Total visitors: 2,665  since 23-02-05

Indian Journal of Biochemistry & Biophysics

 

 

ISSN : 0301-1208

CODEN : IJBBBQ  

VOLUME 42

NUMBER 1

FEBRUARY 2005

 

CONTENTS

Papers

 

Binding of heme to human serum albumin: Steady-state fluorescence, circular dichroism and optical difference spectroscopic studies

7

J K Amisha Kamal and Digambar V Behere*

 

 

 

Amino acids differentially regulate insulin receptor tyrosine kinase and phosphatidyl inositol-3-OH-kinase activities in human monocytes exposed to high glucose concentration

13

 V Srinivasan, M Rajesh, K N Sulochana, C Indra and S Ramakrishnan*

 

 

 

Role of MMP-2 in oxidant-mediated regulation of Ca2+ uptake in microsomes of bovine pulmonary artery smooth muscle

19

Amritlal Mandal, Tapati Chakraborti, Rajdeep Choudhury, Biswarup Ghosh and Sajal Chakraborti*

 

 

 

Effect of iron concentration on the expression and activity of catalase-peroxidases
in mycobacteria

28

Veena C Yeruva, C A S Sivagami Sundaram and Manjula Sritharan*

 

 

 

Isolation and characterization of two N-acetyl-D-lactosamine specific lectins from tubers of Arisaema intermedium Blume and A. wallichianum Hook f.

34

Manpreet Kaur, Jatinder Singh*, Sukhdev Singh Kamboj, Jagmohan Singh, Amandeep Kaur, S K Sood and A K Saxena

 

 

 

An improved method for isolation of photosystem II from marine alga Porphyra yezoensis Udea

41

,Z Q Gao, GC Wang* and C K Tseng

 

 

 

A simplified fluorimetric method for corticosterone estimation in rat serum, tissues and mitochondria

48

Surendra S Katyare* and Jignesh D Pandya

 

 

 

Notes

 

Chlorpyrifos-induced alterations in rat brain acetylcholinesterase, lipid peroxidation and ATPases

54

Anugya Mehta, Radhey S Verma and N Srivastava*

 

 

 

Oxidative stress in paracetamol-induced pathogenesis: (I) Renal damage

59

Premila Abraham

 

 

 

Instructions to Authors

63

               

                *Author for correspondence

 

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 42, February 2005, pp. 7-12

 

Binding of heme to human serum albumin: Steady-state fluorescence, circular dichroism and optical difference spectroscopic studies

J K Amisha Kamal and Digambar V Behere*

 

The binding of monomeric heme to human serum albumin (HSA) was investigated using steady-state fluorescence, circular dichroism (CD) and optical difference spectroscopic (ODS) techniques. The existence of one strong binding site for heme on HSA was confirmed by titrating heme with HSA and following the quenching of tryptophan (Trp214) fluorescence emission intensity that occurred due to energy transfer. Up to around 1:1 stoichiometric ratio of HSA/heme, the quenching was observed to be very strong, however at higher ratios the quenching progressed very weakly. Similarly, the negative CD band centered at ~397 nm, which appeared on adding heme to HSA, increased in intensity on sequential addition of heme up to [heme]/[HSA]=1. Titration of HSA with heme was followed by ODS and the dissociation constant KD = (4.01.0)10-5 M was deduced. Results have been explained on the basis of Michaelis-Menton type of mechanism for the heme binding, in which heme first binds reversibly to His146 at the surface of the protein to form an intermediate complex, followed by irreversible binding to Tyr161 in the interior of the protein.

 

Keywords: human serum albumin (HSA), HSA-heme, methemalbumin (MHA), steady-state fluorescence, circular dichroism, optical difference spectroscopy, two stage binding.

