351
Website address: http://www.bioline.org.br/ib
www.niscom.res.in
Indian Journal of Biochemistry & Biophysics
VOLUME 38
NUMBER 6
DECEMBER 2001
CONTENTS
UDP-galactose 4-epimerase from Escherichia coli: Equilibrium unfolding studies
Suprabha Nayar and Debasish Bhattacharyya*
Limited proteolysis by trypsin influences activity of maize phosphoenolpyruvate
carboxylase
Gururaj B Maralihalli and Anil S Bhagwat*
Equilibrium denaturation of buffalo pituitary growth hormone
Kapil Maithal, H G Krishnamurty and K Muralidhar*
Physico-chemical characterization of growth hormone from water buffaloes
(Bubalus bubalis)
Kapil Maithal, H G Krishnamurty and K Muralidhar*
Buffalo plasma fibronectin : A physico-chemical study
Nizamuddin Ahmed*, Ramesh chandra and H G Raj
Non-coordinate expression of closely linked mouse casein genes
Sisilamma George*, Anthea Springbett, A John Clark and Alan L Archibald
353
361
368
375
384
393
Effect of a water soluble derivative of - tocopherol on radiation response of
Saccharomyces cerevisiae
Rakesh K Singh, Naresh C Verma* and V T Kagiya
399
Effect of -irradiation on H3 histone and DNA in solution
S N Upadhyay
406
Role of liquid membrane phenomenon in biological actions of ACE inhibitors, captopril
and lisinopril
A N Nagappa*, R T Patil, V Pandi and K Ziauddin
Conformational study of peptides containing dehydrophenylalanine : Helical structures
without hydrogen bond
Fateh S. Nandel* , Harpreet Kaur , Nandita Malik, Neelaabh Shankar and
Dharam V S Jain
412
417
Meeting Report
426
Annual Index
429

——————
*Author for correspondence
Indian Journal of Biochemistry & Biophysics
Vol. 38, December 2001, pp. 353- 360
UDP-galactose 4-epimerase from Escherichia coli: Equilibrium
unfolding studies
Suprabha Nayar and Debasish Bhattacharyya*
Indian Institute of Chemical Biology, 4 Raja S.C. Mallick Road, Jadavpur, Calcutta 700 032
Received 12 February 2001; revised and accepted 19 October 2001
UDP-galactose 4-epimerase from Escherichia coli is a homodimer of 39 kDa subunit with
non-covalently bound NAD acting as cofactor. The enzyme can be reversibly reactivated after
denaturation and dissociation using 8 M urea at pH 7.0. There is a strong affinity between the
cofactor and the refolded molecule as no extraneous NAD is required for its reactivation.
Results from equilibrium denaturation using parameters like catalytic activity, circulardichroism, fluorescence emission (both intrinsic and with extraneous fluorophore 1-aniline 8naphthalene sulphonic acid), 'reductive inhibition' (associated with orientation of NAD on the
native enzyme surface), elution profile from size-exclusion HPLC and light scattering have
been compiled here. These show that inactivation, integrity of secondary, tertiary and
quaternary structures have different transition mid-points suggestive of non-cooperative
transition. The unfolding process may be broadly resolved into three parts: an active dimeric
holoenzyme with 50% of its original secondary structure at 2.5 M urea; an active monomeric
holoenzyme at 3 M urea with only 40% of secondary structure and finally further denaturation
by 6 M urea leads to an inactive equilibrium unfolded state with only 20% of residual
secondary structure. Thermodynamical parameters associated with some transitions have been
quantitated. The results have been discussed with the X-ray crystallographic structure of the
enzyme.
Indian Journal of Biochemistry & Biophysics
Vol. 38, December 2001, pp. 361- 367
Limited proteolysis by trypsin influences activity of maize
phosphoenolpyruvate carboxylase
Gururaj B Maralihalli and Anil S Bhagwat*
Molecular Biology and Agriculture Division, Bhabha Atomic Research Centre,
Trombay, Mumbai 400 085
Received 7 March 2001; revised and accepted 13 July 2001
Maize phosphoenolpyruvate carboxylase (PEPC) was rapidly and completely inactivated by
very low concentrations of trypsin at 37C. PEP+Mg2+ and several other effectors of PEP
carboxylase offered substantial protection against trypsin inactivation. Inactivation resulted
from a fairly specific cleavage of 20 kDa peptide from the enzyme subunit. Limited proteolysis
under catalytic condition (in presence of PEP, Mg 2+ and HCO3) although yielded a truncated
subunit of 90 kDa, did not affect the catalytic function appreciably but desensitized the enzyme
to the effectors like glucose-6-phosphate glycine and malate. However, under non-catalytic
condition, only malate sensitivity was appreciably affected. Significant protection of the
enzyme activity against trypsin during catalytic phase could be either due to a conformational
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change induced on substrate binding. Several lines of evidence indicate that the inactivation
caused by a cleavage at a highly conserved C-terminal end of the subunit.
