DNA affinity chromatography of transcription factors.
Explore the source record for details and available documents.
Amino Acid Motifs↗
Biomedical subjects
L A Jurado
Publications and source records attributed to L A Jurado.
DNA affinity chromatography.
DNA-affinity chromatography has been used for the purification of DNA-binding proteins that control various cellular processes. There have been improvements in coupling methods and choice of supports over the years. The procedure for coupling 5'-aminoethyl-(dT)18 to silica activated with N-hydroxysuccinimide and a carbodiimide has been described. Also, the cyanogen bromide mediated coupling of aminoethyl-(dT)18 to Sepharose is described. Determination of (dT)18-coupling to silica and Sepharose is by 5' end-labeling an oligonucleotide containing a (dA)18 stretch of sequence and determining how much hybridizes with the (dT)18 support. Enzymatic synthesis of a double-stranded DNA-silica or Sepharose prevents modification of nucleotide bases. We have explained the use of DNA and RNA templates for template-directed enzymatic synthesis of affinity columns. DNA-affinity chromatography is a powerful method with broad applicability and we are currently extending this technology for purifying transcription factors, polymerases, and nucleases.
Base Sequence↗
Catalytic chromatography.
Catalytic chromatography exploits both specific biological affinity and catalytic specificity to selectively purify enzymes. Two different applications are presented. Purification of EcoRI restriction endonuclease to apparent homogeneity was accomplished in a single step with significantly greater yield and purification than was obtained with affinity chromatography. An attempt to purify the multiple DNA polymerase activities of Escherichia coli was also developed. Five well-resolved peaks of DNA polymerase activity were fractionated. In this new chromatographic mode, the enzyme binds immobilized substrate coupled to a column in the absence of some required cofactor. When the missing cofactor is added, the enzyme converts substrate to product and selectively elutes from the column.
Catalysis↗
DNA affinity chromatography.
Explore the source record for details and available documents.
Base Sequence↗
WBSCR14, a putative transcription factor gene deleted in Williams-Beuren syndrome: complete characterisation of the human gene and the mouse ortholog.
Williams-Beuren syndrome (WBS) is a neurodevelopmental disorder affecting several systems caused by a heterozygous deletion in the chromosomal region 7q11.23. A common interval that includes up to 17 genes reported so far is deleted in the great majority of patients. Elastin haploinsufficiency is responsible for the cardiovascular features, but the specific contribution of other deleted genes to the WBS phenotype remains unknown. We have fully characterised a gene commonly deleted in WBS, WBSCR14, previously reported in a truncated form as WS-bHLH. The WBSCR14 cDNA encodes an 852amino acid protein with a basic helix-loop-helix-leucine-zipper motif (bHLHZip) and a bipartite nuclear localisation signal (BNLS), suggesting a function as a transcription factor. WBSCR14 is expressed as a 4.2kb transcript predominantly in adult liver and at late stages of foetal development. The WBSCR14 locus encompasses 33 kb of genomic DNA with 17 exons. Two intragenic polymorphic dinucleotide repeats have been identified and used to verify hemizygosity in WBS patients. We have also cloned the mouse ortholog and mapped its locus to mouse chromosome 5, in a region of conserved synteny with human 7q11.23. Given that other bHLHZip proteins are dosage sensitive and based on the putative function of WBSCR14 as a transcription factor, hemizygosity at this locus could be involved in some features of WBS.
Adult↗
Identification of a human homolog of the Drosophila rotated abdomen gene (POMT1) encoding a putative protein O-mannosyl-transferase, and assignment to human chromosome 9q34.1.
