scholarly journals Multiple protein kinases from human lymphocytes. Identification enzymes phosphorylating exogenous histon and casein

1975 ◽  
Vol 145 (2) ◽  
pp. 241-249 ◽  
Author(s):  
B E Kemp ◽  
M Froscio ◽  
A Rogers ◽  
A W Murray
Keyword(s):  
Molecular Weight ◽  
Cyclic Amp ◽  
Kinase Inhibitor ◽  
Muscle Protein ◽  
Human Peripheral Blood ◽  
Human Lymphocytes ◽  
Casein Kinase ◽  
Rabbit Muscle ◽  
Sephadex Column ◽  
Histone Kinase

1. Cell-free lysates of human peripheral blood lymphocytes contained two casein kinase activities and two histone kinase activities, which could be separated by chromatography on DEAE-Sephadex. 2. Neither of the casein kinase activities were stimulated by cyclic AMP. The major activity was eluted from DEAE-Sephadex between 0.4 and 0.45M-KCl, had a molecular weight of approx. 130,000 (sucrose density gradients) and was stimulated by KCl (maximum 150mM). It also formed higher-molecular-weight aggregates when centrifuged in sucrose gradients containing 150mM-KCl. The minor activity was not retained by DEAE-Sephadex, had a molecular weight of approx. 50,000 and was not stimulated by KCl. 3. The major histone kinase activity was stimulated by cyclic AMP and was eluted from the DEAE-Sephadex column between 0.05 and 0.2M-KCl. The other activity was not stimulated by cyclic AMP and was insensitive to the rabbit muscle protein kinase inhibitor. 4. Evidence was obtained suggesting that the lymphocyte casein kinases were located primarily in the nuclei.

scholarly journals Histone phosphatase and cyclic nucleotide-stimulated protein kinase from human lymphocytes

1972 ◽  
Vol 129 (5) ◽  
pp. 995-1002 ◽  
Author(s):  
A. W. Murray ◽  
M. Froscio ◽  
B. E. Kemp
Keyword(s):  
Molecular Weight ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Human Peripheral Blood ◽  
Human Lymphocytes ◽  
Enzymic Activity ◽  
Lower Molecular Weight ◽  
Protein Kinase Activity ◽  
Cyclic Monophosphate ◽  
Lower Molecular Weight Species

1. Extracts of human peripheral blood lymphocytes contained a histone phosphatase that catalysed the release of Pi from phosphorylated whole thymus histone. 2. Stimulation of the phosphatase was obtained by concentrations of KCl and NaCl of up to 75mm, and by MgCl2; CaCl2 inhibited the enzymic activity. 3. In the absence of MgCl2, phosphoenol-pyruvate inhibited histone phosphatase activity; this inhibition could be partially reversed by adding MgCl2 to assays. 4. Lymphocyte extracts contained a protein kinase activity which was maximally stimulated by 1μm-cyclic AMP (adenosine 3′:5′-cyclic monophosphate) or by 0.1mm-cyclic GMP (guanosine 3′:5′-cyclic monophosphate). 5. Incubation of the enzyme with histone in the absence of ATP or MgCl2 resulted in the dissociation of the enzyme into a lower-molecular-weight species that was not stimulated by cyclic AMP. This effect could be prevented if ATP and MgCl2 were present in reaction mixtures before histone and enzyme were allowed to interact. 6. Cyclic AMP also dissociated the kinase into a lower-molecular-weight species. 7. In the presence of 1μm-AMP, half-maximal activities were obtained with 0.92mm-MgCl2, 6.0μm-ATP and 0.23mg of whole thymus histone/ml.

scholarly journals Cyclic AMP-independent casein/glycogen synthase kinases from pig polymorphonuclear leucocytes

1981 ◽  
Vol 193 (3) ◽  
pp. 829-837 ◽  
Author(s):  
J. Manuel Pena ◽  
Roser Cussó ◽  
Emilio Itarte
Keyword(s):  
Cyclic Amp ◽  
Kinase Inhibitor ◽  
Glycogen Synthase ◽  
Gel Filtration ◽  
Casein Kinase ◽  
Casein Kinase 2 ◽  
Polymorphonuclear Leucocytes ◽  
Rabbit Muscle ◽  
Casein Kinase 1 ◽  
Glycogen Synthase Kinases

