Indian Journal of Biochemistry & Biophysics

CODEN: IJBBBQ  ISSN: 0301-1208

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VOLUME 46

NUMBER 4

AUGUST 2009

CONTENTS

Papers
Contribution of Ser463 residue to the enzymatic and autoprocessing activities of Escherichia coli g-glutamyltranspeptidase	281
Wen-Hwei Hsu, Ping-Lin Ong, Shih-Chun Chen & Long-Liu Lin*
Molecular cloning and expression analysis of evolutionarily conserved stathmin from Gekko japonicus spinal cord	289
Xiaoxia Jiang, Xiaosong Gu, Yan Liu, Fei Ding, XingXing Gu, Youjuan Huan, Lijie Ren & Yongjun Wang*
Properties of alkaline protease genetically engineered on cell surface of the yeast Yarrowia lipolytica	294
Xiumei Ni, Lixi Yue, Jing Li, Zhenming Chi*, Zhiqiang Liu & Catherine Madzak
Iron-zinc interaction during uptake in human intestinal Caco-2 cell line: Kinetic analyses and possible mechanism	299
Vasuprada Iyengar, Raghu Pullakhandam & K Madhavan Nair*
Homeopathic drugs Natrum sulphuricum and Carcinosin prevent azo dye-induced hepatocarcinogenesis in mice	307
Nandini Bhattacharjee, Pathikrit Banerjee & Anisur Rahman Khuda-Bukhsh*
Identification of α2u-globulin and bound volatiles in the Indian common house rat (Rattus rattus)	319
R Rajkumar R Ilayaraja, C Mucignat, A Cavaggioni & G Archunan*
Characterization of erythrosine B binding to bovine serum albumin and bilirubin displacement	325
Vinodaran M K Mathavan, Boon Kim Boh & Saad Tayyab*
Effect of nickel on root growth and the kinetics of metal ions transport in onion (Allium cepa) root	332
Halide Akbaş*, Feruzan Dane & Çiler Meriç
Notes
Purification and characterization of bacteriocin produced by strain of Lactobacillus brevis MTCC 7539	337
Neha Gautam* & Nivedita Sharma
Serum biochemical markers in rheumatoid arthritis	342
Vasanthi Pallinti, Nalini Ganesan*, M Anbazhagan & G Rajasekhar
Instructions to Authors	345
Announcement	348

——————

*Author for correspondence

AUTHOR INDEX

Akbaş H	332
Anbazhagan M	342
Archunan G	319
Banerjee P	307
Bhattacharjee N	307
Boh B K	325
Cavaggioni A	319
Chen S C	281
Chi Z	294
Dane F	332
Ding F	289
Ganesan N	342
Gautam N	337
Gu X X	289
Gu X	289
Hsu W H	281
Huan Y	289
Ilayaraja R	319
Iyengar V	299
Jiang X	289
Khuda-Bukhsh A R	307
Li J	294
Lin L L	281
Liu Y	289
Liu Z	294
Madzak C	294
Mathavan V M K	325
Meriç Ç	332
Mucignat C	319
Nair K M	299
Ni X	294
Ong P L	281
Pallinti V	342
Pullakhandam R	299
Rajasekhar G	342
Rajkumar R	319
Ren L	289
Sharma N	337
Tayyab S	325
Wang Y	289
Yue L	294

PAPERS

Indian Journal of Biochemistry & Biophysics

Vol. 46, August 2009, pp.281-288

Contribution of Ser463 residue to the enzymatic and autoprocessing
activities of Escherichia coli g-glutamyltranspeptidase

Wen-Hwei Hsu¹, Ping-Lin Ong², Shih-Chun Chen³ and Long-Liu Lin³*

¹Institute of Molecular Biology, National Chung Hsing University, 402-27 Taichung, Taiwan
²Department of Biochemical Science and Technology, ³Department of Applied Chemistry, National Chiayi University,
300 University Road, Chiayi, Taiwan

