ABSTRACT
Arthropod hemocyanins (Hcs) are a family of large extracellular oxygen-transporting proteins with high molecular mass and hexameric or multi-hexameric molecular assembly. This study reports for the first time the isolation and characterization of the structure of an arthropod hemocyanin from crab Eriphia verrucosa (EvH) living in the Black Sea. Its oligomeric quaternary structure is based on different arrangements of a basic 6 × 75 kDa hexameric unit, and four of them (EvH1, EvH2, EvH3, and EvH4) were identified using ion-exchange chromatography. Subunit 3 (EvH3) shows high similarity scores (75.0, 87.5, 91.7, and 75.0 %, respectively) by comparison of the N-terminal sequence of subunit 1 from Cancer pagurus of the North Sea (Cp1), subunits 3 and 6 of Cancer magister (Cm3 and Cm6), and subunit 2 of Carcinus aestuarii (CaSS2), respectively. Moreover, a partial cDNA sequence (1309 bp) of E. verrucosa hemocyanin encoding a protein of 435 amino acids was isolated. The deduced amino acid sequence shows a high degree of similarity with subunits 3, 4, 5, and 6 of C. magister (81-84 %). Most of the hemocyanins are glycosylated, and three putative O-linkage sites were identified in the partial amino acid sequence of EvH at positions 444-446, 478-480, and 547-549, respectively. The higher stability of native Hc in comparison to its subunit EvH4 as determined by circular dichroism (CD) could be explained with the formation of a stabilizing quaternary structure.
Subject(s)
Brachyura/metabolism , Hemocyanins/chemistry , Amino Acid Sequence , Animals , Base Sequence , Binding Sites/genetics , Brachyura/genetics , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Cloning, Molecular , Female , Hemocyanins/genetics , Hemocyanins/isolation & purification , Molecular Sequence Data , Polymerase Chain Reaction , Protein Structure, Quaternary , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
The response of tobacco plants (Nicotiana tabacum L.)--non-transformed and transformed with a metallothionein gene MThis from Silene vulgaris L.--to increase cadmium supply in the nutrient solution was compared. The transgenic plants accumulated significantly more Cd both in the roots and the leaves. Visual toxicity symptoms and disturbance in water balance were correlated with Cd tissue content. Treatment with 300 microM CdCl(2) resulted in inhibition of photosynthesis and mobilization of the ascorbate-glutathione cycle. Treatment with 500 microM CdCl(2) led to irreversible damage of photosynthesis and oxidative stress. An appearance of a new peroxidase isoform and changes in the leaf polypeptide pattern were observed at the highest Cd concentration. The level of non-protein thiols gradually increased following the Cd treatment both in transgenic and non-transformed plants.
Subject(s)
Cadmium/toxicity , Nicotiana/metabolism , Plants, Genetically Modified/metabolism , Soil Pollutants/toxicity , Antioxidants/metabolism , Ascorbic Acid/metabolism , Cadmium/analysis , Cadmium Chloride/pharmacology , Chlorophyll/analysis , Fluorescence , Gene Expression , Glutathione/metabolism , Hydrogen Peroxide/analysis , Lipids/analysis , Metallothionein/genetics , Molecular Weight , Oxidative Stress/drug effects , Photosynthesis/drug effects , Plant Leaves/metabolism , Plant Proteins/analysis , Plant Roots/metabolism , Plants, Genetically Modified/drug effects , Plants, Genetically Modified/genetics , Soil Pollutants/analysis , Nicotiana/drug effects , Nicotiana/geneticsABSTRACT
A new processed pseudogene (rpL32-5C) that belongs to the mouse rpL32 multigene family has been sequenced and compared to previously characterised mammalian rpL32 family members. rpL32-5C has intact direct terminal repeats of 13 nt, a poly A tail of 10 nt and it does not contain conserved promoter elements at its 5' end. The comparison with the L32 cDNA and 4A processed pseudogene shows a deletion of 25 nt in rpL32-5C as well as several nt substitutions that lead to frameshifts and interruption of the ORF. Most of the consistent substitutions occur in the third codon position lending support to the suggestion that all rpL32 processed pseudogenes have been derived from transcripts of the functional gene.
