Purification of a high molecular weight human terminal deoxynucleotidyl transferase.

Terminal deoxynucleotidyltransferase has been purified from lymphoblasts of leukemic patients. The enzyme has a molecular weight of approximately 62,000 as determined by gel filtration and nondenaturing gel electrophoresis and is not dissociated into subunits by sodium dodecyl sulfate. In contrast, the terminal transferase enzyme from calf thymus has a molecular weight of 42,000 as determined by gel filtration, and is dissociated into 2 subunits of Mr 30,000 and 8,000 by sodium dodecyl sulfate. The enzyme has an isoelectric point of 8.2 and kinetic characteristics which are similar to those of calf thymus terminal transferase. The apparent Km for purine nucleotide polymerization at saturating initiator concentration with Mg2+ is 0.2 mM and with Mn2+ is 0.05 mM. Like calf terminal transferase, the reaction velocity is higher in the presence of Mg2+ than Mn2+. ATP inhibits the reaction catalyzed by terminal transferase isolated from human lymphoblasts due to mutual recognition of ATP and dATP by a common site on the enzyme. Preliminary experiments indicate that human terminal transferase may contain a small amount of carbohydrate. This report represents the first purification to near homogeneity of terminal transferase from a tissue source other than calf thymus.     

Expression of human terminal deoxynucleotidyl transferase in Escherichia coli

A cloned DNA fragment related to pT17 containing a partial cDNA sequence of human terminal deoxynucleotidyl transferase was used as a probe to screen for the full length cDNA sequence of the enzyme in a lambda gt11 library constructed from human lymphoblastoid KM-3 cDNA. A recombinant containing a 2068-base pair insert was isolated and recloned into the EcoRI site of the sequencing plasmic pUC-8 as two subclones, pT711 and pT106. DNA sequencing and hybridization studies showed that pT711 contains the pT17 sequence and an additional 172 upstream nucleotides. pT711 represents the coding sequence for the carboxyl half of the terminal transferase protein. pT106, containing a 965-base pair insert, hybridizes to the same mRNA as pT711 on Northern blots and contains an open reading frame that is in phase with the reading frame of the insert in pT711. Amino acid sequencing of the 58-kDa peptide of the calf thymus terminal transferase failed, indicating that the N terminus is blocked. N-Terminal sequencing of a 56-kDa form of the protein produced 24 amino acids corresponding to the translated human cDNA coding sequence starting at residue 398 of the insert in pT106 with 83% homology between bovine and human sequence. The initiation codon is assigned to an ATG sequence at nucleotide 329 of the insert in pT106. Comparison of the translated human terminal transferase sequence with peptides from the calf thymus enzyme showed that the homology between the human and bovine enzyme is better than 90% among 263 amino acids determined. The coding sequences in pT106 and pT711 were recloned into an expression plasmid pUC-19 downstream from the lac promoter and in phase with the coding sequence of the lac Z gene. Lysates of bacteria carrying the reconstructed coding sequence of human terminal transferase contain a fused protein of 60 kDa that reacts with rabbit antibody to terminal transferase on immunoblots and exhibits enzyme activity. Isolation of this fused protein from bacterial lysates with mouse monoclonal antibody to human terminal transferase produces the expected protein of 60 kDa.     

Limited proteolysis of calf thymus terminal deoxynucleotidyl transferase

Controlled, limited proteolysis of homogeneous calf thymus terminal deoxynucleotidyl transferase (EC 2.7.7.31) using immobilized Staphylococcus aureus V-8 protease results in a low molecular weight form of the enzyme which possesses unaltered catalytic activity. Analysis of the products of limited proteolysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that only the large subunit, β, is modified from a molecular weight of 30,500 to 25,500. The small subunit, α, which has a molecular weight of 9500, is unchanged. A shift in the apparent isoelectric pH of the calf enzyme following proteolysis is observed from pI = 8.2 to 7.8. Both forms of the enzyme are homogeneous in the isoelectric focusing gel system, as determined by coincidence of single protein bands with terminal transferase activity on the gel. The specific activities of cleaved and uncleaved terminal transferase proteins, as well as their thermal stabilities, are comparable. These results suggest that the polypeptide domain involved in terminal transferase enzymatic activity can be probed further by novel methods involving limited proteolysis without concomitant loss in enzymatic function.     

