ABSTRACT
Affinity purification of annexin V from human placenta on column with appropriate monospecific antibodies is developed. The procedure permits purification of the protein to a highly purified state by a two stage procedure. The yield of the protein is about 5 mg per 100 g of wet tissue. Because of high homologies between various annexins, it was supposed that this procedure can be also applied for purification of other annexins from other tissues.
Subject(s)
Annexin A5/isolation & purification , Chromatography, Affinity/methods , Placenta/metabolism , HumansABSTRACT
Lectin binding assay was applied to polyacrylamide slices to study the effects of lysozyme on carbohydrate profile of plasma membranes from liver and heart tissues. The investigations were carried out on polyacrylamide slices immobilized with wheat germ agglutinin or Concanavalin A. It was shown that lysozyme changes the carbohydrate profile of plasma membranes of liver and heart cells.
Subject(s)
Cell Membrane/drug effects , Heart/drug effects , Hepatocytes/drug effects , Lectins/metabolism , Muramidase/pharmacology , Acrylic Resins/chemistry , Animals , Carbohydrates/analysis , Cell Membrane/chemistry , Cell Survival/drug effects , Clinical Laboratory Techniques , Concanavalin A/chemistry , Concanavalin A/metabolism , Hepatocytes/ultrastructure , Myocardium/cytology , Myocardium/ultrastructure , Protein Binding , Rats , Tissue Extracts/chemistry , Wheat Germ Agglutinins/chemistryABSTRACT
A simplified procedure for purification of annexin V from human placenta was developed. At first, the protein was separated from other proteins in membrane bound form in the presence of Ca2+, then was extracted with EDTA and purified by affinity chromatography on PAAG-immobilized phosphatidylserine. The purified protein gave a single band with a molecular weight of 35,000 in SDS-PAGE.
Subject(s)
Annexin A5/isolation & purification , Placenta/chemistry , Annexin A5/chemistry , Chromatography, Affinity , Female , Humans , PregnancyABSTRACT
Cu-thioneins isolated from liver and brains are able to transfer their copper atoms to apoforms of neurocuprein 1 and 2. At the same time, the apoform of Cu-thionein is unable to accept copper from holoforms of both neurocupreins.
Subject(s)
Copper/metabolism , Metalloproteins/metabolism , Metallothionein/metabolism , Animals , Brain/metabolism , Cattle , Chromatography, Gel , Electron Spin Resonance Spectroscopy , In Vitro Techniques , Liver/metabolism , RabbitsABSTRACT
Secretory granules obtained from bovine pituitary, atrium and adrenal medulla contain an extremely acidic copper protein resembling by its main physico-chemical and antigenic properties as well as by the ability of its apoform to inhibit dopamine beta-monooxygenase the protein from brain, neurocuprein.
Subject(s)
Cytoplasmic Granules/ultrastructure , Metalloproteins/analysis , Adrenal Medulla/ultrastructure , Animals , Cattle , Chemical Phenomena , Chemistry, Physical , Dopamine beta-Hydroxylase/antagonists & inhibitors , Heart Atria/ultrastructure , Pituitary Gland/ultrastructureABSTRACT
A novel copper-containing protein has been isolated in electrophoretically homogeneous state from bovine brain extracts. Some physico-chemical properties of the protein are determined (molecular weight, copper content, isoelectric point, amino acid composition, optical and EPR spectra), and they are compared with those of neurocuprein, an extremely acidic copper protein isolated earlier from brain. A number of differences between neurocuprein and the novel protein are clearly shown, although certain similarities are also detected.
ABSTRACT
Extremely acidic copper-containing proteins, neurocupreins, were isolated from brains of various mammals (bovine, rabbit, pig and sheep). Neurocupreins from all these sources were found to have similar physico-chemical and antigenic properties. Using the immunological approach, it was shown that neurocuprein is located only in brain cytosol and synaptosomal fractions. Extremely acidic copper-containing proteins were also isolated from soluble and membranous fractions of chromaffin granules from bovine adrenal medulla. The soluble form of the protein from the granules has practically the same physico-chemical and antigenic properties as neurocupreins. The copper protein isolated from membranes of granules has slightly higher molecular weight and somewhat different amino acid composition, although their EPR spectra are identical. However, both copper proteins from chromaffin granules are immunoprecipitated with antibodies to neurocuprein. It is suggested that the membranous form differs from the soluble one in possessing a peptide which prolongs the protein chain without changes in its antigenic properties.
Subject(s)
Antigens/isolation & purification , Brain Chemistry , Chromaffin Granules/analysis , Chromaffin System/analysis , Metalloproteins/isolation & purification , Animals , Cattle , Chemical Phenomena , Chemistry, Physical , Chromatography, Gel , Electron Spin Resonance Spectroscopy , Immunochemistry , Membrane Proteins/isolation & purification , Rabbits , Sheep , Subcellular Fractions/analysis , SwineABSTRACT
The apoform of neurocuprein, the copper protein from brain and chromaffin granules, was found to be a potent inhibitor of the hydroxylating activity of dopamine beta-monooxygenase, whereas the holoform of neurocuprein has no effect on the activity of the enzyme. The inhibiting capacity of neurocuprein may be due to the property of the apoprotein to chelate copper from the enzyme. A role of neurocuprein as an endogenous protein regulator of dopamine beta-monooxygenase is suggested.
Subject(s)
Apoproteins/pharmacology , Dopamine beta-Hydroxylase/antagonists & inhibitors , Metalloproteins/pharmacology , Animals , Brain Chemistry , Catechols/metabolism , Cattle , Chromaffin Granules/analysis , Copper/metabolism , Dose-Response Relationship, Drug , Oxygen Consumption/drug effects , Structure-Activity Relationship , Tyramine/metabolismABSTRACT
The copper-containing protein, neurocuprein, has been obtained in a highly purified state from human brain. Many similarities in properties of neurocupreins from human and bovine brains are noted. These concern molecular weights, copper contents, isoelectric points, amino acid compositions, optical and EPR spectra as well as their reaction with catecholamines. However, it was found that the content of neurocuprein in the whole human brain is significantly (by a factor 2.5-3.0) higher than in bovine brain.
