Lalitha R. Gowda

Arachin derived peptides as selective angiotensin I-converting enzyme (ACE) inhibitors: Structure–activity relationship

Current attention focuses on mechanisms of controlling blood pressure through the inhibition of angiotensin I-converting enzyme (ACE). Bioactive antihypertensive peptides of food origin are increasingly gaining importance as alternates to synthetic drugs in hypertension therapy. The ACE inhibitory property of an enzymatic digest of arachin, the major storage globulin of peanut (Arachis hypogaea) has been demonstrated. The ACE inhibitory activity of a tripeptide (IEY) isolated from these digests has been characterized. Five synthetic structural analogs of this peptide (IEW, IKY, IKW, IEP and IKP) were assembled and their ACE inhibitory activity evaluated. Among these, the tripeptide IKP was a potent competitive inhibitor with an IC(50) of 7+/-1 x 10(-6)M similar to that of the potent whey peptides IPP and VPP. The inhibition data of these peptide analogs have been rationalized through docking simulations using the tACE-lisinopril complex at 2A resolution (PDB: 1086). The best docking poses were located at the tACE catalytic site resembling the mode of inhibition exerted by lisinopril, the synthetic drug. The degree of inhibition by the peptides correlated with the coordination distance between the catalytic Zn(II) and the carbonyl oxygen of the peptide bond between the amino-terminal and middle residue. These studies illustrate that these peptides, like lisinopril, behave as transition state analog inhibitors and are useful in therapeutic intervention for blood pressure management.

Enhanced Hypocholesterolemic Effects of Interesterified Oils Are Mediated by Upregulating LDL Receptor and Cholesterol 7-α- Hydroxylase Gene Expression in Rats

The concentration of LDL cholesterol in plasma is strongly influenced by the amount and type of lipid in the diet. Our studies have shown that positional changes in the fatty acids in blended oil introduced using lipase-catalyzed interesterification differentially modulate circulating LDL levels in rats compared with those observed in rats given a physical blend of oils. To investigate the molecular basis of these differences, transcriptional profiling of genes involved in cholesterol homeostasis was studied after feeding rats with a semipurified diet containing 10% fat from native oils; coconut oil (CNO), rice bran oil (RBO), or sesame oil (SESO); blended (B); CNO+RBO(B) or CNO+SESO(B) and interesterified oil (I); CNO+RBO(I) or CNO+SESO(I) for 60 d. Hepatic LDL receptor (LDL-R) expression significantly increased in rats fed interesterified oils by 100-200% compared with rats fed blended oils and by 400-500% compared with rats fed CNO. Positional alteration in fatty acids of oils used in the diet induced changes in LDL-R expression, which was accompanied by parallel changes in cholesterol-7α-hydroxylase (CYP7A1) and SREBP-2 genes. This suggested that not only the fatty acid type but also its position in the TG of dietary lipids play an important role in maintaining plasma cholesterol levels by suitably modulating gene expression for LDL-R in rat liver.

Utilization of tannery fleshings: Optimization of conditions for fermenting delimed tannery fleshings using Enterococcus faecium HAB01 by response surface methodology

Conditions for fermentation of delimed tannery fleshings--to obtain higher degree of protein hydrolysis and reasonably better antioxidant activity--using Enterococcus faecium HAB01 (GenBank #FJ418568) were optimized. Three independent variables--viz., inoculum level (X1), glucose level (X2) and fermentation time (X3)--were optimized using response surface method considering degree of hydrolysis (DH; %) and total titrable acidity (TTA) as response variables. The optimized conditions were found to be 12.5% (v/w) inoculum, 17.5% (w/w) glucose and 96h of fermentation at 37+/-1 degrees C to obtain a maximum DH%. The usefulness of the predicted model was further validated by considering random combinations of the independent factors. The chemical score of the hydrolysate revealed an excess amount of essential amino acids, viz., arginine and leucine compared to reference protein. The liquor portion had relatively high antioxidant activities, indicating its potential for use as a high value feed ingredient.

Functional expression of horsegram (Dolichos biflorus) Bowman-Birk inhibitor and its self-association.