We have isolated a human gene homologous to Drosophila melanogaster rotated abdomen, rt, a poorly viable recessive mutation causing a clockwise twisted abdomen in affected flies due to defects in embryonic muscle development. The human gene, like rt, encodes a protein with high homology to the yeast mannosyl-transferases (Pmts) and has been named POMT1. POMT1 is expressed as a 3.1-kb transcript in all tissues tested, with highest levels in testis and fetal brain. Alternative splicing of several exons in all tissues predicts the generation of several protein isoforms. The most common mRNA variant encodes a 725-aa protein with 40% identity and 62.5% similarity to rt, as well as 30.5% identity and 54% similarity to yeast Pmts. Computer prediction of protein sorting suggests that the POMT1 product could be an integral protein of the endoplasmic reticulum membrane. Given the strong conservation of protein motifs between POMT1 and the yeast Pmts, POMT1 may function as a mannosyl-transferase involved in O-mannosylation of proteins, being the first of such a class found in mammals. The POMT1 locus has been assigned to human chromosome 9q34.1 by somatic cell hybrids, radiation hybrids, and linkage analysis. On the basis of the rt phenotype, POMT1 could be a candidate for uncharacterized genetic disorders of the muscular system, such as some forms of congenital muscular dystrophy or congenital myopathy.
Amino Acid Sequence↗
Comparison of the reaction progress of calcineurin with Mn2+ and Mg2+.
Activation of calcineurin by Mn2+ and Mg2+ was compared using a heavy atom isotope analogue of the substrate p-nitrophenyl phosphate (pNPP). Heavy atom isotope effects were measured for Mg2+ activation and compared to published results of the isotope effects with Mn2+ as the activating metal. Isotope effects were measured for the kinetic parameter Vmax/Km at the nonbridging oxygen atoms [18(V/K)nonbridge]; at the position of bond cleavage in the bridging oxygen atom [18(V/K)bridge]; and at the nitrogen atom in the nitrophenol leaving group [15(V/K)]. The isotope effects increased in magnitude upon changing from an optimal pH to a nonoptimal pH; the 18(V/K)bridge effect increased from 1.0154 (+/-0.0007) to 1.0198 (+/-0.0002), and the 15(V/K) effect increased from 1.0018 (+/-0. 0002) to 1.0021 (+/-0.0003). The value for 18(V/K)nonbridge is 0. 9910 (+/-0.0003) at pH 7.0. As with Mn2+, the 18(V/K)nonbridge isotope effect indicated that the dianion was the substrate for catalysis, and that a dissociative transition state was operative for the phosphoryl transfer. Comparison to results for Mn2+ activation suggested that chemistry was more rate-limiting with Mg2+ than with Mn2+. Changing the activating metal concentration showed opposite trends with increasing Mg2+ increasing the commitment factor and seemingly making the chemistry less rate-limiting. The influence of viscosity was evaluated as well to gauge the role of chemistry. The activation of calcineurin-catalyzed hydrolysis of pNPP1 by Mg2+ or Mn2+ at pH 7.0 was compared in the presence of viscogens, glycerol and poly(ethylene glycol). Increasing glycerol caused different effects with the two activators. With Mn2+ as the activator, calcineurin activity showed a normal response with kcat and kcat/Km decreasing with viscosity. There was an inverse response with Mg2+ as the activator as values of kcat/Km increased with viscosity. From values of the normalized kcat/Km with Mn2+, the chemistry was found to be partially rate-limiting, consistent with previous heavy atom isotope studies (22). The effect observed for Mg2+ seems consistent with a change in the rate-limiting step for the two different metals at pH 7.0.
Animals↗
Apocalmodulin.
Intracellular Ca2+ is normally maintained at submicromolar levels but increases during many forms of cellular stimulation. This increased Ca2+ binds to receptor proteins such as calmodulin (CaM) and alters the cell's metabolism and physiology. Calcium-CaM binds to target proteins and alters their function in such a way as to transduce the Ca2+ signal. Calcium-free or apocalmodulin (ApoCaM) binds to other proteins and has other specific effects. Apocalmodulin has roles in the cell that apparently do not require the ability to bind Ca2+ at all, and these roles appear to be essential for life. Apocalmodulin differs from Ca2+-CaM in its tertiary structure. It binds target proteins differently, utilizing different binding motifs such as the IQ motif and noncontiguous binding sites. Other kinds of binding potentially await discovery. The ApoCaM-binding proteins are a diverse group of at least 15 proteins including enzymes, actin-binding proteins, as well as cytoskeletal and other membrane proteins, including receptors and ion channels. Much of the cellular CaM is bound in a Ca2+-independent manner to membrane structures within the cell, and the proportion bound changes with cell growth and density, suggesting it may be a storage form. Apocalmodulin remains tightly bound to other proteins as subunits and probably hastens the response of these proteins to Ca2+. The overall picture that emerges is that CaM cycles between its Ca2+-bound and Ca2+-free states and in each state binds to different proteins and performs essential functions. Although much of the research focus has been on the roles of Ca2+-CaM, the roles of ApoCaM are equally vital but less well understood.
