inactivated plastocyanin did not completely inactivate ferricyanide reduction. These data support the conclusion that ferricyanide accepts electrons both before and after plastoquinone in photosystem II.     

Multiconformational states in phosphoglycerate dehydrogenase

Phosphoglycerate dehydrogenase (PGDH) catalyzes the first step in the serine biosynthetic pathway. In lower plants and bacteria, the PGDH reaction is regulated by the end-product of the pathway, serine. The regulation occurs through a V(max) mechanism with serine binding and inhibition occurring in a cooperative manner. The three-dimensional structure of the serine inhibited enzyme, determined by previous work, showed a tetrameric enzyme with 222 symmetry and an unusual overall toroidal appearance. To characterize the allosteric, cooperative effects of serine, we identified W139G PGDH as an enzymatically active mutant responsive to serine but not in a cooperative manner. The position of W139 near a subunit interface and the active site cleft suggested that this residue is a key player in relaying allosteric effects. The 2.09 A crystal structure of W139G-PGDH, determined in the absence of serine, revealed major quaternary and tertiary structural changes. Contrary to the wildtype enzyme where residues encompassing residue 139 formed extensive intersubunit contacts, the corresponding residues in the mutant were conformationally flexible. Within each of the three-domain subunits, one domain has rotated approximately 42 degrees relative to the other two. The resulting quaternary structure is now in a novel conformation creating new subunit-to-subunit contacts and illustrates the unusual flexibility in this V(max) regulated enzyme. Although changes at the regulatory domain interface have implications in other enzymes containing a similar regulatory or ACT domain, the serine binding site in W139G PGDH is essentially unchanged from the wildtype enzyme. The structural and previous biochemical characterization of W139G PGDH suggests that the allosteric regulation of PGDH is mediated not only by changes occurring at the ACT domain interface but also by conformational changes at the interface encompassing residue W139.     

Organelle and translocatable forms of glyoxysomal malate dehydrogenase. The effect of the N-terminal presequence

Many organelle enzymes coded for by nuclear genes have N-terminal sequences, which directs them into the organelle (precursor) and are removed upon import (mature). The experiments described below characterize the differences between the precursor and mature forms of watermelon glyoxysomal malate dehydrogenase. Using recombinant protein methods, the precursor (p-gMDH) and mature (gMDH) forms were purified to homogeneity using Ni2+-NTA affinity chromatography. Gel filtration and dynamic light scattering have shown both gMDH and p-gMDH to be dimers in solution with p-gMDH having a correspondingly higher molecular weight. p-gMDH also exhibited a smaller translational diffusion coefficient (D(t)) at temperatures between 4 and 32 degrees C resulting from the extra amino acids on the N-terminal. Differential scanning calorimetry described marked differences in the unfolding properties of the two proteins with p-gMDH showing additional temperature dependent transitions. In addition, some differences were found in the steady state kinetic constants and the pH dependence of the K(m) for oxaloacetate. Both the organelle-precursor and the mature form of this glyoxysomal enzyme were crystallized under identical conditions. The crystal structure of p-gMDH, the first structure of a cleavable and translocatable protein, was solved to a resolution of 2.55 A. GMDH is the first glyoxysomal MDH structure and was solved to a resolution of 2.50 A. A comparison of the two structures shows that there are few visible tertiary or quaternary structural differences between corresponding elements of p-gMDH, gMDH and other MDHs. Maps from both the mature and translocatable proteins lack significant electron density prior to G44. While no portion of the translocation sequences from either monomer in the biological dimer was visible, all of the other solution properties indicated measurable effects of the additional residues at the N-terminal.     

Mutations of fumarase that distinguish between the active site and a nearby dicarboxylic acid binding site.

Two mutant forms of fumarase C from E. coli have been made using PCR and recombinant DNA. The recombinant form of the protein included a histidine arm on the C-terminal facilitating purification. Based on earlier studies, two different carboxylic acid binding sites, labeled A- and B-, were observed in crystal structures of the wild type and inhibited forms of the enzyme. A histidine at each of the sites was mutated to an asparagine. H188N at the A-site resulted in a large decrease in specific activity, while the H129N mutation at the B-site had essentially no effect. From the results, we conclude that the A-site is indeed the active site, and a dual role for H188 as a potential catalytic base is proposed. Crystal structures of the two mutant proteins produced some unexpected results. Both mutations reduced the affinity for the carboxylic acids at their respective sites. The H129N mutant should be particularly useful in future kinetic studies because it sterically blocks the B-site with the carboxyamide of asparagine assuming the position of the ligand's carboxylate. In the H188N mutation at the active site, the new asparagine side chain still interacts with an active site water that appears to have moved slightly as a result of the mutation.