ATP-agarose Affinity Chromatography Research Articles

Fast Track Approval of Therapeutic Blood Substitutes

Fast Track Approval of Therapeutic Blood Substitutes

The Kinase Homology Domain of Receptor Guanylyl Cyclase C: ATP Binding and Identification of an Adenine Nucleotide Sensitive Site

The role of the kinase homology domain (KHD) in receptor guanylyl cyclases is to regulate the activity of the catalytic guanylyl cyclase domain. The KHD lacks many of the amino acids required for phosphotransfer activity and, therefore, is not expected to possess kinase activity. Guanylyl cyclase activity of the receptor guanylyl cyclase C (GC-C) is modulated by ATP, and computational modeling showed that the KHD can adopt a structure similar to protein kinases, suggesting that the KHD is the site for ATP interaction. A monoclonal antibody, GCC:4D7, raised to the KHD of GC-C, fails to react with GC-C in the presence of ATP and ATP analogues that regulate GC-C catalytic activity, indicating that a conformational change occurs in the KHD on ATP binding. Mapping of the epitope of the antibody through the use of recombinant protein constructs and phage display showed that the epitope for GC-C:4D7 lies immediately C-terminal to a critical lysine residue (Lys516 in GC-C), required for ATP interaction in protein kinases. By employing a novel approach utilizing ATP-agarose affinity chromatography, we demonstrate that the intracellular domain of GC-C and the KHD bind ATP. Mutation of Lys516 to Ala abolishes ATP binding. Thus, this report is the first to show direct ATP binding to the pseudokinase domain of receptor guanylyl cyclase C, as well as to identify dramatic conformational changes that occur in this domain on ATP binding, akin to those seen in catalytically active protein kinases.

Acidic Residues in the Nucleotide-binding Site of the Bacteriophage T7 DNA Primase

DNA primases catalyze the synthesis of oligoribonucleotides to initiate lagging strand DNA synthesis during DNA replication. Like other prokaryotic homologs, the primase domain of the gene 4 helicase-primase of bacteriophage T7 contains a zinc motif and a catalytic core. Upon recognition of the sequence, 5'-GTC-3' by the zinc motif, the catalytic site condenses the cognate nucleotides to produce a primer. The TOPRIM domain in the catalytic site contains several charged residues presumably involved in catalysis. Each of eight acidic residues in this region was replaced with alanine, and the properties of the altered primases were examined. Six of the eight residues (Glu-157, Glu-159, Asp-161, Asp-207, Asp-209, and Asp-237) are essential in that altered gene 4 proteins containing these mutations cannot complement T7 phage lacking gene 4 for T7 growth. These six altered gene 4 proteins can neither synthesize primers de novo nor extend an oligoribonucleotide. Despite the inability to catalyze phosphodiester bond formation, the altered proteins recognize the sequence 5'-GTC-3' in the template and deliver preformed primer to T7 DNA polymerase. The alterations in the TOPRIM domain result in the loss of binding affinity for ATP as measured by surface plasmon resonance assay together with ATP-agarose affinity chromatography.

The 78 kDa glucose-regulated protein (GRP78/BIP) is expressed on the cell membrane, is released into cell culture medium and is also present in human peripheral circulation.

In human rabdomiosarcoma cells (TE671/RD) chronic exposure to 500 nM thapsigargin (a powerful inhibitor of the endoplasmic reticulum Ca2+-ATPases) resulted in the induction of the stress protein GRP78/BIP. Making use of the surface biotinylation method, followed by the isolation of the GRP78 using ATP-agarose affinity chromatography, it was found that a fraction of the thapsigargin-induced GRP78 is expressed on the cell surface. The presence of GRP78 on the membrane of thapsigargin-treated cells was confirmed by fractionation of cell lysates into a soluble and a membrane fraction, followed by Western blot analysis with an anti-GRP78 antibody. It was also found that conspicuous amounts of GRP78 are present in the culture medium collected from thapsigargin-treated cultures. This extracellular GRP78 originates mostly by an active release from intact cells and does not result solely from the leakage of proteins from dead cells. Moreover, small amounts of circulating, free GRP78 and naturally-occurring anti-GRP78 autoantibodies were detected in the peripheral circulation of healthy human individuals.