Purification and immunological characterization of DNA polymerase-alpha from human acute lymphoblastic leukemia cells

DNA polymerase-alpha was purified from the cytosol of blast cells of a patient with acute lymphoblastic leukemia by ammonium sulfate fractionation and successive column chromatographies. The purified enzyme had a specific activity of 2943 units/mg protein with activated calf thymus DNA as a template. The enzyme sediments under high-salt conditions as a homogeneous band at 7.2 S and free from other DNA polymerases (beta, gamma) and terminal deoxynucleotidyl transferase activity. The native molecular weight of the enzyme from gel filtration and glycerol gradient centrifugation was found to be 175 000. The values of Stokes radius (53 A), diffusion coefficient (4.05 x 10(-7) cm2/s) and frictional ratio (1.42) determined by gel filtration suggest that the native enzyme is compact and globular. Antibodies to DNA polymerase-alpha were raised in rabbits. These antibodies, partially purified by 50% ammonium sulfate saturation and Sephadex G-200 chromatography, gave one precipitin band on immunodiffusion and inactivate DNA polymerase-alpha-. This antibody preparation also inhibited, in vitro, the activity of DNA polymerase-alpha from calf thymus, phytohemagglutinin-stimulated normal human lymphocytes, as well as that from other leukemic cells. Thus, DNA polymerase-alpha from calf thymus and human leukemic cells resemble each other in antibody specificity.     

Expression and processing of recombinant human terminal transferase in the baculovirus system

Overproduction of human terminal transferase protein has now been accomplished by cloning the coding sequence of human terminal transferase into a baculovirus, where the expression of terminal transferase is under the control of the polyhedrin protein promoter. Two constructs were made, one producing a protein containing the entire terminal transferase fused to 12 amino acids from the NH2 terminus of the polyhedrin protein, and the other producing 58-kDa human terminal transferase. The terminal transferase levels expressed in cells infected with either recombinant baculovirus are around 10,000 units/10(7) cells at 48 h postinfection, about 200-fold greater than levels expressed in thymus and cultured lymphoblastoid cells. The chimeric polyhedrin/human terminal transferase protein produced in the infected insect cells has a molecular weight of about 60,000 while the nonfused recombinant human terminal transferase is identical in molecular weight to that present in human lymphoblastoid cells. Both forms of recombinant terminal transferase show immunological and enzymatic activity. When infected cells are pulse-labeled with [35S] methionine at 42-45 h postinfection, about 10% of newly synthesized protein is terminal transferase. Both forms of terminal transferase are phosphorylated in recombinant virus-infected cells as demonstrated by pulse-labeling infected cells with 32P-inorganic phosphate and isolation of labeled terminal transferase peptides by immunoprecipitation.     

层析聚焦法分离纯化末端脱氧核苷酰转移酶及其抗血清制备和鉴定

 应用层析聚焦和Oligo(dT)-纤维素亲和层析相结合的方法,从小牛胸腺中分离纯化末端脱氧核苷酰转移酶(TdT)。纯化的TdT聚丙烯酰胺凝胶电泳呈一条区带,SDS-聚丙烯酰胺凝胶电泳分子量为24,000及26,000d的两条区带。此纯化TdT径戊二醛交联法,自身交联后免疫家兔,得到兔抗小牛TdT的单价抗血清,并进行了免疫学鉴定。     

Cell-free translation of the messenger RNA for calf terminal deoxynucleotidyltransferase

Poly(A)-rich RNA has been isolated from calf thymus and translated in vitro in a rabbit reticulocyte translation system. Three peptides with Mr = 58,000, 33,000, and 13,000 were specifically immunoprecipitated from the translation products with calf terminal deoxynucleotidyltransferase antiserum. An oligo(dT)-purified preparation of calf terminal transferase competed with only the Mr = 58,000 peptide in the immunoprecipitation reaction. The anti-terminal transferase serum did not precipitate a Mr = 58,000 peptide from translation products of spleen or liver mRNA, but it did precipitate the Mr = 33,000 and 13,000 peptides from products of spleen mRNA and a Mr = 13,000 peptide from products of liver mRNA. In addition, when an affinity-purified antibody to calf terminal transferase was used, only a Mr = 58,000 peptide was immunoprecipitated from the translation products of calf thymus mRNA, and none was immunoprecipitated from spleen or liver mRNA products. This antibody also precipitated a Mr = 58,000 peptide from the cell lysates of calf thymocytes labeled in vitro with [35S]methionine. These results demonstrate that calf terminal transferase is biosynthesized as a Mr = 58,000 peptide.     

Terminal deoxynucleotidyl transferase in human brain

A DNA polymerase lacking template direction for base selection has been partially purified from human brain. The molecular size, optimum reaction conditions, initiator preferences and chemical inhibitors of the brain enzyme were similar to calf thymus terminal deoxynucleotidyl transferase (TdT). TdT has been found only in the thymus, and now in brain. The possibility exists that its function is related to biological property unique to these two organs.     