Received 25 August 2008; revised 10 June 2009

A serine residue Ser463, required for proper function of E. coli g-glutamyltranspeptidase (EcGGT) was identified by site-directed mutagenesis on the basis of sequence alignment of human, pig, rat, and three bacterial enzymes. Thr-, Asp-, and Lys-substituted variants were overexpressed in E. coli M15 cells and the recombinant proteins were purified to near homogeneity by nickel-chelate chromatography. With the exception of S463T, the other two variants completely lost GGT activity, implying the importance of this residue in EcGGT. Moreover, substitution of Ser463 with either Lys or Asp impaired the capability of autocatalytic processing of the precursor into a- and b-subunit. Computer modeling showed that the critical bonding distance of Gln390 C-Thr391 OG1 was significantly increased in S463D and S463K, indicating that these distance changes might be responsible for the lack of enzyme maturation. Measurements of intrinsic tryptophan fluorescence revealed alteration of the microenvironment of aromatic amino acid residues in S463D and S463K, while circular dichroism (CD) spectra were nearly identical for wild-type and all mutant enzymes. The temperature-dependent signal in the far-UV region for S463T was consistent with that of wild-type enzyme, but S463D and S463K showed a different sensitivity towards temperature-induced denaturation. These results implied that a significant conformational change occurred as a result of Asp- and Lys-substitution.

Keywords: E coli, g-Glutamyltranspeptidase, Site-specific mutagenesis, Autocatalytic processing, Tryptophan emission fluorescence, Circular dichroism

*E-mail: llin@mail.ncyu.edu.tw

Indian Journal of Biochemistry & Biophysics

Vol. 46, August 2009, pp.289-293

Molecular cloning and expression analysis of evolutionarily conserved stathmin from Gekko japonicus spinal cord

Xiaoxia Jiang, Xiaosong Gu, Yan Liu, Fei Ding, XingXing Gu, Youjuan Huan, Lijie Ren and Yongjun Wang*

Key Laboratory of Neuroregeneration, Nantong University, 19 Qixiu Road, Nantong 226007, P.R. China

Received 18 November 2008; revised 19 May 2009

The cDNA encoding stathmin is identified from the brain and spinal cord cDNA library of Gekko japonicus. It contains a 450 bp open-reading-frame, corresponding to a deduced protein of 149 amino acids. At amino acid level, gecko stathmin shares more than 76.4% identities with vertebrate stathmins, and especially, it shares 100% identity with human stathmin, suggesting that the selective pressure must have been extremely high for the conservation of stathmin during the vertebrates including reptile evolution. Reverse transcriptase polymerase chain reaction (RT-PCR) shows that gecko stathmin is ubiquitously expressed in all tissues examined. In situ hybridization reveals that stathmin transcript mainly appear in the gray matter of spinal cord. The change of stathmin expression in spinal cord after tail amputation is examined by semi-quantitative RT-PCR. Stathmin expression increases at 1 day and 3 day after amputation and decreases to the control level at 1 week. However, the expression level increases again at 2 weeks. These suggest that stathmin may be associated with the immune protection of the injury, as well as in the regeneration of spinal cord.

Keywords: Gecko, Stathmin, Spinal cord, Regeneration, Expression

*Email: wyjbs@yahoo.com.cn

Indian Journal of Biochemistry & Biophysics

Vol. 46, August 2009, pp.294-298

Properties of alkaline protease genetically engineered on cell surface of the yeast Yarrowia lipolytica

Xiumei Ni, Lixi Yue, Jing Li, Zhenming Chi*, Zhiqiang Liu and Catherine Madzak⁺

Unesco Chinese Center of Marine Biotechnology, Ocean University of China, Yushan Road, No. 5, Qingdao, China

⁺UMR1238 Microbiologie et Génétique Moléculaire, INRA/CNRS/INAPG, CBAI, BP 01, F-78850 Thiverval-Grignon, France

Received 23 November 2008; revised 22 June 2009

ALP2 gene encoding alkaline protease cloned from Aureobasidium pullulans HN2-3 was ligated into the surface display plasmid and expressed in the cells of the yeast Yarrowia lipolytica. The expressed alkaline protease was immobilized on the yeast cells. The activity of the immobilized enzyme with 6´ His tag was found to be significantly higher than that of without 6´ His tag. The immobilized enzyme showed lower optimal temperature and a lower affinity for azocasein than the free enzyme purified from A. pullulans HN2-3. The thermal stability of the immobilized enzyme enhanced and the pH stability decreased, compared to that of the free enzyme.

