Association of the A subunits of recombinant placental factor XIII with the native carrier B subunits from human plasma.
Interactions of a recombinant human placental protein (rA2) expressed in yeast and considered to be identical to the catalytic A2 subunits of factor XIII, the fibrin stabilizing factor zymogen, were examined with the native carrier subunits (B2) of the factor isolated from human plasma. Nondenaturing electrophoresis and HPLC gel-filtration experiments showed a tight binding of rA2 to B2 for forming an ensemble similar to that of plasma factor XIII (A2B2). In the presence of excess B2, however, some higher ordered oligomers (rA2Bn, where n > 2) were also seen in electrophoresis. The same technique revealed a microheterogeneity in the rA2 preparation; nevertheless, all isoforms could bind to B2. By employing an ELISA procedure for measuring free B2 in mixtures with rA2, an apparent binding constant of 4 x 10(7) M-1 was derived for the association of rA2 with B2. Fluorescence depolarization was used to monitor the heterologous association of rA2 with fluorescein-labeled B2F as well as the dissociation of the rA2B2F structure. The former was characterized by an increase, and the latter by a decrease, in the fluorescence anisotropy of the system. Binding of rA2 to B2F (pH 7.5, mu = 0.315, 37 degrees C) was not influenced by low concentrations of Ca2+ (< or = 30 mM), and rA2B2F proved to be quite stable under these conditions. Much higher concentrations of Ca2+, as well as higher ionic strengths, were required to dissociate this assembly. By contrast, release of B2F from the thrombin-modified rA2'B2F occurred rapidly in the presence of low concentrations of Ca2+ at low ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)
Radek
J T
JT
Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208.
Jeong
J M
JM
Wilson
J
J
Lorand
L
L
D002118:Calcium / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D004586:Electrophoresis; D004797:Enzyme-Linked Immunosorbent Assay; D005176:Factor XIII / Q000737:chemistry* / Q000378:metabolism*; D005338:Fibrin Fibrinogen Degradation Products / Q000494:pharmacology; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D006801:Humans; D009994:Osmolar Concentration; D010920:Placenta / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology
D005338:Fibrin Fibrinogen Degradation Products; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; C011468:fibrinmonomer; D005176:Factor XIII; D013917:Thrombin; D002118:Calcium