DNA Polymerase-Associated Lectin (DPAL) and Its Binding to the Galactose-Containing Glycoconjugate of the Replication Complex

1999 ◽  
Vol 19 (5) ◽  
pp. 433-447
Author(s):  
Thomas J. Kelley ◽  
Tara St. Amand ◽  
Jeremy M. Groll ◽  
Satyajit Ray ◽  
Subhash Basu
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibody ◽  
Dna Polymerase ◽  
Recombinant Dna ◽  
Cultured Cells ◽  
Polyclonal Antibodies ◽  
Immunoblot Analysis ◽  
Post Translational Modification ◽  
Human Neuroblastoma ◽  
Dna Polymerase Α

The highly purified DNA Pol-α from rat prostate tumor (PA-3) and human neuroblastoma (IMR-32) cells appeared to be inhibited by Ricin (RCA-II), and Con-A. Loss of activity (40 to 60%) of a specific form of DNA polymerase from IMR-32 was observed when the cells were treated with tunicamycin [Bhattacharya, P. and Basu, S. (1982) Proc. Natl. Acad. Sci., USA79:1488–1492]. Binding of ConA and RCA to human recombinant DNA polymerase-α showed a specific labile site in the N-terminus [Hsi et al.. (1990) Nucleic Acid Res.18:6231–6237]. The catalytic polypeptide, DNA polymerase-α of eukaryotic origin, was isolated from developing tissues or cultured cells as a family of 180 to 120 kDa polypeptides, perhaps derived from a single primary structure. Immunoblot analysis with a monoclonal antibody (SJK-237-71) indicated that the lower molecular weight polypeptides resulted from either proteolytic cleavage of post-translational modification after specific cleavages. Present results suggest DNA polymerase-α from embryonic chicken brain (ECB) contains an α-galactose-binding subunit which may be involved in developmental regulation of the enzyme. It was shown before that the catalytic subunit of DNA polymerase-α reduces from 186 kDa in 11-day-old ECB to 120 kDa in 19-day-old ECB [Ray, S. et al. Cell Growth and Differentiation2:567–573] by the treatment with methyl-α-galactose. The low molecular weight DNA polymerase activity (120 kDa) can be reconstituted to high molecular weight (Mr = 186 kDa) with an α-galactose binding, 56 kDa lectin-like protein. Polyclonal antibodies raised against the purified lectin were able to precipitate DNA. Pol-α as determined by immunostaining with the polymerase-α-specific monoclonal antibody SJK 132-20, suggesting this is a DNA polymerase associated-lectin (DPAL). RCA-II and GS-I-Sepharose 4B chromatographies resulted in significant purification of DNA-α and a complete separation of polymerase complex and primase.

scholarly journals Effect of Dehydroaltenusin-C12 Derivative, a Selective DNA Polymerase α Inhibitor, on DNA Replication in Cultured Cells

Molecules ◽  
2008 ◽  
Vol 13 (12) ◽  
pp. 2948-2961 ◽  
Author(s):  
Isoko Kuriyama ◽  
Takeshi Mizuno ◽  
Keishi Fukudome ◽  
Kouji Kuramochi ◽  
Kazunori Tsubaki ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Cultured Cells ◽  
Dna Polymerase Α

MOLECULAR CLONING OF PLATELET GPIIb FROM HEL CELLS AND HUMAN MEGAKARYOCYTES

1987 ◽  
Author(s):  
G Uzan ◽  
A Lajmanovich ◽  
M H Prandini ◽  
Ph Frachet ◽  
A Duperray ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Fusion Protein ◽  
Recombinant Dna ◽  
Polyclonal Antibodies ◽  
Northern Blotting ◽  
Cellular Systems ◽  
Hel Cells ◽  
Function Relationship ◽  
Von Willebrand ◽  
Relationship Of

Platelet GP IIb-IIIa is an heterodimer which functions as a receptor for fibrinogen, fibronectin and Von Willebrand factor and is implicated in platelet adhesive reactions. To study the structure function relationship of this glycoprotein, a recombinant DNA approach was initiated. cDNA expression libraries were constructed in » gtll vector, from erythro-leukemia cells (HEL) and megakaryocytes mRNA. The human megakaryocytes were isolated from patients with chronic myeloid leukemia. The HEL library was initially screened with polyclonal antibodies anti GPIIb IIIa. One clone, λIIbI, containing a 1.65 kbp insert reacted with a panel of different polyclonal antibodies anti GPIIb IIIa and a monoclonal antibody anti GPIIb. To further characterize this clone the synthesis of the fusion protein was induced by IPTG. The bacterial protein was then blotted onto nitro cellulose and incubated with antisera anti GPIIb-IIIa. Antibodies that specifically bound with the fusion protein were eluted and tested on platelet membrane extracts. The selected antibodies produced a positive signal at the GPIIb position similar to the signal produced by the monoclonal antibody anti GPIIb on the same membrane extract. Finally on western blotting, a protein of Mr= 170kD reacted with the monoclonal antibody anti GPIIb. λIIbI insert was used to screen the megakaryocyte library and 3 clones, λIIb2,λIIb3 and λIIb4 were isolated. The size of HEL cells and megakaryocytes GPIIb mRNA was estimated by northern blotting. Only one species of 3.9 kb was identified in both cells. The four different clones accounted for 50% of the coding sequence of this mRNA.Sequencing of these cDNAs indicated that the plasmatic domain of GPIIb contains a cystein rich region. The sequence of these clones will allow the study of the adhesines genetic diversity in different cellular systems.