Constitutive heat shock protein 70 (HSC70) expression in rainbow trout hepatocytes: effect of heat shock and heavy metal exposure

The 70-kDa family of heat shock proteins plays an important role as molecular chaperones in unstressed and stressed cells. The constitutive member of the 70 family (hsc70) is crucial for the chaperoning function of unstressed cells, whereas the inducible form (hsp70) is important for allowing cells to cope with acute stressor insult, especially those affecting the protein machinery. In fish, the role of hsc70 in the cellular stress response process is less clear primarily because of the lack of a fish-specific antibody for hsc70 detection. In this study, we purified hsc70 to homogeneity from trout liver using a three-step purification protocol with differential centrifugation, ATP-agarose affinity chromatography and electroelution. Polyclonal antibodies to trout hsc70 generated in rabbits cross-reacted strongly with both purified trout hsc70 protein and also purified recombinant bovine hsc70. Two-dimensional electrophoresis followed by Western blotting confirmed that the isoelectric point of rainbow trout hsc70 was more acidic than hsp70. Using this antibody, we detected hsc70 content in the liver, heart, gill and skeletal muscle of unstressed rainbow trout. Primary cultures of trout hepatocytes subjected to a heat shock (+15 °C for 1 h) or exposed to either CuSO 4 (200 μM for 24 h), CdCl 2 (10 μM for 24 h) or NaAsO 2 (50 μM for 1 h) resulted in higher hsp70 accumulation over a 24-h period. However, hsc70 content showed no change with either heat shock or heavy metal exposure suggesting that hsc70 is not modulated by sublethal acute stressors in trout hepatocytes. Taken together, we have for the first time generated polyclonal antibodies specific to rainbow trout hsc70 and this antibody will allow for the characterization of the role of hsc70 in the cellular stress response process in fish.

Import of a Cytosolic Protein into Lysosomes by Chaperone-mediated Autophagy Depends on Its Folding State

Import of a Cytosolic Protein into Lysosomes by Chaperone-mediated Autophagy Depends on Its Folding State

Identification of a Region of Escherichia coli DnaB Required for Functional Interaction with DnaG at the Replication Fork

The fundamental activities of the replicative primosomes of Escherichia coli are provided by DnaB, the replication fork DNA helicase, and DnaG, the Okazaki fragment primase. As we have demonstrated previously, DnaG is recruited to the replication fork via a transient protein-protein interaction with DnaB. Here, using site-directed amino acid mutagenesis, we have defined the region on DnaB required for this protein-protein interaction. Mutations in this region of DnaB affect the DnaB-DnaG interaction during both general priming-directed and phiX174 complementary strand DNA synthesis, as well as at replication forks reconstituted in rolling circle DNA replication reactions. The behavior of the purified mutant DnaB proteins in the various replication systems suggests that access to the DnaG binding pocket on DnaB may be restricted at the replication fork.

Human stress protein hsp70: overexpression in E coli, purification and characterization.

The gene encoding the stress-inducible member of human heat shock protein hsp70, was expressed in E. coli using the bacteriophage T7 RNA polymerase-based gene expression system. Recombinant hsp70 (R-hsp70) was purified from inclusion bodies after solubilization and refolding, using a combination of ATP-agarose affinity chromatography and ion-exchange chromatography. R-hsp70 was shown to be monomeric and free of its structurally similar E. coli counterpart, DnaK. In addition, R-hsp70 is functional as demonstrated by its ability to bind to peptides and to ATP. The availability of pure, correctly folded R-hsp70 in sufficient quantity will assist in the structural and functional characterization of hsp70. Furthermore, an understanding of the cytoprotective function of hsp70 and its role in immune responses during infections will be facilitated by the availability of pure R-hsp70.

CDNA cloning and efficient mitochondrial import of pre-mtHSP70 from rat liver.

