ABSTRACT
Cytoplasmic protein kinase C (PKC) has been studied in phytohemagglutinin (PHA) activated peripheral blood mononuclear cells (PBMC) and macrophage depleted E+ cell culture. Within 10' after contemporanous addition of PHA and anti HLA class I monoclonal antibody 01.65 (MoAb) PKC is depleted in both cell types. Enzyme activity recovers in the following hours however at 72 hours is at control values in E+ cultures while in PBMC cultures it is still depleted at 68% of the control. Anti HLA class I MoAb induced tritiated lymidine (3H-TdR) incorporation inhibition appears to be related to low levels of PKC activity.
Subject(s)
Antibodies, Monoclonal , Histocompatibility Antigens Class I/immunology , Lymphocyte Activation , Protein Kinase C/metabolism , T-Lymphocytes/enzymology , Cells, Cultured , DNA Replication , Humans , Kinetics , Protein Kinase C/immunology , Rosette Formation , T-Lymphocytes/cytology , T-Lymphocytes/immunologyABSTRACT
Anti HLA Class I Monoclonal Antibody depletes Protein Kinase C (PKC) to 20% of control value in PHA activated human T cells. The effect is reversible: in 24 hours the enzymatic activity returns to 58% of control value. Removal of antibody from the culture medium increases the rate of recovery. Implications of this finding for the modulation by HLA Class I antigens of the proliferative response of T cells to lectins are discussed.
Subject(s)
Antibodies, Monoclonal , HLA Antigens/immunology , Lymphocyte Activation , Phytohemagglutinins/pharmacology , Protein Kinase C/metabolism , T-Lymphocytes/enzymology , Adult , Cells, Cultured , Humans , T-Lymphocytes/drug effectsABSTRACT
Erythrocyte transglutaminase was purified by anion-exchange chromatography, size exclusion and affinity chromatography. Homogeneity was achieved by an additional step of HPLC size-exclusion chromatography. The molecular mass of the purified enzyme was calculated to be 65,000 Da by size-exclusion chromatography and sucrose-gradient centrifugation, and 92,000 Da by SDS-PAGE, thus suggesting a high degree of asymmetry. The amino-acid composition of erythrocyte transglutaminase differed substantially from that of the guinea-pig liver enzyme, notably with respect to the number of histidine, cysteine and acidic amino-acid residues. The enzyme has an absolute requirement for divalent cations for activity: calcium, manganese, and the lanthanides terbium and gadolinium activate the enzyme in decreasing order of efficacy, while no activity is displayed in the presence of magnesium. In the presence but not in the absence of calcium ions, the enzyme is rapidly inactivated by N-ethylmaleimide and by diethylpyrocarbonate suggesting that the cation influences the reactivity of amino acids essential for catalysis. When erythrocyte proteins are employed as amine acceptors in the presence of calcium, the erythrocyte transglutaminase appears to preferentially modify membrane-associated proteins, although, in the absence of calcium ions and exogenous amines, it displays a pH-dependent interaction with soluble proteins.
Subject(s)
Erythrocytes/enzymology , Transglutaminases/blood , Amino Acids/analysis , Cations, Divalent , Chromatography, Affinity , Chromatography, Gel , Chromatography, Ion Exchange , Enzyme Activation , Humans , Kinetics , Molecular Weight , Transglutaminases/isolation & purificationABSTRACT
The guanine nucleotides GTP, GDP and GMP inhibit the activity of erythrocyte transglutaminase (protein-glutamine:amine gamma-glutamyltransferase, EC 2.3.2.13) in a decreasing order of effectiveness. The inhibition is more apparent at low than at saturating levels of calcium ions and is not due to the chelation of Ca2+, but to an interference with the process of activation by the cation. This inhibition is likely to contribute to the latency of erythrocyte transglutaminase in physiological conditions.
Subject(s)
Erythrocytes/enzymology , Guanosine Triphosphate/pharmacology , Transglutaminases/antagonists & inhibitors , Erythrocytes/drug effects , Guanosine Diphosphate/pharmacology , Guanosine Monophosphate/pharmacology , HumansABSTRACT
Incubation of skeletal muscle glycogen phosphorylase with sodium periodate's results into irreversible loss of enzyme activity. The rate of inactivation is influenced by the ionic strength of the medium and by the presence of caffeine, but not by nucleotides. During the reaction, cysteine residues slowly reactive towards DTNB are modified and the coenzyme is released. These results suggest the presence of cysteine residues at the protein site involved in the binding of the phosphate group of pyridoxal phosphate.
