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Bipyramidal crystalline, spheroidal crystalline and amorphous proteins were prepared from following four seeds: white kidney, navy (Phaseolus vulgaris) beans; baby lima, large lima (Phaseolus lunatus) beans. A study of the biological properties of the proteins which exhibit the different microstructures, was carried out. The nature of tryptic inhibitory activity and alpha-amylase inhibitory activity were investigated. All protein showed both trypsin inhibitory and alpha-amylase activities; the kinetics of the alpha-amylase revealed non-competitive mechanism. The crystalline isolates showed lower trypsin inhibitory (TI) and alpha-amylase inhibitory (AI) activities than the amorphous isolates. The extents of tryptic hydrolysis (in vitro) were not related to TI indicating involvement of some other factors, in addition to trypsin inhibitory activity. Electropherograms of SDS-PAGE indicated that the major proteins of P. lunatus beans were more resistant to tryptic hydrolysis than the major fractions of the P. vulgaris. Phytate was found to have an effect on trypsin inhibition as well as on alpha-amylase inhibition; in the latter case, however, phytate complexed to protein was required for the inhibitory effect. There were no relationships between tannin content of the proteins and biological activity. Fractionation of bipyramidal crystalline and amorphous proteins by size exclusion chromatography showed that each isolate contained three fractions having approximate MW of 443,000, 200,000 and 150,000 daltons. The 200,000 MW fraction was a principal fraction. The fraction of 150,000 contained most of the trypsin inhibitory activity, alpha-amylase inhibitory activity was not detected in any of the fractions.

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This study was directed at elucidating the structural and functional characteristics of legume protein isolates with different microstructures. / Protein isolates with different microstructures (crystalline and amorphous) were prepared from four varieties of Phaseolus beans; white kidney bean and navy bean (P. vulgaris), and large lima and baby lima beans (P. lunatus). The protein isolates were subjected to tryptic hydrolysis at various time intervals, and the effect of hydrolysis on the functional properties of the protein isolates was studied, using bovine casein as control. Reversed phase HPLC with UV detection was used to generate peptide maps of the tryptic hydrolysates, and electrospray mass spectrometry was used to characterize the peptide profile of the protein hydrolysates. / There was no difference between the susceptibility of the crystalline and amorphous protein isolates to tryptic hydrolysis, however, the hydrolytic products of the crystalline isolates showed higher solubility and functional properties compared with those of the amorphous isolates. Limited hydrolysis markedly improved the functional properties of both crystalline and amorphous protein isolates. The effect of hydrolysis on the functionality of the bean protein isolates was higher compared with casein. In general the hydrolysates of the bean protein isolates showed higher functional properties than casein and its hydrolysates. / Reversed phase HPLC peptide mapping of the tryptic digests and ESI/MS of the RP-HPLC fractions showed structural and compositional difference between crystalline and amorphous protein isolates prepared from the same bean variety. The results also showed that off-line ESI/MS, and ESI/MS/MS of RP-HPLC fractions of the tryptic hydrolysates could be used for the structural characterization of the protein isolates.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 5 uppsatser.

Aeromonas calmonicida is a Gram negative rod shaped bacterium capable of causing furunculosis in salmonid fish and other chronic and inflammatory diseases in goldfish, carp and also salmonids. The surface layer of A. salmonicida, the A-layer, has been demonstrated to be a major virulence factor for the organism , and its subunit A -protein has been purified and its Structural gene vapA has been cloned. The vapA gene from A. salmonicida strain A450 was subcloned (pSC150) and expressed in Escherichia coli. Its DNA sequence was then determined to consist of 1,506 bp encoding a 502-amino acid residue protein, containing a 21- residue signal peptide and a mature protein of 50,778 Dalton. The A-protein assembled on the cell surface in the form of an S-layer was refractile to trypsin cleavage while trypsin digestion of the purified mature protein revealed a highly resistant 39,400 Dalton N-terminal fragment and a 16,700 Dalton C-terminal fragment with moderate resistance, These trypsin-resistant fragments may form distinct structural domains, consistent with three-dimensional ultrastructural observations. The plasmid pSC150 contained 62 bp of Aeromonas DNA in front of the vapA structural gene. A promoter (P2) was predicted in this region which showed sequence homology to the E. coli c70 promoter. However, prim er extension in the wild type strain A449 showed a transcriptional start site 181 bp upstream from the gene, and thus, another promoter (P1) was shown to be the major promoter. The DNA sequence coding for the untranslated leader mRNA contained two stem-loop structures, a putative small open reading frame spanning the stem-loop structures, and a palindromic sequence which overlaps the predicted ribosome binding site. Northern analyses of A449 vapA mRNA showed that incubation at 15°C produced the highest level of the transcript, and the transcript half-life was 22 m in in cells grown at 15°C compared to 11 min in cells grown at 20°C. DNA gyrase inhibitors nalidixic acid and novobiocin significantly reduced the vapA transcript level. A. salmcnicida 30°C mutants were found to produce significantly reduced levels of A-protein and some of them were shown to have the native insertion elements, ISA1 and ISA2, inserted in the vapA area. These insertion elements have been cloned and sequenced, and also identified in the wild type strains A449 and A450. ISA2 was shown to have sequence similarity to other bacterial insertion elements. Plasmid encoded vapA expression in E. coli was also affected by a downstream gene abcA, which, when deleted from the clone, significantly reduced vapA expression. This reduction could be complemented by the abcA gene carried on a second plasmid. In addition, the lipopolysaceharide (LPS) O-chain deficient phenotype of A449 mutant strain TM4, which has the abcA gene interrupted by ISA1, was also complemented by abcA. DNA sequence analysis showed that the abcA gene coded for a 308 amino acid residue protein, which was confirmed by in vivo and in vitro expression and gene fusion with lacZ, and was localized in the inner membrane fraction of E. coli. At the N-terminal part of the protein, the predicted sequence of AbcA displayed high homology with a bacterial transport protein super family, including a well conserved nucleotide binding sequence. This binding sequence was shown by site-directed mutagenesis to be required for LPS O-chain complementation in TM4. ATP binding activity was confirmed in the purified AbcA-LacZ fusion protein. A leucine zipper-basic region sequence with predominantly α-helical conformation was predicted further downstream, with leucine residues in four of the five heptad repeats and a valine residue in the remaining heptad repeat. / Graduate

Complications of rubella virus infection, including congenital rubella syndrome and the association of rubella virus with joint inflammation, emphasize the need for continued research on rubella virus. The finding that the association of rubella virus infection with joint manifestations is more pronounced with wild strains than with vaccine strains suggested the possibility of strain variation. Several different techniques have been employed in order to compare six rubella virus strains and identify variations in their structural proteins. Differences in biological activities were detected, including the extent of virus production and the ability of various cell types to support replication of rubella virus (tissue tropism). However, the strains were shown to have remarkably similar electrophoretic patterns. Variation appeared to result from alterations in glycosylation. Efforts to isolate the protein components of the two envelope glycoproteins were unsuccessful, and it was therefore not possible to localize variation to either the protein or the carbohydrate components. Future work employing more sensitive methods for examination of fine molecular structure and the correlation of these structures with biological activity will further our understanding of the pathogenesis of rubella virus infection. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

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