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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.