Structure, stability, and chaperone function of alphaA-crystallin: role of N-terminal region.

Small heat shock protein alphaA-crystallin, the major protein of the eye lens, is a molecular chaperone. It consists of a highly conserved central domain flanked by the N-terminal and C-terminal regions. In this article we studied the role of the N-terminal domain in the structure and chaperone function of alphaA-crystallin. Using site directed truncation we raised several deletion mutants of alphaA-crystallin and their protein products were expressed in Escherichia coli. Size exclusion chromatography of these purified proteins showed that deletion from the N-terminal beyond the first 20 residues drastically reduced the oligomeric association of alphaA-crystallin and its complete removal resulted in a tetramer. Chaperone activity of alphaA-crystallin, determined by thermal and nonthermal aggregation and refolding assay, decreased with increasing length of deletion and little activity was observed for the tetramer. However it was revealed that N-terminal regions were not responsible for specific recognition of natural substrates and that low affinity substrate binding sites existed in other part of the molecule. The number of exposed hydrophobic sites and the affinity of binding hydrophobic probe bis-ANS as well as protein substrates decreased with N-terminal deletion. The stability of the mutant proteins decreased with increase in the length of deletion. The role of thermodynamic stability, oligomeric size, and surface hydrophobicity in chaperone function is discussed. Detailed analysis showed that the most important role of N-terminal region is to control the oligomerization, which is crucial for the stability and in vivo survival of this protein molecule.

Cloning and functional expression of an acyl-ACP thioesterase FatB type from Diploknema (Madhuca) butyracea seeds in Escherichia coli.

A cDNA of fatty acyl-acyl carrier protein (ACP) thioesterase (Fat) from developing seed of Madhuca butyracea has been cloned. The deduced amino acid sequence of the cDNA corresponding to the mature polypeptide showed 30-40% and 60-75% identity to the reported FatA and FatB class of plant thioesterases, respectively. This gene, MbFatB, is present as a single copy in M. butyracea genome and the MbFatB protein was detected clearly in seed tissues of this plant but not in that of Indian mustard (Brassica juncea). Heterologous expression of the MbFatB gene driven by different promoters in E. coli wild type and fatty acid beta-oxidation mutant (fadD88) strains resulted production of the recombinant protein with various fusion tags either as biologically inactive (insoluble) or functionally active forms. Expression of functionally active recombinant MbFatB in E. coli affected bacterial growth and cell morphology as well as changed the fatty acid profiles of the membrane lipid and the culture supernatant. Alteration of the fatty acid composition was directed predominantly towards palmitate and to a lesser extent myristate and oleate due to acyl chain termination activity of plant thioesterase in bacteria. Thus, this new MbFatB gene isolated from a non-traditional oil-seed tree can be used in future for transgenic development of oil-seed Brassica, a widely cultivated crop that expresses predominantly oleoyl-ACP thioesterase (FatA) in its seed tissue and has high amount of unwanted erucic acid in edible oil in order to alter the fatty acid profile in a desirable way.

Efficacy of the phosphorylation of synthetic peptides by purified catalytic subunit of PKA (PKAcat) from bovine lens depends on the amino acid sequence of the peptides.

Protein kinase (PK) A catalytic (PKAcat) subunit was purified to homogeneity from bovine lens using a 100-kDa cut-off membrane filtration followed by different chromatographic procedures. The molecular weight of PKAcat was found to be 41 kDa. The kinase phosphorylates histone IIIs and other synthetic modified peptides of VRKRTLRRL with different amino acid environment. The extent of phosphorylation depends not only on the presence of Ser or Thr (phosphorylating residues) but also on other surrounding amino acid residues. Although some peptides compete in phosphorylating histone, they are not very significant. The result suggests that the extent of phosphorylation depends on the amino acid residue(s) surrounding phosphorylable residue(s) on the peptide.

STa-induced translocation of protein kinase C from cytosol to membrane in rat enterocytes.

