Biomedical subjects

P Santhoshkumar

Publications and source records attributed to P Santhoshkumar.

7 recordsLinked to original sources

PubMed2003

A peptide sequence-YSGVCHTDLHAWHGDWPLPVK [40-60]-in yeast alcohol dehydrogenase prevents the aggregation of denatured substrate proteins.

The structural and functional characteristics of a yeast alcohol dehydrogenase (ADH) peptide (YSGVCHTDLHAWHGDWPLPVK, residues 40-60) have been studied in detail. The peptide is hydrophobic in nature, binds the hydrophobic probe bis-ANS, and is mostly present in a random coil conformation. It shows chaperone-like activity by preventing dithiothreitol (DTT)-induced aggregation of insulin at 27 degrees C, oxidation-induced aggregation of gamma-crystallin at 37 degrees C, and aggregation of thermally denatured ADH and beta(L)-crystallins at 52 degrees C. However, the ADH peptide does not solubilize protein aggregates as do surfactants. Substitution of Pro for His in the ADH peptide leads to diminished anti-aggregation activity. Further, analysis of ADH incubated at 47 degrees C suggests that a significant portion of the enzyme remains as soluble inactive protein with negligible conformational change. Therefore, we propose that the residues 40-60 in native protein may be an intramolecular chaperone site of yeast ADH.

Alcohol Dehydrogenase↗

PubMed2002

Effect of trifluoroethanol on the structural and functional properties of alpha-crystallin.

Alpha crystallin is an eye lens protein with a molecular weight of approximately 800 kDa. It belongs to the class of small heat shock proteins. Besides its structural role, it is known to prevent the aggregation of beta- and gamma-crystallins and several other proteins under denaturing conditions and is thus believed to play an important role in maintaining lens transparency. In this communication, we have investigated the effect of 2,2,2-trifluoroethanol (TFE) on the structural and functional features of the native alpha-crystallin and its two constituent subunits. A conformational change occurs from the characteristic beta-sheet to the alpha-helix structure in both native alpha-crystallin and its subunits with the increase in TFE levels. Among the two subunits, alphaA-crystallin is relatively stable and upon preincubation prevents the characteristic aggregation of alphaB-crystallin at 20% and 30% (v/v) TFE. The hydrophobicity and chaperone-like activity of the crystallin subunits decrease on TFE treatment. The ability of alphaA-crystallin to bind and prevent the aggregation of alphaB-crystallin, despite a conformational change, could be important in protecting the lens from external stress. The loss in chaperone activity of alphaA-crystallin exposed to TFE and the inability of peptide chaperone--the functional site of alphaA-crystallin--to stabilize alphaB-crystallin at 20-30% TFE suggest that the site(s) involved in subunit interaction and chaperone-like function are quite distinct.

Animals↗

PubMed2001

Phe71 is essential for chaperone-like function in alpha A-crystallin.

Experiments with mini-alphaA-crystallin (KFVIFLDVKHFSPEDLTVK) showed that Phe(71) in alphaA-crystallin could be essential for the chaperone-like action of the protein (Sharma, K. K., Kumar, R. S., Kumar, G. S., and Quinn, P. T. (2000) J. Biol. Chem. 275, 3767-3771). In the present study we replaced Phe(71) in rat alphaA-crystallin with Gly by site-directed mutagenesis and then compared the structural and functional properties of the mutant protein with the wild-type protein. There were no differences in molecular size or intrinsic tryptophan fluorescence between the proteins. However, 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid interaction indicated a higher hydrophobicity for the mutant protein. Both wild-type and mutant proteins displayed similar secondary structure during far UV CD experiments. Near UV CD signal showed a slight difference in the tertiary structure around the 285-295 region for the two proteins. The mutant protein was totally inactive in suppressing the aggregation of reduced insulin, heat-denatured citrate synthase, and alcohol dehydrogenase. However, a marginal suppression of beta(L)-crystallin aggregation was observed when mutant alphaA-crystallin was included. These results suggest that Phe(71) contributes to the chaperone-like action of alphaA-crystallin. Therefore we conclude that the 70-88-region in alphaA-crystallin, identified by us earlier, is the functional chaperone site in alphaA-crystallin.

Amino Acid Sequence↗

PubMed2001

Analysis of alpha-crystallin chaperone function using restriction enzymes and citrate synthase.

