ABSTRACT
The uptake of radiocopper by HepG2 cells is a saturable, temperature-dependent and cellular energy-independent process with a Vmax of 7.1 +/- 0.2 pmoles min-1 mg protein-1 and an estimated Km of 3.3 +/- 0.5 microM. The rate of copper uptake is reduced at an equimolar concentration of albumin and is unaffected by zinc at a 10-fold molar excess. Approximately 70% of the newly incorporated radiocopper binds to membranes and organelles, while 30% is recovered in the cytosol. The soluble fraction can be resolved into two copper-binding protein peaks. Incubation of HepG2 with nonisotopic copper results in displacement of radiocopper associated with the proteins contained in the lower molecular weight peak. Exposure of the cells to cycloheximide inhibits the incorporation of the isotope into this fraction.
Subject(s)
Carcinoma, Hepatocellular/metabolism , Copper/metabolism , Biological Transport , Cell Line , Humans , Liver Neoplasms , Radioisotopes , Serum Albumin, Bovine , Subcellular Fractions/metabolism , Temperature , Time FactorsABSTRACT
We studied the amino acid sequence of canine hepatic lysosomal copper protein obtained from Bedlington terriers affected by inherited copper toxicosis. The primary structure was determined by manual Edman degradations and carboxypeptidase Y digestions of peptides generated by cleavage of the S-carboxyamidomethylated and S-aminoethylated protein with trypsin. Although the amino terminus was blocked and heterogeneous, the protein showed extensive sequence homology to mammalian metallothioneins. In particular, all cysteinyl residues were conserved, in agreement with their function as metal ligands. The microheterogeneity observed in the amino-terminal part of the molecule indicated the presence of two isoforms in canine liver like those found in most other mammals studied so far.
Subject(s)
Liver/ultrastructure , Lysosomes/analysis , Metallothionein/analysis , Amino Acid Sequence , Animals , Chromatography, High Pressure Liquid , Copper/poisoning , Dog Diseases/genetics , Dog Diseases/metabolism , Dogs , Horses , Humans , Mice , Peptide Fragments/analysis , Trypsin/metabolismABSTRACT
Sections of paraffin-embedded specimens of liver obtained from Bedlington terriers were stained with rhodanine for copper and examined and graded by two pathologists. Their results correlated well with each other, as well as with the results of quantitative determinations of hepatic copper content. Copper toxicosis was established by cytochemistry in 20 of 21 specimens, indicating a sensitivity of 95%. The method is highly specific--none of the 19 specimens obtained from unaffected dogs displayed copper-containing granules suggestive of copper toxicosis. Copper cytochemistry appears to be a satisfactory substitute for chemical analysis of hepatic copper content.
Subject(s)
Copper/metabolism , Dog Diseases/diagnosis , Metal Metabolism, Inborn Errors/veterinary , Age Factors , Animals , Cytoplasmic Granules/analysis , Dog Diseases/genetics , Dog Diseases/metabolism , Dogs , Female , Histocytochemistry , Liver/metabolism , Liver/pathology , Liver/ultrastructure , Male , Metal Metabolism, Inborn Errors/diagnosis , Metal Metabolism, Inborn Errors/genetics , Metal Metabolism, Inborn Errors/metabolism , RhodanineABSTRACT
The mode of transmission of copper toxicosis, previously reported to be associated with progressive hepatic disease in certain Bedlington Terriers, has been studied by means of 5 matings involving affected and unaffected Bedlington Terriers and dogs of different breeds. The abnormally large concentration of hepatic copper underlying the disorder was found to follow an autosomal recessive pattern of inheritance.
Subject(s)
Copper/poisoning , Dog Diseases/genetics , Animals , Dogs , Female , Genes, Recessive , Male , PedigreeABSTRACT
Highly purified streptococcal nicotinamide adenine dinucleotide glycohydrolase (NADase) was obtained by utilizing disodium tetrathionate to protect the enzyme by blocking the sulfhydryl groups of streptococcal proteinase. This was followed by two-step ion-exchange chromatography. The pure enzyme, demonstrated as a single band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, had a specific activity of 11,200 NADase units per mg of protein and was devoid of hemolytic activity. NADase had a molecular weight of about 55,000 as determined by gel filtration, by summation of amino acid residues, and by sodium dodecyl sulfate/gel electrophoresis. The purified enzyme had optimal activity at pH 7.3 and at 40 C and a calculated Km of 5.1 times 10- minus 4 mM. It was inhibited by alpha-iodoacetamide.
Subject(s)
N-Glycosyl Hydrolases , NAD+ Nucleosidase , Streptococcus pyogenes/enzymology , Amino Acids/analysis , Ammonium Sulfate , Chemical Precipitation , Chromatography, DEAE-Cellulose , Chromatography, Gel , Electrophoresis, Polyacrylamide Gel , Enzyme Inhibitors , Hydrogen-Ion Concentration , Iodoacetamide/pharmacology , Molecular Weight , NAD+ Nucleosidase/isolation & purification , NAD+ Nucleosidase/metabolism , Peptide Hydrolases/metabolism , Streptolysins/metabolism , Temperature , Tetrathionic AcidSubject(s)
Bacterial Proteins/metabolism , Cholesterol/blood , Erythrocytes/metabolism , Hemolysin Proteins/metabolism , Receptors, Drug/drug effects , Saponins/metabolism , Streptolysins/metabolism , Animals , Bacillus cereus , Binding Sites , Cell Membrane/drug effects , Cell Membrane/metabolism , Chromatography, Ion Exchange , Erythrocytes/cytology , Erythrocytes/drug effects , Hemolysis , Microscopy, Electron , Plants , Sulfhydryl Compounds , Sulfhydryl Reagents/pharmacologyABSTRACT
Streptococcal nicotinamide adenine dinucleotide glycohydrolase (NADase) with a molecular weight of about 55,000 and an isoelectric pH of 8.55 was isolated from crude streptolysin O (SLO) preparations. NADase differed from SLO in size, charge, and immunological behavior. Streptococcal NADase is considered to have no role in the hemolytic process because it has no hemolytic activity; conversely, partially purified SLO showed no NADase activity. The hemolytic activity of crude SLO was completely inhibited by anti-tetanolysin, whereas the NADase activity in the same reaction mixture was unaffected. Experiments involving double diffusion in agar also demonstrated immunological nonidentity of the two proteins.