Escherichia coli heat stable enterotoxin (STa) binds to isolated rat intestinal epithelial cells and triggers a cascade reaction including increase of intracellular calcium levels ([Ca(2+)](i)) and membrane bound protein kinase C (PKC) activity. In response to STa, the cytosolic PKC activity falls from 110 to 35 nmol with increase of membrane bound PKC activity from 15 to 78 nmol. Furthermore, the increase of PKC activity induced by STa treatment was always preceded by an increase in [Ca(2+)](i). Cytosolic [Ca(2+)](i) was significantly higher (161 nM) in STa treated cells as compared to untreated cells (51.3 nM). In addition, immunoblot performed on extracts of STa treated rat enterocytes with a monoclonal antibody against PKC alpha showed a prominent band of PKC alpha. Translocation of PKC alpha could be blocked by dantrolene, a drug which inhibits the mobilisation of [Ca(2+)](i) from the intracellular store. Our results, therefore, provide evidence for the role of [Ca(2+)](i) in STa treated cells for the translocation of PKC alpha from cytosol to membrane.

Purification, characterization and partial amino acid sequencing of a 70 kD inhibitor protein of Na+,K+-ATPase from goat testis cytosol.

A protein isolated from goat testis cytosol is found to inhibit Na+,K+-ATPase from rat brain microsomes. The inhibitor has been purified by ammonium sulphate precipitation followed by hydroxyapatite column chromatography. The purified fraction appears as a single polypeptide band on 10% SDS-PAGE of approximate molecular mass of 70 kDa. The concentration at which 50% inhibition (I50) occurs is in the nanomolar range. The inhibitor seems to bind Na+,K+-ATPase reversibly at ATP binding site in a competitive manner with ATP, but away from ouabain binding site. It does not affect p-nitrophenyl-phosphatase activity. The inhibitor is found to inhibit the phosphorylation step of the Na+,K+-ATPase. The enhancement of tryptophan fluorescence and changes in CD pattern suggest conformational changes of Na+,K+-ATPase on binding to the inhibitor. Amino acid sequence of the trypsinised fragments show some homology with aldehyde reductase.

Protein A-activated rat splenic lymphocyte proliferation involves tyrosine kinase-phospholipase C-protein kinase C pathway.

Protein A (PA) of Staphylococcus aureus was long been known for its affinity towards the Fc domain of immunoglobulin G. It is now well established that PA is a potent biological response modifier showing simultaneously antitumor, antitoxic, and anticarcinogenic properties. This bacterial protein was also observed to stimulate production of cytokines. But the molecular mechanism(s) of immunocyte activation by PA still remained essentially unknown. In this report, we demonstrate a hitherto undescribed role of PA as a signal inducer in rat splenic lymphocytes. Our studies describe that PA induces transition of G0/G1 to S and G2/M phases of cell cycle, thus ultimately stimulating splenic lymphocyte proliferation. It has also been revealed that PA binds to rat splenic lymphocytes in a dose dependent manner and stimulates proliferation via tyrosine kinase-phospholipase C (PLC)-Ca2+-dependent protein kinase C (PKC) pathway. These observations will be of valid help in correlating the immunostimulatory activities of PA with the molecular mechanism(s) of its action.

Interaction of chlorpromazine with low molecular mass ion-transporting ATPase modulator proteins from rat brain cytosol.

Two low molecular mass proteins (13 kDa which inhibits Na+,K(+)-ATPase and 12 kDa which modulates Ca2+, Mg(2+)- and Ca(2+)-ATPases), purified from rat brain cytosol form complexes with chlorpromazine (CPZ) on incubation. The conformational characteristics of the proteins and their complex have been studied by comparing the fluorescence and CD spectra. The tryptophan fluorescence data show that the inhibitor-CPZ complex does not quench the fluorescence of NA+,K(+)-ATPase significantly. CD spectra indicate that the structure of the inhibitor is changed on formation of the complex. The inhibitor-CPZ complex significantly changes the conformation of Na+,K(+)-ATPase. The regulator protein-CPZ complex does not have any appreciable effect on Ca2+, Mg(2+)- and Ca(2+)-ATPase activities. The Trp-fluorescence of Ca2+,Mg(2+)- and Ca(2+)-ATPase are not significantly affected in presence of the complex. CD spectra indicate that the structure of the regulator is abruptly affected on formation of the complex. The conformations of Ca2+,Mg(2+)- and Ca(2+)-ATPases are found to be altered in presence of the complex.