IPC Code: G01J 3/00

 

 

 

Indian Journal of Biochemistry & Biophysics

 Vol. 42, February 2005, pp. 13-18

 

Amino acids differentially regulate insulin receptor tyrosine kinase and phosphatidyl inositol-3-OH-kinase activities in human monocytes exposed to high glucose concentration

V Srinivasan1,2, M Rajesh1,5, K N Sulochana1,3, C Indra1,4  and S Ramakrishnan1*

 

Chronic hyperglycemia and insulin resistance are the common factors involved in the development of vascular complications in diabetes mellitus (DM) patients. Since insulin signaling pathway has been shown to be regulated by nutritional supplements, in the present study, we investigated the possible effects of free amino acids, such as lysine, arginine and alanine and their mixture in modulating the insulin receptor tyrosine kinase (IRTK) and phosphatidyl inositol-3-OH-kinase (PI3K) activities and on the changes in actin dynamics in monocytes (MC), exposed to high glucose concentration (25 mM). IRTK and PI3K activities were markedly decreased in MC, incubated with 25 mM glucose. However, on treatment with amino acids, only lysine was effective in augmenting IRTK and PI3K activities in a dose-dependent manner. Arginine had marginal effect in promoting these activities. Equimolar mixture of amino acids showed marginal effect of augmenting only IRTK activity. Alanine had no effect. The F-actin filaments showed grossly diminished organization in the cells treated with 25 mM glucose alone, as assessed by specific binding to phalloidin-FITC, when compared with cells treated with 5 mM glucose. On the other hand, a significant improvement in the F-actin organization was observed in the cells co-incubated with 25 mM glucose and lysine. A possible molecular mechanism is the antiglycating effect of amino acids. The signal transduction starts with binding of ATP to lysine at position 1030 in the b sub unit of the receptor. This lysine (1030) may be protected by the added lysine or to some extent arginine from glycation and loss of function. In summary, our findings suggest that the amino acids apart from their antiglycating property can also modulate/influence the activities of pivotal enzymes that are upstream in the insulin-mediated signal transduction pathway and bring down glucose.

 

Keywords: Diabetes mellitus, insulin receptor tyrosine kinase, phosphatidyl inositol-3-OH-kinase, lysine and other amino acids, monocytes, glucose, insulin signaling pathway, actin dynamics, F-actin organization

IPC Code: C 12 Q 1/54

 

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 42, February 2005, pp. 19-27

 

Role of MMP-2 in oxidant-mediated regulation of Ca2+ uptake in microsomes of bovine pulmonary artery smooth muscle

Amritlal Mandala, Tapati Chakrabortia, Rajdeep Choudhurya , Biswarup Ghosha and Sajal Chakrabortia*

 

