Indian Journal of Biochemistry & Biophysics

CONTENTS

Indian Journal of Biochemistry & Biophysics

Vol 40, February 2003, pp. 5-13

Relaxation of arterial smooth muscle by water-soluble

derivatives of coenzyme Q (ubiquinone)

R Bindu1, B V Venkataraman1, Aparna V S Rao2 and T Ramasarma2*

1Department of Pharmacology, St. John's Medical College, Bangalore 560 034

2Department of Biochemistry and Solid State & Structural Chemistry Unit,

Indian Institute of Science, Bangalore 560 012, India

Received 16 September 2002; revised 22 October 2002

Treatment of coenzyme Q with ozone, permanganate, and ferrous sulfate in presence of ascorbate or hydrogen peroxide yielded water-soluble degradation products, possibly having truncated side-chain and modified ring.These derivatives, but not the intact lipid-quinone, showed relaxation of phenylephrine-contracted rat arterial rings. Representative samples of these also decreased blood pressure when injected into the femoral vein in the rat.These effects offer an explanation for the hypotensive action of exogenous coenzyme Q regardless of its side-chain length.

Indian Journal of Biochemistry & Biophysics

Vol 40, February 2003, pp.  14-21

Crystal structure of human seminal diferric lactoferrin

at 3.4 Å resolution

Janesh Kumar1, Wolfgang Weber2, Sabine Münchau2, Savita Yadav1, S Bhaskar Singh1, K Saravanan1,

M Paramasivam1, Sujata Sharma1, Punit Kaur1, A Bhushan1, A Srinivasan1, Christian Betzel3 and T P Singh1*

1Department of Biophysics, All India Institute of Medical Sciences, New Delhi 110029, India

2Universitätsklinikum Hamburg-Eppendorf, IMBM, Martinistrasse 52, D-20246 Hamburg, Germany

3Institute of Medical Biochemistry and Molecular Biology, C/o DESY, Geb 22a Notkestrasse 85, 22603, Hamburg, Germany

Received 29 October 2002; revised 16 January 2003

Lactoferrin was purified from human seminal fluid obtained from the semen bank. The purified samples were saturated with Fe3+ and crystallized by microdialysis method. The crystals belong to orthorhombic space group P212121 with a = 55.9 Å, b = 97.2 Å, c = 156.1 Å and Z = 4. The structure was determined with molecular replacement method and refined to an R factor of 18.7% for all the data to 3.4 Å resolution. The overall structure of seminal lactoferrin is similar to human colostrum lactoferrin. The amino acid sequence of seminal lactoferrin shows that it has one amino acid less than human colostrum lactoferrin and the structure of its N-terminal region is far more ordered than other lactoferrins. The structure of the iron-binding site and its immediate surroundings indicate well defined features.

Indian Journal of Biochemistry & Biophysics

Vol 40, February 2003, pp.  22-26

Radioprotective property of polysaccharide in Tinospora cordifolia

Mahesh Subramanian, Gajanan J Chintalwar and Subrata Chattopadhyay*

Bio-Organic Division, Bhabha Atomic Research Centre, Mumbai 400 085, India

Received 14 June 2002; revised 6 September 2002

Radioprotective activity of a polysaccharide preparation from the Indian medicinal plant, Tinospora cordifolia Miers has been established using Saccharomyces cerevisiae X2180 strain as the in vivo test model. The entire activity could be attributed to the radical scavenging capacity of the preparation, as it did not enhance the expression of the protective enzymes, catalase and superoxide dismutase in the yeast cells.

Indian Journal of Biochemistry & Biophysics

Vol 40, February 2003, pp.  27-30

Kinetics and mechanism of protection of adenine from sulphate radical anion by caffeic acid under anoxic conditions

