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PubMed2000

Thermodynamic stability of HLA-B*2705. Peptide complexes. Effect of peptide and major histocompatibility complex protein mutations.

Designing synthetic vaccines from class I major histocompatibility complex (MHC)-binding antigenic peptides requires not only knowledge of the binding affinity of the designed peptide but also predicting the stability of the formed MHC-peptide complex. In order to better investigate structure-stability relationships, we have determined by circular dichroism spectroscopy the thermal stability of a class I MHC protein, HLA-B*2705, in complex with a set of 39 singly substituted peptide analogues. The influence of two anchoring side chains (P3 and P9) was studied by peptide mutation and appropriate site-directed mutagenesis of the HLA-B*2705 binding groove. The side chain at P9 is clearly the one that contributes the most to the thermal stability of the MHC-peptide complexes, as destabilization up to 25 degrees C are obtained after P9 mutation. Interestingly, structure-stability relationships do not fully mirror structure-binding relationships. As important as the C-terminal side chain are the terminal ammonium and carboxylate groups. Removal of a single H-bond between HLA-B27 and the terminal peptide moieties results in thermal destabilization up to 10 degrees C. Depending on the bound peptide and the location of the deleted H-bond, the decrease in the thermal stability of the corresponding complex is quantitatively different. The present study suggests that any peptidic amino acid at positions 3 and 9 promotes refolding of the B27-peptide complex. Once the complex is formed, the C-terminal side chain seems to play an important role for maintaining a stable complex.

Base Sequence↗
PubMed2001

Identification of another actin-related protein (Arp) 2/3 complex binding site in neural Wiskott-Aldrich syndrome protein (N-WASP) that complements actin polymerization induced by the Arp2/3 complex activating (VCA) domain of N-WASP.

Neural Wiskott-Aldrich syndrome protein (N-WASP) is an essential regulator of actin cytoskeleton formation via its association with the actin-related protein (Arp) 2/3 complex. It is believed that the C-terminal Arp2/3 complex-activating domain (verprolin homology, cofilin homology, and acidic (VCA) or C-terminal region of WASP family proteins domain) of N-WASP is usually kept masked (autoinhibition) but is opened upon cooperative binding of upstream regulators such as Cdc42 and phosphatidylinositol 4,5-bisphosphate (PIP2). However, the mechanisms of autoinhibition and association with Arp2/3 complex are still unclear. We focused on the acidic region of N-WASP because it is thought to interact with Arp2/3 complex and may be involved in autoinhibition. Partial deletion of acidic residues from the VCA portion alone greatly reduced actin polymerization activity, demonstrating that the acidic region contributes to Arp2/3 complex-mediated actin polymerization. Surprisingly, the same partial deletion of the acidic region in full-length N-WASP led to constitutive activity comparable with the activity seen with the VCA portion. Therefore, the acidic region in full-length N-WASP plays an indispensable role in the formation of the autoinhibited structure. This mutant contains WASP-homology (WH) 1 domain with weak affinity to the Arp2/3 complex, leading to activity in the absence of part of the acidic region. Furthermore, the actin comet formed by the DeltaWH1 mutant of N-WASP was much smaller than that of wild-type N-WASP. Partial deletion of acidic residues did not affect actin comet size, indicating the importance of the WH1 domain in actin structure formation. Collectively, the acidic region of N-WASP plays an essential role in Arp2/3 complex activation as well as in the formation of the autoinhibited structure, whereas the WH1 domain complements the activation of the Arp2/3 complex achieved through the VCA portion.

Actin-Related Protein 2↗
PubMed2001

Complete reconstitution of human IkappaB kinase (IKK) complex in yeast. Assessment of its stoichiometry and the role of IKKgamma on the complex activity in the absence of stimulation.