Horsegram (Dolichos biflorus), a protein-rich leguminous pulse, native to Southeast Asia and tropical Africa, contains multiple forms of Bowman-Birk inhibitors. The major Bowman-Birk inhibitor from horsegram (HGI-III) was cloned and functionally expressed in Escherichiacoli (rHGI), which moved as a dimer in solution similar to the natural inhibitor. The biochemical characterization of rHGI also points to its close resemblance with HGI-III not only in its structure but also in its inhibitory characteristics. To explore the electrostatic interactions involved in the dimerization, a site-directed mutagenesis approach was used. The role of reactive site residue K24 and the C-terminal Asp in the structure and stability of the dimer was accomplished by mutating K24 and D75/76. The mutants produced in this study confirm that the self-association of HGI-III is indeed due to the electrostatic interaction between K24 of one monomer and D75/76 of the second monomer, in agreement with our previous data. The functional expression of a Bowman-Birk inhibitor minus a fusion tag serves as a platform to study the structural and functional effects of the special pattern of seven conserved disulphide bridges.

The unique enzymatic function of field bean (Dolichos lablab)D-galactose specific lectin: a polyphenol oxidase.

The polyphenol oxidase (PPO) of field bean (Dolichos lablab) is a tetramer made up of two subunits of mass 29,000 and 31,000xa0Da. The amino acid sequence of the tryptic peptides showed approximately 90% sequence identity to the d-galactose specific legume lectins. The haemagglutinating activity of a pure and homogenous preparation of PPO measured using human erythrocytes was 1261xa0HAU mg−1 protein and was inhibited by d-galactose. Purification by galactose-sepharose chromatography also indicated that the PPO and haemagglutinating activities were associated with a single protein. Crude extracts of other legumes did not exhibit PPO activity, yet cross reacted with anti-PPO antibodies. This dual function protein with PPO and haemagglutinating activity is unique to field bean. The two activities are independent of each other occurring at different loci on the protein. These observations further evidence and strengthen the assumption that galactose specific legume lectins have enzymatic function. Both PPO and lectins are proteins that play a vital role in the defense mechanism of plants. The complementarity of these two simultaneous and independent powerful defense mechanisms exhibited by a single protein renders it a candidate gene for the development of inbuilt plant protection.

A novel catalysis by porcine pepsin in debranching guar galactomannan.

BACKGROUNDnPepsin (porcine stomach mucosa, E.C. 3.4.23.1), an acid protease catalyzes the hydrolysis (debranching) of guar galactomannan (GG), a co-polymer of mannose and galactose residues thereby showing its non-specific catalysis towards glycosidic substrates.nnnRESULTS AND CONCLUSIONSnUse of non-specific inhibitors, chemical modification agents and peptide mapping of native and GG--bound pepsin upon proteolytic digestion with Staphylococcus aureus V8 protease revealed the involvement of Asp(138) residue in the catalysis, which was confirmed by computational modelling studies.nnnGENERAL SIGNIFICANCEnHere we show a novel mode of catalysis (other than proteolysis) by porcine pepsin with a different active site residue.

Molecular engineering of a small trypsin inhibitor based on the binding loop of horsegram seed Bowman-Birk inhibitor

Context: The Bowman-Birk inhibitors (BBIs) are currently investigated with renewed interest due to their therapeutic properties in cancer and other inflammatory disease treatment. The molecular mass of the BBI is a limitation, as sufficient amounts of the inhibitor do not reach the organs outside the gastrointestinal tract when administered orally. Method: The anti-tryptic domain of HGI-III of horsegram (Dolichos biflorus) was cloned using the vector pET-20b (+) and expressed in E. coli BL21 (DE3) pLysS. Results: Kinetic analysis of this anti-tryptic peptide (recombinant trypsin inhibitory domain (rTID)) reveals that it is a potent inhibitor of trypsin and human tryptase. The Ki (3.2u2009±u20090.17u2009×u200910−8 M) establishes a very high affinity to bovine trypsin. rTID inhibited human lung tryptase (IC50 3.78u2009±u20090.23u2009×u200910−7 M). The rTID is resistant to the digestive enzymes found in humans and animals. Conclusion: These properties propagate further research on the use of rTID as a therapeutic for cancer and other related inflammatory diseases.