M Sudha Swaraga and M Adinarayana

Department of Chemistry, Osmania University, Hyderabad 500 007, India

Received 23 May 2002; revised 5 November 2002

The rates of photo-oxidation of adenine in the presence of peroxydisulphate (PDS) have been determined by measuring the absorbance of adenine at 260.5 nm spectrophotometrically. The rates and the quantum yields () of oxidation of adenine by sulphate radical anion (SO4• ) have been determined in the presence of different concentrations of caffeic acid. Increase in the concentration of caffeic acid is found to decrease the rate of oxidation of adenine suggesting that caffeic acid acts as an efficient scavenger of SO4•  and protects adenine from it; SO4•  competes for adenine as well as for caffeic acid. From competition kinetics, the rate constant of SO4•  with caffeic acid has been calculated to be 1.24  0.2  1010 mol-1dm3s-1. The quantum yields of photo-oxidation of adenine have been calculated from the rates of oxidation of adenine and the light intensity absorbed by PDS at 254 nm, the wavelength at which PDS is activated to SO4•  . The results of experimentally determined quantum yields (exptl) and the quantum yields calculated (cl) by assuming that caffeic acid acts only as a scavenger of SO4•  radicals show that exptl values are lower than cl values. The  values, which are experimentally found quantum yield values at each caffeic acid concentration and corrected for SO4•  scavenging by caffeic acid, are also found to be greater than exptl values. These observations suggest that the adenine radicals are repaired by caffeic acid, in addition to scavenging of sulphate radical anions.

Indian Journal of Biochemistry & Biophysics

Vol 40, February 2003, pp.  31-39

Purification, characterization and chemical modification studies on a translation inhibitor protein from Luffa cylindrica

Ranjit C Singh, Anis Alam# and Vinod Singh*

Hormone Biochemistry Laboratory, Institute of Self-Organising Systems and Biophysics,

#Immunology Laboratory, Department of Biochemistry, North-Eastern Hill University,

Permanent Campus, Shillong 793 022, Meghalaya, India

Received 20 May 2002

A ribosome-inactivating protein (RIP), luffin has been isolated from the seeds of Luffa cylindrica of Cucurbitaceae family by ammonium sulfate fractionation followed by cation exchange and gel-filtration chromatography. Extensive physico-chemical, immunological and biological characterizations were carried out on luffin and compared with that of gelonin. The molecular mass of luffin was 28 kDa as determined by gel-filtration chromatography and SDS-PAGE. The -NH2 group(s) of luffin were sequentially modified by N-succinimidyl 6-[3-(2-pyridyldithio) propionamido] hexanoate (LC-SPDP), N-succinimidyl-3-(2-pyridylthio)propionate (SPDP) and 2-iminothiolane (2IT) and their effect on immunoreactivity and ribosome inactivating property was evaluated. Modification of single amino group resulted in about 80% inhibition of immunoreactivity and more than 90% loss of protein synthesis inhibition activity. Modification of 2-3 amino groups further hampered both immunoreactivity and protein-synthesis inhibition property LC-SPDP modification played more pronounced effects on immunoreactivity and RIP activity than that of SPDP. However, 2IT modification retained both the immunoreactivity and RIP activity of luffin-LC-SPDP substantially. SPDP showed more pronounced effect on immunoreactivity and RIP activity as compared to 2IT. Therefore, it seems that the positive charge on lysine residues plays an important role in immunological as well as protein synthesis inhibitory effect of luffin.

Indian Journal of Biochemistry & Biophysics

Vol 40, February 2003, pp. 40-45

Characterization of tyrosinase and accompanying laccase

from Amorphophallus campanulatus

Pallavi S Paranjpe1, Meena S Karve2 and Subhash B Padhye1*

1Department of Chemistry and 2Biochemistry, University of Pune, Pune 411007, India

Received 13 March 2002; revised 22 November 2002

Tyrosinase and laccase activities were detected in the corm of Amorphophallus campanulatus after extraction with ethanol followed by ammonium sulphate precipitation (20-60%) and dialysis against 10 mM Na2HPO4 buffer at pH 7.0. Tyrosinase was found to be the predominant enzyme exhibiting mono- and di-phenolase activities, specificity for L-DOPA as substrate, optimum pH being 6.0, optimum temperature at 40C and Km at 1.05 mM. Laccase showed substrate specificity for p-phenylenediamine (p-PD), Km at 2.7 mM, optimum pH being 5.0 and was inactivated above 40C. Three isoforms of tyrosinase were detected on SDS-PAGE with apparent molecular mass ~127, 31 and 27 kDa respectively. On staining sections of A. campanulatus with L-DOPA as substrate and 3-methyl benzothiazolinone hydrazone (MBTH) for colour development, tyrosinase was detected in the intercellular spaces of the plant tissue. The cytosolic region did not show any colour indicating the absence of the enzyme.

