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Indian Journal of Biochemistry & Biophysics

 

 

ISSN : 0301-1208

CODEN : IJBBBQ  

VOLUME 40

NUMBER 4

AUGUST 2003

 

CONTENTS

 

Minireview

 

N-Glycolylneuraminic acid conjugates: Implications of their absence in mammalian biochemistry

217

Daniel W Mwangi and Devi D Bansal*

 

Papers

 

Disruption of distal interactions of Arg 262 and of substrate binding to Ser 52 affect catalysis of sheep liver cytosolic serine hydroxymethyltransferase

226

Venkatakrishna Rao Jala, M Ambili, V Prakash, N Appaji Rao and H S Savithri*

 

Nimesulide affects antioxidant status during acute lung inflammation in rats

238

Kiranjit K Sohi and Krishan L Khanduja*

 

Effect of oxidative stress on the spermatogenic process and hsp70 expressions in
mice testes

246

Parminder Kaur and M P Bansal*

 

Kinetic attributes of rat liver microsomal adenosine 5’ triphosphate phosphohydrolase (ATPase)

252

Subhash D Katewa and Surendra S Katyare*

 

Protective role of selenium status on T3/T4 kinetics in rats under hyperlipidemia

260

Sanjiv Dhingra, Uma Singh and M P Bansal*

 

Effect of terminal achiral and chiral residues on the conformational behaviour of poly DZPhe and analysis of various interactions

265

Fateh S Nandel* and Harpreet Kaur

 

Notes

 

Purification and partial characterization of cytochrome c552 from Halobacterium salinarium

274

K Sreeramulu

 

Beneficial effects of tender coconut water against isoproterenol induced toxicity on heart mitochondrial activities in rats

278

P Anurag and T Rajamohan*

 

Instructions to Authors

281

 

——————

*Author for correspondence

 

 

 

Call for Paper

 

Indian Journal of Biochemistry and Biophysics (IJBB), has its own niche in the competitive field of biochemistry where there are about 460 journals published from all over the world. Started since 1964, IJBB publishes original articles in the following areas: structure-function relationships of biomolecules, biomolecular recognition, protein-protein and protein-DNA interactions; novel DNA structures and their biological implications, conformational studies, computer simulation, protein folding; enzymes structure; membrane biochemistry, antigen-antibody binding, receptors, transport, carrier proteins, drug targeting, drug design; neurochemistry, ion channels, signal transduction, cell-cell communication; glycobiology, cell cycle control; hormones, vitamins, growth factors, catalytic mechanisms, coenzymes, regulation, intermediary metabolism; ageing apoptosis, oncogenes, molecular basis of genetic diseases, disease processes, host-virus interactions, viral assembly and structure; toxicology; plant and microbial biochemistry; and surface forces, micelles and microemulsions, colloids, electrical phenomena, etc. in biological systems.

IJBB is a peer-reviewed journal with active support from academicia and other eminent experts in the field associated with the journal either as board members, referees or as authors. IJBB has a circulation of 560 and is covered by many abstracting/indexing services, such as: Anal Abstr, Anim Bread Abstr, Biotech Abstr, Biol Abstr, Chem Abstr, Curr Adv Curr Cont, Excerp Med, Dairy Sci Abstr, Food Sci & Tech Abstr, Helminthol Abstr, Ind Sci Abstr, Nutr Abstr, Sci Cit Ind, Rev Appl Entomol, Rev Plant Path, Vet Bull, Trop Dis Bull, etc.

IJBB invites quality research papers and minireviews from active researchers in the above mentioned subject fields. Please find “Instructions to Authors” published at the end of this issue (August ’03) for kind adherence. Please visit us online at http://www.niscair.res.in

 

EDITOR

 


 

Minireview

Indian Journal of Biochemistry & Biophysics

Vol. 40, August 2003, pp. 217-225

 

 

N-Glycolylneuraminic acid conjugates: Implications of their absence in mammalian biochemistry

Daniel W Mwangi and Devi D Bansal*

 

 

