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Indian Journal of Biochemistry & Biophysics

 

VOLUME 39

NUMBER 1

FEBRUARY 2002

 
CONTENTS

 

Minireview

 

On the importance of backbone loop and peptide flip in the Walker sequence in
F1-ATPase action

5

T Ramasarma* and C Ramakrishnan

 

 

Papers

 

Crystallization and structure determination of goat lactoferrin at 4.0 resolution: A new form of packing in lactoferrins with a high solvent content in crystals

16

Pravindra Kumar, Savita Yadav and Tej P Singh*

 

 

Constant variation in structure and function of geometrical isomers of acitretin under natural light

22

Akira Murayama, Takakazu Suzuki, Minoru Iwamoto and Sridhar Rao Kunchala

 

 

Chemical modification of catalytic site of lipase from wheat germ: Altered structure-activity profile

28

K N Gopalakrishna, P Ramesh Kumar and V Prakash*

 

 

Compositional correlation and codon usage studies in Buchnera aphidicola

35

S K Gupta, T K Bhattacharyya and T C Ghosh*

 

 

Affinity purification and characterization of a seed lectin from Crotalaria medicaginea

49

Navjot Kaur, Jatinder Singh* and Sukhdev Singh Kamboj

 

 

Inactivation of maize NADP-malic enzyme by Cu2+-ascorbate

55

S E Pinto, S R Rao( and A S Bhagwat

 

Solubilization of m-opioid receptors enriched from bovine brain membranes

60

KVH Sastry*, B Yadgiri, J M Reddy and M K Janardanasarma

 

 

Notes

 

Related affinities of different mononucleosides and mononucleotides towards Pr (III) and Nd (III) in aquated organic solvents explored through 4f-4f transition spectral analysis

66

Sudhindra N Mishra, Suveerkumar C M, Manish C Patel, Prashant N Bhatt and Jignasu P Mehta*

 

 

Announcement

70

 

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 39, February 2002, pp. 5- 15

 

On the importance of backbone loop and peptide flip in the
Walker sequence in F1-ATPase action

T Ramasarma and C Ramakrishnan

 

The Walker sequence, GXXXXGKT, present in all the six subunits of F1-ATPase exists in a folded form, known as phosphate-binding loop (P-loop). Analysis of the Ramachandran angles showed only small RMS deviation between the nucleotide-bound and nucleotide-free forms. This indicated a good overlap of the backbone loops.

The catalytic b-subunits (chains D, E and F) showed significant changes in the Ramachandran angles and the side chain torsion angles, but not the structural a-subunits (chains A, B and C). Most striking among these are the changes associated with Val160 and Gly161 corresponding to a flip in the peptide unit between them when a nucleotide is bound (chains D or F compared to nucleotide-free chain E).

The conformational analysis further revealed a hitherto unnoticed hydrogen bond between amide-N of the flipped Gly161 and terminal phosphate-O of the nucleotide. This assigns a role for this conserved amino acid, otherwise ignored, of making an unusual direct interaction between the peptide backbone of the enzyme protein and the incoming nucleotide substrate. Significance of this interaction is enhanced, as it is limited only to the catalytic subunits, and also likely to involve a mechanical rotation of bonds of the peptide unit. Hopefully this is part of the overall events that link the chemical hydrolysis of ATP with the mechanical rotation of this molecule, now famous as tiny molecular motor.

Indian Journal of Biochemistry & Biophysics

Vol. 39, February 2002, pp. 16- 21

 

 

Crystallization and structure determination of goat lactoferrin at 4.0 resolution: A new form of packing in lactoferrins with a high solvent content in crystals

Pravindra Kumar, Savita Yadav and Tej P Singh

 

Lactoferrin was purified from fresh samples of goat colostrums, saturated with Fe3+ and CO32- ions and crystallized by microdialysis method. The crystals belong to orthorhombic space group P212121 with a=104.6 , b=153.8 , c=155.1 and Z=4. The quality of crystals was poor, thus the intensity data were restricted to 4.0 resolution only. The structure was determined by molecular replacement method using diferric buffalo lactoferrin as a model. The solution clearly indicated the presence of one molecule in the asymmetric unit, which corresponds to a Vm value of 7.1 3/Da. The structure was refined with stringent constraints to an R-factor of 0.246 using all the reflections 15,870 to 4.0 resolution. The overall structure of goat lactoferrin is essentially similar to those of buffalo and bovine lactoferrins. However, the iron-binding environment in goat lactoferrin is somewhat different, in which 2 CO32- ions have low occupancies. The solvent content of approximately 84% was very high in the present case which explains the fragility of the crystals of goat lactoferrin. In a way, it is very surprising that the crystals grow at all, although crystals with solvent as high as 89% have been reported.

