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Indian Journal of Biochemistry & Biophysics VOLUME 37 NUMBER 4 AUGUST 2000 CONTENTS Development of oLH-S-S-gelonin conjugate bearing gelonin on -subunit of oLH Vinod Singh* A comparative study on serum levels of testosterone and SHBG in carcinomas of breast and uterine cervix A Ray, S L D Naik, S Katiyar, A Kumar, N S Murthy, S Sharma, A K Bahadur, S T Pasha, S A Husain and B K Sharma* 205 210 Formation of site and conformation specific antibodies to centchroman, a new non-steroidal contraceptive drug Apurva K Srivastava* and P K Grover 216 Purification and characterization of serum 2-globulin binding protein from Alocasia macrorhiza tuber B Shivananda Nayak and B Shivaraj* 227 Effect of glyphosate on the activity of DAHP synthase isozymes in callus cultures of groundnut (Arachis hypogaea L.) selected in vitro Mukesh Jain and Neera Bhalla-Sarin* 235 Nutrient associated changes in plasma membrane H +-ATPase activity of permeabilized Candida albicans cells Nikhat Manzoor, Bushra Rashid, M Amin and Luqman A Khan* 241 Dye-mediated photodynamic inactivation of Bacillus subtilis cells: Involvement of singlet oxygen and superoxide radicals Alok Dube*, Harsha Bansal and Pradeep K Gupta 245 X-band electron paramagnetic resonance spectra of pig serum albumin-copper(II) and pig serum albumin-copper(II)-amino acid systems R N Patel* and K B Pandeya 251 Metal ion mediated inhibition of firefly bioluminescence: A possibility via a quaternary complex T Sudhaharan and A Ram Reddy* 256 Molecular structure-activity relationship study of some non-steroidal anti-inflammatory agents using electrostatic potential mapping C Gopi Mohan and P C Mishra* 268 ___________ *Author for correspondence AUTHOR INDEX Amin M 241 Bahadur A K Bansal H 210 245 Dube A 245 Grover P K Gupta P K 216 245 Husain S A 210 Jain M 235 Katiyar S Khan L A Kumar A 210 241 210 Manzoor N Mishra P C Mohan C G Murthy N S 241 268 268 210 Naik S L D Nayak B S 210 227 Pandeya K B Pasha S T Patel R N 251 210 251 Rashid B Ray A Reddy A R 241 210 256 Sarin N B Sharma B K Sharma S Shivaraj B Singh V Srivastava A K Sudhaharan T 235 210 210 227 205 216 256 Indian Journal of Biochemistry & Biophysics Vol. 37, August 2000, pp. 205- 209 Development of oLH-S-S-gelonin conjugate bearing gelonin on subunit of oLH Vinod Singh* Hormone Biochemistry Laboratory, Institute of Self Organising Systems and Biophysics, North-Eastern Hill University, Permanent Campus, Shillong 793 022, Meghalaya, India Received 14 September 1999; accepted 28 December 1999 An attempt was made to cross-link the oLH of ovine luteinizing hormone (oLH) to the ribosome inactivating protein, gelonin in order to develop an effective hormonotoxin for selective targeting to specific cells in the gonads. Three different molar ratios of oLH and N-succinimidyl-3-(2pyridyldithio)propionate (SPDP) were used to activate the epsilon -NH2 groups of oLH. The oLH-SPDP derivatives recombine to oLH and the purified recombinants retained substantial receptor binding, steroidogenic activity and immunoreactivity to native oLH. The purified (gel-filtration) oLH-S-Sgelonin conjugates were allowed to recombine to oLH, but an RP-HPLC analysis indicated that recombination did not take place. The failure to recombine may be due to: (i) the site of -NH2 activation by SPDP may be different in oLH from native oLH; (ii) the activation site may be in close proximity to the recombination site, which facilitates the recombination of -subunit but failed to reassociate to oLHS-S-gelonin conjugate and (iii) the introduction of gelonin (30 kDa basic protein) might have induced some steric hindrance for oLH to recombine to the oLH site which might have been masked in oLHS-S-gelonin conjugates. Indian Journal of Biochemistry & Biophysics Vol. 37, August 2000, pp. 210- 215 A comparative study on serum levels of testosterone and SHBG in carcinomas of breast and uterine cervix A Ray1, S L D Naik1, S Katiyar1, A Kumar1, N S Murthy1, S Sharma1, A K Bahadur†, ‡ S T Pasha ,S A Husain$ and B K Sharma* of Cytology and Preventive Oncology (ICMR), †Department of Radiotherapy, Maulana Azad Medical College, New Delhi 110 002; ‡Division of Biochemistry and Biotechnology, National Institute of Communicable Diseases, Delhi 110 054 and $Department of Biosciences, Jamia Millia Islamia, New Delhi 110 025 Received 26 August 1999; revised 3 February 2000 1*Institute Serum levels of testosterone and sex-hormone binding globulin (SHBG) were measured in 31 patients with breast carcinoma and 33 patients with carcinoma of