NISCAIR Online Periodicals Repository Collection: IJBB Vol.38(5) [October 2001]

Role of positive charge of lysine residue on ribosome-inactivating property of gelonin

Title: Role of positive charge of lysine residue on ribosome-inactivating property of gelonin

Authors: Singh, Ranjit C; Alam, Anis; Singh, Vinod

Abstract: The report that gelonin cross-linked with monoclonal antibodies with the use of 2-iminothiolane (2-IT) exhibited higher cytotoxicity than the conjugates prepared with the use of N-succinimidyl-3-(2-pyridylthio) propionate (SPDP) alone, has prompted us to investigate the effect of ɛ-NH₂ group modification with 2-IT on the ribosome-inactivating property (RIP) of gelonin. The purified gelonin was modified with 2-IT at a different molar ratio and their effects on immunoreactivity and ribosome- inactivating property were compared with those of N-succinimidyl 6-[3-(2-pyridyldithio) propionamido ] hexanoate (long chain-SPDP) and SPDP modified gelonin derivatives. Modification of single amino group with 2-IT results in about 25-50% inhibition of immunoreactivity and 60-70% loss of protein synthesis inhibition activity. Modification of 2-3 amino groups further hampers both immunoreactivity and protein synthesis inhibition property of gelonin. Both the long chain-SPDP with SPDP modifications showed more pronounced effects on immunoreactivity and RIP activity as compared to the similar ratio of 2-IT modification(s). It may, therefore, be concluded that the positive charge plays an important role in the immunological as well as the protein synthesis inhibitory effect of gelonin .

Page(s): 309-312

An alternative approach for screening active Bam HI variants: Overexpression in T-7 RNA polymerase based system

Title: An alternative approach for screening active Bam HI variants: Overexpression in T-7 RNA polymerase based system

Authors: Acharya, Asha S; Roy, Kunal B

Abstract: The type II restriction endonuclease. BamHI , has been overexpressed in E. coli by cloning the BamHI gene in frame with an E. coli Ribosome Binding Site (RBS) under the T7 promoter of an E. coli expression vector pRSET A. The expression level of BamHI endonuclease using this construct was found to be higher than that reported of the overexpressing clone pAEK 14. Our overexpressing clone, pAABRw in BL21 cell in presence of BamHI methylase in pMAP6 following induction with IPTG yields about 9.2x10⁶ units per gram wet cell paste. In vivo activity of the recombinant endonuclease could be confirmed by the SOS induction assay in JH 139 cells even in the absence of T7 polymerase and cognate BamHI methylase because of leaky expression in E. coli. This provides an alternate way to screen the active endonuclease and its variants.

Page(s): 303-308

Mercury induced modifications in the stereochemistry of the active site through Cys-73 in a serine protease - Crystal structure of the complex of a partially modified proteinase K with mercury at 1.8 Å resolution

Title: Mercury induced modifications in the stereochemistry of the active site through Cys-73 in a serine protease - Crystal structure of the complex of a partially modified proteinase K with mercury at 1.8 Å resolution

Authors: Gourinath, S; Degenhardt, M; Eschenburg, S; Moore, K; Delucas, L J; Betzel, Ch; Singh, T P

Abstract: Proteinese K(PK) isolated from Tritirachium album Limber was crystallized with HgCl₂ in excess. under microgravity conditions. The intensity data were collected at 4°C to 1.8 Ǻ resolution and the final R-factor after refinement for all the reflections was 0. 164. Mercury has been found at two sites with partial occupancies (0.4 and 0.6) which are at distances of 2.48 Ǻ and 2.58 Ǻ respectively from Cys-73 Sγ. The Cys-73 in the enzyme structure is located close to the active site residue. His-69. This region is completely buri ed and is not accessible to the solvent. It is rather tightly packed. Therefore. the binding of mercury distorts the stereochemistry of the neighbouring residues including those be longing to the catalytic tri ad. As a result of this. the Oγ of Ser-224 is displaced by 0.6 Ǻ which causes the inactivation of proteinase K by increasing the H-bond distance to 3.7 Ǻ between Ser-224 Oγ and His-69 Nε2.

Page(s): 298-302

Molecular modelling of epitope presentation using membrane protein OmpC

Title: Molecular modelling of epitope presentation using membrane protein OmpC

Authors: Sujatha, S; Arockiasamy, A; Krishnaswamy, S; Usha, R

Abstract: Three-dimensional models of the chimeric S. typhi OmpC protein carrying an epitope from rotavirus VP4 capsid protein on either of two exposed loops ( fourth and sixth) were constructed separately, using computer-aided homology modelling. The theoretical model of S. typhi OmpC was used as a template. The monomers were initially energy minimized. The trimers were generated for both ihe chimeric S. typhi OmpC proteins and the structures were optimized after several cycles of minimization. The surface accessibility calculations for the resulting models show that epitope recognition should be more effective in the fourth loop than in the sixth loop. in accordance with the experimental results on the immunogenic nature of the rotaviral epitope inserted into the two putative loops of S. typhi OmpC.

Page(s): 294-297