The IkappaB kinase (IKK) complex, composed of two catalytic subunits (IKKalpha and IKKbeta) and a regulatory subunit (IKKgamma), is the key enzyme in activation of nuclear factor kappaB (NF-kappaB). To study the mechanism and structure of the complex, we wanted to recombinantly express IKK in a model organism that lacks IKK. For this purpose, we have recombinantly reconstituted all three subunits together in yeast and have found that it is biochemically similar to IKK isolated from human cells. We show that there is one regulatory subunit per kinase subunit. Thus, the core subunit composition of IKKalpha.beta.gamma complex is alpha(1)beta(1)gamma(2), and the core subunit composition of IKKbeta.gamma is beta(2)gamma(2). The activity of the IKK complex (alpha+beta+gamma or beta+gamma) expressed in yeast (which lack NF-kappaB and IKK) is 4-5-fold higher than an equivalent amount of IKK from nonstimulated HeLa cells. In the absence of IKKgamma, IKKbeta shows a level of activity similar to that of IKK from nonstimulated HeLa cells. Thus, IKKgamma activates IKK complex in the absence of upstream stimuli. Deleting the gamma binding domain of IKKbeta or IKKalpha prevented IKKgamma induced activation of IKK complex in yeast, but it did not prevent the incorporation of IKKgamma into IKK and large complex formation. The possibility of IKK complex being under negative control in mammalian cells is discussed.

Blotting, Western↗
PubMed2004

The Fanconi anemia core complex forms four complexes of different sizes in different subcellular compartments.

Fanconi anemia (FA) is an autosomal recessive disease marked by congenital defects, bone marrow failure, and cancer susceptibility. FA cells exhibit a characteristic hypersensitivity to DNA crosslinking agents such as mitomycin C. The molecular mechanism for the disease remains elusive, but at least 6 FA proteins are known to be part of what is termed the FA core complex. We used affinity pulldown of FLAG-FANCA to pull down the FA complex from whole-cell extracts. Mass spectroscopy detected previously reported FA-binding proteins, including FANCA, FANCC, FANCG, cdc2, and GRP94, thus validating the approach. We further describe a method of purification of the FA core complex in an effort to find novel complex components and biochemical activity to define the function of the complex. By using conventional chromatographic fractionation of subcellular preparations, we report: (i) the FA core complex exists in a cytoplasmic form at 500-600 kDa; (ii) a larger, 750-kDa cytoplasmic form is seen only at mitosis; (iii) a nuclear form achieves a size of 2 megaDaltons; and (iv) a distinct 1-megaDalton FA core complex exists bound to chromatin that contains phosphorylated FANCA after undergoing DNA damage. We are continuing our analysis using mass spectroscopy in an effort to characterize novel binding proteins. These data will help define the biochemical role of the FA core complex in normal cell physiology as well as in the development of the FA disease state.

CDC2 Protein Kinase↗
PubMed2005

A mammalian chromatin remodeling complex with similarities to the yeast INO80 complex.

The mammalian Tip49a and Tip49b proteins belong to an evolutionarily conserved family of AAA+ ATPases. In Saccharomyces cerevisiae, orthologs of Tip49a and Tip49b, called Rvb1 and Rvb2, respectively, are subunits of two distinct ATP-dependent chromatin remodeling complexes, SWR1 and INO80. We recently demonstrated that the mammalian Tip49a and Tip49b proteins are integral subunits of a chromatin remodeling complex bearing striking similarities to the S. cerevisiae SWR1 complex (Cai, Y., Jin, J., Florens, L., Swanson, S. K., Kusch, T., Li, B., Workman, J. L., Washburn, M. P., Conaway, R. C., and Conaway, J. W. (2005) J. Biol. Chem. 280, 13665-13670). In this report, we identify a new mammalian Tip49a- and Tip49b-containing ATP-dependent chromatin remodeling complex, which includes orthologs of 8 of the 15 subunits of the S. cerevisiae INO80 chromatin remodeling complex as well as at least five additional subunits unique to the human INO80 (hINO80) complex. Finally, we demonstrate that, similar to the yeast INO80 complex, the hINO80 complex exhibits DNA- and nucleosome-activated ATPase activity and catalyzes ATP-dependent nucleosome sliding.