Members of the 70-kD heat shock protein family have been found in all free-living organisms investigated and in major compartments of eukaryotic cells where they are essential to a wide range of functions, including protein folding and targeting. We have isolated a mitochondrial homolog (mtHSP70) from rat liver using ATP agarose affinity chromatography. Its identity was confirmed on the basis of immunological analysis and Ca(2+)-dependent autophosphorylation. Using protein sequence obtained from the amino termius and nine endo Lys-C peptide fragments, we have employed oligonucleotides to isolate a full-length cDNA clone. The open reading frame encodes a protein of 679 amino acids and calculated M(r) 73,913 daltons. The sequence has a high degree of identity with other members of the HSP70 family, including Escherichia coli DnaK (51%), Saccharomyces cerevisiae SSC1p (65%), the constitutive cytosolic HSP70 from rat, HSC70 (46%), and the rat endoplasmic reticulum isoform, BiP, (49%). The cDNA encodes a precursor protein with a 46-amino-acid signal peptide that is absent from the protein isolated from rat liver. The protein also shows a high degree of identity (98%) with a protein isolated from mouse and human tissues (PBP74, Domanico et al., 1993; mortalin, Wadhwa et al., 1993a; CSA, Michikawa et al., 1993a); however, the intracellular localization of these proteins is uncertain. We show that the precursor of mtHSP70 is efficiently imported into isolated mitochondria from rat liver and processed from 74 kD to the mature 69-kD protein.

The 65-kDa cytosolic protein associated with warm temperature acclimation in goldfish, Carassius auratus

Goldfish Carassius auratus were acclimated to either 10 or 30°C for a minimum of 5 weeks. A 65-kDa protein specific to warm-temperature-acclimated fish was extracted from the gel with 70% formic acid after two-dimensional electrophoresis of the muscle cytoplasmic protein fraction. The 65-kDa protein thus prepared to homogeneity was used to raise specific antibodies in rabbit by conventional methods. The antibody produced exhibited specific reaction with a protein having the same molecular weight from brain and liver tissue, suggesting that the 65-kDa protein is a ubiquitous cytosolic component in warm-acclimated goldfish. When water temperature was increased from 20 to 30°C over a 20-h period, a prominent amount of the 65-kDa protein was observed in muscle tissue extracts within 5 days of additional rearing; this was demonstrated by immunoblotting with the specific antibody. The N-terminal amino acid sequence of the 65-kDa protein was determined as Asp-Glu-Pro-Gln-Gly-His-Gln-His (or Asp)-Glu-Leu, differing from that of a family of known heat-shock proteins having about 70 kDa in molecular mass (hsp 70). No interaction between ATP and the 65-kDa protein revealed by ATP-agarose affinity chromatography further confirmed the different properties of the 65-kDa protein from those of hsp 70.

Regulation of phosphofructokinase and the control of cryoprotectant synthesis in a freeze-avoiding insect

Phosphofructokinase (PFK) from larvae of the freeze-avoiding gall moth Epiblema scudderiana was purified 711-fold using ATP-agarose affinity chromatography to a final specific activity of 23 U/mg protein. The native molecular mass of the enzyme was 420 000 ± 20 000 Da. The enzyme showed an optimum pH of 8.13 ± 0.21 at22 °Cand 8.19 ± 0.11 at5 °C. Arrhenius plots of PFK activity showed a sharp break at 9 °C. S0.5 values for fructose 6-phosphate showed positive thermal modification, decreasing with decreasing assay temperature; the opposite was true for ATP-Mg2+. PFK was activated by fructose 2,6-bisphosphate, AMP, and inorganic phosphate; activator effects were temperature-dependent. The enzyme was inhibited by ATP-Mg2+, citrate-Mg2+, and phosphoenolpyruvate. The positive effects of low temperature on enzyme kinetic properties would promote PFK activity to channel glycolytic carbon flow into the production of glycerol during cold-stimulated cryoprotectant synthesis.

