Indian Journal of Biochemistry & Biophysics

CODEN: IJBBBQ    ISSN: 0301-1208

VOLUME 39

NUMBER 5

OCTOBER 2002

 
CONTENTS

 

Minireviews

 

Proteomics - A new player in the post-genomic era

291

  Kapil Maithal

 

 

 

Essential fatty acids in maternal and infant nutrition

303

  C Gopalan* and Rita Patnaik

 

 

 

Papers

 

Contrasting effects of mutating active site residues, Aspartic acid 64 and Histidine 187 of Escherichia coli uracil-DNA glycosylase on uracil excision and interaction with an inhibitor protein

 

 

312

  Priya Handa, Narottam Acharya, Ramappa K Talawar, Sudipta Roy and

  Umesh Varshney*

 

 

 

Molten globule intermediates of human serum albumin in low concentration of urea

318

  B K Muralidhara and V Prakash*

 

 

 

Conformational preferences of two peptides DYASL and DYA from haemagglutinin of influenza virus and their possible role in the initiation of protein folding

 

 

325

    C Ratna Prabha

 

 

 

Purification and characterization of 3-phosphoglycerate kinase from Ehrlich ascites carcinoma cells

 

332

  Kasturi Mukherjee, Swapna Ghosh, Manju Ray* and Subhankar Ray

 

 

 

Co-immobilization of lipase, glycerol kinase, glycerol-3-phosphate oxidase and peroxidase onto alkylamine glass beads through glutaraldehyde coupling

 

342

  Vandana Kalia and C S Pundir*

 

 

 

Notes

 

Purification and properties of invertase from the flowers of Woodfordia fruticosa

347

  M K B Weersaooriya* and H P Yatawara

 

 

 

 

Contd.

 

 

Quantitative structure-activity relationship study on N-(pyridin-4-yl)-(indol-3-yl)alkylamides as antiallergic agents

 

351

  P Singh*, B K Sharma and R Kumar

 

 

——————

*Author for correspondence

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 39, October 2002, pp. 303-311

 

Proteomics-A new player in the post-genomic era

Kapil Maithal

Received 27 June 2002; accepted 9 August 2002

In the post-genomic era the concept of personalized medicine and molecular medicine emphasizes the utility of the proteomics approach. Proteomics is the global analysis of cellular proteins and complements the genomics approach. Proteins, in principle do all the work of the cell and ultimately dictate all biological processes and the cellular fate. Proteomics uses a combination of sophisticated techniques including two-dimensional (2D) gel electrophoresis, image analysis, mass spectrometry, amino acid sequencing and bioinformatics to identify and characterize proteins. This review aims at providing the various approaches and pitfalls associated with this technique and gives a brief overview of the utility of this approach in the area of biomedical research.

 

Indian Journal of Biochemistry & Biophysics

Vol. 39, October 2002, pp. 291-302

 

Essential fatty acids in maternal and infant nutrition

C Gopalan* and Rita Patnaik

Received 13 May 2002; accepted 2 July 2002

 

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 39, October 2002, pp. 312-317

 

Contrasting effects of mutating active site residues, Aspartic acid 64 and
Histidine 187 of Escherichia coli uracil-DNA glycosylase on uracil excision and interaction with an inhibitor protein

Priya Handa, Narottam Acharya, Ramappa K Talawar, Sudipta Roy and Umesh Varshney*

Received 29 May 2002; revised 22 July 2002

 

Uracil, a promutagenic base, arises in DNA by spontaneous deamination of cytosine or by the malfunctioning of DNA polymerases. To maintain the genomic integrity, cells possess a highly conserved base excision repair enzyme, uracil-DNA glycosylase (UDG). UDGs have a notably high turnover number and strict specificity for uracil in DNA. UDGs are inhibited by a small proteinaceous inhibitor, Ugi, which acts as a transition state substrate mimic. Crystal structure studies have identified the residues crucial in catalysis, and in their interaction with Ugi. Here, we report on the mutational analyses of D64 (D64H and D64N) and H187 (H187C, H187L and H187R) in the active site pocket of Escherichia coli UDG. The mutants were compromised in uracil excision by ~200-25,000 fold when compared to the native protein. In contrast, our analysis of the in vivo formed UDG-Ugi complexes on urea gels shows that D64 and H187 contribute minimally to the interaction of the two proteins. Thus, our findings provide further evidence to the primary function of D64 and H187 in catalysis.

Indian Journal of Biochemistry & Biophysics

Vol. 39, October 2002, pp. 318-324

 

Molten globule intermediates of human serum albumin in
low concentration of urea

B K Muralidhara and V Prakash*

Received 14 December 2001; revised and accepted 11 May 2002

Interaction of non-electrolytes such as urea with proteins especially at lower concentrations is opening-up newer concepts in the understanding of protein stability and folding in proteomics. In this study, the secondary and tertiary structural characteristics and thermal stability of human serum albumin at lower concentrations of urea have been monitored. The protein attains a molten globule like structure at concentration urea below 2 M. This structural state also shows an increase in the a-helical content as compared to the native state. At concentrations of urea above 2 M, human serum albumin starts unfolding, resulting in a three-state transition with two mid points of transitions at around 4 M and 7 M urea concentrations. The characteristics of the partially folded intermediates are discussed with respect to the three component system analyses. Preferential hydration dominates over preferential interaction at lower concentration of urea (up to 2.5 M) and at higher concentration, the preferential interaction overtakes preferential hydration in a competitive manner. Formation of structural intermediates at lower concentration of urea is hypothesized as a general phenomenon in proteins and fits in with the observation with preferential interaction parameters by Timasheff and co-workers in the case of lysozyme and ribonuclease at different pH values.