Treatment of bovine pulmonary artery smooth muscle microsomes with tert-butylhydroperoxide (t-buOOH) (300 M) markedly stimulated matrix metalloproteinase-2 (MMP-2) activity and enhanced Ca2+-ATPase activity and ATP-dependent Ca2+ uptake. Pre-treatment with vit. E (1 mM) and tissue inhibitor of metalloproteinase-2 (TIMP-2) (50 g/ml) prevented t-buOOH-induced stimulation of MMP-2 activity, Ca2+-ATPase activity and ATP-dependent Ca2+ uptake. In contrast, Na+-dependent Ca2+ uptake was inhibited by t-buOOH and the inhibition was reversed by vit. E (1 mM) and TIMP-2 (50 g/ml). However, t-buOOH-triggered changes in MMP-2 activity, and ATP- and Na+-dependent Ca2+ uptake were not reversed upon pre-treatment of the microsomes with a low concentration of 5 g/ml of TIMP-2, which on the contrary reversed MMP-2 (1 g/ml)-mediated alteration on these parameters. The inhibition of Na+-dependent Ca2+ uptake by MMP-2 under t-buOOH treatment overpowered the stimulation of ATP-dependent Ca2+ uptake in the microsomes. Combined treatment of the microsomes with low doses of MMP-2 (0.5 g/ml) and t-buOOH (100 mM) augmented Ca2+-ATPase activity and ATP-dependent Ca2+ uptake, but inhibited Na+-dependent Ca2+ uptake, compared to that elicited by either MMP-2 (0.5 g/ml) or t-buOOH (100 M). Pre-treatment with TIMP-2 (50 g/ml) reversed the effects of MMP-2 (0.5 g/ml) and/or t-buOOH (100 mM). Although pre-treatment with 5 g/ml of TIMP-2 reversed the effects produced by MMP-2 (0.5 g/ml), but it did not inhibit the responses elicited by t-buOOH (300 M) or t-buOOH (100 mM) plus MMP-2 (0.5 mg/ml) in the microsomes. Treatment with TIMP-2 (5 mg/ml) inhibited MMP-2 (1 mg/ml) activity (assessed by [14C]-gelatin degradation), whereas treatment of t-buOOH (300 M) with TIMP-2 (5 g/ml) abolished the inhibitory effect of TIMP-2 (5 g/ml) on MMP-2 (1 g/ml) activity (assessed by [14C]-gelatin degradation). Overall, these results suggested that t-buOOH inactivated TIMP-2, the ambient inhibitor of MMP-2, leading to activation of the ambient proteinase, MMP-2 which subsequently stimulated Ca2+-ATPase activity and ATP-dependent Ca2+ uptake, but inhibited Na+-dependent Ca2+ uptake, resulting in a marked decrease in Ca2+ uptake in the microsomes.

 

Keywords: Pulmonary artery smooth muscle, microsomes, oxidant, tert-butylhydroperoxide, antioxidant, vitamin E, matrix metalloproteinase-2, tissue inhibitor of metalloproteinase-2, Ca2+-ATPase, ATP-dependent Ca2+ uptake, Na+-dependent Ca2+ uptake.

IPC Code: C12N 9/00

 

 

 

 Indian Journal of Biochemistry & Biophysics

Vol. 42, February 2005, pp. 28-33

 

Effect of iron concentration on the expression and activity of catalase-peroxidases in mycobacteria

Veena C Yeruva, C A S Sivagami Sundaram and Manjula Sritharan*

 

Mycobacterial catalases are known to exist in different isoforms. We studied the influence of iron concentration on the expression and activity of the different isoforms in Mycobacterium bovis BCG, M. smegmatis, M. fortuitum, M. kansasii and M. vaccae by growing them under iron-sufficient (4 μg Fe/mL) and iron-deficient (0.02 μg Fe/ml) conditions. Upon iron deprivation, significant differences were observed in the catalase/peroxidase activities in both quantitative spectrophotometric assays and in the activity staining in native gels. Notable feature was that the peroxidase activity showed a significant decrease upon iron deprivation in all the mycobacteria, except M. vaccae. Peroxidase activity in all the mycobacteria, irrespective of the iron status was susceptible to heat inactivation. However, the isoforms of catalase showed differences in their heat stability, indicating possible structural differences in these proteins. For example, M. bovis BCG expressed a heat labile catalase under iron-sufficient conditions, while a heat stable catalase band of similar mobility was expressed under iron-deprivation conditions. The study clearly indicates that iron plays an important role in the regulation of expression of the different isoforms of the catalase-peroxidases.

 

Keywords: catalase-peroxidases, mycobacteria, iron.

IPC Code: C12R 1/32

 

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 42, February 2005, pp. 34-40

 

Isolation and characterization of two N-acetyl-d-lactosamine specific lectins from tubers of Arisaema intermedium Blume and  A.wallichianum Hook f.