N-glycolylneuraminic acid (Neu5Gc) is one of the two most common forms of sialic acids present in glycoproteins and glycolipids of mammalian tissues. It is synthesized from the most ubiquitous sialic acid, N-acetylneuraminic acid (Neu5Ac) in a hydroxylation reaction catalysed by the enzyme Neu5Ac hydroxylase. Though Neu5Gc conjugates are prevalent in many tissues of mammals, they are absent in glycolipids and only trace amounts are present in glycoproteins of the brain and central nervous system. In humans Neu5Ac is the main sialic acid as Neu5Ac hydroxylase is inactive due to mutation of its gene. The importance of sialic acids in biochemical phenomena and the distinct roles played by specific forms of these amino sugars is adequately reflected in functional studies of selectin and sialoadhesin families of adhesion molecules. The absence of Neu5Gc, therefore, in tissues of humans and brain of mammals has raised interest, especially with regard to its impact on biochemical differences evident between humans and other mammals. It is suggested that though Neu5Gc conjugates are important in cellular interactions, their presence in brain and the central nervous system is deleterious to the latter’s normal functions. Their interaction with other cellular components to form supramolecular associations is indicated that may have a bearing on major biochemical differences, a few of which are presently evident between humans and other mammals.

 

Key words: Cell adhesion molecules, glycolipids, glycoproteins, N-acetylneuraminic acid, N-acetylneuraminic acid hydroxylase; N-glycolylneuraminic acid, tumour, inflammatory disease, sialic acid

 

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 40, August 2003, pp. 226-237

 

 

Disruption of distal interactions of Arg 262 and of substrate binding to Ser 52
affect catalysis of sheep liver cytosolic serine hydroxymethyltransferase

Venkatakrishna Rao Jala#, M Ambili, V Prakash+, N Appaji Rao and H S Savithri*

 

 

The crystal structure of human liver cytosolic recombinant serine hydroxymethyltransferase (hcSHMT) suggested that Ser53 and Arg 263 could participate in the reaction catalyzed by SHMT. The mutation of Arg262 (corresponding to Arg263 in hcSHMT) to ‘A’ in sheep liver cytosolic SHMT (scSHMT) resulted in a 5-fold increase in Km for l-Ser and a 5-fold decrease in kcat compared to scSHMT. Further, in R262A SHMT-glycine complex, the peak at 343 nm (geminal diamine) was more pronounced, compared to wild-type enzyme. Stopped-flow studies showed that the rate constant for the formation of glycine-geminal diamine for R262A SHMT was also decreased. The rate of reaction, concentration of spectral intermediates, fluorescence excitation maximum of glycine geminal diamine and interaction with methoxyamine were altered in R262A SHMT. Although Arg263 in hcSHMT is located outside the PLP binding pocket, it positions Tyr73 for interaction with PLP, by forked H-bonding with the carbonyl groups of main chain residues, Asn71 and Lys72 of the other subunit of the tight dimer. Mutation of Arg262 to Ala and the consequent alteration in orientation of PLP leads to decreased catalytic efficiency. Ser53 (in hcSHMT) is in hydrogen bonding distance to one of the carboxylate oxygens of the amino acid substrate, which also interacts with Tyr83 and Arg402. Replacement of Ser53 with Cys (using ‘O’ software program) in the structure of hcSHMT resulted in disruption of these interactions, whereas replacement with Ala (S53A) only weakened the substrate interactions. There was a 10-fold increase in Km and 20-fold decrease in catalytic activity efficiency for S52C SHMT, whereas S52A SHMT retained 20% of the activity without change in Km for serine. These results suggest that S52 affects substrate binding and catalysis.

 

Key words: Active site geometry, distal interactions, geminal diamine, glycine-external aldimine, pyridoxal-5¢-phosphate, serine hydroxymethyltransferase (SHMT), site-directed mutagenesis

 

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 40, August 2003, pp. 238-245

 

 

Nimesulide affects antioxidant status during acute lung inflammation in rats

Kiranjit K Sohi and Krishan L Khanduja*

 

 

Antiradical activity of nimesulide, a commonly used COX-2 specific inhibitor, was estimated in vitro by 1, 1 diphenyl-2-picrylhydrazyl (DPPH) assay, nitroblue tetrazolium reduction assay and lipid peroxidation assay, respectively. The biochemistry of antioxidant functions of nimesulide was also investigated under control and inflammatory conditions, caused by intratracheal instillation of lipopolysaccharide (LPS). Pro-inflammatory conditions generally end up in oxidative insults, which have been suggested to be the cause of multiple organ failure in inflammation. A primary defense, constituted of antioxidant enzymes, against this oxidative damage has evolved in the body. In this study, male Wistar rats were orally administered with nimesulide (9 mg/kg/twice daily for 1 week), followed by intratracheal instillation with 2 mg of LPS and after 18 hr, antioxidant defense system and lipid peroxidation were measured in liver, lungs and kidneys. Nimesulide pretreatment was found to protect the tissue from enhanced levels of lipid peroxidation, and also stimulated the levels of glutathione-S-transferase (GST) in liver and glutathione reductase in kidneys. Surprisingly, nimesulide oral feeding also significantly suppressed superoxide dismutase (SOD) activity in all the three organs. Although, in our study, nimesulide proved to be an inducer of GST (a marker for chemoprevention) and a scavenger of superoxide anions at higher concentrations (> 250 mM), but the relevance of suppression of SOD enzyme activity, which may contribute to the drug’s toxic effects cannot be ignored. The work suggests that further long term studies are needed to confirm nimesulide as a safe drug.

