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J Spychala

Publications and source records attributed to J Spychala.

30 records · Page 2Linked to original sources
PubMed1993

Altered properties of human T-lymphoblast soluble low Km 5'-nucleotidase: comparison with B-lymphoblast enzyme.

Soluble low Km 5'-nucleotidases have been purified from human cultured T- and B-lymphoblasts to compare their properties and to examine the mechanism of different rates of nucleotide dephosphorylation. The enzyme from B-lymphoblasts (MGL-8) was 4385-fold purified with a specific activity of 114 mumol/min/mg, while the enzyme from T-lymphoblasts (CEM, MOLT-4) was 4355-fold purified with a specific activity of 35 mumol/min/mg. The activity of both enzymes have an absolute requirement for Mg++. The B-cell enzyme has maximum activity with Mg2+ > Mn2+ > Co2+, while the T-cell enzyme had maximum activity with Co2+ > Mn2+ > Mg2+. The optimum activity was at pH 7.4-9.0 for the B-cell enzyme and pH 9.0 for the T-cell enzyme. Substrate specificity was the same for both enzymes with the following relative Vmax values: CMP > UMP > dUMP > dCMP > dAMP > IMP > GMP > dIMP > dGMP. The Km values for AMP and IMP were 12 and 25 microM for the B-cell enzyme, and 7.0 and 12 microM for the T-cell enzyme. ATP and ADP are competitive inhibitors of these enzymes with apparent Ki values of 100 and 20 microM for the B-cell enzyme, and 44 microM and 8 microM for the T-cell enzyme, respectively. The apparent molecular mass by gel filtration column chromatography is 145 kD for the B-cell enzyme and 72 kDa for the T-cell enzyme. The subunit molecular masses by Western blots are 69.2 kD for both enzymes. These properties suggest that the B-lymphoblast enzyme is identical or similar to the enzyme from human placenta. However, the T-cell enzyme has some different properties. We conclude that these differences plus a lower content of low Km 5'-nucleotidase in T-cells may account for the decreased ability of T-lymphoblasts to dephosphorylate nucleotides and may contribute to the selective cytotoxicity of deoxyribonucleosides for T-lymphoblasts as compared to B-lymphoblasts.

5'-Nucleotidase↗
PubMed1992

Famotidine, the new antiulcero-genic agent, a potent ligand for metal ions.

Potentiometric, polarographic, and spectroscopic results obtained for Cu2+ and Ni(2+)-famotidine systems clearly indicated that this anti-ulcerogenic drug is a very potent chelating agent able to coordinate cupric ion that was at pH below 2. This drug exhibits excellent histamine H2 receptor blocking effects and its effective coordination to metal ions may have significant biological implications. Famotidine is found to be a very effective ligand for Ni2+ ions also.

Anti-Ulcer Agents↗
PubMed1991

Regulatory properties of AMP deaminases from rat tissues.

1. Phosphocellulose column chromatography under double gradient conditions (phosphate and KCl) revealed two forms of AMP deaminase in rat heart and brain and a single form in the liver and skeletal muscle. 2. Kinetically all purified AMP deaminases were classified into two categories: those, which elute from the column at lower KCl and Pi concentrations, display low S0.5 value are only moderately affected by MgATP, MgGTP and Pi; and those which elute at higher KCl and Pi concentrations, display high S0.5 values and are strongly regulated by allosteric effectors. 3. Physiological significance of the occurrence of two kinetic forms of AMP deaminase in some tissues is discussed.

AMP Deaminase↗
PubMed1989

AMP and IMP dephosphorylation by soluble high- and low-Km 5'-nucleotidases.

