Biomedical subjects
M Kopelman
Publications and source records attributed to M Kopelman.
Patient information recall in a rheumatology clinic.
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Comparative studies of human and chicken retinol-binding proteins and prealbumins.
Microheterogeneity of retinol-binding proteins of human plasma and urine, and of chicken plasma was studied by polyacrylamide gel electrophoresis. All three protein systems were found microheterogenous. Incorporation of retinol into the protein preparations on the one hand, and depletion of these proteins from retinol on the other hand, enabled us to clarify the extent to which the presence or absence of the ligand affects the apparent heterogeneity. Upon electrophoresis, each of the native proteins displayed two pairs of protein zones. It appeared that within each pair the fast moving band corresponded to aporetinol-binding protein which upon binding of retinol was converted to a holoprotein with a slightly lower mobility. However, it did not seem that proteins of one pair were converted to proteins of the second pair upon binding of retinol, substantiating ghe microheterogenous character of this protein system. A rapid, two step procedure for isolation of prealbumins from plasma is described. The method which consists of DEAE-cellulose chromatography follwed by preparative electrophoresis was utilized to separate human and chicken prealbumins. Routine dodecyl sulphate electrophoresis resulted in partial dissociation of human prealbumin but in no dissociation of the chicken protein. More drastic treatments prior to electrophoresis were needed to effect complete disruption of both proteins into subunits.
The interaction between retinol-binding proteins and prealbumins studied by fluorescence polarization.
The interaction between retinol-binding proteins and prealbumins of human and chicken was studied by fluorescence polarization techniques. The binding affinity between chicken plasma retinol-binding protein and chicken prealbumin was essentially the same as between the respective human proteins. Human urine retinol-binding protein displayed a similar affinity, though possibly slightly smaller than that of the human plasma protein, toward human prealbumin. Retinol-binding proteins and prealbumins of human and chicken have been found to cross-interact displaying an affinity similar to that displayed by the proteins of the same species. Solution of a binding equation which assumes identical, independent sites, indicated that the number of binding sites on prealbumin for retinol-binding protein is somewhat less than 2 with the human system, and in the neighborhood of 4 with the chicken system. A possible interpretation suggests that prealbumin possesses four identical binding sites for retinol-binding protein, one for each subunit, but that the binding is of a negative cooperative nature. A major share of the negative cooperativity is likely to result from steric hindrance induced by already bound retinol-binding protein molecules, which have a sizable volume compared to the volume of the prealbumin molecule. The cooperativity is likely to be more pronounced with the human system. Rotational relaxation times derived from Perrin plots suggest that 1:1 molecular complexes of retinol-binding proteins with prealbumins have a compact structure.
Binding affinities of retinol and related compounds to retinol binding proteins.
Fluorimetric titrations were used to determine apparent dissociation constants of the all-trans isomers of retinol, retinoic acid, retinyl acetate and retinyl palmitate to human-retinol binding protein and chicken-retinol binding protein. Enhancement of the fluorescence of retinol and retinyl acetate when bound to the protein was utilized to establish the binding affinity of these compounds. With retinoic acid which is essentially a non-fluorescent compound, quenching of protein fluorescence due to energy transfer to the bound ligand from tryptophanyl residues served to determine the binding affinity. The various ligands display 1:1 molecular complexes with both types of retinol binding proteins. Retinol, retinoic acid and retinyl acetate were found to have similar binding affinities to both species of carrier proteins: For retinol K'd=1.9 X 10(-7) M with human-retinol binding protein and K'd=1.5 X 10(-7) M with chicken-retinol binding protein; for retinoic acid K'd-2.1 X 10(-7) M with human-retinol binding protein; for retinyl acetate and K'd=2.2 X 10(-7) M with chicken-retinol binding protein; for retinyl acetate K'd=2.2 X 10(-7) M with human-retinol binding protein and K'd=1.7 X 10(-7) M with chicken-retinol binding protein. Retinyl palmitate appeared to have weak association with either of the two retinol binding proteins, if at all. The above results suggest that both human and chicken retinol binding proteins behave similar with respect to the binding of the ligands. Non-polar interactions probably play a primary role in the binding and effects of functional groups and charges are of secondary importance.