Indian Journal of Biochemistry & Biophysics
Vol. 38, December 2001, pp. 368- 374
Equilibrium denaturation of buffalo pituitary growth hormone
Kapil Maithal1,2, H G Krishnamurty1 and K Muralidhar2,*
1Department
of Chemistry, University of Delhi, Delhi 110007, India
2Department
of Zoology, University of Delhi, Delhi 110007, India
Received 1 September 2000; accepted 15 January 2001
To understand the structural properties of buffalo growth hormone (buGH), the equilibrium
denaturation using guanidinium chloride (GdmCl) was carried out and was monitored by
ultraviolet absorption spectroscopy, intrinsic fluorescence spectroscopy, far UV-circular
dichroism and size-exclusion chromatography. The normalized denaturation transition curves for
each of the above methods were not coincident, showing that buGH does not follow a simple two
state folding mechanism. Further, size-exclusion chromatography also showed the presence of an
associated intermediate during the unfolding of buGH. It was observed that in buGH,
denaturation resulted in an initial disruption of the tertiary structure, whereas the secondary
structure and the degree of compactness were disrupted at a higher concentration of the
denaturant. This suggests that buGH follows the hierarchical model of protein folding.
Indian Journal of Biochemistry & Biophysics
Vol. 38, December 2001, pp. 375- 383
Physico-chemical characterization of growth hormone from
water buffaloes (Bubalus bubalis)
Kapil Maithal1,2, H G Krishnamurty1 and K Muralidhar2,*
1Department
of Chemistry, University of Delhi, Delhi 110 007, India
2Department
of Zoology, University of Delhi, Delhi 110 007, India
Received 8 September 2000; accepted 15 January 2001
A purified preparation of growth hormone from pituitaries of water buffaloes (Bubalus bubalis) has
been extensively characterized with regard to physico-chemical properties. The molecular size of buffalo
GH (buGH) by electrospray ionization mass spectroscopy (ES-MS) was found to be 21394.00  8.44Da
and its stokes radius was determined as 2.3 nm. Size heterogeneity in buffalo GH was checked both by
electrophoresis and molecular sieve chromatography using 125I-labelled buffalo GH. Similar size
heterogeneity was found in standard preparations of ovine and bovine growth hormones. Isoelectric
focussing and chromatofocussing indicated charge heterogeneity in buffalo GH preparation. Major charge
isoforms having pI of 7.2, 7.7 and minor forms in the pI range of 5.7 to 7.0 were found. Lectin
chromatography on Concanavalin A matrix showed that less than 1% of buffalo GH was glycosylated.
Heterogeneity in NH2-terminal sequence was also observed, with alanine, phenylalanine and methionine
as the NH2-terminal residues as checked by dansyl and DABITC methods. Estimation of tryptophan
residue indicated that a single tryptophan residue was present. Ellman’s method showed presence of two
disulfide bridges per mole of buffalo GH. Intrinsic fluorescence spectrum of buffalo GH exhibited 
emission maximum at 337 nm. UV-CD spectrum showed that almost 48% of the secondary structure of
buGH was constituted by -helicity. The TM of buGH as determined by differential scanning calorimetric
(DSC) studies was found to be 63C.