Indian Journal of Biochemistry & Biophysics

Vol 40, February 2003, pp. 46-50

Changes in NMR relaxation times in soybean and wheat seeds equilibrated at different temperatures and relative humidity

P Krishnan1,*#, S Nagarajan2 and A V Moharir1

1Division of Agricultural Physics, 2Nuclear Research Laboratory, Indian Agricultural Research Institute, New Delhi 110 012

Received 6 May 2002; revised 16 July 2002

The relationship between equilibration injury and equilibration dependence of the transverse relaxation time (T2) measurements was examined using NMR in two different seed species (sensitive-soybean and tolerant-wheat) differing in their sensitivity to seed equilibration conditions. The T2 values of both seed species declined with high temperature (45oC) and low RH (5.5-1%) and, also with high temperature (45oC) and high RH (74.5-100%) conditions. A comparison of injury based on electrolyte leakage, seed germination percentage and T2 indicated that membrane permeability increased both at high temperature (45oC) and low RH (5.5-1%) and high temperature (45oC) and high RH (74.5-100%) seed equilibration conditions. There was an increase in T2 until 11.5% and 5.5% RH in soybean and wheat species respectively, followed by a decline. Loss of seed viability during equilibration at very low RH (5.5-1%) at 45oC, and similarly at high RH (74.5-100%) at 45oC indicates that the changes in T2 are probably due to the loss of membrane injury.

Indian Journal of Biochemistry & Biophysics

Vol 40, February 2003, pp. 51-58

Computational analysis of evolutionary divergence

of scorpion toxins

R K Upadhyay

Laboratory of Human Genetics, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110 067, India

Received 24 September 2002; revised 27 December 2002

Conserved and consensus sequences of several members of scorpion toxin families have been analyzed. Multiple sequence alignment define the highly conserved residues that play important structural role in formation of toxin sub-groups. Scorpion toxins have characteristic feature in signature pattern {(GA)}- K-C {(LIVM)}-X (2)- K- C- C-X-C) }, where lysine and cysteine residues are well conserved in the polypeptide. The toxins show two major groups, one with conserved active region GKCMNKGKC and second with CTPK. There is variability in position of lysine and arginine in different toxins. Most of the scorpion toxins contain six conserved cysteines involved in disulphide bonds. From the evolutionary analysis it is concluded that Leiurus is a primitive ancestral species from which probably various toxins have evolved during the evolution.

Indian Journal of Biochemistry & Biophysics

Vol 40, February 2003, pp. 59-61

EPR studies on Fc receptor-mediated changes in

lymphocyte membrane

Rajesh K Gupta, Abhay H Pande†, Sumati and Krishnan Hajela*

School of Life Sciences, Devi Ahilya Vishwavidyalaya, Vigyan Bhawan, Khandwa Road Campus,

Indore 452 001 (MP), India

Received 8 July 2002; revised 26 August 2002

Biophysical evidence has been presented for the interaction of human lymphocyte membrane Fc receptors with aggregated IgG by severely restricting the rotational mobility of the cell surface proteins, as well as membrane lipids. Decrease in membrane fluidity was more prominent with aggregated IgG since the multivalency of Fc regions in aggregated IgG cross-linked cell surface Fc receptor.

Indian Journal of Biochemistry & Biophysics

Vol 40, February 2003, pp.  62-

Purification and properties of glucose

6-phosphate dehydrogenase from turkey erythrocytes

Hayrullah Yılmaz1, Mehmet Çiftçi2, 3,*, Şükrü Beydemir2, Ebubekir Bakan4 and  Ö İrfan Küfrevioğlu2

1Dicle University, Faculty of Education, Department of Chemistry

2Atatürk University, Arts and Science Faculty, Department of Chemistry

3Atatürk University, Biotechnology Application and Research Center

4Atatürk University, Medicinal Faculty, Department of Biochemistry

Received 25 February 2002; revised 28 October 2002

Glucose 6-phosphate dehydrogenase (G6PD) was purified from turkey erythrocytes by ammonium sulphate precipitation and followed by ADP Sepharose affinity gel chromatography. The yield was 49.71% and specific activity of the enzyme was found to be 44.16 EU/mg protein. By gel filtration the molecular mass was found to be 75 kDa. The enzyme had an optimum pH at 9.0, and optimum temperature at 50oC. Km and Vmax for NADP+ and glucose 6- phosphate (G6-P) as substrates were also determined and effects of inhibitors such as ATP, NADH and NADPH were examined.