the uterine cervix along with 30 healthy (control) women. Amongst the patients of breast cancer, significantly higher level of testosterone and a low level of SHBG were found in comparison with the other two groups. The study has revealed that testosterone and SHBG may have an indirect role in breast cancer, probably through their influences on the amount of bioavailable estrogen. Indian Journal of Biochemistry & Biophysics Vol. 37, august 2000, pp. 216- 226 Formation of site and conformation specific antibodies to centchroman, a new non-steroidal contraceptive drug Apurva K Srivastava* and P K Grover Pharmacokinetics and Metabolism Division, Central Drug Research Institute, Lucknow 226 001, India Received 14 September 1998; revised 28 January 2000 Centchroman [Ormeloxifene, 3,4-trans-2, 2-dimethyl-3-phenyl-4-(p-pyrrolidino ethoxy)phenyl-7-methoxy-chroman (2a)] is a new non steroidal oral contraceptive for women. In an attempt to develop sensitive immunoassay to measure centchroman concentration in biological fluids, we describe production of antibodies against four hapten derivatives of centchroman, and evaluation of efficacy of these antibodies in developing sensitive and specific immunoassay of centchroman. Antigens were made by coupling four hapten derivatives, 3,4-trans-2,2-dimethyl-3-phenyl-4-(ppyrrolidinoethoxy)phenyl-7-carboxymethoxyl-chroman(2c),3,4-trans-2,2-dimethyl-3-phenyl-4-(p-inoylethoxy)phenyl7-methoxychroman (3a), 3, 4-trans-2,2-dimethyl-3-(m-hydroxyl)phenyl-4-(p-pyrro- lidinoethoxy)phenyl-7-methoxychroman (5a), and 3,4-trans-2,2-dimethyl-3-phenyl-4-(3’-amino,4’-pyrrolidinoethoxy)phenyl-7-methoxy-chroman (6a), to bovine serum albumin (BSA). Three enzyme tracers, made by coupling centchroman derivatives (2c, 3a, and 5a) to peroxidase (HRP), were utilized to monitor four antisera. Four homologous and heterologous enzyme immunoassay formats were developed and specificity of each antiserum was analyzed against anticipated metabolite of centchroman. Antiserum raised against centchroman derivative (6a) was found to be most specific, showing <4% crossreactivity with a putative metabolite, 7-desmethyl centchroman (2b). This antiserum, when used in solid phase EIA format, exhibited sensitivity of 250 pg/ml, total assay variance of CV<14%, and analytical recoveries between 82106%. Comparison of the EIA with a RIA, which was developed by using same antibody, showed a close correlation (r=0.9, n=40). Specificity of all antisera, as determined by the respective enzyme immunoassays, followed an uncharacteristic pattern of crossreactivity towards cis centchroman. Especially, antiserum generated against an immunogen (5b) showed high crossreaction with cis centchroman. Evidence provided in this study showed that immunogen (5b) produced a sub population of antibodies to a flipped conformation of the drug, which conformationally looks similar to cis centchroman. These conformation specific antibodies accounted for high crossreactivity towards cis centchroman. In conclusion, we describe the formation of conformation specific antibodies and the significance of site of attachment of centchroman derivative to carrier protein on the specificity of antibodies towards anticipated metabolites. Furthermore, by using antiserum generated in this study, sensitive immunoassays were developed and validated for the measurement of centchroman in serum. Indian Journal of Biochemistry & Biophysics Vol. 37, August 2000, pp.227- 234 Purification and characterization of serum 2-globulin binding protein from Alocasia macrorhiza tuber B Shivananda Nayak and B Shivaraj* Department of Biochemistry, Kasturba Medical College, Manipal 576 119, Karnataka, India Received 2 August 1999; revised 6 December 1999 A protein capable of precipitating serum 2-globulin was purified from Alocasia macrorhiza tuber. The protein, designated as Alocasia protein was heat labile and was found to exist in four isomeric forms, each having four subunits. The serum 2-globulin binding activity of the Alocasia protein was not altered by the action of proteolytic enzymes like trypsin, chymotrypsin and pepsin. Unlike the naturally occurring lectins, the Alocasia protein failed to agglutinate erythrocytes, leukocytes and the microbial organisms. In addition, different sugars and sugar derivatives did not prevent the complex formation between the Alocasia protein and 2-globulin of serum. Divalent metal ions and SH agents did not affect the activity of the protein. Preliminary studies indicated that haptoglobin and 2-macroglobulins were the major, if not the exclusive