ATPases Associated with Diverse Cellular Activitie↗
PubMed2002

Sequence complexity profiles of prokaryotic genomic sequences: a fast algorithm for calculating linguistic complexity.

MOTIVATION: One of the major features of genomic DNA sequences, distinguishing them from texts in most spoken or artificial languages, is their high repetitiveness. Variation in the repetitiveness of genomic texts reflects the presence and density of different biologically important messages. Thus, deviation from an expected number of repeats in both directions indicates a possible presence of a biological signal. Linguistic complexity corresponds to repetitiveness of a genomic text, and potential regulatory sites may be discovered through construction of typical patterns of complexity distribution. RESULTS: We developed software for fast calculation of linguistic sequence complexity of DNA sequences. Our program utilizes suffix trees to compute the number of subwords present in genomic sequences, thereby allowing calculation of linguistic complexity in time linear in genome size. The measure of linguistic complexity was applied to the complete genome of Haemophilus influenzae. Maps of complexity along the entire genome were obtained using sliding windows of 40, 100, and 2000 nucleotides. This approach provided an efficient way to detect simple sequence repeats in this genome. In addition, local profiles of complexity distribution around the starts of translation were constructed for 21 complete prokaryotic genomes. We hypothesize that complexity profiles correspond to evolutionary relationships between organisms. We found principal differences in profiles of the GC-rich and other (non-GC-rich) genomes. We also found characteristic differences in profiles of AT genomes, which probably reflect individual species variations in translational regulation. AVAILABILITY: The program is available upon request from Alexander Bolshoy or at http://csweb.haifa.ac.il/library/#complex.

Algorithms↗
PubMed1996

Further study on association of 5SrRNA-L5 protein complex and methionyl-tRNA to methionyl-tRNA synthetase in the macromolecular aminoacyl-tRNA synthetase complex.

To obtain direct evidence for the attachment of 5SrRNA-ribosomal L5 protein particles (5SRNP) and methionine-tRNA (tRNA(met)) to methionyl-tRNA synthetase (MetRS) in the macromolecular aminoacyl-tRNA synthetase (ARS) complex of rat liver, a MetRS-5SRNP-tRNA(met) complex was dissociated from the macromolecular ARS complex fraction by n-octyl-beta-D-glucoside (Method I) or by omega-aminooctyl agarose (Method II) chromatography. The dissociated MetRS complex fraction was purified by gel filtration followed by tRNA-Sepharose chromatography using partially purified tRNA(met) in Method I, and by hydrophobic interaction chromatography in Method II. In both methods, final Superdex200 chromatography showed that MetRS activity was present in the region corresponding to the molecular weight of the MetRS-5SRNP-tRNA(met) complex (M(r) 200,000). One main protein band corresponding to the molecular weight of MetRS was observed on SDS-PAGE of the final product, which was concentrated by lyophilizing after dialysis against water. Using serum albumin as an inhibitor of adhesion of L5 to the microconcentrators which was used to concentrate the final product, a distinct L5 band was detected on SDS-PAGE, the intensity of which was comparable to that of the MetRS band. Northern blot analysis of RNA prepared from the tRNA-Sepharose fraction showed the presence of 5SrRNA. Dot blot analysis using an antibody against ribosomal protein L5 showed that L5 was present in the Superdex200 fractions prepared by both methods. The MetRS specific activities in MetRS complex fractions incubated without tRNA increased during the purification procedures, indicating that endogeneous tRNA(met) exists stably in the MetRS complex. 5SRNP and 5SrRNA markedly enhanced the MetRS activity in the MetRS complex, indicating that 5SRNP(A) plays a role as a positive effector of MetRS.

Amino Acyl-tRNA Synthetases↗
PubMed2003

Molecular structures of the inclusion complexes beta-cyclodextrin-1,2-bis(4-aminophenyl)ethane and beta-cyclodextrin-4,4'-diaminobiphenyl; packing of dimeric beta-cyclodextrin inclusion complexes.

