ABSTRACT
Dipeptidyl peptidase II (DPPII) from bovine kidney cortex and lung was purified to the electrophoretically homogeneous state. The molecular and catalytic characteristics of the enzyme were determined. It was revealed that DPPII preparations possess adenosine deaminase (ADA) activity at all purification steps. For the first time, the ADA-binding ability of DPPII has been shown similar to the well-known ADA-binding enzyme, DPPIV. The dissociation constant of the DPPII-ADA complex was estimated using a resonant mirror biosensor (80 nM), fluorescence polarization (60 nM), and differential spectroscopy (36 nM) techniques. The data demonstrate that DPPII can form a complex with ADA, but with one order of magnitude higher dissociation constant than that of DPPIV (7.8 nM).
Subject(s)
Adenosine Deaminase/metabolism , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/metabolism , Multiprotein Complexes/metabolism , Adenosine Deaminase/isolation & purification , Animals , Cattle , Dipeptidyl Peptidase 4/metabolism , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/isolation & purification , Humans , Kidney Cortex/enzymology , Lung/enzymology , Protein BindingABSTRACT
The interaction of adenosine deaminase (adenosine aminohydrolase, ADA) from bovine spleen with inhibitors--erythro-9-(2-hydroxy-3-nonyl)adenine, erythro-9-(2-hydroxy-3-nonyl)-3-deazaadenine, and 1-deazaadenosine--was investigated. Using selective chemical modification by diethyl pyrocarbonate (DEP), the possible involvement of His residues in this interaction was studied. The graphical method of Tsou indicates that of six His residues modified in the presence of DEP, only one is essential for ADA activity. Inactivation of the enzyme, though with low rate, in complex with any of the inhibitors suggests that the adenine moiety of the inhibitors (and consequently, of the substrate) does not bind with the essential His to prevent its modification. The absence of noticeable changes in the dissociation constants of any of the enzyme-inhibitor complexes for the DEP-modified and control enzyme indicates that at least the most available His residues modified in our experiments do not participate in binding the inhibitors--derivatives of adenosine or erythro-9-(2-hydroxy-3-nonyl)adenine.
Subject(s)
Adenosine Deaminase Inhibitors , Adenosine Deaminase/metabolism , Diethyl Pyrocarbonate/chemistry , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Adenosine Deaminase/chemistry , Animals , Binding Sites , Cattle , Histidine/metabolism , Hydrogen-Ion Concentration , Kinetics , Spleen/enzymology , Substrate SpecificityABSTRACT
Antibodies to the soluble form of the copper-containing enzyme, peptidyl-glycine-alpha-amidating monooxygenase isolated from secretory granules of bovine pituitary anterior lobes were found to belong to immunoglobulin G1. The antibodies were used to study the subcellular distribution of the enzyme in this tissue, and positive tests were found only for granular and cytosol fractions. The antibodies do not crossreact with other copper-containing systems of secretory granules, such as neurocuprein and dopamine-beta-monooxygenase. It was shown that the antibodies give the crossreaction with the enzyme isolated from secretory granules of bovine pituitary anterior lobes, cardiac atria, pancreas and adrenal medulla, indicating the antigenic identity of the enzyme from secretory granules of different glands.
Subject(s)
Antibodies/immunology , Mixed Function Oxygenases/immunology , Multienzyme Complexes , Adrenal Medulla/enzymology , Animals , Cattle , Cross Reactions , Cytoplasmic Granules/enzymology , Heart Atria/enzymology , Immunodiffusion , Pancreas/enzymology , Pituitary Gland, Anterior/enzymologyABSTRACT
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
The procedure for the isolation of two water soluble copper-containing proteins from the white and gray matter of bovine brain is described. One of the proteins, cerebrocuprein I, is superoxide dismutase; and three molecular forms of this enzyme are to be found in brain. The other protein present in gray and white matter is devoid of superoxide dismutase and amine oxidase activities. The amino acid composition, molecular weight, isoelectric point and copper content of this protein were determined. The effect of some agents, pH and thermal treatment of the optical and EPR spectra of the protein were also studied. The copper of the protein may be removed and the holoprotein reconstituted again from apoprotein and copper. The results obtained led to the conclusion that in brain a new copper protein is discovered, which is named neurocuprein.