proteins that are responsible for interaction with the purified Alocasia protein. Indian Journal of Biochemistry & Biophysics Vol. 37, August 2000, pp. 235- 240 Effect of glyphosate on the activity of DAHP synthase isozymes in callus cultures of groundnut (Arachis hypogaea L.) selected in vitro Mukesh Jain† and Neera Bhalla-Sarin* School of Life Sciences, Jawaharlal Nehru University, New Delhi 110 067, India Received 31 May 1999; revised 10 September 1999 Two isozymes of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHP synthase or DS, EC 4.1.2.15) from the callus cultures of Arachis hypogaea L. were resolved by DEAE cellulose column chromatography and characterized using selective assays based on divalent cation requirements and regulation by the end-product amino acids. The total extractable activity of DAHP synthase did not show any change in the glyphosate sensitive and tolerant cell lines when compared to 5-enolpyruvylshikimate3-phosphate synthase (EPSP synthase, EC 2.5.1.19), the known target of inhibition by glyphosate. However, the activities of both, DAHP synthase and EPSP synthase were inhibited by glyphosate in vitro. The inhibitory effects of glyphosate on the activities of (a) EPSP synthase and DAHP synthase, and (b) isozymes of DAHP synthase, have been compared. The data indicate that the Co 2+ dependent isozyme of DAHP synthase is a putative target for the action of glyphosate in A. hypogaea. Indian Journal of Biochemistry & Biophysics Vol. 37, August 2000, pp. 241- 244 Nutrient associated changes in plasma membrane H+-ATPase activity of permeabilized Candida albicans cells Nikhat Manzoor, Bushra Rashid, M Amin and Luqman A Khan* Centre For Biosciences, Jamia Millia Islamia, New Delhi 110 025, India Received 22 April 1999; accepted 23 May 2000 Nutrient stimulated H+ extrusion by cells and spheroplasts of Candida albicans and its underlying mechanism has been investigated. Glucose and glutamic acid stimulated extrusion by a factor of 6 and 1.4 respectively in cells, but had no effect on spheroplasts. Proline showed no H+ stimulation in cells, however, it stimulated spheroplasts 1.5 fold. ATPase activity of toluene-ethanol-Triton-X-100 (TET) permeabilized cells decreased by 20% following exposure to glucose and proline and 30% following exposure to glutamic acid. Permeabilized spheroplasts of Candida albicans showed drastic reduction in ATPase activity by 40%, 70% and 85% following exposure to glucose, proline and glutamic acid respectively. Externally added F-actin fully restored activity in the case of permeabilized cells and partly with spheroplasts suggesting that ATPase interacts with other membrane proteins during nutrient stimulation of cells. Indian Journal of Biochemistry & Biophysics Vol. 37, August 2000, pp. 245- 250 Dye-mediated photodynamic inactivation of Bacillus subtilis cells: Involvement of singlet oxygen and superoxide radicals Alok Dube*, Harsha Bansal and Pradeep K Gupta Laser Programme, Centre for Advanced Technology, Indore 452013, India Received 7 May 1999; accepted 16 January 2000 Studies were carried out on the photodynamic inactivation of Bacillus subtilis cells by He-Ne laser irradiation in presence of methylene blue and toluidine blue. Electron paramagnetic resonance (EPR) measurements show decrease in the cell membrane fluidity due to photodynamic damage of the lipophilic compartments. Study of cell damage in presence of sodium azide, D2O and glutathione as well as measurements on singlet oxygen/free radical generation by EPR show that with toluidine blue, singlet oxygen is the major reactive species leading to cell membrane damage. No free radical generation was observed in toluidine blue mediated photodamage. However, with methylene blue our observations suggest that the photodamage arises due to both singlet oxygen mediated membrane damage and intracellular damage by superoxide radical. Indian Journal of Biochemistry & Biophysics Vol. 37, August 2000, pp. 251- 255 X-band electron paramagnetic resonance spectra of pig serum albumincopper(II) and pig serum albumin-copper(II)-amino acid systems R N Patel* and K B Pandeya Department of Chemistry, A P S University, Rewa (M P) 486 003, India Received 18 March 1999; accepted 29 March 2000 EPR spectra of the binary, PSA-copper(II) (1:1 and 2:1) and ternary systems, PSA-copper(II)-amino acid (1:1:1) have been studied. In binary system, two distinct EPR features have been observed, one of the features (towards the low field), showing broad and overlapping signals is attributed to non-specific bonding of copper(II) to