The present investigation is part of an ongoing study on the influence of the long end-functonalized guest molecules DBA and BNZ in the crystal packing of beta-cyclodextrin (betaCD) dimeric complexes. The title compounds are 2:2 host:guest complexes showing limited host-guest hydrogen bonding at the primary faces of the betaCD dimers. Within the betaCD cavity the guests exhibit mutual pi...pi interactions and between betaCD dimers perpendicular NH...pi interactions. The DBA guest molecule exhibits one extended and two bent conformations in the complex. The BNZ guest molecule is not planar inside betaCD, in contrast to the structure of BNZ itself, which indicates that the cavity isolates the molecules and forbids the pi...pi stacking of the aromatic rings. NMR spectroscopy studies show that in aqueous solution both DBA and BNZ form strong complexes that have 1:1 stoichiometry and structures similar to the solid state ones. The relative packing of the dimers is the same in both complexes. The axes of two adjacent dimers form an angle close to 20 degrees and have a lateral displacement approximately 2.45 A, both of which characterize the screw-channel mode of packing. Although the betaCD/BNZ complex indeed crystallizes in a space group characterizing the latter mode, the betaCD/DBA complex crystallizes in a space group with novel dimensions not resembling any of the packing modes reported so far. The new lattice is attributed to the three conformations exhibited by the guest in the crystals. However, this lattice can be transformed into another, which is isostructural to that of the betaCD/BNZ inclusion complex, if the conformation of the guest is not taken into account.

Aminobiphenyl Compounds↗
PubMed1991

X-ray studies on crystalline complexes involving amino acids and peptides. XXIII. Variability in ionization state, conformation and molecular aggregation in the complexes of succinic acid with DL- and L-lysine.

Crystalline complexes of succinic acid with DL- and L-lysine have been prepared and analysed by X-ray diffraction. DL-Lysine complex: C6H15N2O2+.-1/2C4H4O4(2-).1/2C4H6O4, Mr = 264.2, P1, a = 5.506 (4), b = 8.070 (2), c = 14.089 (2) A, alpha = 92.02 (1), beta = 100.69 (3), gamma = 95.85 (3) degrees, Z = 2, Dx = 1.44 g cm-3, R = 0.059 for 2546 observed reflections. Form I of the L-lysine complex: C6H15N2O2+.C4H5O4-, Mr = 264.2, P1, a = 5.125 (2), b = 8.087 (1), c = 8.689 (1) A, alpha = 112.06 (1), beta = 99.08 (2), gamma = 93.77 (2) degrees, Z = 1, Dm = 1.34 (3), Dx = 1.34 g cm-3, R = 0.033 for 1475 observed reflections. Form II of the L-lysine complex: C6H15N2O2+.1/4C4H4O4(2-).-1/4C4H6O4.1/4(C4H5O4.. .H...C4H4O4)2-, Mr = 264.2, P1, a = 10.143 (4), b = 10.256 (2), c = 12.916 (3) A, alpha = 105.00 (2), beta = 99.09 (3), gamma = 92.78 (3) degrees, Z = 4, Dm = 1.37 (4), Dx = 1.38 g cm-3, R = 0.067 for 2809 observed reflections. The succinic acid molecules in the structures exhibit a variety of ionization states. Two of the lysine conformations found in the complexes have been observed for the first time in crystals containing lysine. Form II of the L-lysine complex is highly pseudosymmetric. In all the complexes, unlike molecules aggregate into separate alternating layers. The basic element of aggregation in the lysine layer in the complexes is an S2-type head-to-tail sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1993

Influence of complex structure on the biodegradation of iron-citrate complexes.