Antigenic relationships in calf thymus and human leukemic cell terminal deoxynucleotidyl transferase.

Antibody to purified terminal deoxynucleotidyl transferase (nucleosidetriphosphate : DNA deoxy-nucleotidylexotransferase, E.C. 2.7.7.31) from calf thymus was prepared in rabbits using terminal deoxynucleotidyl transferase crosslinked to bovine serum albumin. These antibodies, partially purified by 60% ammonium sulfate precipitation and Sephadex G-200 column chromatography, produced one precipitation band with calf thymus terminal deoxynucleotidyl transferase on immunodiffusion. This antibody preparation also inhibited the in vitro activity of terminal deoxynucleotidyl transferase from calf thymus, acute leukemic lymphoblasts and Molt-4 cells but not that of DNA polymerases alpha, beta and psi from these cells     

Terminal deoxynucleotidyltransferase. Alignment of alpha- and beta-subunits of the core enzyme along the primary translation product.

Terminal deoxynucleotidyltransferase exists in multiple Mr forms, all apparently generated from a single polypeptide of 62kDa. On isolation and purification, the smallest catalytically active protein of this enzyme consists of two subunits, alpha (12kDa) and beta (30kDa). Recently a complementary-DNA nucleotide sequence has been reported for a portion of the enzyme from human lymphoblast. We have pinpointed the locations of the alpha- and beta-subunits within the elucidated nucleotide sequence. From these data, the portions of the nucleotide sequence coding for the catalytically important area of the transferase can be estimated. Here the amino acid sequence of a number of tryptic peptides from calf alpha- and beta-subunits is presented. Because of the striking homology between the amino acid sequence of the calf enzyme and that predicted for human lymphoblast enzyme, it is possible for us to conclude that the alpha-subunit was generated from the C-terminus of the precursor protein and the beta-subunit was non-overlapping and proximal.     

Identification of the serine residue phosphorylated by protein kinase C in vertebrate nonmuscle myosin heavy chains

Two-dimensional mapping of the tryptic phosphopeptides generated following in vitro protein kinase C phosphorylation of the myosin heavy chain isolated from human platelets and chicken intestinal epithelial cells shows a single radioactive peptide. These peptides were found to comigrate, suggesting that they were identical, and amino acid sequence analysis of the human platelet tryptic peptide yielded the sequence -Glu-Val-Ser-Ser(PO4)-Leu-Lys-. Inspection of the amino acid sequence for the chicken intestinal epithelial cell myosin heavy chain (196 kDa) derived from cDNA cloning showed that this peptide was identical with a tryptic peptide present near the carboxyl terminal of the predicted alpha-helix of the myosin rod. Although other vertebrate nonmuscle myosin heavy chains retain neighboring amino acid sequences as well as the serine residue phosphorylated by protein kinase C, this residue is notably absent in all vertebrate smooth muscle myosin heavy chains (both 204 and 200 kDa) sequenced to date.     

The plasma membrane H+‐ATPase from maize roots is phosphorylated in the C‐terminal domain by a calcium‐dependent protein kinase

A protein kinase activity associated with maize root plasma membranes was partially purified and characterized. Biochemical properties, such as calcium dependence, inhibition by calmodulin antagonists, and absence of calmodulin stimulation, indicated that the enzyme belongs to the calcium‐dependent protein kinase (CDPK) family. By means of an in‐gel phosphorylation assay the molecular mass of active polypeptides was determined: two bands of 55 and 51 kDa became labelled. The same proteins were also immunodecorated by monoclonal antibodies against soybean CDPK. Results from in vitro assays demonstrated that maize H⁺‐ATPase was a suitable substrate for this protein kinase and that the phosphorylation site was located at the C‐terminal domain of the enzyme. This result was confirmed by using as substrate in phosphorylation assays the isolated C‐terminal domain of the H⁺‐ATPase expressed in Escherichia coli as a glutathione‐transferase fusion protein.     