Three distinct 5'-phosphomonoesterase activities were isolated from soluble fractions of human placenta, cultured human T and B lymphoblasts, and rat liver using 5'-AMP-sepharose 4B affinity chromatography. We define these activities as "low-Km" 5'-nucleotidase, "high-Km" 5'-nucleotidase, and nonspecific phosphatase. High-Km 5'-nucleotidase was eluted with 0.5 M NaCl, low-Km 5'-nucleotidase was eluted with 10 mM ADP, and nonspecific phosphatase was not retained on the column. We have found significant variability in the relative content of high- to low-Km activities in the tissues studied with the ratios ranging from 5.5 to 264. The properties were studied after further purification. The molecular mass of the low-Km enzymes ranged from 72.5 to 209 kDa, optimum pH ranged from 7.4 to 9.0, Km for AMP ranged from 7 to 15 microM, and Km for IMP ranged from 10 to 26 microM. The molecular mass of the high-Km enzymes ranged from 182 to 210 kDa, pH optimum was at 6.5, Km for AMP ranged from 3.0 to 9.4 mM, and the Km for IMP ranged from 0.3 to 0.5 mM. The data indicate that the soluble low- and high-Km 5'-nucleotidase coexist in the mammalian cells and tissues studied. These observations suggest a complex system for the regulation of nucleoside 5'-monophosphate dephosphorylation.

5'-Nucleotidase↗
PubMed1989

The application of affinity chromatography for the separation of "high Km" and "low Km" 5'-nucleotidase and other AMP metabolizing enzymes.

AMP-sepharose 4B has been widely used as a general ligand affinity chromatography for purification of AMP deaminase, 5'-nucleotidase, adenosine kinase and other adenine nucleotide metabolizing enzymes. Since these enzymes generally differ in their kinetic properties related to the values of Km for AMP and analogous compounds, it was assumed that there may be a specific elution pattern of some of the enzymes which would enable sequential elution from the column during a single run. Using 0.5 M NaCl, 10 mM ATP and 5 mM adenosine as eluting agents, it was possible to separate on AMP-sepharose column AMP deaminase "high Km" and "low Km" 5'-nucleotidase and adenosine kinase. Adenylate kinase, adenosine deaminase and nonspecific phosphatase did not bind to the column. Using human placental extract, AMP deaminase, "high Km" and "low Km" 5'-nucleotidase and adenosine kinase were purified 2.8, 2.9, 105 and 1240 fold, respectively. AMP deaminase and "high Km" 5'-nucleotidase were further separated using phosphocellulose column chromatography and the final purification was 227 and 143 fold, respectively. The specific activities of purified enzyme preparations were 9.1, 1.0, 0.4 and 0.5 mumols/min/mg protein of AMP deaminase, "high Km" 5'-nucleotidase and adenosine kinase, respectively. This approach provides a rapid method for initial purification of these enzymes from crude soluble extracts.

5'-Nucleotidase↗
PubMed1989

Evidence for "low Km" and "high Km" soluble 5'-nucleotidases in human tissues and rat liver.

Two kinetically distinct purine 5'-phosphomono-esterase activities were isolated from soluble fractions of human placenta, cultured human T- and B-lymphoblasts and rat liver using AMP-sepharose chromatography. We have defined these activities as "high Km" and "low Km" 5'-nucleotidase. The relative content of "high Km" and "low Km" activities in the tissues studied ranged from 2 to 264. The optimum pH of "low Km" 5'-nucleotidases ranged from 7.4 to 9.0, Km for AMP from 7 to 15 uM and for IMP from 10 to 26 uM. ATP and ADP were inhibitors of "low Km" enzymes with the apparent Ki values of 55 to 20 uM and 8 to 20 uM for ATP and ADP, respectively. "High Km" 5'-nucleotidases had an optimum pH at 6.5, Km for IMP of 0.3 to 0.5 mM and Km for AMP of 1.0 to 9.4 mM. "High Km" enzymes were activated by ATP with A0.5 values, of 1.7 to 2.3 mM at 100 microM IMP. The data indicate that soluble "low Km" and "high Km" 5'-nucleotidases coexist in mammalian cells and fulfill different functions. These observations suggest a complex system for the regulation of AMP and IMP dephosphorylation.

5'-Nucleotidase↗
PubMed1987

Developmental forms of human skeletal muscle AMP-deaminase.

Chromatography on phosphocellulose revealed the existence of two well-separable forms of skeletal muscle AMP-deaminase in the tissue extracts of 11- and 16-week-old human fetuses. One of these forms elutes from the column at the same salt concentration as the muscle isozyme found in the skeletal muscle extract from adult man, and seems to have similar kinetic properties. The second form, which was found only in vestigial amounts in adult human tissue extract, represents different kinetic properties and seems to be a form characteristic for the fetal period of ontogenesis.