Keywords: Yeast surface display, Alkaline protease, Aureobasidium pullulans, Yarrowia lipolytica, Marine yeast

*Email: zhenming@sdu.edu.cn

Indian Journal of Biochemistry & Biophysics

Vol. 46, August 2009, pp.299-306

Iron-zinc interaction during uptake in human intestinal Caco-2
cell line: Kinetic analyses and possible mechanism

Vasuprada Iyengar, Raghu Pullakhandam and K Madhavan Nair*

Micronutrient Research Group, Biophysics Division, National Institute of Nutrition, Indian Council of Medical Research,
Jamai Osmania, Hyderabad 500 604, India

Received 07 January 2009; revised 02 July 2009

Iron and zinc interact at the enterocyte during absorption, but the mechanism(s) remain elusive. The aim was, therefore, to understand the mechanism of interaction using kinetic analyses of iron and zinc uptake, individually and in combination under normal and altered cellular mineral concentrations in human intestinal Caco-2 cell line. Striking differences in kinetic parameters were observed between iron and zinc uptake. Iron uptake followed a two-component model, while zinc uptake followed a three-component model. Iron uptake had a K_m of 3.6 µM and V_max of 452 pmol/mg protein/min, while zinc uptake had a K_m of 42 µM and V_max of 3.09 pmol/mg protein/min. Zinc dose-dependently inhibited iron uptake through mixed-inhibition but iron marginally increased zinc uptake. Cellular zinc repletion doubled iron uptake and eliminated inhibition, but zinc depletion decreased iron uptake. Iron pre-treatment had no effect on zinc uptake. Based on these results, a two-transporter model of iron uptake, comprising the apical iron uptake transporter divalent metal ion transporter-1
(DMT-1) and an unknown putative transporter was derived. This model for DMT-1 was verified by immunoblotting. These results implied that cellular zinc status profoundly influenced iron uptake and its interactions with zinc during uptake. DMT-1 might not simultaneously transport iron and zinc, providing a mechanistic basis for observed interactions.

Keywords: Caco-2 cells, DMT-1, Interactions, Iron, Kinetics, Zinc

*E-mail: nairthayil@hotmail.com

Indian Journal of Biochemistry & Biophysics

Vol. 46, August 2009, pp.307-318

Homeopathic drugs Natrum sulphuricum and Carcinosin prevent azo dye-induced hepatocarcinogenesis in mice

Nandini Bhattacharjee, Pathikrit Banerjee and Anisur Rahman Khuda-Bukhsh*

Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741 235, India

Received 12 January 2009; revised 02 June 2009

The study was undertaken to examine whether Carcinosin-200 (Car-200) could provide additional ameliorative effect, if used intermittently with Natrum sulphuricum-30 (Nat Sulph-30) against hepatocarcinogenesis induced by chronic feeding of p-dimethylaminoazobenzene (p-DAB) and phenobarbital (PB) in mice (Mus musculus). Mice were randomly divided into seven sub-groups: (i) normal untreated; (ii) normal + succussed alcohol; (iii) p-DAB (0.06%) + PB (0.05%); (iv) p-DAB + PB + succussed alcohol, (v) p-DAB + PB + Nat Sulph-30, (vi) p-DAB + PB + Car-200, and (vii) p-DAB + PB + Nat Sulph-30 + Car-200. They were sacrificed at 30, 60, 90 and 120 days for assessment of genotoxicity through cytogenetical end-points like chromosome aberrations, micronuclei, mitotic index and sperm head anomaly and cytotoxicity through assay of widely accepted biomarkers and pathophysiological parameters. Additionally, electron microscopic studies and gelatin zymography for matrix metalloproteinases (MMPs) were conducted in liver at 90 and 120 days. Results showed that administration of Nat Sulph-30 alone and in combination with Car-200 reduced the liver tumors with positive ultra-structural changes and in MMPs expression, genotoxic parameters, lipid peroxidation, γ-glutamyl transferase, lactate dehydrogenase, blood glucose, bilirubin, creatinine, urea and increased GSH, glucose-6-phosphate dehydrogenase, superoxide dismutase, catalase, glutathione reductase activities and hemoglobin, cholesterol, and albumin levels. Thus, intermittent use of Car-200 along with Nat Sulph-30 yielded additional benefit against genotoxicity, cytotoxicity, hepatotoxicity and oxidative stress induced by the carcinogens during hepatocarcinogenesis.