PURPOSE: To compare the abilities of [alpha]A-crystallin, [alpha]B-crystallin, and mini-[alpha]A-crystallin (a synthetic peptide chaperone representing the functional unit of [alpha]A-crystallin) to protect against heat-induced inactivation of citrate synthase (CS) and restriction enzymes, SmaI and NdeI. METHODS: Restriction enzymes, SmaI and NdeI were heated at different temperatures in the presence of various amounts of molecular chaperones and tested for their ability to cleave plasmid DNA. The aggregation of CS was measured at 43 degrees C while the loss in activity was monitored at 37 degrees C in the presence of various crystallins. RESULTS: Restriction enzyme activities were protected by the crystallin subunits up to 37 degrees C for SmaI and 43 degrees C for NdeI. However, the mini-[alpha]A-crystallin was unable to protect endonuclease activity. The crystallin subunits and the peptide chaperone were able to suppress thermal aggregation of CS at 43 degrees C, but failed to stabilize its activity at 37 degrees C. CONCLUSIONS: The ability of [alpha]-crystallin subunits to stabilize denaturing proteins varies from enzyme to enzyme as evidenced by the inactivation of CS and protection of SmaI and NdeI activity in the presence of [alpha]-crystallin subunits. Additionally, our results show that there could be more than one site in [alpha]A-crystallin responsible for its chaperone-like action. By addition of crystallin subunits to restriction enzymes prior to or during storage, transport, or assay would maintain or improve their activity thereby decreasing their cost.

Animals↗

PubMed1999

In vitro sequestration of two organophosphorus homologs by the rat liver.

Bromophos (Bp) and ethylbromophos (EBp) are two structurally homologous organophosphorus insecticides (OP) which show a 24-fold difference in their toxicity to the laboratory rat (LD50--2215 and 91 mg/kg b.w., respectively). The role of rat liver in the sequestration of the OP oxons was studied based on carboxylesterase (CaE) inhibition in vitro. Bromoxon (Bo) and ethylbromoxon (EBo) were greater inhibitors of rat hepatic CaE than brain acetylcholinesterase (AChE) with IC50 values at nanomolar and picomolar levels, respectively. The capacity of the liver to sequester OPs was determined by measuring AChE inhibition pre-incubated with or without liver homogenate. AChE inhibition by Bo decreased with increasing concentration of liver tissue, whereas it was unaffected in the case of EBo. The results imply that liver tissue contains binding sites, which sequester Bo thereby reducing the number of OP molecules available to inhibit AChE. Although CaE inhibition leads to sequestration, other binding sites in the liver may have a significant role in determining the toxicity of OPs. Differential sequestration of the OPs by hepatic tissue, therefore, could be important in understanding the role of differential saturation of the target molecules, which has a bearing on differential toxicity.

Acetylcholinesterase↗

PubMed1996

Neurotoxicity and pattern of acetylcholinesterase inhibition in the brain regions of rat by bromophos and ethylbromophos.

Bromophos (Bp) and ethylbromophos (EBp) are two structurally homologous organophosphorus (OP) insecticides which show wide differences in their toxicity as well as neurotoxic symptoms in the laboratory rat. EBp is 24-fold more toxic (LD50 = 91 +/- 14 mg/kg body wt) than Bp (LD50 = 2218 +/- 195 mg/kg body wt) and only EBp produced characteristic tremors and lacrimation. In vivo cholinesterase inhibition was in the order plasma > erythrocytes > brain. Experiments with equitoxic and equimolar doses showed that EBp is a more potent anticholinesterase compound than Bp. Since IC50 values for the brain AChE were similar for both OPs, the target enzyme sensitivity was not a major factor in their differential toxicity. In vitro reactivation of serum ChE was significantly higher in the case of EBp than that of Bp. AChE in the brain regions showed differential inhibition in vivo. The brain stem AChE inhibition was least by Bp, whereas it was highest in the case of EBp. Both the OPs produced high AChE inhibition in the hippocampus. Differential inhibition of AChE in the brain regions and its consequent effects may be important factors in the differential neurotoxicity of OPs.

Animals↗

PubMedDate not supplied

Differential in vivo inhibition of the foetal and maternal brain acetylcholinesterase by bromophos in the rat.

Bromophos, an organophosphorus compound, is known to cross the placental barrier. The response of the foetal brain acetylcholinesterase (AChE) to in vivo Bromophos exposure is not known. This study measured the in vivo time-course of cholinesterase (ChE) inhibition and recovery in rat maternal serum, brain, and foetal brain after administration of a single acute oral dose of Bromophos (500 mg/kg b.w.) on Day 18 of pregnancy. ChE inhibition in all tissues started as early as 2 h and reached a maximum at 16 h post-exposure. Foetal brain AChE was inhibited least and the first to recover followed by maternal brain and serum. The in vivo ID50 values for the ChE inhibition by Bromophos were 2.02, 205.0, 952.92 mg/kg b.w. and the in vitro IC50 values were 119.12, 115.17, 112.14 microM for the maternal serum, brain, and foetal brain, respectively. The IC50 values show that maternal serum, brain, and foetal brain are equisensitive to Bromophos. However, the ID50 values suggest that they have differential in vivo sensitivity to Bromophos. The foetal brain seems to be protected against the AChE inhibition by Bromophos, probably by detoxication in the maternal, placental, and foetal compartments.

Acetylcholinesterase↗