 

Indian Journal of Biochemistry & Biophysics

Vol. 39, February 2002, pp. 22- 27

 

 

Constant variation in structure and function of geometrical isomers of acitretin under natural light

Akira Murayama, Takakazu Suzuki, Minoru Iwamoto and Sridhar Rao Kunchala

 

Acitretin, a beneficial retinoid, was shown to undergo constant structural interconversions among its geometrical isomers (all-trans-acitretin, 9-cis-acitretin, 13-cis-acitretin, 9, 13-di-cis-acitretin, etc.) by photoisomerization under natural light. The photoisomerization was zero order reaction with an apparent velocity of 410-7 M/min under illumination by white fluorescent lamps (1, 200 lx). An equilibrium mixture of the geometrical isomers (all-trans-acitretin 20%, 9-cis-acitretin 15%, 13-cis-acitretin 30%, 9, 13-di-cis-acitretin 15%, and unidentified compounds 20%) was formed at around 30 min. Equilibrium mixtures with similar composition were obtained by photoisomerization reactions starting from other geometrical isomers. Geometrical isomers of acitretin thus formed, showed different effects to induce differentiation of human acute promyelocytic leukemia cells (HL-60 cells): activity of all-trans-acitretin (ED50, 3.210-6M), 9-cis-acitretin (ED50, 2.310-5M), 13-cis-acitretin (ED50, 1.110-5M), 9, 13-di-cis-acitretin (ED50, 2.610-6M). 9-cis-Acitretin acted synergistically with all-trans-acitretin, 13-cis-acitretin and 9, 13-di-cis-acitretin on HL-60 cells. On the other side, all-trans-acitretin, 13-cis-acitretin and 9, 13-di-cis-acitretin acted additively. Geometrical isomers of acitretin showed different effects on differentiation of human epidermal keratinocytes; expression of keratinocyte differentiation markers, keratin 1 and keratin 10, were suppressed more strongly by 9-cis-acitretin and 13-cis-acitretin as compared to all-trans-acitretin or 9, 13-di-cis-acitretin.

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 39, February 2002, pp. 28- 34

 

 

Chemical modification of catalytic site of lipase from wheat germ: Altered structure-activity profile

 

K N Gopalakrishna, P Ramesh Kumar and V Prakash

 

Wheat germ lipase (WGL) was inactivated by chemical modification of histidine, serine and carboxyl groups of Asp/Glu residues with diethyl pyrocarbonate (DEPC), phenyl methyl sulfonyl fluoride (PMSF) and 1-ethyl-3-(3-dimethylaminopropyl) carbodi-imide (EDC), respectively. Loss of activity of WGL was concentration dependent of the inhibitor and at 30 mM PMSF most of the activity of the enzyme was lost. The stoichiometry of modification showed one mole of histidine, serine and two moles of carboxyl groups modified per mole of protein. Kinetic measurements indicated that the inhibition of the enzyme was competitive in nature. The modified enzyme was further characterized by far UV-circular dichroic measurements of the secondary structure and fluorescence spectroscopy. PMSF-modified enzyme showed decreased thermal stability, whereas no change was observed in DEPC-modified enzyme as evidenced by differential scanning calorimetry. These studies indicate that histidine, serine and Asp/Glu residues play an important role in the catalytic function of WGL. The mechanism of loss of activity is due to minor conformational change in the microenvironment of the active site rather than the gross conformational change of the molecule itself.

 

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 39, February 2002, pp.35- 48

 


Compositional correlation and codon usage studies in Buchnera aphidicola

S K Gupta, T K Bhattacharyya and T C Ghosh

 

Compositional distributions in three different codon positions as well as codon usage biases of all available DNA sequences of Buchnera aphidicola genome have been analyzed. It was observed that GC levels among the three codon positions is I>II>III as observed in other extremely high AT rich organisms. B. aphidicola being an AT rich organism is expected to have A and/or T at the third positions of codons. Overall codon usage analyses indicate that A and/or T ending codons are predominant in this organism and some particular amino acids are abundant in the coding region of genes. However, multivariate statistical analysis indicates two major trends in the codon usage variation among the genes; one being strongly correlated with the GC contents at the third synonymous positions of codons, and the other being associated with the expression level of genes. Moreover, codon usage biases of the highly expressed genes are almost identical with the overall codon usage biases of all the genes of this organism. These observations suggest that mutational bias is the main factor in determining the codon usage variation among the genes in B. aphidicola.