Interaction of terminal transferase with single-stranded DNA

A 58-kDa monomer of terminal transferase was isolated from calf thymus using a monoclonal antibody affinity column. The enzymatic activity was comparable to that of the 44-kDa alpha beta dimer isolated by conventional methods. Binding of the two enzyme forms to single-stranded DNA was monitored by fluorescence. The site size of both forms was approximately 11 +/- 2 nucleotides. Binding of the 44-kDa alpha beta dimer to polydeoxyadenosine was examined under several conditions. The cooperativity parameter increased from about 90 in the presence of Mg2+ to 300-400 in the absence of Mg2+. The observed dissociation constant of 3-5 microM was essentially independent of salt concentration, whereas the intrinsic dissociation constant decreased about 5-fold in the presence of Mg2+. The binding parameters of the 58-kDa monomer were independent of buffer composition and were similar to those of the 44-kDa alpha beta dimer in the presence of Mg2+. These results indicate that the additional 14-kDa peptide sequences present in the high molecular mass monomer form are not part of the DNA-binding site of terminal transferase.     

Characterization of recombinant human nicotinamide mononucleotide adenylyl transferase (NMNAT), a nuclear enzyme essential for NAD synthesis

Nicotinamide mononucleotide adenylyl transferase (NMNAT) is an essential enzyme in all organisms, because it catalyzes a key step of NAD synthesis. However, little is known about the structure and regulation of this enzyme. In this study we established the primary structure of human NMNAT. The human sequence represents the first report of the primary structure of this enzyme for an organism higher than yeast. The enzyme was purified from human placenta and internal peptide sequences determined. Analysis of human DNA sequence data then permitted the cloning of a cDNA encoding this enzyme. Recombinant NMNAT exhibited catalytic properties similar to the originally purified enzyme. Human NMNAT (molecular weight 31932) consists of 279 amino acids and exhibits substantial structural differences to the enzymes from lower organisms. A putative nuclear localization signal was confirmed by immunofluorescence studies. NMNAT strongly inhibited recombinant human poly(ADP-ribose) polymerase 1, however, NMNAT was not modified by poly(ADP-ribose). NMNAT appears to be a substrate of nuclear kinases and contains at least three potential phosphorylation sites. Endogenous and recombinant NMNAT were phosphorylated in nuclear extracts in the presence of [gamma-(32)P]ATP. We propose that NMNAT's activity or interaction with nuclear proteins are likely to be modulated by phosphorylation.     

猪胸腺末端脱氧核苷酰转移酶的分离纯化及性质的研究

 本文报道了一种较简便的从猪胸腺中分离纯化末端脱氧核苷酰转移酶(TdT)的方法。经一次磷酸纤维素柱层析,使TdT与DNA聚合酶分离;经三次柱层析,可获得SDS-电泳纯,分子量约60K的产品。其酶学性质与牛胸腺TdT相似。     

Polypeptide structure of human terminal transferase

The polypeptide structure of terminal transferase purified from human lymphoblasts was examined with an immunoblot procedure using rabbit anti-calf thymus terminal transferase antibodies. Two doublets of bands of M_r 58-56,000 and M_r 44-42,000 are the major immunoreactive polypeptides. Only the M_r 44-42,000 polypeptides can be efficiently renatured $\text{in}\text{situ}$ after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Controlled degradation with trypsin produces fully active enzyme containing the α and β polypeptides typical of the low molecular weight terminal transferase, suggesting that the different forms of purified terminal transferase may arise by proteolysis of the M_r 58,000 polypeptide.     

Affinity chromatography of terminal deoxynucleotidyl tranferase from calf thymus and human leukemic cells on a solid-phase immunoadsorbent

A solid-phase immunoadsorbent specific for terminal deoxynucleotidyl transferase has been prepared. The enzyme from calf thymus and acute lymphoblastic leukemia cells binds to columns of this material. Bound enzyme can be eluted in an active form. Selective and rapid purification of terminal deoxynucleotidyl transferase from crude extracts of cells containing this enzyme can be achieved by this method since the immunoadsorbent has no affinity for other cellular DNA polymerases.     

Immunological reagents for comparisons of DNA polymerase-alpha and DNA polymerase-beta

Antibodies to homogeneous calf thymus DNA polymerase-beta and calf thymus DNA polymerase-alpha preparations were raised in rabbits. The antiserum against calf thymus DNA polymerase-beta cross-reacts with all vertebrate DNA polymerase-beta preparations tested, but does not cross-react with trypanosome DNA polymerase-beta, DNA polymerase-gamma, terminal transferase, yeast DNA polymerases, and Escherichia coli DNA polymerase I. The antibodies against calf thymus DNA polymerase-alpha cross-react with DNA polymerase-alpha from mouse, human, and chicken, but do not cross-react with DNA polymerase-alpha from sea urchin embryos and Drosophila embryos, DNA polymerase-beta, DNA polymerase-gamma, terminal transferase, yeast DNA polymerases, and E. coli DNA polymerase I.