 

Indian Journal of Biochemistry & Biophysics

Vol. 39, October 2002, pp. 325-331

 

Conformational preferences of two peptides DYASL and DYA from haemagglutinin of influenza virus and their possible role in the initiation of protein folding

C Ratna Prabha

 

Received 8 March 2002; revised 26 June 2002; accepted 19 July 2002

The conformational preferences of two peptides DYASL and DYA from haemagglutinin of influenza virus were studied using PCILO programme. This was done to understand the possible role of DYAS in the initiation of protein folding and to understand the contribution of the fourth residue serine in the formation of turn. Our results indicate that this sequence shows an inherent preference for turn conformation, with a stabilizing Asx turn. DYA with NH group in the C-terminal protection models a type I β turn more closely than DYAS, because serine has a weak potential for turn conformation.

Indian Journal of Biochemistry & Biophysics

Vol. 39, October 2002, pp. 332-341

 

Purification and characterization of 3-phosphoglycerate kinase from Ehrlich ascites carcinoma cells

Kasturi Mukherjee, Swapna Ghosh, Manju Ray* and Subhankar Ray

Received 30 May 2002; revised and accepted 29 July 2002

3-Phosphoglycerate kinase (3-PGK) has been purified to apparent homogeneity from Ehrlich ascites carcinoma (EAC) cells by (NH4)2SO4 precipitation, gel filtration and ion-exchange chromatography. The enzyme has been partially characterized and compared with the characteristics of this enzyme of other normal and malignant cells. The EAC cell 3-PGK is composed of a single subunit of 47 kDa. It has a broad pH optimum (pH 6.0-7.5) for its enzymatic activity. The apparent Km values of 3-phosphoglycerate (3-PGA) and ATP for 3-PGK have been found out to be 0.25 mM and 0.1 mM respectively. Similar to 3-PGK of other cells, the EAC enzyme requires either Mg2+ or Mn2+ for full activity; the optimum concentrations of Mg2+ and Mn2+ are 0.8 mM and 0.5 mM respectively. When ATP and 3-PGA act as substrates, ADP, the reaction product of 3-PGK-catalyzed reaction has been found to inhibit this enzyme. Kinetic studies were made on the inhibition of ADP in presence of the substrates ATP and 3-PGA. Attempts to hybridize 3-PGK and glyceraldehyde-3-phosphate dehydrogenase of EAC cells by NAD or glutaraldehyde were unsuccessful.

 

Indian Journal of Biochemistry & Biophysics

Vol. 39, October 2002, pp. 342-346

 

Co-immobilization of lipase, glycerol kinase, glycerol-3-phosphate oxidase and peroxidase onto alkylamine glass beads through glutaraldehyde coupling

Vandana Kalia and C S Pundir*

Received 28 January 2002; revised and accepted 12 June 2002

 

A method for co-immobilizing lipase from porcine pancreas, glycerol kinase (GK) from Cellulo­monas spp., glycerol-3-phosphate oxidase (GPO) from Aerococcus viridans and per­oxidase from horseradish onto zirconia-coated alkylamine glass beads through glutaraldehyde coupling has been described. The co-immobilized enzymes retained 71.4% of initial specific activity with a conjugation yield of 43.6 mg/g support. The optimum pH and Km for triolein in­creased, while Vmax was decreased slightly, but incubation temperature for maximum activ­ity remained unaltered after co-immobilization. The co-immobilized enzymes showed increased thermal and storage stabilities in cold, compared to their native form. Among the various metal salts tested, only CuSO4 caused inhibition of both free and co-immobilized enzymes. The co-immobilized en­zymes showed better suitabil­ity over mixture of individually immobilized enzymes in deter­mination of serum tri­glyceride.

 

Indian Journal of Biochemistry & Biophysics

Vol. 39, October 2002, pp. 347-350

 

Purification and properties of invertase from the flowers of Woodfordia fruticosa

M K B Weersaooriya* and H P Yatawara

Received 23 October 2001; revised 3 July 2002

Invertase (b-fructofuranosidase, EC 3.2.1.26) was purified from the flowers of Woodfordia fruticosa, which is used to prepare certain fermented Ayurvedic drugs. The enzyme was purified to near homogeneity as judged by native PAGE with a yield of 10.7%, using (NH4)2SO4 fractionation, followed by gel filtration through Sepharose 4B and DEAE cellulose chromatography at pH 6.8 and 4.42. The molecular mass of the purified enzyme as determined by elution through Sepharose 4B gel column was found to be ~ 280 kDa. SDS-PAGE of the purified enzyme showed that the enzyme is composed of three subunits with molecular mass of 66, 43 and 40 kDa. The enzyme showed a broad pH optimum between 4.0-7.0. Optimum assay temperature was 37ºC and above 45ºC, the enzyme activity slowly declined and inactivated around 80ºC. The apparent Km value of the enzyme for sucrose was
160 mM.

 

Indian Journal of Biochemistry & Biophysics

Vol. 39, October 2002, pp. 351-355

 

Quantitative structure-activity relationship study on N-(pyridin-4-yl)-(indol-3-yl) alkylamides as antiallergic agents

P Singh*, B K Sharma and R Kumar

               Received 3 October 2001; revised and accepted 5 March 2002

 

The antihistamine activity of N-(pyridin-4-yl)-(indol-3-yl) alkylamides has been analyzed using Fujita-Ban and Hansch approaches. The analyses have helped to ascertain the role of different substituents in explaining the antiallergic actions of these analogues. From both approaches it is revealed that the small size substituents at R and R2 and non-hydrogen bond acceptor substituent at R improve histamine antagonist activity of a compound. Likewise, a small incision such as –CH2CONH– serving as the spacer between pyridinyl and indolyl rings and a bigger substituent like 4-FBn at R1 are also desirable for inhibitory activity.