Characteristics of an Hsp70 Homolog Localized in Higher Plant Chloroplasts That Is Similar to DnaK, the Hsp70 of Prokaryotes

Members of the 70-kD heat-shock protein (Hsp70) family are important cellular factors that are thought to mediate protein folding and assembly. A chloroplast-localized Hsp70 homolog (Chsp70) was recently identified based on its similarity to DnaK, the Hsp70 homolog of Escherichia coli (D. Amir-Shapira, T. Leustek, B. Dalie, H. Weissbach, N. Brot [1990] Proc Natl Acad Sci USA 87: 1749-1752). To learn more about the function of Chsp70, we purified the protein from Spinacia oleracea chloroplasts by ATP-agarose affinity chromatography. A single, 75,000-D protein was isolated which becomes phosphorylated on a threonine residue when incubated with [gamma-32P]ATP and 10 mM Ca2+, a property similar to DnaK. Chloroplast fractionation and immunoblot analysis showed that Chsp70 is a soluble stromal protein. Chsp70-specific antiserum was used to clone a partial cDNA that shows greater homology with Hsp70 from prokaryotes than with cytoplasmic Hsp70 from eukaryotes. The antiserum and cDNA were used to study Chsp70 expression. Following heat shock of spinach seedlings at 37 degrees C, Chsp70 synthesis increase 12-fold, the level of Chsp70 mRNA increases 5-fold, and the level of Chsp70 protein increases less than 2-fold. Chsp70 is constitutively expressed in all spinach tissues, indicating that it is likely to be localized in all plastid types. The highest levels occur in seeds, leaves, florets, and seedlings grown in the light. Lower levels occur in roots, stems, and etiolated seedlings.

A mitochondrial heat shock protein from Crithidia fasciculata

MCP72 is a mitochondrial hsp70 protein from the trypanosomatid Crithidia fasciculata. An MCP72 cDNA clone was isolated from a C. fasciculata cDNA library by screening with antiserum specific for the homologous protein of Trypanosoma cruzi [9]. The MCP72 cDNA encodes a polypeptide of 663 amino acids which is 84% identical to the Trypanosoma cruzi protein and 56% identical to the Escherichia coli hsp70 protein DnaK. MCP72 is less similar to other hsp70 proteins. Native MCP72 was purified to homogeneity by ATP-agarose affinity chromatography. Comparison of its N-terminal amino acid sequence with that deduced from the cDNA sequence shows that 20 amino acid residues had been cleaved from the N-terminus; this sequence probably represents a mitochondrial import signal which is cleaved during translocation into the mitochondrion. Fluorescene microscopy, using antibodies specific for MCP72, indicates that the protein is concentrated in a region of the mitochondrial matrix which surrounds the kinetoplast.

Lys-197 and Asp-414 are critical residues for binding of ATP/Mg2+ by rat brain inositol 1,4,5-trisphosphate 3-kinase.

Rat brain inositol 1,4,5-trisphosphate (InsP3) 3-kinase A was expressed in Escherichia coli in order to identify the amino acid residues involved in substrate ATP/Mg2+ binding. Two amino acid regions that are conserved in the catalytic domain of InsP3 3-kinase isoenzymes A and B had characteristics consistent with two ATP/Mg(2+)-binding motives. Site-directed mutagenesis was performed on residues Lys-197, Lys-207 and Asp-414 to generate three mutant enzymes, referred to as C5 K197I, C5 K207I and C5 D414N. Comparison of the wild-type and mutant proteins with regard to enzymic activity revealed that C5 K197I exhibited 10% of control enzyme activity, C5 D414N was totally inactive and C5 K207I was fully active. The reduced levels of enzyme activity for C5 K197I and C5 D414N were correlated with an altered ability of the mutant enzymes to bind ATP/Mg2+, as determined by ATP-agarose affinity chromatography. Neither Ca2+/calmodulin binding nor InsP3 binding appeared to be affected. Mutant C5 K207I showed the same characteristics as the wild-type enzyme. Taken together, these results strongly indicated (i) that amino acid residues Lys-197 and Asp-414 are necessary for InsP3 3-kinase activity and form part of the ATP/Mg(2+)-binding domain, and (ii) that amino acid residues Lys-197, Lys-207 and Asp-414 are not involved in either InsP3 binding or enzyme stimulation by Ca2+/calmodulin.