albumin and other feature (towards higher field), showing sharp intense signals is attributed to specific bonding. The change from non-specific to specific bonding is favoured by increase in pH as well as by increase in protein concentration. Observed pH for transition from nonspecific binding is 10.00 < pH < 11.30. A better resolution of nitrogen hyperfine couplings are obtained for both binary, PSA-Cu(II) (1:1 and 2:1) and ternary PSA-Cu(II)-amino acid (1:1:1) systems. Seven and more than seven nitrogen superhyperfine lines are observed in g region of binary and ternary systems respectively. Indian Journal of Biochemistry & Biophysics Vol. 37, August 2000, pp. 256- 267 Metal ion mediated inhibition of firefly bioluminescence: A possibility via a quaternary complex T Sudhaharan and A Ram Reddy Department of Chemistry, Nizam College, Hyderabad 500 001, A P, India Received 13 January 1999; revised 26 July 1999 D(-) Luciferin, interacts with different metal ions to produce colourless soluble salts with absorption spectra broader, intense and red shifted as compared to those of the parent compound. The equilibrium constants for the luciferin-metal ion system vary in the order, depository divalent transition metal ions > alkali metal ions. The equilibrium constants for the ternary complexes formed between metal ions and a mixture of luciferin and luciferase are larger than that of binary complexes but vary in the same order. Steady state fluorometric titration's of luciferin further confirmed its complexation with metal ions. The single absorption maximum of firefly luciferase at 278 nm originating from tyrosine was split into a doublet in presence of transition metal ions. The absorption maximum at lower wavelength is attributed to the H-bond raptured free tyrosine denatured conformation of the luciferase while the longer wavelength band to tyrosine-transition metal ion complex. Difference spectra of luciferase metal ion complex yielded change in the molar extinction coefficients from which the number of tyrosine molecules exposed to aqueous solution by the perturbant metal ions are evaluated following the Donovan model. The number of tyrosine molecules exposed to the aqueous medium as a result of conformational change in the enzyme are 4, 3, 3, 2 and 3 by Hg2+, Mn2+, Co2+, Cd2+ and Cs+ respectively. The denaturation constants calculated for the luciferase-metal ion complexes vary between 0.152 and 0.570 and follow the order of Hg2+>Cs+>Cd2+>Co2+>Mn2+. Steady state fluorescence data reveal that the metal ions quench the fluorescence of enzyme by complexation with the side chain residues of the excited state tyrosine. Profound change in the UV CD spectrum of luciferin and luciferase in presence of metal ions was attributed to the conformational change in the substrate and enzyme. Thus the inhibition of luciferase activity in the firefly bioluminescence by metal ions is attributed to the quaternary complex formed between metal ion-luciferin-luciferase and ATP near or around the active site of the enzyme. Indian Journal of Biochemistry & Biophysics Vol. 37, August 2000, pp. 268- 272 Molecular structure-activity relationship study of some non-steroidal anti-inflammatory agents using electrostatic potential mapping C Gopi Mohan* and P C Mishra† † * Department of Physics, Banaras Hindu University, Varanasi 21 005, India Molecular Biophysics Unit, Indian Institute of Science, Bangalore-560 012, India and Received 4 October 1999; revised 13 March 2000 A series of 6,11-dihydro-11-oxodibenz[b,e]oxepin-2-acetic acids (DOAA) which are known to be anti-inflammatory agents were studied. The geometries of some of the molecules obtained from X-ray crystallography were used in the calculations as such while the geometries of their derivatives were obtained by local, partial geometry optimization around the sites of substitution employing the AM1 method, keeping the remaining parts of the geometries the same as those in the parent molecules. Molecular electrostatic potential (MEP) mapping was performed for the molecules using optimized hybridization displacement charges (HDC) combined with Löwdin charges, as this charge distribution has been shown earlier to yield near ab initio quality results. A good correlation has been found between the MEP values near the oxygen atoms of the hydroxyl groups of the carboxy groups of the molecules and their anti-inflammatory activities. The result is broadly in agreement with the model proposed earlier by other authors regarding the structure-activity relationship for other similar molecules.