Unsaturated fatty acid-activated protein kinase (PKx) from goat testis cytosol.

The cytosolic fraction of goat cauda epididymis possesses a protein kinase (PKx) activity which is stimulated by a number of unsaturated fatty acids of which arachidonic acid is the best activator in absence of cAMP or Ca(2+). Phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and diacylglycerol have no effect either alone or in combination. The membrane fraction does not show any appreciable kinase activity even after detergent treatment. PKx migrates as a single band of apparent molecular mass of 116 kDa on 10% SDS-PAGE after sequential chromatographic separation on DEAE-cellulose, phenyl-Sepharose, high-Q anion exchange and protamine-agarose affinity column. PKx phosphorylates histone H1, histone IIIs and protamine sulfate, but not casein. However, the best phosphorylation was obtained with a substrate based on PKC pseudosubstrate sequence (RFARKGSLRQKNV). The kinase phosphorylates two endogenous cytosolic proteins of 60 and 68 kDa. Ser residues are primarily phosphorylated although a low level of phosphorylation is observed on Thr residues also. Ca(2+) and Mn(2+) inhibit PKx activity in the micromolar range. Staurosporine is found to inhibit the PKx activity to a significant level at sub-nanomolar concentration. Lyso-phosphatidylcholine and certain detergents at very low concentrations (<0.05%) stimulate enzyme activity to some extent. The immuno-crossreactivity study with antibody against different PKC isotypes suggests that the protein kinase under study is not related to any known PKC family. Even the antibody against PKN (a related protein kinase reported in rat testis found to be activated by arachidonic acid) does not cross-react with this protein kinase. Hence we believe that the protein kinase (PKx) reported here is different even from the PKN of rat testis. The phosphorylation of endogenous proteins by the protein kinase may be involved in cell regulation including fertility regulation and signal transduction.

The effect of binding of chlorpromazine and chloroquine to ion transporting ATPases.

The inhibition of ion transporting ATPases (Na+,K+-ATPase, Ca2+,Mg2+- and Ca2+-ATPase) by two amphiphilic drugs e.g. chlorpromazine (antipsychotic) and chloroquine (antimalarial) are found to be competitive in nature in vitro with respect to the substrate. Two binding sites - high and low affinity are found to exist on all the three ATPases toward these drugs as evident from the plot of F/F0 vs. different drug concentrations of tryptophan fluorescence of the enzymes. Circular dichroism analysis suggest that binding of drugs to the high affinity site does not involve any change in conformation of ATPase molecules which occur only when drug binds to the low affinity sites. The drug binding sites and possible effect on conformational change of ATPase molecules of these two drugs have been described in this report.

A 75-kDa Na+,K+-ATPase competitive inhibitor protein isolated from rat brain cytosol binds to a site different from the ouabain-binding site.

A Na+,K+-ATPase inhibitor protein has been purified to homogeneity from rat brain cytosol by ammonium sulphate precipitation, DEAE anion-exchange chromatography and hydroxyapatite adsorption column chromatography. The purified protein migrates as a single polypeptide band of 75 kDa on 7.5% SDS/PAGE. Amino acid composition data shows the presence of a high number of acidic amino acids in the molecule in relation to the pI value of 4.6. The inhibitor binds Na+,K+-ATPase reversibly and blocks ATP binding sites at micromolar concentrations with an I50 of approximately 700 nm. As a result, formation of the phosphorylated intermediate of Na+,K+-ATPase is hindered in the presence of the inhibitor. It does not affect p-nitrophenylphosphatase activity. Tryptophan fluorescence studies and CD analysis suggest conformational changes of Na+,K+-ATPase on binding to the inhibitor.

Mimosa pudica apyrase requires polysaccharide and Ca2+ for the activity.