1. Two cyclic AMP-independent casein/glycogen synthase kinases were purified from pig polymorphonuclear leucocytes by chromatography on phosphocellulose followed by affinity chromatography on casein–Sepharose 4B or gel filtration on Bio-Gel A-1.5m. When the affinity step was used, the specific activities were 86 and 43units/mg of protein for casein kinase 1 and 2, respectively, whereas these values were 94 and 90units/mg of protein when the gel-filtration step was used. 2. These kinases differ as follows: (a) the molecular weight of casein kinase 1 (38000) is very much lower than that of casein kinase 2 (185000); (b) the Km for casein (0.46mg/ml) and Ka for Mg2+ (0.3mm) of casein kinase 1 are lower than those of casein kinase 2 (0.90mg/ml and 1.7mm respectively); (c) KCl stimulates the phosphorylation of casein by casein kinase 1, whereas it inhibits phosvitin phosphorylation by this enzyme; on the contrary, the effect of KCl on casein kinase 2 is very similar with either casein or phosvitin as substrate; (d) although both kinases phosphorylate rabbit muscle glycogen synthase I, the ratio of glycogen synthase to casein phosphorylation by casein kinase 1 is about 4-fold greater than that by casein kinase 2. Furthermore, 32P incorporation into glycogen synthase promoted by casein kinase 1 (3.6mol of 32P/mol of 85000-dalton subunit) is twice that observed with casein kinase 2 (1.8mol of 32P/mol of 85000-dalton subunit). Such a phosphorylation results in a decrease in the glucose 6-phosphate-independence ratio of glycogen synthase to 10–15 with casein kinase 1 and to 35–45 with casein kinase 2. 3. The activity of both kinases is neither stimulated by cyclic AMP, Ca2+ and calmodulin nor inhibited by cyclic AMP-dependent protein kinase inhibitor protein. 4. No phosphorylation kinase activity was observed with casein kinase 1 and 2 at either pH6.8 or 8.2 in the presence of Ca2+. 5. Activities of both kinases on casein and glycogen synthase decreased in parallel when incubated at 50°C.

scholarly journals Purification and partial characterization of a high hemagglutinating chitin-binding lectin from Aponogeton natans tubers

2021 ◽  
Author(s):  
Shashikanth Dara ◽  
Harikrishna Naik Lavudi ◽  
Venkateswara Rao ◽  
Nanibabu Badithi ◽  
Seshagirirao Kottapalli
Keyword(s):  
Molecular Weight ◽  
Cell Lines ◽  
Cytotoxic Effect ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Human Lymphocytes ◽  
Single Step ◽  
List Type ◽  
Peripheral Blood Mononuclear ◽  
Binding Lectin

AbstractA novel chitin-binding lectin was isolated from the tubers of a plant Aponogeton natans from the monocot family Aponogetonaceae, designated as ANTL (Aponogeton natans tuber lectin). The lectin agglutinated both untreated and trypsin-treated rabbit erythrocytes, as well as human blood cells of groups A, B and O with different specificities. Lectin activity is inhibited by the oligomers of N-acetylglucosamine. ANTL is a dimeric glycoprotein with molecular weight of ∼66 KDa and has two identical sub-units of 33 KDa. The carbohydrate percent is 8.2% of the total lectin. The lectin was thermo stable up to 50°C with broad pH optima (pH 4–10). ANTL is found to be potent mitogen for normal murine and human lymphocytes at the concentration as low as 1 µg/ml. Cytotoxic studies of the lectin on human U 266 cell lines has revealed that there is 50% decrease in the proliferation. The confirmation of both the hemagglutination and mitogenic proliferation activity suggests that ANTL is a Chitin-binding lectin with diverse functions. The pharmacological relevance of ANTL as a potent mitogen with some cytotoxic effect in certain cell lines are reported for the first time.HighlightsA novel chitin-binding lectin was purified from the tuber extracts of Aponogeton natans (ANTL) in a single step on chitin column by affinity chromatography.ANTL is a dimeric glycoprotein with a molecular weight of ∼66 KDa with high hemagglutination activity towards rabbit erythrocytes.The cytotoxic effect of ATNL on human cell lines U266 has shown 50 % inhibition of their proliferation.ANTL displayed potent mitogenic response towards murine and human peripheral blood mononuclear cells.

Rat Liver Nuclear Protein Kinases

1975 ◽  
Vol 53 (3) ◽  
pp. 354-363 ◽  
Author(s):  
P. R. Desjardins ◽  
C. C. Liew ◽  
A. G. Gornall
Keyword(s):  
Ionic Strength ◽  
Cyclic Amp ◽  
Dehydrogenase Activity ◽  
Kinase Activity ◽  
Nuclear Material ◽  
Casein Kinase ◽  
Casein Kinases ◽  
Liver Nuclei ◽  
Histone Kinase ◽  
Low Ionic Strength