Manpreet Kaur, Jatinder Singh*, Sukhdev Singh Kamboj, Jagmohan Singh,

Amandeep Kaur, S K Sooda and A K Saxenab

 

Two new lectins were purified from the tubers of Arisaema intermedium Blume and A. wallichianum Hook. f. (family: Araceae) by affinity chromatography on asialofetuin-linked amino activated silica beads. The bound lectins were eluted with 0.1 M glycine-HCl, pH 2.5. They gave a single band corresponding to subunit Mr 13.4 kDa in SDS-PAGE, pH 8.3. On gel filtration chromatography, the lectins showed a Mr of 51.2 kDa, suggesting a homotetrameric structure. Both the lectins gave a single peak on size exclusion HPLC and cation-exchange columns and a single band on PAGE, pH 4.5. However, like other monocot lectins, they gave multiple bands in isoelectric focusing and at PAGE 8.3. The lectins were inhibited by N-acetyl-D-lactosamine (LacNAc), a disaccharide and asialofetuin, a complex desialylated serum glycoprotein. They had no requirement for divalent metal ions i.e., Ca2+ and Mn2+ for their activity and were found to be mitogenic towards human lymphocytes. A. intermedium showed antiproliferative effect against various human cancer cell lines in vitro.

 

Keywords:   Araceae, Arisaema, tuber, N-acetyl-d-lactosamine, asialofetuin, lectin, mitogenic, antiproliferative effect, human lymphocytes, human cancer cell lines, carbohydrate specificity, denaturing agents.

IPC Code:    C 07K 14/42, G 01N 33/53

 

 

 

Indian Journal of Biochemistry & Biophysics

 Vol. 42, February 2005, pp. 41-47

 

An improved method for isolation of photosystem II from marine alga Porphyra yezoensis Udea

Z Q Gao1, 2, G C Wang1, * and C K Tseng1

 

An improved method for isolation and characterization of photosystem (PS)II particles from thylakoid membranes of gametophytes of a marine alga Porphyra yezoensis Udea is reported. Thylakoid membranes were isolated using ultracentrifugation and differential speeds centrifugation and were further purified by the first sucrose density gradient centrifugation (SDGC). PSII particles with high 2, 6-dichloroindophenol (DCIP) photo-reduction activity were isolated by the second SDGC from the thylakoid membranes. Absorption and fluorescence spectra of the thylakoid membranes and PSII particles were recorded and their polypeptides composition was studied. Thylakoid membranes obtained by the above two methods showed similar spectral properties and polypeptides composition. PSII particles, in addition to common extrinsic proteins found in PSII of other plants, contained cyt c-550, a 20 kDa protein, along with two new proteins (14 kDa and 16 kDa).

 

Keywords: Porphyra yezoensis Udea, thylakoid membranes, PSII, ultracentrifugation, differential speeds centrifugation, SDS-PAGE, absorption spectra, fluorescence spectra

IPC Code: B 01D 21/00

 

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 42, February 2005, pp. 48-53

 

A simplified fluorimetric method for corticosterone estimation in rat serum, tissues and mitochondria

Surendra S Katyare* and Jignesh D Pandya

 

A simplified procedure has been developed for the extraction and estimation of corticosterone from rat serum, tissues and mitochondria. The suitably diluted samples were treated with freshly prepared chloroform: methanol mixture (2:1, v/v) and then extracted directly with the chloroform. Almost quantitative recoveries (90% and above) were obtained with the present method, compared to poor recoveries (65-81%) and variable results obtained by earlier procedure. Quantification of corticosterone content in tissues, such as liver, brain and heart, and in the mitochondria indicated significant concentration of corticosterone in tissues and mitochondria, as compared to the serum. The presence of corticosterone in the mitochondria suggests that the hormone may play a role in regulation of mitochondrial gene expression and/or their turnover.