Mimosa pudica Linn leaves with pulvini contain unique isoforms (I and II) of Apyrase enzyme (EC 3.6.1.5). The activity of isoform I depends on divalent cation Mn2+. This isoform is associated noncovalently with the polysaccharide, containing mainly of galactose and arabinose sugars. The apparent molecular mass of these 2 isoforms are 36 and 34 Kd respectively. The association of the polysaccharide with the isoform I has been found to be Ca2+ dependent which is endogenously present in this isoform. Removal of Ca2+ and polysaccharide from the enzyme (isoform I) leads to an inactivation. The enzyme activity can be restored when both Ca2+ and endogenous polysaccharide fraction were added at an optimal molar ratio of Ca2+:protein of 7:1. The endogenous polysaccharide can be replaced by the standard arabinogalactan. No other sugar or polysaccharide except the arabinogalactan can restore the apyrase activity. Calcium mediates a conformational change in the protein which helps in association of polysaccharide as evidenced from fluorometric and far UV-CD studies to restore the enzymic activity. Neither any interaction of the polysaccharide with the protein is detected in absence of Ca2+ nor the enzyme activity could be recovered under such condition.

Purification and functional characterization of a low-molecular-mass Ca2+,Mg2+- and Ca2+-ATPase modulator protein from rat brain cytosol.

A low-molecular-mass modulator protein having a molecular mass of about 12 kDa has been purified from rat brain cytosol following gel filtration and FPLC/Mono Q anion-exchange chromatographic separation. A number of protein fractions were obtained from an FPLC column when eluted with a 0.1 M NaCl hold gradient. One fraction (peak no. 5) was found to stimulate Ca2+,Mg2+-ATPase but inhibit Ca2+-ATPase isolated from goat spermatozoa. The S50 (concentration producing 50% stimulation) and I50 were found to be in the nanomolar range. The modulator seems to bind to Ca2+, Mg2+- or Ca2+-ATPase at a site distal from the ATP binding site. The binding to both the ATPases is reversible and non-competitive in nature. The inhibitory activity is found to depend significantly on -SH or -NH2 group(s) of the modulator, whereas no appreciable dependency of the stimulatory effect was apparent. The study indicates that the modulator is not a glycoprotein. CD analysis suggests that the protein exists as an unordered secondary structure. An immuno-cross-reactivity study with specific antibody and inhibition by thapsigargin suggests that the Ca2+,Mg2+- and Ca2+-ATPases from goat testes microsomal membranes are two isoforms of the sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase (SERCA) family. The modulator does not contain any Trp molecules, as evident from Trp fluorescence analysis. Amino acid analysis shows that glycine, serine, derivatives of tyrosine and phenylalanine are the predominant amino acids. The data suggest that the modulator is a negatively charged protein and is a good tool for distinguishing the regulation of Ca2+,Mg2+- and Ca2+-ATPase activities.

Purification and functional characterization of a low-molecular-mass Na+, K+-ATPase inhibitor protein from rat brain cytosol.

A number of low-molecular mass (12-13 kDa) Na+, K+-ATPase inhibitor proteins have been purified from rat brain cytosol by gel filtration followed by FPLC fractionation on a Mono Q anion-exchange column. Eight peaks were obtained using 0.1 M NaCl eluent of which one peak was found to be the most potent inhibitor of Na+, K+-ATPase. The molcular mass of the inhibitor was about 13 kDa on 16.5% SDS/PAGE. The concentration at which 50% inhibition (I50) was found was in the nanomolar range. The inhibitor seems to bind to Na+, K+-ATPase at a site distal from the ATP-binding site. The binding to the ATPase is non-competitive. The CD analysis suggests an unordered secondary structural element. It also inhibits p-nitrophenyl phosphatase activity from rat brain with comparable I50 value to that for Na+, K+-ATPase. The protein does not contain any Trp as evident from Trp fluorescence and amino acid analysis. Amino acid analysis shows that glycine and serine, derivatives of tyrosine and phenylalanine are the predominant amino acids. The data suggests that it is a negatively charged protein in which the contribution of the hydrophobic part is 27%.

Na(+)-K(+)-ATPase activity of different regions of rat brain in response to immunization and bone marrow cytokine treatment.