 

Key words: Nimesulide, antioxidant defense, superoxide anions, lipid peroxidation

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 40, August 2003, pp. 246-251

 

 

Effect of oxidative stress on the spermatogenic process and hsp70 expressions in mice testes

Parminder Kaur and M P Bansal*

 

 

The effect of oxidative stress on the process of spermatogenesis in terms of hsp70 expression was studied. For creating different oxidative stressed mice, three selenium (Se) levels viz., deficient (group I), adequate (group II) and excess (group III) were fed for 8 weeks in a yeast-based diet. After completion of diet feeding, Se level was significantly decreased in group I and significantly increased in group III, as compared to group II. Glutathione peroxidase (GSH-Px) activity was significantly decreased in both liver and testis in group 1 animals; however, the activity was comparable in groups II and III. Significant increase in the testis glutathione-S-transferase (GST) activity was observed in group I. No change was seen in group III, when compared to group II. Histological analysis of testis revealed a significant decrease in the germ cell population in group I, as compared to group II, with a predominant effect on spermatid and mature sperm numbers. In group III, displacement of germ cell population was observed. ELISA assays for hsp70 level showed increase in group I as compared to group II, whereas no significant change was observed in group III, as compared to group II. Immuno-histochemical analysis revealed intense localization of hsp70 only in spermatid and sperm cells. The expression in groups II and III was homogeneous with slightly increased expression around lumen in group III. The data indicate that excessive oxidative stress in Se deficient group, affects the spermatogenesis process, especially affecting the mature sperm number which in turn leads to infertility.

 

Key words: selenium, testis, hsp70, oxidative stress

 

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 40, August 2003, pp. 252-259

 

 

Kinetic attributes of rat liver microsomal adenosine 5’ triphosphate phosphohydrolase (ATPase)

Subhash D Katewa and Surendra S Katyare*

 

 

The kinetic properties of the rat liver microsomal ATPase, with respect to Na+, K+ and ATP requirements were examined. Presence of Na+ and K+ or both hardly caused any stimulation of the enzyme activity. The Km values for Na+ and K+ were substantially low (0.32 and 0.05 mM, respectively), compared to those reported for the Na+, K+ ATPases from different tissues. Substrate kinetics studies revealed that in the absence of Na+ and K+, ATP is an activator of the enzyme. The enzyme displayed increased activity with increase in the energy of activation in the absence of Na+ and K+. The activity was partially inhibited by ouabain only in the presence of Na+ and K+. The results suggest that the liver microsomal enzyme is not a Na+, K+ ATPase, but has requirement of monovalent cations for the regulation of its activity. Also, the b3 subunit of the enzyme has a Km lowering effect.

 

Key words: Liver microsomal ATPase, Na+/K+ dependence, a1b3 subunits, substrate kinetics, ouabain inhibition.

 

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 40, August 2003, pp. 260-264

 

 

Protective role of selenium status on T3/T4 kinetics in rats under hyperlipidemia

Sanjiv Dhingra, Uma Singh and M P Bansal*

 

 

The effect of high fat diet (HFD) on thyroid hormones (T3/T4) and protective role of selenium (Se) were studied in rats. Se levels in serum and liver decreased significantly, whereas glutathione peroxidase (GSH-Px) in liver and lipid levels (cholesterol and triglycerides) in serum increased after 1, 2 and 3 months of HFD feeding in comparison to controls in all the three Se status i.e. deficient (0.02 ppm), adequate (0.2 ppm) and excess (1 ppm) groups. Levels of T3/T4 decreased significantly on HFD feeding, as compared to respective controls in all the groups. Within the deficient group, as Se deficiency progressed, T3/T4 levels decreased after 2 and 3 months in comparison to 1 month. A significant increase was observed in T3/T4 concentration on feeding 1 ppm (excess) Se supplemented diet, in comparison to adequate group. Also, in 1 ppm Se supplemented group as the Se deposition increased i.e. after 2 and 3 months, levels of T3/T4 increased significantly. So, the present study indicates that Se supplementation up to 1 ppm normalizes the T3 and T4 concentrations or regulates the hypothyroidism induced by hyperlipidemia.