Keywords: Biomarkers, Genotoxicity, Homeopathy, Hepatocarcinogenesis, Mice, Carcinosin-200, Natrum sulphuricum-30

*E-mail: prof_arkb@yahoo.co.in; khuda-bukhsh_48@rediffmail.com

Indian Journal of Biochemistry & Biophysics

Vol. 46, August 2009, pp.319-324

Identification of α2u-globulin and bound volatiles in the Indian
common house rat (Rattus rattus)

R Rajkumar¹,^#R Ilayaraja¹, C Mucignat², A Cavaggioni² and G Archunan¹*

¹Center for Pheromone Technology, Department of Animal Science, School of Life Sciences,

Bharathidasan University, Tiruchirappalli-620 024, India

²Department of Human Anatomy and Physiology, University of Padova, Via Marzolo 3, 35131, Italy

Received 29 January 2009; revised 13 May 2009

The α2u-globulin (α2u) is a pheromone carrier urinary protein believed to be relevant for sexual communication among rats and is characterized in laboratory rats. In the present study 17 kDa protein and the bound pheromones were characterized in a population of wild-type Indian common house rat (Rattus rattus). The protein was purified by two runs of Sephadex G-50 chromatography and analyzed with SDS-PAGE with MALDI-TOF/MS. The results of MASCOT search identified the protein as an α2u and suggested a role for binding pheromones. To confirm the protein bound volatiles, purified α2u was extracted with dichloromethane and volatile molecules were detected using of gas chromatography linked to mass spectrometry (GC-MS). 1-Chlorodecane was detected as the predominant compound and 2-methyl-N-phenyl-2- propenamide, hexadecane and 2,6,11-trimethyl decane as the minor compounds. The simple method of protein purification and the identification of bound volatiles may help in designing efficient pheromone-based rat traps.

Keywords: α2u-Globulin, Major urinary protein, Mass spectrometry, Pest control, Pheromone-binding protein, Rattus rattus, Urine

*E-mail: garchu56@yahoo.co.in

Indian Journal of Biochemistry & Biophysics

Vol. 46, August 2009, pp.325-331

Characterization of erythrosine B binding to bovine serum albumin
and bilirubin displacement

Vinodaran M K Mathavan, Boon Kim Boh and Saad Tayyab*

Biomolecular Research Group, Biochemistry Programme, Institute of Biological Sciences, Faculty of Science,
University of Malaya, 50603 Kuala Lumpur, Malaysia

Received 16 March 2009; revised 26 June 2009

The interaction of erythrosine B (ErB), a commonly used dye for coloring foods and drinks, with bovine serum albumin (BSA) was investigated both in the absence and presence of bilirubin (BR) using absorption and absorption difference spectroscopy. ErB binding to BSA was reflected from a significant red shift of 11 nm in the absorption maximum of ErB (527 nm) with the change in absorbance at λ_max. Analysis of absorption difference spectroscopic titration results of BSA with increasing concentrations of ErB using Benesi-Hildebrand equation gave the association constant, K as 6.9 ´ 10⁴ M^-1. BR displacing action of ErB was revealed by a significant blue shift in the absorption maximum, accompanied by a decrease in absorbance difference at λ_max in the difference spectrum of BR-BSA complex upon addition of increasing concentrations of ErB. This was further substantiated by fluorescence spectroscopy, as addition of increasing concentrations of ErB to BR-BSA complex caused a significant decrease in fluorescence at 510 nm. The results suggest that ErB binds to a site in the vicinity of BR binding site on BSA. Therefore, intake of ErB may increase the risk of hyperbilirubinemia in the healthy subjects.