We have shown that nuclei isolated by two methods contain grossly different amounts of cyclic AMP-dependent histone kinase activity. Repeated washing of the isolated nuclei with a low ionic strength buffer removed the majority of the cyclic AMP-dependent histone kinase and cyclic AMP binding activity. Nuclear cyclic AMP-dependent histone kinase activity accounted for only 0.42% of the total cytoplasmic enzyme activity. Similarly, the lactate dehydrogenase activity associated with liver nuclei represented only 0.07% of the total cytoplasmic activity. The isolated liver nuclei contained only 0.27% of the total homogenate glutamate dehydrogenase activity and 1.7% of the total homogenate glucose-6-phosphatase activity. The cyclic AMP-dependent histone kinase behaves as a cytoplasmic rather than a nuclear enzyme. We have also shown that using crude extracts, one can achieve separation of the two nuclear casein kinases, NI and NII, on sucrose density gradients in the presence of 0.5 M NaCl. Nuclear casein kinases NI and NII had sedimentation coefficients of 3.0 and 5.3 S, respectively, in the presence of 0.5 M NaCl. Under conditions of low ionic strength, all of the casein kinase activity in the crude nuclear extract sedimented as one peak with a sedimentation coefficient of 7.3 S. The aggregation–disaggregation which occurred in the crude extract was reversible and was mainly due to the aggregative and disaggregative properties of casein kinase NII The two nuclear casein kinases have different affinities for chromatin. When nuclei were disrupted in a hypotonic solution and extracted with a buffer containing 0.14 M NaCl, casein kinase NII could be completely extracted from the viscous nuclear material. Although a significant amount of casein kinase NI was extracted by the buffer containing 0.14 M NaCl, re-extraction of the nuclear material with a buffer containing 0.5 M NaCl yielded substantial amounts of casein kinase NI, and a final extraction with a buffer containing 1.0 M NaCl yielded measurable amounts of casein kinase NI. No casein kinase NII activity could be detected in the 0.5 M and 1.0 M NaCl extracts.

P1, P5-Bis-(5′-adenosyl)pentaphosphate: Is this Adenylate Kinase Inhibitor Substrate for Mitochondrial Processes?

1977 ◽  
Vol 32 (9-10) ◽  
pp. 786-791 ◽  
Author(s):  
Josef Köhrle ◽  
Joachim Lüstorff ◽  
Eckhard Schlimme
Keyword(s):  
Adenylate Kinase ◽  
Kinase Inhibitor ◽  
Adenine Nucleotide ◽  
Nucleoside Diphosphate Kinase ◽  
Liver Mitochondria ◽  
Inhibitory Effect ◽  
Rat Liver Mitochondria ◽  
Intermembrane Space ◽  
Rabbit Muscle ◽  
Inner Mitochondrial Membrane

Abstract 1. P1, P5-Bis-(5′-adenosyl)pentaphosphate (Ap5A) inhibits “soluble” adenylate kinase even when this enzyme is an integral part of the complete mitochondrion. The Ki is 10-5м , i. e. about two orders of magnitude higher than the inhibitor constants determined for the purified adenylate kinase of rabbit muscle and an enzyme preparation separated from the mitochondrial intermembrane space. The weaker inhibitory effect is due to a lower accessibility of the enzyme.2. As to be expected Ap5A which is of the “multisubstrate analogue”-type does not affect mito­ chondrial nucleoside diphosphate kinase.3. Though Ap5A owns the structural elements of both ATP and ADP it is not a substrate of the adenine nucleotide carrier, i.e. neither it is exchanged across the inner mitochondrial membrane nor specifically bound.4. Ap5A is not metabolized by rat liver mitochondria.

scholarly journals Investigation of the Immune Modulatory Potential of Zinc Oxide Nanoparticles in Human Lymphocytes

Nanomaterials ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 629
Author(s):  
Helena Moratin ◽  
Pascal Ickrath ◽  
Agmal Scherzad ◽  
Till Jasper Meyer ◽  
Sebastian Naczenski ◽  
...  
Keyword(s):  
Zinc Oxide ◽  
Zinc Oxide Nanoparticles ◽  
Immune Modulation ◽  
Human Peripheral Blood ◽  
Human Lymphocytes ◽  
Drug Carriers ◽  
Physiological Model ◽  
Peripheral Blood Lymphocytes ◽  
Major Influence ◽  
Oxide Nanoparticles

Zinc oxide nanoparticles (ZnO-NP) are commonly used for a variety of applications in everyday life. In addition, due to its versatility, nanotechnology supports promising approaches in the medical sector. NP can act as drug-carriers in the context of targeted chemo- or immunotherapy, and might also exhibit autonomous immune-modulatory characteristics. Knowledge of potential immunosuppressive or stimulating effects of NP is indispensable for the safety of consumers as well as patients. In this study, primary human peripheral blood lymphocytes of 9 donors were treated with different sub-cytotoxic concentrations of ZnO-NP for the duration of 1, 2, or 3 days. Flow cytometry was performed to investigate changes in the activation profile and the proportion of T cell subpopulations. ZnO-NP applied in this study did not induce any significant alterations in the examined markers, indicating their lack of impairment in terms of immune modulation. However, physicochemical characteristics exert a major influence on NP-associated bioactivity. To allow a precise simulation of the complex molecular processes of immune modulation, a physiological model including the different components of an immune response is needed.

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