AMP Deaminase↗
PubMed1987

The influence of subcutaneously administered lead(II) acetate on the concentrations of copper, iron, and zinc in the blood, kidney, liver, and spleen of rats.

The concentrations of copper, iron, lead, and zinc in the blood, kidney, liver and spleen were determined before and after subcutaneous administration of lead(II) acetate (100 mg Pb kg-1 body weight) to male albino Wistar rats. The control rats had the following concentrations (microgram g-1 dry weight) of Cu, Fe, Pb and Zn: blood, 4.5, 3200, 2.1, 182; kidney, 36, 585, 120, 92; liver, 11.1, 720, 14.3, 124; spleen, 4.5, 420, 7.0, 76. After administration of lead, rats were sacrificed after 12, 24, 48, 72 and 96 h. The Pb concentration in the blood remained constant for the first 24 h at the level of the control group (2.1 micrograms g-1) and had decreased to half that level at 96 h. The lead concentrations peaked in the organs at 110-142% of those in the control group and had decreased at 96 h to levels considerably below those in the control group. The concentrations of Cu, Fe and Zn increased in the three organs to values 117-161% found for the control group. At 96 h the concentrations of Cu, Fe and Zn in the spleen had returned to levels of the control group; the concentrations in the liver were 112-153%, and in the blood 89-93% of those of the control group. In the kidney the iron (110%) and the zinc (126%) concentrations at 96 h were higher than the control values, whereas the copper concentration was the same as the control value. The concentrations in the blood were least affected. The most drastic changes were observed in the liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

High substrate affinity and GTP insensitive AMP deaminase from frog liver.

1. Relatively high activity of AMP deaminase (2.5 mumol/min/g wet wt of tissue) estimated at 70 microM AMP concentration was found in frog liver. 2. The enzyme was purified to homogeneity, its subunit and native protein molecular weights were about 70,000 and 330,000 respectively. The specific activity of the purified enzyme was 44 mumol/min/mg of protein at 70 microM AMP and Km for AMP was 0.7 mM. 3. The enzyme was rather specific for AMP, several structural analogues of 5'AMP were deaminated at the rate not exceeding 5% of the rate of AMP deamination. 4. Frog liver AMP deaminase was activated by monovalent cations, highest rate of reaction was observed in the presence of potassium, rhubidium and sodium ions. The enzyme was activated by ADP and ATP and inhibited by inorganic phosphate but was not influenced by GTP.

AMP Deaminase↗
PubMed1979

Comparative studies on muscle AMP-deaminase--II. Regulation by monovalent cations, ATP and orthophosphate of the enzyme from hen, frog and pikeperch muscle.

1. Michaelis constants, maximum velocity and pH-dependence of the reaction catalysed by homogeneous AMP-deaminase preparations from hen, frog and pikeperch skeletal muscle were compared, as well as the influence of monovalent cations, ATP and inorganic phosphate. 2. ATP was found to activate the enzymes in the absence of K+ and at optimum (150 mM) KCl concentration. 3. Absolute dependence on potassium ions and considerable dependence of Km and Vmax on the kind of monovalent cation present in the medium were found for pikeperch enzyme.

AMP Deaminase↗
PubMedDate not supplied

Tumor-promoting functions of adenosine.

Tumor growth is a multifactorial process that, in addition to mutations leading to dysregulated expression of oncogenes and tumor suppressive genes, requires specific conditions that provide a supportive physiological environment at the primary and metastatic sites of the disease. Adenosine is one of the factors potentially contributing to tumor growth that thus far has not received adequate attention, despite evidence for a broad range of cytoprotective, growth-promoting, and immunosuppressive activities. Adenosine accumulates in solid tumors at high concentrations, and has been shown to stimulate tumor growth and angiogenesis and to inhibit cytokine synthesis, adhesion of immune cells to the endothelial wall, and the function of T-cells, macrophages, and natural killer cells. However, the mechanisms whereby adenosine accumulates in cancer and the specific effects that result from this accumulation are not well understood. This article surveys the available evidence that supports an important role of adenosine in cancer.

5'-Nucleotidase↗