 

Keywords: Corticosterone, fluorimetric estimation, serum, tissues, mitochondria

IPC Code: C 07 J 1/00

 

 

 

Indian Journal of Biochemistry & Biophysics

 Vol. 42, February 2005, pp. 54-58

 

Chlorpyrifos-induced alterations in rat brain acetylcholinesterase, lipid peroxidation and ATPases

Anugya Mehta, Radhey S Verma and N Srivastava*

 

The effect of chlorpyrifos (O, O-diethyl-3, 5, 6-trichloro-2-pyridyl phosphorothionate, CPF) exposure on acetylcholinesterase (AChE) activity, lipid peroxidation and different ATPases activities was studied in rats. CPF caused significant inhibition of synaptosomal AChE activity in different regions of brain (fore, mid and hind) and inhibition ranged from 36 to 82% in rats receiving 20-100 mg CPF/kg body wt for 3 days. It also produced oxidative stress, resulting in marked increase in peroxidative damage of membrane lipids in a dose-dependent manner. The levels of malondialdehyde (MDA) and 4-hydroxy-2-nonanal (4-HNE), two major end products of lipid peroxidation were significantly increased in all the regions of brain. Increase in MDA levels was 66%, 117% and 172% in fore brain, 70%, 108% and 170% in mid brain and 40%, 110% and 169% in hind brain of rats given 20, 50 and 100 mg CPF/kg body wt for 3 days. The maximum increase in 4-HNE levels in all the three regions of brain was observed in the animals receiving CPF 100 mg/kg body wt. Na+/K+, Mg2+ and Ca2+-ATPases were inhibited to different extents in fore-, mid- and hind brain regions of rats given 20, 50 and 100 mg/kg body wt CPF for 3 days. Highest inhibition in the activity of Na+/K+-ATPase observed more than 90% in mid and hind-brain. Mg2+-ATPase in hind brain showed inhibition up to 97%. Inhibition in Ca2+-ATPase activity was also ranged from 22-94% in synaptosomes at different doses of CPF.

 

Keywords: Chlorpyrifos, oxidative stress, lipid peroxidation, ATPases, acetylcholinesterase activity, rat brain

IPC Code: C 12N 9/00

 

 

 

Indian Journal of Biochemistry & Biophysics

 Vol. 42, February 2005, pp. 59-62

 

Oxidative stress in paracetamol-induced pathogenesis: (I) Renal damage

Premila Abraham

 

The effect of administration of paracetamol (1 g/kg body wt) on oxidative damage to proteins and lipids in the kidney was studied at various time intervals in adult male Wistar rats. Iindicators of oxidative stress, such as protein thiol, protein carbonyl content and lipid peroxide levels were assayed along with thiol-dependent enzyme activities, glutamine synthase and glyceraldehyde-3-phosphate dehydrogenase. Paracetamol-induced renal damage after 4 hr of administration was evidenced by elevation in plasma creatinine levels and the presence of acute tubular necrosis on histological examination of the kidney. No significant change in any other parameters was observed, except for decreased glutathione level. An increase in lipid peroxide level was observed at 24 hr after treatment. The results suggest that oxidative stress may not play a causative role, but contribute to the pathogenesis of paracetamol-induced renal damage.

 

Keywords: lipid peroxidation, protein thiol, protein carbonyl, paracetamol , renal damage

IPC Code: A 61 K 31/167

 

 

AUTHOR INDEX

 

Abraham P

59

Katyare S S

48

Sood S K

34

 

 

Kaur A

34

Srinivasan V

13

Behere D V

7

Kaur M

34

Sritharan M

28

 

 

 

 

Srivastava N

54

Chakraborti S

19

Mandal A

19

Sulochana K N

13

Chakraborti T

19

Mehta A

54

Sundaram C A S S

28

Choudhury R

19

 

 

 

 

 

 

Pandya J D

48

Tseng C K

41

Gao Z Q

41

 

 

 

 

Ghosh B

19

Rajesh M

13

Verma R S

54

 

 

Ramakrishnan S

13

 

 

Indra C

13

 

 

Wang G C

41

 

 

Saxena A K

34

 

 

Kamal J K A

7

Singh Jagmohan

34

Yeruva V C

28

Kamboj S S

34

Singh J

34