scholarly journals Detection of the growth fraction in colorectal tumours by a monoclonal antibody against DNA polymerase α

1990 ◽  
Vol 61 (3) ◽  
pp. 390-393 ◽  
Author(s):  
A Yamaguchi ◽  
S Takegawa ◽  
T Ishida ◽  
G Nishimura ◽  
M Kato ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Dna Polymerase ◽  
Growth Fraction ◽  
Dna Polymerase Α ◽  
Colorectal Tumours

Identification of high molecular weight peptides in bovine adrenomedullary chromaffin granules using monoclonal antibodies to a preproenkephalin A fusion peptide

1989 ◽  
Vol 2 (2) ◽  
pp. 119-129 ◽  
Author(s):  
S. Jackson ◽  
B. A. Spruce ◽  
D. M. Glover ◽  
B. P. Glynn ◽  
P. J. Lowry
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibodies ◽  
High Molecular Weight ◽  
High Performance ◽  
Polyclonal Antibodies ◽  
Immunoblot Analysis ◽  
Fusion Peptide ◽  
Chromaffin Granules ◽  
Immunoreactive Band ◽  
Adrenal Chromaffin

ABSTRACT Two mouse monoclonal antibodies (PE-1 and PE-2) raised to a β-galactosidase—preproenkephalin A(69– 207) fusion peptide recognize pro-enkephalin A (pro-enk-A) peptides of 33–5 kDa isolated from bovine adrenal chromaffin granules. The preliminary characterization of the high molecular weight adrenomedullary pro-enk-A peptides recognized by PE-1 and PE-2 is described. The high molecular weight peptides were resolved after Sephadex G-50 chromatography and high-performance liquid chromatography (HPLC) into three components (peaks I, II and III). Immunoblot analysis showed each HPLC peak to be heterogeneous. Peak I contained PE-1-and PE-2-immunoreactive peptides of 33, 29, 24 and 22 kDa; peak II contained a peptide of 22 kDa recognized by PE-2, and peptides of 24 and 22 kDa recognized by PE-1; peak III contained a PE-2-immunoreactive peptide of 15 kDa and PE-1-immunoreactive peptide of 18 kDa. Using polyclonal antibodies to peptide F and methionineenkephalin-Arg6-Gly7-Leu8 (MetEnk-RGL), the 22 kDa band cross-reacted with both MetEnk-RGL and peptide F antibodies, whilst the 24 kDa band was shown to possess predominantly MetEnk-RGL immunoreactivity. The 15 kDa (PE-2-immunoreactive) band was recognized by the peptide F but not the MetEnk-RGL antibody, whereas the polyclonal antibodies did not recognize the 18 kDa (PE-1-immunoreactive) band. We propose that the immunological and size characteristics of some of these peptides (29, 24/22, 15 kDa) suggest their similarity to the peptides of predicted molecular mass 23·3, 18·2 and 12·6 kDa previously found in bovine adrenal medulla. The results also indicate the existence of high molecular weight pro-enk-A peptides shortened at the N-terminus. The use of an immunoradiometric assay designed to measure the proenk-A-derived 18·2 kDa peptide using PE-2 and an affinity purified and radioiodinated MetEnk-RGL IgG has supported these findings.

scholarly journals Production of monoclonal antibodies against a novel glycoprotein synthesized and secreted by dog thyroid C-cells.

1992 ◽  
Vol 40 (4) ◽  
pp. 541-553 ◽  
Author(s):  
Y Kameda
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibody ◽  
Mammalian Species ◽  
Immunoblot Analysis ◽  
Secretory Protein ◽  
C Cells ◽  
Reaction Products ◽  
Medullary Thyroid ◽  
Thyroid C Cells ◽  
Thyroid Glands

A monoclonal antibody (MAb) that reacted only with thyroid C-cells was raised against cell suspensions from dog thyroid glands, to examine a glycoprotein secreted by C-cells. After chronically-induced hypercalcemia and administration of an anti-thyroid drug, reaction products for the antibody markedly decreased in C-cells, coinciding with alterations in calcitonin immunoreactivity. The antigen recognized by the MAb appears to be a secretory protein. The MAb reacted with C-cells from a wide variety of mammalian species, including rats, mice, hamsters, cattle, cats, rabbits, and monkeys. Furthermore, tumor cells of human medullary thyroid carcinoma, which is derived from C-cells, were immunoreactive to the MAb. Exceptionally, C-cells from guinea pigs and pigs were not stained with the MAb. No crossreactivity was observed in any of the dog tissues examined. Immunoblot analysis demonstrated that the MAb recognized a single prominent band at a molecular weight of approximately 79,000. The 79 KD band reacted with various digoxigenin-labeled lectins, including GNA, DSA, SNA, and MAA; it is a glycoprotein containing mannose, N-acetylglucosamine, and sialic acid. Dog thyroid C-cells were also densely stained with these lectins. The results indicate that thyroid C-cells synthesize and secrete a specific glycoprotein in addition to peptide hormones.

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