Indian Journal of Biochemistry & Biophysics
Vol. 38, December 2001, pp. 384- 392
Buffalo plasma fibronectin : A physico-chemical study
Nizamuddin Ahmeda*, Ramesh Chandraa and H G Rajb
aDr
B R Ambedkar Center for Biomedical Research, University of Delhi, Delhi 110 007;
bDepartment
of Biochemistry, V.P. Chest Institute, University of Delhi, Delhi 110 007, India
Received 13 November 2000; revised and accepted 30 October 2001
Plasma fibronectin (FN) of buffalo (Babulis babulis) was purified to apparent homogeneity,
using gelatin-Sepharose and heparin-Sepharose affinity columns. It was found to have two
subunits of molecular mass 246 kDa and 228 kDa, on SDS-gel. Its immunological crossreactivity with anti-human plasma FN was confirmed by Western blotting. The amino acid
composition was found to be similar to that of human and bovine plasma FNs. Buffalo plasma
FN contained 2.23% neutral hexoses and 1.18% sialic acids. No titrable sulfhydryl group could
be detected in the absence of denaturant. Reaction with DTNB indicated 3.4 sulfhydryl groups
in the molecule, whereas BDC-OH titration gave a value of 3.8 SH groups in buffalo plasma
FN. Stoke's radius, intrinsic viscosity, diffusion coefficient and frictional ratio indicated that
buffalo plasma FN did not have a compact globular conformation at physiological pH and ionic
strength. Molecular dimensions (average length, 120 nm; molar mass to length ratio, 3950 nm -1
and mean diameter, 2.4 nm) as revealed by rotary shadowing electron microscopy further
supported the extended conformation of buffalo plasma FN. These results show that buffalo
plasma FN has similar properties as that of human plasma FN.
Indian Journal of Biochemistry & Biophysics
Vol. 38, December 2001, pp. 393- 398
Non-coordinate expression of closely linked mouse casein genes
Sisilamma George†, Anthea Springbett*, A John Clark* and Alan L Archibald*
Department of Veterinary Biochemistry, College of Veterinary and Animal Sciences, Mannuthy, Trichur,
680 651, India
*Roslin Institute (Edinburgh), Roslin, Midlothian, EH25 9PS, Scotland, UK
Received 13 February 2001; revised and accepted 6 June 2001
Expression levels of five mouse casein genes were analysed in the mammary gland of
virgin, pregnant and lactating mice. We have already shown that the five murine casein genes
are arranged in the order, ---- in a tandem array, very close to each other in a 250 kb
DNA fragment of mouse genome. Northern blot analysis showed that, of the calcium-sensitive
casein genes, the casein gene is expressed only during lactation unlike the andcasein
genes which are expressed during pregnancy and lactation. Even though the  and  genes
exhibited a co-ordinated expression pattern from mid to the later stages of pregnancy, the
mRNA levels varied considerably (60, 90 and 100% respectively) by the onset of lactation. The
mRNA level of the calcium-insensitive casein gene increased from mid-pregnancy but at a
lower rate and reached ~60% by the first day of lactation. Considering the locations and
351
closeness of the casein genes, a non-coordinate expression profile is exhibited by the mouse
casein genes, particularly the casein gene.
Indian Journal of Biochemistry & Biophysics
Vol. 38, December 2001, pp. 399- 405
Effect of a water soluble derivative of -tocopherol on radiation
response of Saccharomyces cerevisiae
Rakesh K Singh, Naresh C Verma* and V T Kagiya$
Radiation Biology Division, Bhabha Atomic Research Centre, Mumbai 400 085, India
$Health
Research Foundation, Kyoto, Japan
Received 5 March 2001; revised 20 June 2001; accepted 18 September 2001
The radioprotection conferred by a highly water soluble glucose derivative of -tocopherol,
namely, 2-(-D-glucopyranosyl) methyl-2,5,7,8-tetramethylchroman-6-ol (TMG) in
Saccharomyces cerevisiae was studied. Cells grown in standard YEPD-agar medium and
irradiated in the presence of TMG showed a concentration dependent higher survival up to 10
mM of TMG in comparison to cells irradiated in distilled water. Treatment of TMG to cells
given either before or immediately after irradiation but not during irradiation, had no effect on
their radiation response. S. cerevisiae strain LP1383 (rad52) which is defective in
recombination repair showed enhanced radioresistance only when subjected to irradiation in
presence of TMG. Cells of rad52 strain grown in the medium containing TMG showed a
radiation response similar to that of cells grown in the medium without TMG. The nature of
TMG dependent enhanced radioresistance was studied by scoring the mutations in the strain D7, which behaved like wild type strain in complete medium, at trp and ilv loci. Our study
indicated that TMG confers radioresistance in S. cerevisiae possibly by two mechanisms viz.
(i), by eliminating radiation induced reactive free radicals when the irradiation is carried out in
the presence of TMG and (ii), by activating an error – prone repair process involving RAD52
gene, when the cells are grown in the medium containing TMG.
