Glyceraldehyde-3-phosphate dehydrogenase binds to homologous and heterologous single-stranded but not double-stranded DNA. Binding to RNA, poly(A) and poly(dA-dT) has also been observed. Enzyme binding to these nucleic acids leads to the formation of an insoluble complex which can be sedimented at low speed. The interaction of glyceraldehyde-3-phosphate dehydrogenase with DNA is strongly inhibited by NAD and NADH but not by NADP. Adenine nucleotides, which inhibit the dehydrogenase activity by competing with NAD for its binding site (Yang, S.T. and Deal, W.C., Jr. (1969) Biochemistry 8, 2806--2813), also inhibit enzyme binding to DNA, whereas glyceraldehyde-3-phosphate and inorganic phosphate are non-inhibitory. These results suggest that DNA interacts through the NAD binding sites of glyceraldehyde-3-phosphate dehydrogenase. In accordance with this idea, it was found that DNA also binds to lactate dehydrogenase, an enzyme containing a similar dinucleotide binding domain, and that this binding is inhibited by NADH. A study of the base specificity of the DNA-glyceraldehyde-3-phosphate dehydrogenase interaction using dinucleoside monophosphates shows that inhibition of DNA binding by the dinucleotides requires the presence of a 3'-terminal adenosine and is greater when the 5'-terminus contains a pyrimidine instead of a purine. These results suggest that the dinucleotides bind at the NAD site of the dehydrogenase and that the enzyme would interact preferentially with PypA dinucleotides present in the nucleic acid.

Animals↗

PubMed1979

Thrombotic obstruction of the Björk-Shiley aortic valve prosthesis. Report of four cases.

We present four cases of late thrombotic obstruction of the Björk-Shiley aortic valve prosthesis. The incidence of this complication reported in the literature to vary from 0.7% to 5%. In our experience of a total of 623 Björk-Shiley aortic valve replacements, we observed this complication in only 4 patients (0.6%). Two of them had poorly controlled anticoagulation therapy. The clinical presentation was subacute in all four patients. Surgical treatment, thrombectomy and debridement, was performed in all of them. The diagnosis was made upon abrupt and progressive onset of dyspnoea, physical examination data and echocardiographic and radioscopic findings. Angiocardiographic studies were needed in two patients.

Adult↗

PubMed1978

Characterization of two poly(A) polymerases from cultured hamster fibroblasts.

The enzymatic and physiochemical properties of poly(A) polymerases IIA and IIB from cultured hamster fibroblasts were investigated. The enzymes show an absolute requirement for Mn2+ as the divalent ion. Although Mg2+ alone is inactive, maximum activity is observed in the presence of both Mn2+ and Mg2+. An optimal pH of approx. 8 is found for polymerases IIA and IIB. The enzymes, however, differ somewhat in the pH curves as well as in the Mn2+ and Mg2+ concentration curves. Poly(A) polymerases IIA and IIB have an isoelectric point of about 6 and a sedimentation coefficient of 3.5--4 S. The molecular weights, obtained by gel filtration chromatography, are 145 000 and 155 000 for enzymes IIA and IIB, respectively. Poly(A) polymerases IIA and IIB can utilize a variety of natural and synthetic RNAs as well as DNA as primers. Poly(A) polymerase IIA is saturated at much lower concentrations of primer than enzyme IIB. On the other hand, the chain length of the product synthesized by polymerase IIA is independent of the primer concentration, whereas, with polymerase IIB, the length of the product decreases when the concentration of RNA is increased.

Adenosine Triphosphate↗

PubMed1978

Binding of microtubule proteins to DNA: specificity of the interaction.

Tubulin is detected among the DNA-binding proteins when an extract from fibroblasts is chromatographed on DNA-cellulose. Further purification of the colchicine-binding activity shows that purified tubulin from fibroblasts does not bind to DNA. Depolymerized brain microtubule proteins show a high affinity for DNA. The fraction bound is composed of tubulin and microtubule-associated proteins. Experiments with fractionated microtubule proteins indicate that tubulin-free microtubule associated proteins bind to DNA, while tubulin free of microtubule-associated proteins does not. Microtubule-associated proteins bind better to eukaryotic than to phage DNA suggesting a specificity of the interaction.

Animals↗

PubMed1977

Identification of the mammalian DNA-binding protein P8 as glyceraldehyde-3-phosphate dehydrogenase.

The DNA-binding protein P8 from transformed hamster fibroblasts (line NIL-1-hamster sarcoma virus) has been purified to homogeneity by DNA-cellulose and phosphocellulose chromatography. The molecular weight of dissociated P8 is 36000, the same as that reported for the subunits of glyceraldehyde-3-phosphate dehydrogenase, and the mobility of these proteins in polyacrylamide gels is identical. The amino acid composition of P8 is very similar to that of glyceraldehyde-3-phosphate dehydrogenase. When assayed for glyceraldehyde-3-phosphate dehydrogenase activity the P8 preparation had a specific activity of 54.6 units/mg, a value comparable to that of the crystalline enzyme from several sources. Furthermore, serum prepared against P8 crossreacts with glyceraldehyde-3-phosphate dehydrogenase from hamster muscle. These results show that P8 is glyceraldehyde-3-phosphate dehydrogenase. The interaction of P8 from transformed fibroblasts and glyceraldehyde-3-phosphate dehydrogenase from hamster and rabbit muscle with DNA has been studied using a Millipore filtration technique. These proteins have affinity for single-stranded DNA but not for double-stranded DNA.