Autophosphorylation of the Pea Mitochondrial Heat-Shock Protein Homolog

Highly purified mitochondria isolated from 14-day-old pea (Pisum sativum L., cv Little Marvel) seedlings contain a homolog of the 70,000 molecular weight heat-shock protein. The amount of this heat-shock cognate (Hsc70) was not reduced by limited proteolysis of intact mitochondria or by preparation of mitoplasts, indicating that the protein is located within the matrix compartment. Pea mitochondrial Hsc70 binds to immobilized ATP and reacts on western blots with anti-tomato Hsc70 antiserum. When a mitochondrial matrix fraction was incubated with [gamma-(32)P]ATP, there was phosphorylation of Hsc70. The extent of phosphorylation was increased by including calcium chloride in the reactions. Phospho amino acid analysis of purified mitochondrial Hsc70, phosphorylated in the calcium-stimulated reaction, revealed only phosphothreonine. Pea mitochondrial Hsc70, purified by a combination of ATP-agarose affinity chromatography and gel permeation chromatography, was labeled when incubated with ATP plus calcium, suggesting autophosphorylation rather than phosphorylation by an associated kinase. In analogy to mammalian cells and yeast, it is likely that mitochondrial Hsc70 acts as a molecular chaperone, and it is possible that phosphorylation plays a role in chaperone function.

Purification of Hepatic N-Hydroxyarylamine Sulfotransferases and Their Regulation by Growth Hormone and Thyroid Hormone in Rats1

From liver cytosols of male Sprague-Dawley rats, two N-hydroxyarylamine sulfotransferases (HASTs) which sulfate N-hydroxy-2-acetylaminofluorene and a phenolsulfotransferase (PST-I) have been purified about 290-, 690-, and 210-fold, respectively, by the use of DEAE-anion exchange, Blue-Sepharose CL-6B, DEAE-HPLC, and ATP-agarose affinity chromatography. All three enzymes showed almost the same molecular weight of 33 kDa on SDS-polyacrylamide gel electrophoresis. In Western blots using antibody raised against HAST I, the two HASTs, but not PST-I, were detected. The content of total HAST in male rats was estimated as 4-5 micrograms/mg cytosolic protein, about 4 times higher than that in female rats. Direct comparison of the DEAE-HPLC elution profiles of cytosol showed that HAST I and HAST II were male-dominant and male-specific, respectively, in their expression in the livers. Hypophysectomy decreased the level of HAST by 60% in male rats, but had no obvious effect in female rats. Intermittent injection of growth hormone to mimic the male secretory pattern raised the content in hypophysectomized rats of both sexes close to that in intact male rats, while the continuous infusion of growth hormone to mimic the female secretory pattern showed limited effects. In addition, the administration of triiodothyronine stimulated HAST in hypophysectomized rats of both sexes, and the extent of stimulation was nearly the same as observed in the male-type growth hormone treatment. PST-I, in contrast to HAST, showed no clear sex-related difference in hepatic content, and was not apparently affected by growth hormone or triiodothyronine.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of the ATP-binding domain of vaccinia virus thymidine kinase.

Although small in size (20 kDa), the vaccinia virus (VV) thymidine kinase protein (EC 2.7.1.21 TK) is a relatively complex enzyme which must contain domains involved in binding both substrates (ATP and thymidine) and a feedback inhibitor (dTTP), as well as sequences directing the association of individual protein monomers into a functional tetrameric enzyme. Alignment of predicted amino acid sequences of the thymidine kinase genes from a variety of sources was used to identify highly conserved regions as a first step toward locating potential regions housing essential domains. A conserved domain (domain I) near the amino terminus of VV TK protein had characteristics consistent with a nucleotide-binding site. Analysis of the nucleotide substrate specificity of VV TK indicated that ATP acts as the major phosphate donor for thymidine phosphorylation while GTP, CTP, and UTP were inefficient substrates. Site-directed mutagenesis was performed on domain I to generate 11 mutant enzymes. Comparison of the wild-type and mutant proteins with regard to enzyme activity revealed that two of the mutant enzymes, T18 and S19, exhibited enhanced enzyme activity (3.73-fold and 1.35-fold, respectively) relative to the control. The other mutations introduced led to greatly reduced levels of enzyme activity which correlated with a reduced or altered ability of the mutant enzymes to bind ATP as determined by ATP-agarose affinity chromatography. Wild-type VV TK bound to an ATP affinity column could also be eluted with dTTP. Glycerol gradient separation of wild-type TK in the presence or absence of dTTP indicated that dissociation of the tetrameric complex was not the means by which enzymatic inhibition was achieved. Taken together, these results suggest that (i) domain I (amino acids 11-22) of the VV TK corresponds to the ATP-binding site, and (ii) that dTTP is able to interfere with ATP binding, either directly or indirectly, and thereby inhibit enzymatic activity without dissociating the native enzyme.