Indian Journal of Biochemistry & Biophysics
Vol. 38, December 2001, pp. 406- 411
Effect of -irradiation on H3 histone and DNA in solution*
S N Upadhyay
Radiation Biology Department, Institute of Nuclear Medicine & Allied Sciences, Lucknow Road, Delhi
110 054
Received 3 October 2000; revised 26 October 2001
Three methods, namely, absorbance of colour by reaction with Folin–Ciocalteau reagent,
UV absorbance and fluorescence intensity measurements for detection of H3 histone in 0.15 M
standard saline citrate (SSC) solution were compared. Maximum sensitivity was found with the
Folin-Ciocalteau method. Effect of varying pH and of - radiation on H3 histone and on
interaction of H3 histone with DNA were studied. For this, solutions of H3 histone in SSC, in
0.9% NaCl, H3 histone + DNA in 0.9% NaCl were subjected to varying pH (1-10) and radiation (dose 10-50 Gy) and max and Amax were monitored. From the molar ratios of histone
and DNA in the complex, it was observed that at  -radiation dose of 50 Gy and pH 8.54, there
was a depletion of 6-8 g/ml of histone from the histone-DNA complex.
Indian Journal of Biochemistry & Biophysics
Vol. 38, December 2001, pp. 412- 416
Role of liquid membrane phenomenon in biological actions of ACE
inhibitors, captopril and lisinopril
A N Nagappa*, R T Patil, V Pandi and K Ziauddin
Department of Pharmacology, SCS College of Pharmacy, Harapanahalli, 583 131, India
Received 3 September 2000; accepted 2 February 2001
The liquid membrane phenomenon in angiotensin converting enzyme (ACE) inhibitors
namely, captopril and lisinopril has been studied. Hydraulic permeability data have been
obtained to demonstrate the existence of the liquid membrane in series with a supporting
membrane generated by the ACE inhibitors. Data on the transport of the relevant permeants in
presence of the liquid membrane formed by ACE inhibitors indicate that liquid membrane
phenomenon is likely to play a significant role in the action of ACE inhibitors.
Indian Journal of Biochemistry & Biophysics
Vol. 38, December 2001, pp. 417- 425
Conformational study of peptides containing
dehydrophenylalanine : Helical structures without hydrogen bond
Fateh S Nandel*, Harpreet Kaur, Nandita Malik, Neelaabh Shankar and Dharam V S Jain 1
*Department of Biophysics, 1Department of Chemistry, Panjab University, Chandigarh 160014, India
Received 31 January 2001; revised and accepted 6 August 2001
The conformational behaviour of ZPhe has been investigated in the model dipeptide Ac Phe-NHMe
and
in
the
model
tripeptides
Ac-X-ZPhe-NHMe
with
X=Gly,Ala,Val,Leu,Abu,Aib and Phe and is found to be quite different. In the model
tripeptides with X=Ala,Val,Leu,Abu,Phe the most stable structure corresponds to 1=-30o,
1=120o and 2=2=30o. This structure is stabilized by the hydrogen bond formation between
C=O of acetyl group and the NH of the amide group, resulting in the formation of a 10membered ring but not a 310 helical structure. In the peptides Ac-Aib-ZPhe-NHMe and Ac(Aib-ZPhe)3-NHMe, the helical conformers with = 30o, = 60o for Aib residue and ==
30o for ZPhe are predicted to be most stable. The computational studies for the positional
preferences of ZPhe residue in the peptide containing one ZPhe and nine Ala residues reveal
the formation of a 310 helical structure in all the cases with terminal preferences for ZPhe. The
conformational behaviour of Ac-(ZPhe)n-NHMe with n4 is predicted to be very labile. With
n > 4, degenerate conformational states with , values of 0  90 adopt helical structures
which are stabilized by carbonyl-carbonyl interactions and the N-H- interactions between the
amino group of every ZPhe residue with one C-C edge of its own phenyl ring. The results are
in agreement with the experimental finding that screw sense of helix for peptides containing
ZPhe residues is ambiguous in solution. The helical structures stabilized by hydrogen bond
formation are found to be atleast 3kCalmol-1 less stable. Conformational studies have also been
carried out for the peptide Ac-(EPhe)6-NHMe and the peptide Ac-Ala-(ZPhe)6-NHMe
containing Ala residue at the N-terminal. The N-H- interactions are absent in peptide Ac(EPhe)6-NHMe.
Z
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Indian Journal of Biochemistry & Biophysics
Vol. 38, December 2001, pp. 426- 428
TRendys Meeting, 2001
Indian Journal of Biochemistry & Biophysics
Vol. 38, December 2001, pp. 429- 440
Annual Index