Amino Acids↗

PubMed1976

Synthesis of a class of DNA-binding proteins in synchronized untransformed and virus-transformed cells.

The synthesis of a classs of proteins with affinity for denatured DNA has been studied in synchronized cultures of the established hamster fibroblast line HIL-1 and its virus-transformed derivative NIL-1-hamster sarcoma virus. It has been found that the synthesis of a DNA-binding protein (P6, molecular weight 50 000) in synchronized untransformed NIL-1 cells follows a pattern different from that observed in the transformed cells. The protein is low in stationary cultures of NIL-1 and NIL-1-hamster sarcoma virus but increases after the cells are stimulated to grow, although the time of maximal P6 synthesis relative to cellular DNA synthesis is different in NIL-1 and NIL-1-hamster sarcoma virus. In contrast, the pattern of synthesis of two other DNA-proteins (P7 and P8') in essentially identical in synchronized untransformed and transformed cells. P7 is very low in resting cultures of both types of cells, but greatly increases early after the cells are stimulated to divide, while P8'is hish in stationary cultures and decreases slightly after the initiation of cellular DNA synthesis has occurred.

Cell Division↗

PubMed1976

Subcellular distribution of DNA-binding proteins from cultured hamster fibroblasts.

The distribution between nuclei and cytoplasm of DNA-binding proteins from growing NIL cells was studied. To obtain the subcellular fractions, cell monolayers or cells previously detached from the culture dish were treated with non-ionic detergent N onidet P-40. Proteins with affinity for DNA were isolated from nuclear or cytoplasmic fractions by chromatography on DNA-cellulose columns and were further analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The results show that P8, one of the major components in the 0.15 M NaC1-eluted proteins, is found predominantly in the cytoplasmic fractions, whereas P6, the other main protein peak in this eluate, is more prominent in the nuclear fraction. Among the other proteins eluted at 0.15 M NaC1 from the DNA-cellulose column, P5 and P5' are detected in both nuclear and cytoplasmic fractions. All the other proteins in the 0.15 M NaC1 eluate are present almost exclusively in the cytoplasmic fraction. On the other hand, most of the proteins with higher affinity for DNA, eluted from the column at 2 M NaC1, are present in the nuclear fraction, although they are also detected in the cytoplasm in amounts similar to those observed in the nuclei.

Animals↗

PubMed1975

Deoxyribinucleic acid-binding proteins in virus-transformed cell lines.

The synthesis of proteins with affinity for DNA has been studied in clones of a Syrian hamster cell line (NIL) and subclones of this line transformed by polyoma virus (NIL-Py) or hamster sarcoma virus (NIL-HSV). The results show that the synthesis of DNA-binding proteins in NIL and in its virus-transformed derivatives NIL-Py and NIL-HSV is very similar in exponentially growing cells, but in dense culture there is a very significant difference in the level of a protein (P8), which is much higher in the transformed lines than in untransformed NIL. The high levels of P8 in dense transformed cells have been observed in all the clones of transformed cells examined, indicating that this behavior of P8 is related to transformation and not simply due to a fortuitous clonal selection from the NIL. Experiments with synchronized cells indicate that the time of maximal P8 synthesis relative to cellular DNA synthesis in NIL-HSV precedes that observed in NIL cells. P8 has a molecular weight of 30,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and is present in large amounts in the transformed cells in dense culture, where it makes up 0.5 to 1% of the total soluble protein.

Agglutination Tests↗

PubMed1975

Poly(A) polymerases in normal and virus-transformed cells.

The presence of poly(A) polymerase(s) has been studied in a clone of the established hamster embryo fibroblast line (NIL), and in a subclone of this line transformed by an RNA tumour virus, hamster sarcoma virus, (NIL-HS VIRUS). The results suggest the existence of three distinct poly(A) polymerases, designated I, IIA and IIB, in dense cultures of NIL and NIL-HS virus cells. Forms IIA and IIB have also been found in exponentially growing NIL and NIL-HS virus cells. Poly(A) polymerase I has not been detected in growing NIL cells, while growing and resting NIL-HS virus contain comparable amounts of this enzyme. The poly(A) polymerases differ in chromatographic behaviour and in their requirements for divalent cations. They are highly specific for ATP and require the presence of a primer. Cellular RNA or poly(A), but not the oligoribonucleotide (Ap)4A, can be utilized as primers. The products of the reactions have been identified as poly(A) chains (20-50 nucleotides long) by alkaline degradation and by their resistance to pancreatic RNAase.

Adenine Nucleotides↗