The bone marrow cytokine (Bio-Immuno Modulator, BIM or BM-Fr1) has been suggested to correct immunoincompetence by modulating brain Na(+)-K(+)-ATPase. The brain region affected and mechanism of action of BM-Fr1 are unknown, however. Here we report that immunization of immunocompetent rats indirectly inhibited Na(+)-K(+)-ATPase activity (59%) in the left cerebral lobe (LC) and irrespective of BM-Fr1 treatment, stimulation of the enzyme was observed in the LC at the peak of immune response. BM-Fr1 treatment, which corrected immunoincompetence in malnourished rats, also modulated a different LC Na(+)-K(+)-ATPase profile to that seen in immunocompetent animals. Immunogen and BM-Fr1 seem to exert their influence in brain via a cytosolic inhibitor protein of Na(+)-K(+)-ATPase. Thus we suggest that (1) BM-Fr1 plays an important role in immune homeostatasis by modulating Na(+)-K(+)-ATPase activity of LC and (2) Na(+)-K(+)-ATPase is not the receptor for either immunogen or BM-Fr1.

Alteration of the microenvironment in plasma membranes of rat enterocytes after Escherichia coli heat stable enterotoxin treatment: effect on protein kinase C activity.

Plasma membranes isolated from Escherichia coli heat stable enterotoxin (STa) treated rat enterocytes were studied in respect to protein kinase C activity and fluidity change. Pretreatment of enterocytes with STa increased the membrane bound protein kinase C activity about 5 fold as compared to control. STa treatment made the membrane more fluid as evident from a higher phospholipid/cholesterol ratio and greater unsaturated fatty acid levels. Moreover, the phase transition temperature of the STa treated membrane appeared to be significantly lower than that of the corresponding control membrane, thereby further indicating a rise in fluidity of the membrane in the former case. Our results, therefore, suggested that following STa enterotoxin treatment an appropriate fluid environment in the rat intestinal cell membrane was essential for the activation of protein kinase C.

Differential effect of bone marrow protein on brain ATPase of immunized control and malnourished rats.

After sheep erythrocyte (SRBC) immunization in balanced-diet-fed (BDF) rats, the brain microsomal ATPase activity for the first 48 h was suppressed compared with preimmunization level, then stimulated at the peak of the immune response. In contrast, rats malnourished by deprivation of vitamin B complex and ascorbic acid and with significantly lower pre- and postimmunization enzyme activity than the BDF rats, had stimulated enzyme activity only on immunization, and their immune response was found to be suppressed. A bone marrow-derived bioimmunomodulator (BIM) revived the immunocompetence of the malnourished immunized animals, and brain ATPase activity in these animals after BIM injection followed a pattern similar to that of the immunized BDF rats. We suggest that, after SRBC immunization under the influence of BIM, suppression followed by stimulation of the brain microsomal ATPase might be related to immune response.

Enteropathogenic bacteria in river Ganges in Varanasi.

A bacteriological study of the river Ganges in Varanasi was carried out from December 1985 to November 1987. In all 407 water samples were collected, 335 from bathing ghats and 72 from sewage openings and were examined for the presence of enteropathogenic bacteria. Vibrio cholerae 0-1 (1.72%), Non 0-1 Vibrio cholera (3.69%), Vibrio fluvialis (0.74%), Aeromonas sp. (0.49%), Plesiomonas sp. (0.25%), Salmonella sp. (0.98%) and Shigella sp. (1.23%) were isolated from both ghats and sewage openings along with Pseudomonas sp., Proteus sp., E. coli., etc. This indicates that water of the river Ganges is potentially hazardous to the health of the bathers and is not suitable for drinking. The discharge of raw sewage into the river and its diversion beyond the city, downstream, by installation of pumps have failed to improve the quality of water.

An Na+/K(+)-ATPase inhibitor protein from rat brain cytosol.

A protein isolated from rat brain cytosol is found to inhibit Na+/K(+)-ATPase in rat brain and kidney and H+/K(+)-ATPase from toad gastric mucosa, but has no effect on Ca2+,Mg(2+)-ATPase and Ca(2+)-ATPase isolated either from rat testis or goat spermatozoa. The inhibitor has been partially purified by ammonium sulphate precipitation followed by gel-filtration through Sephadex G-100. The inhibitor seems to bind at or close to the ATP binding site of Na+/K(+)-ATPase, such that the binding of the inhibitor to ATPase is reversible and competitive in nature with respect to the substrate. Optimum inhibition is observed at around the phase transition temperature of brain Na+/K(+)-ATPase and the inhibitory activity is only partially dependent on -SH or -NH2 group(s) of the inhibitor protein.