 

Indian Journal of Biochemistry & Biophysics

Vol. 39, February 2002, pp. 49- 54

 

 

Affinity purification and characterization of a seed lectin
from Crotalaria medicaginea

Navjot Kaur, Jatinder Singh and Sukhdev Singh Kamboj

 

Purification of lectin from the seeds of Crotalaria medicaginea Lamk by affinity chromatography on asialofetuin-linked amino activated silica, yielded a single band on non-denatured PAGE at pH 4.5 and 8.3 and, a single peak on HPLC size exclusion and cation exchange columns. The molecular mass of the native C. medicaginea lectin was determined to be 125 kDa by gel filtration. In SDS-PAGE, the lectin migrated as a single band of Mr 31.6 kDa under reducing and non-reducing conditions, indicating that it is a tetramer of apparently identical subunits. It agglutinated red blood cells (RBCs) from rabbit and human ABO blood groups. It also reacted with RBCs from rat, sheep, goat and guinea pig but after desialylation with neuraminidase. The hemagglutination activity of the lectin was inhibited by d-galactose and its derivatives. Amino acid analysis showed that lectin was rich in aspartic and glutamic acid and, did not contain sulphur containing amino acids. The lectin is a glycoprotein having 1.41% of neutral sugars. It is labile at temperature above 60oC. It needs divalent cations for its activity, as a loss of activity was observed on removal of Ca2+ and Mn2+. Denaturing agents like urea, thiourea and guanidine-HCl have no effect on its activity.

 

Indian Journal of Biochemistry & Biophysics

Vol. 39, February 2002, pp. 55- 59

 

 

 

Inactivation of maize NADP-malic enzyme by Cu2+-ascorbate

 

S E Pinto, S R Rao and A S Bhagwat

 

Maize malic enzyme was rapidly inactivated by micromolar concentrations of cupric nitrate in the presence of ascorbate at pH, 5.0. Ascorbate or Cu2+ alone had no effect on enzyme activity. The substrate L-malate or NADP individually provided almost total protection against Cu2+-ascorbate inactivation. The loss of enzyme activity was accompanied by cleavage of the enzyme. The cleaved peptides showed molecular mass of 55 kDa, 48 kDa, 38 kDa, and 14 kDa. Addition of EDTA, histidine and imidazole provided protection. The results of protection experiments with sodium azide, DABCO and catalase suggested that reactive oxygen species were generated resulting in loss of enzyme activity. This was further supported by experiments showing that the rate of enzyme inactivation was higher in D2O than in water. It is suggested that maize malic enzyme is modified by reactive oxygen species like singlet oxygen and H2O2 generated by Cu2+-ascorbate system and the modified amino acid residue(s) may be located at or near the substrate-binding site of the enzyme

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 39, February 2002, pp 60- 65

 

 

 

Solubilization of μ-opioid receptors enriched from bovine brain membranes

K V H Sastry, B Yadgiri, J M Reddy and M K Janardanasarma

 

Solubilization is the most critical step in the purification of opioid receptors as these proteins are highly sensitive to detergents and get inactivated even with very mild detergents. Membranes enriched with -opioid receptors from bovine corpus striatum were solubilized by various methods to obtain the active soluble receptor suitable for affinity purification. Solubilization by digitonin resulted in marginal yields. CHAPS in presence of NaCl could extract active receptor into the solution. The detergent and NaCl were removed by either polyethylene glycol precipitation or by desalting on Sephadex G50. The polyethylene glycol precipitation resulted in the formation of liposomes into which the receptor protein was incorporated. Liposome formation was not observed in desalting method and the recovery of the receptor was partial.

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 39, February 2002, pp 66- 69

 

 

Related affinities of different mono- nucleosides and mono nucleotides towardsPr (III) and Nd (III) in aquated organic solvents explored through 4f-4f transition spectral analysis

 

Sudhindra N Mishra, Suveerkumar C M, Manish C Patel, Prashant N Bhatt and Jignasu P Mehta

 

Spectroscopic properties of Pr (III) and Nd (III)-mono nucleosides and nucleotides in aquated organic solvents have been investigated through absorption difference and comparative absorption spectroscopy involving 4f-4f transitions. Absorption intensity analysis, has shown that minor coordination changes in structure and chemical make-up of these structurally related ligands induced substantial changes in the intensities of 4f-4f bands and their perturbation has been reflected through oscillator strength, Judd-Ofelt intensity parameters (Tl; l = 2, 4, 6). The results show that different nucleosides and nucleotides bind Pr (III) / Nd (III) with different degrees of relative affinities.