 

Key words: hyperlipidemia, hypothyroidism, lipid, T3/T4 kinetics, selenium status, rats

 

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 40, August 2003, pp. 265-273

 

 

Effect of terminal achiral and chiral residues on the conformational behaviour of poly DZPhe and analysis of various interactions

Fateh S Nandel* and Harpreet Kaur

 

 

Conformational properties of the peptides containing (DZPhe)6 with achiral (DAla, Gly) and chiral (Ala, Leu) residues at both the N- and C-terminal positions have been studied with a view to design a peptide with desired helical screw sense. In all the peptides, the lowest energy conformational state corresponds to F = 0o and Y = + 90o or – 90o or both ± 90o. These structures are characterized by rise per residue of 1.94 Å; rotation per residue of 114o and 3.12 residues per turn and are stabilized by: (i) carbonyl-carbonyl interactions with the carbonyl oxygen of ith residue and carbonyl carbon atom of the carbonyl group of ith+1 residue; and (ii) N-H….p interactions between the amino group of DZPhe and its own aromatic moiety. The Ala/Leu residues at the N-terminus further stabilized the structure, through C-H….p interactions with the farthest edge of the aromatic ring of ith+3 DZPhe residue. For peptides Ac-L-Ala/L-Leu-(DZPhe)6-NHMe, the low energy left handed helical structure (~2.5 Kcalmol-1 higher in energy) state corresponds to F = -30o, Y = 120o for L-residue and F = Y = 30o for DZPhe residues and is in good agreement with the X-ray crystallography results for the peptide Boc-L-Ala-(DZPhe)4-NHMe crystals grown from acetonitrile/ethanol mixture. Computational results suggest that the peptides Ac-D-Ala/D-Leu-(DZPhe)6-NHMe adopt a right handed helical structure in polar solvents with F = 30o, Y = -120o for D-residues and F = Y = -30o for DZPhe residues. Both in the left handed and right handed structures, the carbonyl oxygen of acetyl group is involved in 10-membered hydrogen bonded ring formation with NH of 3rd DZPhe residue whereas DZPhe residues backbone adopts a 310 helix structure. Computational results also suggest that the conformational state with F = 0o and Y = 90o can be realized by keeping D-Ala or D-Leu at the C-terminal. There is hardly any effect of achiral residues Gly/DAla on the conformational behaviour of poly-DZPhe.

 

Key words: poly-dehydrophenylalanine, conformation, chiral amino acid residue effect, design of peptide, interactions.

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 40, August 2003, pp. 274-277

 

 

 

Purification and partial characterization of cytochrome c552 from Halobacterium salinarium

K Sreeramulu

 

 

Cytochrome c552 was purified to near homogenity and partially characterized from Halobacterium salinarium JWS mutant, devoid of carotenoid pigments. The purification involved the extraction of membranes with 1% Triton X-100, followed by butylagarose, DEAE-Sepharose CL6B and hydroxyapatite column chromatography. The fold of purification was 16. The purified cytochrome showed maximum absorption at 552 nm. The molecular mass determined by SDS-PAGE was found to be 14.1 kD.

 

Key words: Cytochrome c552, Halobacterium salinarium, purification, characterization

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 40, August 2003, pp. 278-280

 

 

Beneficial effects of tender coconut water against isoproterenol induced toxicity on heart mitochondrial activities in rats

P Anurag and T Rajamohan*

 

 

Myocardial infarction induced by isoproterenol results in increased mitochondrial lipid peroxidation, decreased activities of carnitine acetyl transferase, acyl CoA dehydrogenase, TCA cycle enzymes and decreased rate of palmitate oxidation into CO2 and ATP formation in the heart in rats. Isoproterenol-induced rats when fed with tender coconut water (West coast tall variety, 5-6 months age) showed improved activities of these mitochondrial enzymes and higher rate of palmitate oxidation into CO2 and ATP production. These results indicate that intake of TCW has a significant beneficial effect on mitochondrial activities.