Purification and characterization of rat liver minoxidil sulphotransferase

Minoxidil (Mx), a pyrimidine N-oxide, is used therapeutically as an antihypertensive agent and to induce hair growth in patients with male pattern baldness. Mx NO-sulphate has been implicated as the agent active in producing these effects. This paper describes the purification of a unique sulphotransferase (ST) from rat liver cytosol that is capable of catalysing the sulphation of Mx. By using DEAE-Sepharose CL-6B chromatography, hydroxyapatite chromatography and ATP-agarose affinity chromatography, Mx-ST activity was purified 240-fold compared with the activity in cytosol. The purified enzyme was also capable of sulphating p-nitrophenol (PNP) at low concentrations (less than 10 microM). Mx-ST was purified to homogeneity, as evaluated by SDS/PAGE and reverse-phase h.p.l.c. The active form of the enzyme had a molecular mass of 66,000-68,000 Da as estimated by gel exclusion chromatography and a subunit molecular mass of 35,000 Da. The apparent Km values for Mx, 3'-phosphoadenosine 5'-phosphosulphate and PNP were 625 microM, 5.0 microM and 0.5 microM respectively. However, PNP displayed potent substrate inhibition at concentrations above 1.2 microM. Antibodies raised in rabbits to the pure enzyme detected a single band in rat liver cytosol with a subunit molecular mass of 35,000 Da, as determined by immunoblotting. The anti-(rat Mx-ST) antibodies also reacted with the phenol-sulphating form of human liver phenol sulphotransferase, suggesting some structural similarity between these proteins.

Phosphatidylinositol kinase activity of a plasma membrane-associated calcium-activated protein kinase from pea

A purified protein kinase preparation from pea is shown to contain phosphatidylinositol kinase activity. The preparation, produced by membrane solubilisation and ATP-agarose affinity chromatography, contains proteins of 18 kDa (ppl8, a calcium activated autophosphorylating protein-serine kinase), 67 kDa and 48 kDa and exhibits the phosphorylation of an endogenous lipid when supplied with [γ- 32P]ATP. This product is tentatively identified as lysophosphatidylinositol phosphate. When supplied with [γ- 32P]ATP and exogenous phosphatidylinositol, the production of phosphatidylinositol phosphate is observed.

Reduced coronary vasoconstrictor activity of hemoglobin solutions purified by ATP-agarose affinity chromatography

Stroma-free hemoglobin (Hb) solutions are being developed as blood substitutes. We previously described coronary vasoconstrictor activity of Hb solutions prepared by conventional methods. In the present study we assessed the constrictor activity of unmodified and covalently modified Hb solutions purified by ATP-agarose affinity chromatography. The starting material was a red cell lysate, partially purified by ultra-filtration. Coronary constrictor activity was measured as increased perfusion pressure in isolated rabbit hearts perfused at constant coronary flow rate with buffer containing various concentrations of added Hb. The starting material increased perfusion pressure by 35 ± 7 mmHg at 50 mg/dl. Purified Hb, retained by the affinity column, increased perfusion pressure by only 18 ± 2 mmHg at 50 mg/dl. Hemoglobin covalently linked to ATP or pyridoxal phosphate, then purified by affinity chromatography, also had less constrictor activity than the starting material. Thus, a substance, removed by affinity chromatography but not by conventional purification, contributes to the vasoconstrictor activity of Hb solutions.