Keywords: Absorption spectroscopy, Bilirubin displacement, Bovine serum albumin, Erythrosine B

*E-mail: saadtayyab2004@yahoo.com

Indian Journal of Biochemistry & Biophysics

Vol. 46, August 2009, pp.332-336

Effect of nickel on root growth and the kinetics of metal ions transport in onion (Allium cepa) root

Halide Akbaş^1*, Feruzan Dane² and Çiler Meriç²

¹Department of Chemistry, ²Department of Biology, Trakya University,
Faculty of Arts and Sciences, 22030, Edirne, Turkey

Received 14 October 2008; revised 25 June 2009

The effect of different concentrations of nickel nitrate (0.25, 0.50, 1.00 and 2.00 mM) uptake by the roots, on root growth of onion (Allium cepa) and the transport of Ni²⁺, Fe²⁺, Mn²⁺, Zn²⁺, K⁺, Na⁺ and H⁺ ions were investigated spectrophotometrically. The uptake of Ni²⁺, Fe²⁺, Mn²⁺ and Zn²⁺ was monitored by flame atomic absorption spectrometry with a 24-h period for 7 days and the amounts of K⁺ and Na⁺ were determined in solutions by flame photometer. The mineral content of the solution, instead of the root material was measured. Ni²⁺ ions showed inhibitory effect on the root growth at all concentrations during the entire treatment. The EC₅₀ (effective concentration that reduced root growth by 50%) was found at 0.25 mM Ni²⁺. No significant change in inhibitory effect was observed after at 0.50 mM Ni²⁺ concentrations. A large amount of Ni²⁺ was translocated into the roots. The kinetics of metal ion transport followed a pseudo-first order reaction in all metal ion concentrations. Ni²⁺, Zn²⁺ Fe²⁺ Mn²⁺ and H⁺ ions transferred together into plant, but Na⁺ and K⁺ ions transferred to the solution from the plant. The amount of H⁺ in the solution decreased at all Ni²⁺ concentrations.

Keywords: Allium cepa L, Atomic absorption spectrometry, Flame photometer, Nickel, Phytotoxic effect

*Email: hakbas34@yahoo.com

NOTES

Indian Journal of Biochemistry & Biophysics

Vol. 46, August 2009, pp.337-341

Purification and characterization of bacteriocin produced by strain of Lactobacillus brevis MTCC 7539

Neha Gautam* and Nivedita Sharma

Microbiology Research Laboratory, Department of Basic Sciences, Dr. Y.S. Parmar University of Horticulture and Forestry,
Nauni-173 230, Solan (HP) India

Received 07 November 2008; revised 29 June 2009

Bacteriocin, an antimicrobial agent having potential for food biopreservation was purified from Lactobacillus brevis (a safe food-grade bacteria isolated from Vari Kandal, a traditional fermented food of Himachal Pradesh by adopting a novel repeated washing method. Its purity was confirmed by SDS-PAGE and Native-PAGE. The relative molecular mass of bacteriocin was 93.74 kD, while specific activity and recovery were 35.52 folds and 17.13%, respectively. It showed high thermal stability and was active over wide range of pH and exhibited sensitivity to trypsin.

Keywords: Bacteriocin, Lactibacillus brevis

*Email: neha_mbg@yahoo.com; nivea_64@yahoo.co.in

Indian Journal of Biochemistry & Biophysics

Vol. 46, August 2009, pp.342-344

Serum biochemical markers in rheumatoid arthritis

Vasanthi Pallinti¹, Nalini Ganesan^1*, M Anbazhagan² and G Rajasekhar³

¹Department of Biochemistry, ²Clinical Laboratory, ³Department of Rheumatology

Sri Ramachandra Medical College and Research Institute, Sri Ramachandra University, Porur, Chennai 600116

Received 19 September 2008; revised 09 July 2009

Rheumatoid arthritis (RA) characterized by local and systemic effects of inflammation has a wide range of biochemical markers implicated directly or indirectly to its pathogenesis. In the present study, homocysteine, cortisol, adenosine deaminase (ADA), ferritin, malondialdehyde (MDA) and α-tocopherol in serum of RA patients and healthy individuals were estimated to assess if they contribute to the disease process. The markers of disease activity such as erythrocyte sedimentation rate (ESR) and rheumatoid factor (RF) were also measured. The study group included a total of 45 subjects, including 30 RA patients and the rest being healthy individuals. RA group showed a significant increase in the levels of homocysteine, ADA and MDA, and a significant decrease in α-tocopherol compared to the healthy individuals. However, cortisol and ferritin levels did not show any significant change. Also, there was no significant correlation between the studied serum markers and markers of disease activity. Our results indicate that these biochemical markers contribute independently to the pathogenesis of RA.

Keywords: Homocysteine, Adenosine deaminase, Cortisol, Oxidative stress, Rheumatoid arthritis

*Email: nalinisrmc@hotmail.com