ABSTRACT
A soluble 5'-nucleotidase from pig thyroid was purified over 110-fold by chromatography on phosphocellulose, (NH4)2SO4 precipitation and gel filtration on Sephadex G-150. The purified 5-nucleotidase was free of non-specific phosphatases. The enzyme had optimum pH at 6.5 and hydrolysed preferentially IMP and GMP. The Km values were 0.66 and 1.0 mM for IMP and GMP, respectively. The enzyme also hydrolysed other nucleotides and showed the following relative Vmax:IMP>CMP>AMP>UMP.Mg2+ was necessary for the enzyme activity.
Subject(s)
5'-Nucleotidase/isolation & purification , 5'-Nucleotidase/metabolism , Thyroid Gland/enzymology , Animals , Chromatography, Gel , Chromatography, Ion Exchange , Hydrogen-Ion Concentration , Kinetics , Magnesium Chloride , Sodium Chloride , Substrate Specificity , SwineABSTRACT
Phospholipids were separated from 5'-nucleotidase in thyroid plasma membranes by Sephadex G-200 gel filtration. After removal of lipids 5'-nucleotidase was still active and reassociation of the enzyme with phospholipids had a little effect on the increase of enzyme activity. Arrhenius plot of the 5'-nucleotidase activity in native thyroid plasma membranes clearly exhibited a break at 28 degrees C. Biphasic nature of Arrhenius plot showed that the enzyme activity was influenced by physical state of membrane bilayer, although phospholipids were not obligatory cofactor for this enzyme.
Subject(s)
5'-Nucleotidase/metabolism , Phospholipids/physiology , Thyroid Gland/enzymology , 5'-Nucleotidase/chemistry , Animals , Cell Membrane/enzymology , Chromatography, Thin Layer , Enzyme Activation , Lipid Bilayers/chemistry , Substrate Specificity , Swine , Temperature , Thyroid Gland/chemistryABSTRACT
The effect of Triton X-100, sodium deoxycholate and saponin upon the solubilization of 5'-nucleotidase, the membrane enzyme derived from pig thyroid was studied. Triton X-100 at the concentration of 0.1% did not cause enzyme solubilization, whereas at the concentration of 1.0% it caused only partial release of the former from the membranes. Saponin (1.0% concentration) brought about a marked (about threefold) increase in the enzyme activity which resulted from the exposing active loci of the enzyme, however it did not cause total solubilization of it. 1% sodium deoxycholate increased the enzyme activity by 5 times and also caused its almost total solubilization (over 90%). The results indicate the 5'-nucleotidase is strongly bound to the cell membranes and the non-ionic detergents like Triton X-100 and saponin do not fit for the solubilization of this enzyme.
Subject(s)
5'-Nucleotidase/metabolism , Detergents/pharmacology , Thyroid Gland/enzymology , Animals , Deoxycholic Acid/pharmacology , Enzyme Activation/drug effects , In Vitro Techniques , Octoxynol , Polyethylene Glycols/pharmacology , Saponins/pharmacology , Solubility , Swine , Thyroid Gland/drug effectsABSTRACT
The activity of 5'-nucleotidase, AMP deaminase, adenosine deaminase, acid phosphatase, alkaline phosphatase and nucleotide pyrophosphatase was assayed in human thyroid glands. The 5'-nucleotidase activity was higher than that of AMP deaminase which suggested that AMP undergoes degradation primarily as a result of dephosphorylation in thyroid tissue. A high acid phosphatase activity was noted as compared to that of alkaline phosphatase activity. In toxic goitre the increase in adenosine deaminase and acid phosphatase was observed together with the decrease in pyrophosphatase activity.
Subject(s)
AMP Deaminase/metabolism , Acid Phosphatase/metabolism , Adenosine Deaminase/metabolism , Alkaline Phosphatase/metabolism , Nucleoside Deaminases/metabolism , Nucleotidases/metabolism , Nucleotide Deaminases/metabolism , Pyrophosphatases/metabolism , Thyroid Gland/enzymology , 5'-Nucleotidase , Autopsy , Humans , Kinetics , Thyroid Gland/pathologyABSTRACT
A homogeneous preparation of EF-2 from Guerin tumour cells was obtained. Its Mr (68 000), pI (6.5), optimum pH (7.0) and amino acid composition are very close to those of rat liver elongation factor. EF-2 from Guerin tumour cells is active in the heterologous liver - tumour system, although half as effective as in the homologous system.