The biodegradation of iron-citrate complexes depends on the structure of the complex formed between the metal and citric acid. Ferric iron formed a bidentate complex with citric acid, [Fe(III) (OH)(2) cit] involving two carboxylic acid groups, and was degraded at the rate of 86 muM h. In contrast, ferrous iron formed a tridentate complex with citric acid, [Fe(II) cit], involving two carboxylic acid groups and the hydroxyl group, and was resistant to biodegradation. However, oxidation and hydrolysis of the ferrous iron resulted in the formation of a tridentate ferric-citrate complex, [Fe(III)OH cit], which was further hydrolyzed to a bidentate complex, [Fe(III)(OH)(2) cit], that was readily degraded. The rate of degradation of the ferrous-citrate complex depended on the rate of its conversion to the more hydrolyzed form of the ferric-citrate complex. Bacteria accelerated the conversion much more than did chemical oxidation and hydrolysis.

Journal Article↗
PubMed2004

RNA polymerase II (Pol II)-TFIIF and Pol II-mediator complexes: the major stable Pol II complexes and their activity in transcription initiation and reinitiation.

Protein purification and depletion studies were used to determine the major stable forms of RNA polymerase II (Pol II) complexes found in Saccharomyces cerevisiae nuclear extracts. About 50% of Pol II is found associated with the general transcription factor TFIIF (Pol II-TFIIF), and about 20% of Pol II is associated with Mediator (Pol-Med). No Pol II-Med-TFIIF complex was observed. The activity of Pol II and the purified Pol II complexes in transcription initiation and reinitiation was investigated by supplementing extracts depleted of either total Pol II or total TFIIF with purified Pol II or the Pol II complexes. We found that all three forms of Pol II can complement Pol II-depleted extracts for transcription initiation, but Pol II-TFIIF has the highest specific activity. Similarly, Pol II-TFIIF has a much higher specific activity than TFIIF for complementation of TFIIF transcription activity. Although the Pol II-TFIIF and Pol II-Med complexes were stable when purified, we found these complexes were dynamic in extracts under transcription conditions, with a single polymerase capable of exchanging bound Mediator and TFIIF. Using a purified system to examine transcription reinitiation, we found that Pol II-TFIIF was active in promoting multiple rounds of transcription while Pol II-Med was nearly inactive. These results suggest that both the Pol II-Med and Pol II-TFIIF complexes can be recruited for transcription initiation but that only the Pol II-TFIIF complex is competent for transcription reinitiation.

Cell Extracts↗
PubMed2004

Mechanism of recruiting Sec6/8 (exocyst) complex to the apical junctional complex during polarization of epithelial cells.

Sec6/8 (exocyst) complex regulates vesicle delivery and polarized membrane growth in a variety of cells, but mechanisms regulating Sec6/8 localization are unknown. In epithelial cells, Sec6/8 complex is recruited to cell-cell contacts with a mixture of junctional proteins, but then sorts out to the apex of the lateral membrane with components of tight junction and nectin complexes. Sec6/8 complex fractionates in a high molecular mass complex with tight junction proteins and a portion of E-cadherin, and co-immunoprecipitates with cell surface-labeled E-cadherin and nectin-2alpha. Recruitment of Sec6/8 complex to cell-cell contacts can be achieved in fibroblasts when E-cadherin and nectin-2alpha are co-expressed. These results support a model in which localized recruitment of Sec6/8 complex to the plasma membrane by specific cell-cell adhesion complexes defines a site for vesicle delivery and polarized membrane growth during development of epithelial cell polarity.

Animals↗
PubMed2005

Domain interactions within the Ski2/3/8 complex and between the Ski complex and Ski7p.

The Ski complex (composed of Ski3p, Ski8p, and the DEVH ATPase Ski2p) is a central component of the 3'-5' cytoplasmic mRNA degradation pathway in yeast. Although the proteins of the complex interact with each other as well as with Ski7p to mediate degradation by exosome, a 3'-exonuclease complex, the nature of these interactions is not well understood. Here we explore interactions within the Ski complex and between the Ski complex and Ski7p using a directed two-hybrid approach combined with coimmunoprecipitation experiments. We also test the functional significance of these interactions in vivo. Our results suggest that within the Ski complex, Ski3p serves as a scaffold protein with its C terminus interacting with Ski8p, and the sub-C terminus interacting with Ski2p, while no direct interaction between Ski2p and Ski8p was found. Ski7p interacts with the Ski complex via its interaction with Ski8p and Ski3p. In addition, inactivating the Ski complex by mutating conserved residues in the DEVH helicase motif of Ski2 did not abrogate its interaction with Ski7p, indicating that Ski2p function is not necessary for this interaction.