Amino Acid Sequence↗
Activation of calcineurin by the trivalent metal terbium.
As a possible probe for metal activation of calcineurin, Tb3+ was tested for effects on calcineurin activity. Calcineurin was activated by Tb3+ with the following kinetic parameters estimated: k(cat) = 0.78 +/- 0.02 sec(-1), Km(pNPP) = 32.6 +/- 1.8 mM, and K(act)(Tb3+) = 0.08 +/- 0.03 mM. Terbium luminescence was demonstrated in the presence of the heterodimer of calcineurin and exploited to localize the binding of exogenous metal to the enzyme active site. Exogenous Mn2+ reduced luminescence, although the affinity of calcineurin for Tb3+ seemed to be greater. Putative active-site ligands, such as para-nitrophenol and a synthetic peptide from the autoinhibitory region, reduced the luminescence of terbium. Collectively, these data suggested that Tb3+ was binding directly at the active site of calcineurin, with the corollary that exogenous activating metal (Mn2+) binds at the active site of the enzyme. These data support the hypothesis that activating, exogenous divalent metal participates directly in catalysis.
Animals↗
Trypanosoma cruzi phospho enol pyruvate carboxykinase (ATP-dependent): transition metal ion requirement for activity and sulfhydryl group reactivity.
We studied the transition metal ion requirements for activity and sulfhydryl group reactivity in phospho enol pyruvate carboxykinase (PEP-carboxykinase; ATP:oxaloacetate carboxylase (transphosphorylating), EC 4.1.1.49), a key enzyme in the energy metabolism of the protozan parasite Trypanosoma (Schizotrypanum) cruzi. As for other PEP-carboxykinases this enzyme has a strict requirement of transition metal ions for activity, even in the presence of excess Mg2+ ions for the carboxylation reaction; the order of effectiveness of these ions as enzyme activators was: Co2+ > Mn2+ > Cd2+ > Ni2+ >> Fe2+ > VO2+, while Zn2+ and Ca2+ had no activating effects. When we investigated the effect of the varying type or concentration of the transition metal ions on the kinetic parameters of the enzyme the results suggested that the stimulatory effects of the transition metal center were mostly associated with the activation of the relatively inert CO2 substrate. The inhibitory effects of 3-mercaptopicolinic acid (3MP) on the enzyme were found to depend on the transition metal ion activator: for the Mn(2+)-activated enzyme the inhibition was purely non-competitive (Kii = Kis) towards all substrates, while for the Co(2+)-activated enzyme the inhibitor was much less effective, produced a mixed-type inhibition and affected differentially the interaction of the enzyme with its substrates. The modification of a single, highly reactive, cysteine per enzyme molecule by 5,5'-dithiobis (2-nitro-benzoate) (DTNB) lead ton an almost complete inhibition of Mn(2+)-activated T. cruzi PEP-carboxykinase; however, in contrast with the results of previous studies in vertebrate and yeast enzymes, the substrate ADP slowed the chemical modification and enzyme inactivation but did not prevent it. PEP and HCO3- had no significant effect on the rate or extent of the enzyme inactivation. The kinetics of the enzyme inactivation by DTNB was also dependent on the transition metal activator, being much slower for the Co(2+)-activated enzyme than for its Mn(2+)-activated counterpart. When the bulkier but more hydrophobic reagent N-(7-dimethylamino-4-methylcoumarinyl)maleimide (DACM) was used the enzyme was slowly and incompletely inactivated in the presence of Mn2+ and ADP afforded almost complete protection from inactivation; in the presence of Co2+ the enzyme was completely resistant to inactivation. Taken together, our results indicate that the parasite enzyme has a specific requirement of transition metal ions for activity and that they modulate the reactivity of a single, essential thiol group, different from the hyperreactive cysteines present in vertebrate or yeast enzymes.
Adenosine Diphosphate↗