Adaptor Proteins, Signal Transducing↗
PubMedDate not supplied

Inhibition of the apolipoprotein B mRNA editing enzyme-complex by hnRNP C1 protein and 40S hnRNP complexes.

The apolipoprotein (apo) B mRNA can be modified by a posttranscriptional base change from cytidine to uridine at nucleotide position 6666. This editing of apo B mRNA is mediated by a specific enzyme-complex of which only the catalytic subunit APOBEC-1 (apo B mRNA editing enzyme component 1) has been cloned and extensively characterized. In this study, two-hybrid selection in yeast identified hnRNP C1 protein to interact with APOBEC-1. Recombinant hnRNP C1 protein inhibited partially purified apo B mRNA editing activity from rat small intestine and bound specifically to apo B sense RNA around the editing site. The inhibition of apo B mRNA editing by hnRNP C1 protein was not due to masking of the RNA substrate as the mutant protein M104 spanning the RNA-binding domain of hnRNP C1 protein bound strongly to the apo B RNA, but did not inhibit the editing reaction. The apo B mRNA editing enzyme-complex of rat liver nuclear extracts sedimented in sucrose density gradients around 22-27S, but did not contain hnRNP C1 protein that was found exclusively within 40S hnRNP complexes. The removal of 40S hnRNP complexes increased the activity of the 22-27S editing enzyme-complex. Adding back 40S hnRNP complexes with hnRNP C1 protein resulted in an inhibition of the 22-27S apo B mRNA editing enzyme-complex, while addition of 18S fractions had no effect. In conclusion, hnRNP C1 protein identified by two-hybrid selection in yeast is a potent inhibitor of the apo B mRNA editing enzyme-complex. The abundant hnRNP C1 protein, which is contiguously deposited on nascent pre-mRNA during transcription and is involved in spliceosome assembly and mRNA splicing, is a likely regulator of the editing of apo B mRNA which restricts the activity of APOBEC-1 to limited and specific editing events.

APOBEC-1 Deaminase↗
PubMed1976

Complex of D-glyceraldehyde-3-phosphate dehydrogenase with Cu2+ ion. I. The apoenzyme-Cu complex.

D-Glyceraldehyde-3-phosphate dehydrogenase binds one Cu2+ ion per subunit, which results in the formation of a specific metal-protein complex. This complex exhibits a sharp absorption band around 370 nm, and a broad, small absorption band in the 600-700 nm region. The shape of the absorption spectrum of Cu-GAPD complex in the visible range depends on the anionic composition of the solution. The Cu-GAPD complex is stable in solutions containing phosphate or pyrophosphate anions, but undergoes a slow change in sulfate-, and a rapid change in chloride-containing solutions. The Cys-149 residue of the enzyme is essential for the formation of the Cu-GAPD complex. The sharp absorption at 370 nm presumably corresponds to a charge transfer interaction between the sulfur and the metal ion. Similar absorption bands are shown by the Cu-complexes of papain and thiol-alcalase enzymes. It is assumed that in addition to the reactive thiol groups an imidazole residue in the active site of these enzymes is also involved in the complex formation, and the specific Cu-GAPD Cu-papain and Cu-thiol-alcalase complexes contain a Cys-Cu-His chelate structure.

Animals↗
PubMed2003

Synthesis and antibacterial activity of cephradine metal complexes: part I complexes with magnesium, calcium, chromium and manganese.

Cephradine is included among the first generation cephalosporins, which is active against a wide range of Gram-positive and Gram-negative bacteria including penicillinase-producing Staphylococci. Since the presence of complexing ligand may affect the bioavailability of a metal in the blood or tissues, therefore, in order to study the probable interaction of cephradine with essential and trace elements present in human body, cephradine has been reacted with cobalt, copper, zinc and cadmium metal halides in L:M ratio of 2:1 in methanol and the products recrystallized from suitable solvents to pure crystals of consistent melting points. Infrared and ultraviolet studies of these complexes were carried out and compared with ligand. Magnetic susceptibility studies of these complexes were also carried out showing their paramagnetic behavior. From the infra red studies and elemental analysis of the complexes it has been shown that the drug molecule serves as a bidentate ligand coordinating through both its carboxylate at C-3 and beta -lactam nitrogen and the metal having a square planar or octahedral geometry. To evaluate the changes in microbiological activity of cephradine after complexation, antibacterial studies were carried out by observing the changes in MIC (minimum inhibitory concentration) of the complexes and compared with the parent drug by measuring the zone of inhibition of complexes against both Gram-positive and Gram-negative organisms. For MIC observation, serial dilution method was employed and zone series were determined by disk diffusion method. Our investigations reveal that formation of complexes results in decrease in antibacterial activity of cephradine and MIC values are increased.

Journal Article↗
PubMed2005

Synthesis and antibacterial activity of cephradine metal complexes : part II complexes with cobalt, copper, zinc and cadmium.

Cephradine, the first generation cephalosporin, is active against a wide range of Gram-positive and Gram-negative bacteria including penicillinase-producing Staphylococci. Since the presence of complexing ligand may affect the bioavailability of a metal in the blood or tissues, therefore, in order to study the probable interaction of cephradine with essential and trace elements present in human body, cephradine has been reacted with cobalt, copper, zinc and cadmium metal halides in L:M ratio of 2:1 in methanol and the products recrystallized from suitable solvents to pure crystals of consistent melting points. Infrared and ultraviolet studies of these complexes were carried out and compared with ligand. Magnetic susceptibility studies of these complexes were also carried out showing their paramagnetic behavior. From the infra red studies and elemental analysis of the complexes, it has been shown that the drug molecule serves as a bidentate ligand coordinating through both its carboxylate at C-3 and beta-lactam nitrogen and the metal having a square planar or octahedral geometry. To evaluate the changes in microbiological activity of cephradine after complexation, antibacterial studies were carried out by observing the changes in MIC (minimum inhibitory concentration) of the complexes and compared with the parent drug by measuring the zone of inhibition of complexes and compared with the parent cephalosporin against both Gram-positive and Gram-negative organisms. For MIC observation, serial dilution method was employed and zone series were determined by disk diffusion method. Our investigations reveal that formation of complexes results in decrease in antibacterial activity of cephradine and MIC values are increased.

Anti-Bacterial Agents↗
PubMed1991

RNA polymerase II elongation complex. Elongation complexes purified using an anti-RNA antibody do not contain initiation factor alpha.

Gene expression in eukaryotes can be regulated by controlling the efficiency of transcript elongation by RNA polymerase II. The composition of the elongation complex is, however, poorly understood. Previous work has identified DNA sequences which block RNA polymerase II transcription and factors which stimulate RNA chain elongation. Here, I have purified elongation complexes arrested at discrete template locations. Complexes were rapidly and efficiently precipitated from in vitro transcription reactions using a monoclonal antibody that binds RNA. The isolated complexes remained transcriptionally active. This technique enables the facile manipulation of transcription elongation complexes. Using this approach, I show that transcription initiation factor alpha is not associated with a RNA polymerase II elongation complex. Since others have shown that alpha associates stoichiometrically with DNA, RNA polymerase II, and other required factors in an initiation complex, this work suggests that alpha departs from the transcription complex after nucleotides are required but before extensive RNA chain synthesis. In this regard alpha resembles the bacterial promoter-recognition factor sigma.

Animals↗